CHROMOTAGRAPHY

B y Nuriya Khan

Explain the theoretical principles upon which

chromatographic methods are based

Chromatography is used for the separation identification and measurement of the chemical components in mixtures

There are a variety of chromotagraphical techniques

All of them depend on the components of a mixture being carried at different rates through a stationary phase by a mobile phase

Adsorption

Chemisorption

Physical adsorption

Desorption

Partition

When a solute is added to a pair of immiscible liquids it may dissolve in both of them In this case the solute will distribute itself between the two solvents It may well be more soluble in one solvent It may well be more soluble in one solvent than the other It is found that the ratio of the two concentrations is constant

[concentration of solute in solvent 1] [concentration of solute in solvent 2] = k

In partition chromatography many extraction are performed in succession in one operation the solutes are partitioned between the stationary phase and the mobile phase

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Explain the theoretical principles upon which

chromatographic methods are based

Chromatography is used for the separation identification and measurement of the chemical components in mixtures

There are a variety of chromotagraphical techniques

All of them depend on the components of a mixture being carried at different rates through a stationary phase by a mobile phase

Adsorption

Chemisorption

Physical adsorption

Desorption

Partition

When a solute is added to a pair of immiscible liquids it may dissolve in both of them In this case the solute will distribute itself between the two solvents It may well be more soluble in one solvent It may well be more soluble in one solvent than the other It is found that the ratio of the two concentrations is constant

[concentration of solute in solvent 1] [concentration of solute in solvent 2] = k

In partition chromatography many extraction are performed in succession in one operation the solutes are partitioned between the stationary phase and the mobile phase

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Chromatography is used for the separation identification and measurement of the chemical components in mixtures

There are a variety of chromotagraphical techniques

All of them depend on the components of a mixture being carried at different rates through a stationary phase by a mobile phase

Adsorption

Chemisorption

Physical adsorption

Desorption

Partition

When a solute is added to a pair of immiscible liquids it may dissolve in both of them In this case the solute will distribute itself between the two solvents It may well be more soluble in one solvent It may well be more soluble in one solvent than the other It is found that the ratio of the two concentrations is constant

[concentration of solute in solvent 1] [concentration of solute in solvent 2] = k

In partition chromatography many extraction are performed in succession in one operation the solutes are partitioned between the stationary phase and the mobile phase

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Adsorption

Chemisorption

Physical adsorption

Desorption

Partition

When a solute is added to a pair of immiscible liquids it may dissolve in both of them In this case the solute will distribute itself between the two solvents It may well be more soluble in one solvent It may well be more soluble in one solvent than the other It is found that the ratio of the two concentrations is constant

[concentration of solute in solvent 1] [concentration of solute in solvent 2] = k

In partition chromatography many extraction are performed in succession in one operation the solutes are partitioned between the stationary phase and the mobile phase

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Partition

When a solute is added to a pair of immiscible liquids it may dissolve in both of them In this case the solute will distribute itself between the two solvents It may well be more soluble in one solvent It may well be more soluble in one solvent than the other It is found that the ratio of the two concentrations is constant

[concentration of solute in solvent 1] [concentration of solute in solvent 2] = k

In partition chromatography many extraction are performed in succession in one operation the solutes are partitioned between the stationary phase and the mobile phase

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

In partition chromatography many extraction are performed in succession in one operation the solutes are partitioned between the stationary phase and the mobile phase

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

The stationary phase stays in place inside the column or in the fibres of the paper If the stationary phase is packed into a column it usually consists of solid particles or a viscous liquid coated onto a solid surface

The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analyte

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture1A schematic representation of the process of a chromatographic separation

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Chromatographical Methods

Liquid Chromatography

Gas Chromatography

Ion Exchange

Gel permeation Chromatography

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Column Chromatography

The diagram on the following slide illustrates

separation of solutes in a solution by column

chromatography The stationary phase is an

inert solid

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Apparatus setup of column-chromatography

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

A solution of the analyte is poured on to the top of the column and the components are adsorbed at the top of the column The mobile phase is a second solvent called the eluant which carries the components of the mixture through the stationary phase This mixture is permitted to trickle through the column

