pharmacognosy- 1 phg 222 - psau · mikhail tswett, russian, 1872-1919 botanist in 1906 tswett used...

Prof. Dr. Amani S. Awaad

Professor of PharmacognosyPharmacognosy Department,

College of Pharmacy Salman Bin Abdulaziz

University,

Al-Kharj. KSA.

Email: [email protected]

Pharmacognosy- 1

PHG 222

Chromatography

1- what is Chromatograph

2- classification of chromatography

3- State the definition of TLC

4-Explain the phases used in TLC

5-List the materials & methods used in TLC

6-List the application of TLC

Mikhail Tswett, Russian, 1872-1919

Botanist

In 1906 Tswett used to chromatography to separate plant pigments

He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column

Chroma means “color” and grapheinmeans to “write”

History

Chromatography

Chromatography has application in every branch of the physical and biological sciences

12 Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital role

Chromatography

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases

one of which is stationary (stationary phase) while the other (the mobile phase) moves through it in a definite direction.

The chromatographic process occurs due to differences in the distribution constant of the individual sample components.

Chromatography

IUPAC definition of chromatography

Chromatography

Is a technique used to separate and identify the components of a mixture.

Works by allowing the molecules present in the

mixture to distribute themselves between a

stationary and a mobile medium.

Molecules that spend most of their time in the mobile

phase are carried along faster.

Other definitions

• Use – to separate and identify components of mixtures.

• All use the principle of “partition” - affinity between two phases, to separate mixtures of substances.

• Substances will move with the mobile phase at different rate depending upon their Partition or Distribution coefficient.

• Compounds with greatest affinity for mobile phase travel further.

• The fraction with greater affinity to stationary phase travels slower and shorter while that with less affinity travels faster and longer.

• The separation is based on Differential partitioning between the mobile and stationary phases

The general principle

Chromatography

Components:

Chromatography

supporting medium: a solid surface on

which the stationary phase is bound or

coated

stationary phase: a layer or coating on the

supporting medium that interacts with the

analytes (a solid or a liquid)

mobile phase: a solvent that flows

through the supporting medium (a gas or a

liquid)

Chromatography cont.…

Classification

Different methods

were attempted for

classification of

chromatography


Classification I-Classification according the techniques :

In this method they classified it according to the methods for

holding the stationary phase

A- Planar chromatography

In this type of chromatography the stationary phase is used in

the form of layer. Plane chromatography is further classified

into:

1- Thin layer chromatography (TLC): the stationary phase is a

thin layer supported on glass, plastic or aluminium plates.

2- Paper chromatography (PC): the stationary phase is a thin

film of liquid supported on an inert support.

B- Column chromatography (CC)

The stationary phase is held in to a tube made of glass or metal

1- Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminium plates.

2- Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert support.

3- Column chromatography (CC): stationary phase is packed in a glass column.


Classification

II-Classification according to the packing of the stationary phase:


Classification

III-Classification according to mechanism of separation

The mechanism of separation depends

mainly on the nature of the stationary

phase. Based on separation mechanisms

chromatography can be classified into:

1- Adsorption Chromatography

It is the oldest and most common type of

chromatography. The stationary phase is a solid

with adsorption power. Mixture components will

be adsorbed on the surface of the stationary

phase with different powers and that account for

separation. Silica gel is the most common

stationary phase in adsorption chromatography.


Classification

III-Classification according to mechanism of separation

2- Partition Chromatography:

The stationary phase is a liquidforming a thin film on an inert solidacts as support. The stationary liquidis usually more polar than the mobileliquid. The two liquids must beimmiscible with each other.

Cellulose powder and wet silica gel areexamples of supports in partitionchromatography that carry film ofwater act as stationary phase. Partitionchromatography is preferable overadsorption when dealing with polarcompounds.


Classification III-Classification according to mechanism of separation

3- Ion Exchange Chromatography:

It is used for separation of charged molecules.

The stationary phase is an ion exchange resinto which a cationic or anionic groups arecovalently bonded.

Ions of opposite charges (counter ions) in themobile phase will be attracted to the resin andcompete with the components of the mixturefor the charged group on the resin.

Both the mixture components and the mobilephase must be changed.

Mixture of Alkaloids (compounds with positivecharges) can be separated on anionicexchanger, while mixture of organic acids(negative charges) can be separated usingcationic exchanger. Both types are used fordesalination of water.