Each solute is partitioned between the adsorbent and the eluant The least strongly adsorbed solutes are desorbed first by the eluant and carried further down the column before being readsorbedWhen fresh eluant reaches the process is repeated carrying it further down the column

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Paper Chromatography

A solution of the mixture to be separated is applied to a strip of chromatography paper The solvents used include water ethanol butanol

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 2 Apparatus setup of paper-chromatography

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

As the solvent rises through the paper it meets the sample and the component bands spread out The separation is stopped when the solvent has travelled nearly the top of the paper The distance travelled by the solvent front is measures Then for each solute the retardation factor Rf is calculated by

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Solvent front

Solute

Starting Point

Rf value= xy

Picture 3 The Rf value

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

The stationary phase is the water or other

solvent that is adsorbed as a film on the surface

of the paper The mobile phase is the second

solvent

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Thin Layer Chromatography

Another version of liquid chromatography is the

thin layer chromatography (TLC) The solid

adsorbent eg silica gel or calcium sulphate is

made into a thick paste with water and spread

evenly over a glass plate

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 4 Apparatus setup of Thin Layer Chromatography

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

The particle size of the stationary phase is

smaller in thin layer chromatography than in

paper chromatography As a result the

separations are much more efficient and more

reproducible Often separations can be achieved

in a few centimetres and coated microscope

slides are frequently used for TLC

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Gas Chromatography

The mobile phase is a gas

The liquid is spread on the surface of inert solid

particles which pack a long (5-10m) narrow (2-

10mm bore) column

The injection chamber is 50-100 above the

temperature of the column

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 5 Apparatus set-up of Gas Chromatography

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Factors affecting Gas Chromatography

Volatility of compound

Polarity of compounds

Column temperature

Column packing polarity

Flow rate of the gas through the column

Length of the column

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

In gas chromatography the mobile phase is a gas The liquid which forms the stationary phase is spread out on the surface of solid particles which are packed into a column

Each component is partitioned between the vapour phase and the liquid phase

A detector monitors the components as they leave the column

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 6 Thermal Conductivity Detector

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Explain the terms retention factor (Rf) and retention time

visualising agent solvent front

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Retention Factor

The ratio of the amount of solute in the stationary phase Nsp compared to the amount of solute in the mobile phase Nmp

k = NspNmp

The retention factor is most commonly used in

describing GC equilibria and is a strong function of film thickness (k increases as film thickness increases)

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 7 A graph showing Detector Signal vs Time

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Rf value

The thin-layer chromatography parameter that

defines the position of the analyse band on the

plate

Rf= (distance of the analyte band from initial spot)(distance to the solvent front from the initial spot)

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Retention Time(Tr)

The time required for a solute to travel from

injection to the detection for set instrument

conditions the value for tr is designated as

occurring at the peak maximum

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Visualizing Reagent

In thin-layer chromatography a visualization

reagent is used to make an analyte band appear

on a plate The reagent used can be compound-

or class specific or general( sorbent contains a

fluorescent label that is quenched by the

presence of the analyte)

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Solvent Front

Solvent Front is defined as the front line of the eluent

Where the eluent is defined as liquid or gas entering a chromatographic bed and used to effect a separation by elution

Picture 8 A demonstration of solvent front

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Describe the basic steps involved in separating and

quantifying the components of a mixture

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Steps Involved in separating the components of a mixture

First each compound leaves the column in the form of a symmetrical bell-shaped band or peak

Second each band emerges from the column at a characteristic time that can be used to identity the compound just as a melting point can be used for the qualitative analysis of an organic compound This retention tR is measured from the time of sample injection to the time the band maximum leaves the column

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

A third characteristic feature is the difference in

retention times between adjacent bands

Fourthly each band is characterized by a band

width tw as shown for band B in the previous

diagram Tangent are drawn to each side of the

band and extended to touch the baseline

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Name examples of commonly used stationary phases

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Cellulose-Paper Form

The use of paper as a chromatographic medium is usually regarded as a typical partition system

The stationary phase is water held by adsorption on cellulose molecules

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 9 Cellulose-Paper form

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Silica Gel

Silica Gel is slightly odd in that although it is

porous and its pore size certainly influences its

performance as a stationary phase

It operates fundamentally as an adsorbent not

as a molecular sieve

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 10 Picture showing Silica Gel

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Alumina

Alumina is a powerful adsorbent It can

hydrogen bond through hydroxyl groups formed

on its surface by hydration attract by dipole-

dipole and dipole-induced dipole attraction

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 11 Picture showing fine alumina

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Carry out simple experiment to separate the components of mixturesusing papercolumn and thin layer chromatographic techniques

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Column Chromatography Experiment

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Chemicals and other materials silica gel 60 (Merck) petroleum ether acetone NaCl CaCO3

Na2SO4

fresh leaves

Apparatus and glass wares glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the

outlet separation funnel 500 mL separator funnel 100 mL powder funnel 5 measuring cylinders 25 mL beaker 100 mL beaker 600 mL 9 Erlenmeyer flask 100 mL volumetric pipette 20 mL pipette bulb mortar amp pestle glass rod cork ring swan-neck lamp

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Hazards and safety precautions

Petroleum ether is volatile and very flammable Petroleum ether presents a high fire risk The toxicity of petroleum ether varies according to its composition Many of the components are of quite low toxicity but some formulations may contain chemicals that are suspected carcinogens Avoid ingestion and inhalation

Acetone is highly flammable Irritating to eyes

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Method Extraction of the leaf pigments

Using a pestle fresh leaves are grinded in a mortar containing 22 mL

of acetone 3 mL of petrol ether and a spatula tip-ful of CaCO3

The pigment extract is filtered The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10 aqueous NaCl solution

The separating funnel is shaken carefully When the layers have separated the lower layer is allowed to drain into a beaker This phase is thrown away The upper layer is washed 3-4 times with 5 mL of dest water

Afterwards the extract is placed in an Erlenmeyer flask and is dried

with about 4 spatula tips of Na2SO4 The liquid is carefully decanted

into a flask

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 12 showing apparatus during experiment

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Results and Discussion

The mobile phase slowly flows down through the silica gel

column by gravity leaving behind zones of colour - the

chromatogram The theory of column chromatography is

analogous to that of thin-layer chromatography The

different components in the sample mixture pass through

the column at different rates due to differences in their

partioning behaviour between the mobile liquid phase and

the stationary phase

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Thin-Layer Chromatography

Experiment

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Test solution a mixture of 7 dyes dissolved in water Erythrosine Brilliant Black BN Fast Red E Naphthol Red S Yellow Orange S Ponceau 4R Tartrazine Reference solutions Yellow Orange S Brilliant Black each dissolved in water Developing solvent 25 sodium citrate solution ammonia 25 2-propanol (20 5 3)

The developing solvent must be freshly prepared

Apparatus and materials developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm) Fertigfolie POLYGRAMreg CEL 300 plate (Macherey Nagel) glass capillaries (1 microL)

Chemicals

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Hazards and safety precautions

Concentrated ammonia solution is extremely damaging to eyes Even contact with dilute ammonia solution can lead to serious eye damage Harmful if swallowed or inhaled and in contact with skin

2-Propanol is highly flammable Safety goggles and protective gloves required

The developing solvent should be prepared in a laboratory fume hood

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Method

Using a soft pencil a line is drawn approximately 15 cm from the bottom of the plate

The spotting points are numbered (123)

At the spotting points 1 and 3 the reference solutions are applied onto the plate at the spotting point 2 the dye mixture

Using capillaries approx 025 microL of the dye solutions are applied to the TLC plate

The capillaries fill themselves quickly when dipped into organic sample solutions

Before emptying the submerged end of the capillary is rolled horizontally on filter paper

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

The clean upper end of the capillary is placed on the layer vertically and carefully vertically so that the capillary empties itself and carefully to avoid damage to the layer Easy application of samples is allowed with a spotting guide

When the solvent is completely evaporated (approx 10 min) from the plate the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom

The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract line)

The TLC chamber is covered

The TLC plate is allowed to remain undisturbed When the solvent front has reached three quarters of the length of the plate the plate is removed from the developing chamber and the position of the solvent front is immediately marked

The solvent on the plate is allowed to evaporate

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 13 A demonstration of apparatus during experiment

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Results and discussion Background

Qualitative analysis of separated components in TLC is based on a comparison of rates of migration The retention factor Rf value is used to characterize and compare components of various samples

The Rf value is defined as follows

In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent The composition of the mobile phase and the temperature must remain constant

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Paper Chromatography experiment

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Apparatus water and rubbing alcohol coffee filter (or filter paper water soluble food colors water-soluble marking pens

andor Skittlestrade candy and Q-Tipstrade clear glasses or other containers

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Method

Cut the coffee filters into strips about 1 ldquo wide Fill one glass about 1rdquo full of water the other about 1rdquo full of rubbing

alcohol Toward the end of the filter strips draw a line with a black watercolour

marking pen or 2 or 3 primary colours of food colouring or use a Q-Tiptrade to rub off colour from candies and rub onto the filter paper (May take several times to get a dense spot of colour) Let them dry

Place that end of the papers in the glasses (donrsquot let the dot of colour touch the water or alcohol) and watch the water soak in and travel up the papers (this is called capillary action which is how water goes up a tree trunk into the branches) As it does it will dissolve the colours which are carried up the paper The lighter ones will be carried higher than the others

After the dots have been completely dissolved remove the papers from the liquid and allow them to dry Observe the results and compare the differences between the two liquids

Try variations using different kinds of paper different markers with different colours and adding vinegar to the water Compare results

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Picture 14 A demonstration of Paper Chromatography

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Results

1 What did you see What colours were actually in the black ink

2 Which colours were carried furthest (the lighter colours) Which remained lowest (the darker colours)

3 Which colour is the lightest in weight (those lightest in colour) The heaviest in weight (the darker colours)

4 What pattern was there to the change

5 What is happening when the colours move up the paper (the molecules of colour are being dissolved by the water and carried with the water up the paper)

6 What causes the colours to separate (the different colours have different affinities for clinging to the paper and those that cling hardest

to the cellulose in the paper will stop first and those that cling the weakest will travel further up the filter paper before stopping)

7 Predict what might happen with different coloursTry it again Do you get the same results

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Cite the wide applications of chromatographic methods of

separation

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Forensic testing

These days (GC) is one of the primary analytical techniques used in every forensic laboratory GC is widely used by forensic scientists ndash from analysis of body fluids for the presence of illegal substances to testing of fibre and blood from a crime scene and to detect residue from explosives Yet scientists from Ohio University explored another application of gas chromatography with differential mobility spectrometry as a low cost onsite detection method for ignitable liquids

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Purification of Natural Products

Reverse Phase- High Performance Liquid Chromatography) has been increasingly used to provide tens of grams to kilograms of high purity material in pharmaceutical product development However even with the development of Flash Chromatography as an alternative these purification techniques are struggling to cope with the throughput demands that the compounds being developed and requiring purification are causing primarily due to solubility issues

By using high performance counter current chromatography instruments chemists are achieving high purity (gt95) and high crude sample masses per injection at low solvent usage (18 grams of sample injected per litre of solvent usage) The reason for this is that chemists are able to use a liquid stationary phase which offers far superior loading capacity and the advantage of loading the crude sample in either the mobile or stationary phases or a mixture of the two These options help eliminate many if not all solubility issues

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Pesticide Analysis

Residue analysis different environmental samples such as water soil and plant matter are examined for pesticide residues Analytical methods and special analytical equipment are employed to provide an accurate identification of these organic environmental pollutants and finally to determine their concentration in microg dm-3of water or microg kg-1of soil or plant matter The substances examined also known as analyses have to be extracted from the sample using an organic solvent or solvent mixture in the first analytical step At the same time these analytes must be available as authentic standard solutions for comparison when determining the identity and concentration of the pesticide in the sample Since different organic solvents are also used here these are indispensable versatile aids

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Reference wwwchemsocorg wwwChemquidecouk wwwa-levelchemistrycouk wwwdemochemhtm A-Level ChemistryCheltenhamNelson Thorne Limited2000 Advanced Chemistry London Oxford University Press2000

Past Paper solutions

2006 U1 P1 Q8 (All Parts)

8 a (i) This is the time required for a solute to travel from injection to detection for a set of instrument conditions The value for tr is designated as occurring at the peak max

(ii) The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analysis The mobile phase (solvent) moves through the column or over the paper It carries the components of the analysis

(iii) Stationary phase Polyethylene glycol acetate (PEG-5) Mobile Phase Nitrogen gas

b (i) Recall that here the solute is the red dye Sample 1

Sample 2

Note that the answer is given to 3 significant figures as the data is given to 3 significant figures

(ii) The same red dye is used to make both types of ink Reasons are 1 The Rf values is the same for both red spots in the 2 samples

2 The both sample give spots of the same colours suggesting that the dyes are the same

2003 U1 Q9 P1 a The general principle is that components of a mixture are carried at

different rates through a stationary phase by a mobile phase

bThe mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analysis (cape exam report 2003)

Eg Ethanol can be used as the mobile phase in paper chromatography

cThe stationary phase stays in place inside the column or in the fibres of the paper If packed into a column it usually consists of solid particles or a viscous liquid onto a solid surface

Eg Alumina can be used as the stationary phase in adsorption chromatography

d(i) A is a mixture of 3 components X Y and Z where Y and Z are present in approximately equal amounts and X is present in a much lower amount X and Y are easier to separate than Y and Z (using ΔT2) Band width shows that separation is best for X

(ii) Y was eluted before Z because it has a lower affinity for the stationary phase that is it is not as strongly held by the stationary phase

• Chromotagraphy
• Explain the theoretical principles upon which chromatographic m
• Slide 3
• Adsorption
• Partition
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Column Chromatography
• Apparatus setup of column-chromatography
• Slide 12
• Paper Chromatography
• Picture 2 Apparatus setup of paper-chromatography
• Slide 15
• Slide 16
• Slide 17
• Thin Layer Chromatography
• Slide 19
• Slide 20
• Gas Chromatography
• Slide 22
• Factors affecting Gas Chromatography
• Slide 24
• Slide 25
• Explain the terms retention factor (Rf) and retention time vi
• Retention Factor
• Slide 28
• Rf value
• Retention Time(Tr)
• Visualizing Reagent
• Solvent Front
• Describe the basic steps involved in separating and quantifying
• Steps Involved in separating the components of a mixture
• Slide 35
• Name examples of commonly used stationary phases
• Cellulose-Paper Form
• Slide 38
• Silica Gel
• Slide 40
• Alumina
• Slide 42
• Carry out simple experiment to separate the components of mixtu
• Column Chromatography Experiment
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Slide 49
• Thin-Layer Chromatography Experiment
• Slide 51
• Slide 52
• Slide 53
• Slide 54
• Slide 55
• Results and discussion
• Paper Chromatography experiment
• Slide 58
• Slide 59
• Slide 60
• Results
• Cite the wide applications of chromatographic methods of separa
• Forensic testing
• Purification of Natural Products
• Pesticide Analysis
• Reference
• Past Paper solutions
• Slide 68
• Slide 69

2006 U1 P1 Q8 (All Parts)

8 a (i) This is the time required for a solute to travel from injection to detection for a set of instrument conditions The value for tr is designated as occurring at the peak max

(ii) The mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analysis The mobile phase (solvent) moves through the column or over the paper It carries the components of the analysis

(iii) Stationary phase Polyethylene glycol acetate (PEG-5) Mobile Phase Nitrogen gas

b (i) Recall that here the solute is the red dye Sample 1

Sample 2

Note that the answer is given to 3 significant figures as the data is given to 3 significant figures

(ii) The same red dye is used to make both types of ink Reasons are 1 The Rf values is the same for both red spots in the 2 samples

2 The both sample give spots of the same colours suggesting that the dyes are the same

2003 U1 Q9 P1 a The general principle is that components of a mixture are carried at

different rates through a stationary phase by a mobile phase

bThe mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analysis (cape exam report 2003)

Eg Ethanol can be used as the mobile phase in paper chromatography

cThe stationary phase stays in place inside the column or in the fibres of the paper If packed into a column it usually consists of solid particles or a viscous liquid onto a solid surface

Eg Alumina can be used as the stationary phase in adsorption chromatography

d(i) A is a mixture of 3 components X Y and Z where Y and Z are present in approximately equal amounts and X is present in a much lower amount X and Y are easier to separate than Y and Z (using ΔT2) Band width shows that separation is best for X

(ii) Y was eluted before Z because it has a lower affinity for the stationary phase that is it is not as strongly held by the stationary phase

• Chromotagraphy
• Explain the theoretical principles upon which chromatographic m
• Slide 3
• Adsorption
• Partition
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Column Chromatography
• Apparatus setup of column-chromatography
• Slide 12
• Paper Chromatography
• Picture 2 Apparatus setup of paper-chromatography
• Slide 15
• Slide 16
• Slide 17
• Thin Layer Chromatography
• Slide 19
• Slide 20
• Gas Chromatography
• Slide 22
• Factors affecting Gas Chromatography
• Slide 24
• Slide 25
• Explain the terms retention factor (Rf) and retention time vi
• Retention Factor
• Slide 28
• Rf value
• Retention Time(Tr)
• Visualizing Reagent
• Solvent Front
• Describe the basic steps involved in separating and quantifying
• Steps Involved in separating the components of a mixture
• Slide 35
• Name examples of commonly used stationary phases
• Cellulose-Paper Form
• Slide 38
• Silica Gel
• Slide 40
• Alumina
• Slide 42
• Carry out simple experiment to separate the components of mixtu
• Column Chromatography Experiment
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Slide 49
• Thin-Layer Chromatography Experiment
• Slide 51
• Slide 52
• Slide 53
• Slide 54
• Slide 55
• Results and discussion
• Paper Chromatography experiment
• Slide 58
• Slide 59
• Slide 60
• Results
• Cite the wide applications of chromatographic methods of separa
• Forensic testing
• Purification of Natural Products
• Pesticide Analysis
• Reference
• Past Paper solutions
• Slide 68
• Slide 69

2003 U1 Q9 P1 a The general principle is that components of a mixture are carried at

different rates through a stationary phase by a mobile phase

bThe mobile phase which is the solvent moves through the column or over the paper and is either a liquid or a gas It carries the components of the analysis (cape exam report 2003)

Eg Ethanol can be used as the mobile phase in paper chromatography

cThe stationary phase stays in place inside the column or in the fibres of the paper If packed into a column it usually consists of solid particles or a viscous liquid onto a solid surface

Eg Alumina can be used as the stationary phase in adsorption chromatography

d(i) A is a mixture of 3 components X Y and Z where Y and Z are present in approximately equal amounts and X is present in a much lower amount X and Y are easier to separate than Y and Z (using ΔT2) Band width shows that separation is best for X

(ii) Y was eluted before Z because it has a lower affinity for the stationary phase that is it is not as strongly held by the stationary phase

• Chromotagraphy
• Explain the theoretical principles upon which chromatographic m
• Slide 3
• Adsorption
• Partition
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Column Chromatography
• Apparatus setup of column-chromatography
• Slide 12
• Paper Chromatography
• Picture 2 Apparatus setup of paper-chromatography
• Slide 15
• Slide 16
• Slide 17
• Thin Layer Chromatography
• Slide 19
• Slide 20
• Gas Chromatography
• Slide 22
• Factors affecting Gas Chromatography
• Slide 24
• Slide 25
• Explain the terms retention factor (Rf) and retention time vi
• Retention Factor
• Slide 28
• Rf value
• Retention Time(Tr)
• Visualizing Reagent
• Solvent Front
• Describe the basic steps involved in separating and quantifying
• Steps Involved in separating the components of a mixture
• Slide 35
• Name examples of commonly used stationary phases
• Cellulose-Paper Form
• Slide 38
• Silica Gel
• Slide 40
• Alumina
• Slide 42
• Carry out simple experiment to separate the components of mixtu
• Column Chromatography Experiment
• Slide 45
• Slide 46
• Slide 47
• Slide 48
• Slide 49
• Thin-Layer Chromatography Experiment
• Slide 51
• Slide 52
• Slide 53
• Slide 54
• Slide 55
• Results and discussion
• Paper Chromatography experiment
• Slide 58
• Slide 59
• Slide 60
• Results
• Cite the wide applications of chromatographic methods of separa
• Forensic testing
• Purification of Natural Products
• Pesticide Analysis
• Reference
• Past Paper solutions
• Slide 68
• Slide 69