COO- NH4

+ Resin Resin COO- NH4

+

Counter counter ions ions Anionic exchanger Cationic exchanger

H+

H+

OH-

OH-



4- Molecular Exclusion ( Size Exclusion )Chromatography:

is a chromatographic method in which molecules in

solution are separated by their size, and in some

cases molecular weight. It is usually applied to

large molecules or macromolecular complexes

such as proteins and industrial polymers.

Typically, when an aqueous solution is used to

transport the sample through the column, the

technique is known as gel-filtration

chromatography,

when an organic solvent is used as a mobile phase

gel permeation chromatography,

Sephadex is an example for stationary phase

http://en.wikipedia.org/wiki/Gel_permeation_chromatography



5- Zone Electrophoresis:

In CE methods, analytes migrate

through electrolyte solutions

under the influence of an electric

field.

Analytes can be separated

according to ionic mobility,

additionally they may be

concentrated by means of

gradients in conductivity and pH

Sample application

Bands of negativey

charged compoundsBands of postivey

charged compounds



6- Affinity Chromatography:

It uses the affinity of

proteins to specific ligands

such as enzymes. The

ligand is attached to

suitable polysaccharide

polymer such as cellulose

- agarose – dextran

7- Chiral Chromatography

In this type we can separate

enantiomers – we used chiral

stationary phase that react with

one enantiomer more then the

other so separation takes place


Classification IV-Classification according to Mobile phase

1- Liquid Chromatography (LC):

The mobile phase is liquid. In case of

separation by adsorption the stationary phase is

solid so it is called: Liquid-Solid

Chromatography (LSC).

If separation occurs through partition the

stationary phase is liquid so it is called: Liquid

-Liquid Chromatography (LLC).

2- Gas Chromatography (GC)

Where the mobile phase is inert gas nitrogen or

helium. Again if the stationary phase is solid it is

called: Gas–Solid Chromatography (GSC). When

stationary phase is liquid it is called: Gas-Liquid

Chromatography (GLC).

In this regard chromatography is classified into:


Classification V- According To Purpose Of Use:

1- Analytical Chromatography:

Chromatography can be used for analytical work and also to

obtain pure materials from mixtures

a- Qualitative Chromatography

In this case Chromatography can be used to:

1- Confirm the absence or probable presence of certain

constituent in the sample under investigation

2- Give an idea about the complexity of the mixture and

the least number of compounds present.

3- Check purity and identity of any compound.

4- Establish a (finger print ) pattern for extracts , volatile

oils or pharmaceutical preparations. These finger prints

can be then used to check the identity and purity in the

future.

5- Monitor both column chromatography and organic

chemical reactions.


Classification V- According To Purpose Of Use:

1- Analytical Chromatography:

Chromatography can be used for analytical work and also to obtain pure materials

from mixtures

b- Quantities Chromatography

The development of modern instruments enable the

use of chromatography to determine the amount of

any component in a mixture as absolute amount or

relative to another component HPLC/ GC/ HPTLC

can be used for there applications.

2- Preparative application:

This was the first and is the main application of

chromatography. The technique was developed

primarily for this purpose. Chromatography is

used to obtain reasonable quantities of pure

compounds from mixtures.


A- Planar chromatography

Plane chromatography

includes two types:

1- Thin layer

chromatography (TLC)

2- Paper chromatography

(PC).

Thin layer chromatography (TLC)



TLC is generally used as a qualitative analytical

technique, such as checking the purity of a compound

or determining the number of components in a

mixture or column chromatographic fraction.

In addition.

TLC is useful for determining the best solvents for a

column chromatographic separation.

It can also be used for an initial check on the identity

of an unknown sample (by spotting the plate with a

known compound as well as with the sample).

With calibration, TLC can be used as a quantitative

technique. Preparative work can be carried out with

special thick-layered TLC plat



Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase.

The stationary phase:

is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate.

The mobile phase:

is a developing liquid which travels up the stationary phase, carrying the samples with it.

Components of the samples will separate on the stationary phase according to

how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.


Thin layer chromatography Cont…

Types supportive surfaces

TLC plate is a sheet of glass, metal(Aluminum), or plastic

which is coated with a thin layer of a solid adsorbent

Coating materials

1-Adsorption:a- silica gel (silicic acid). b- Alumina (Aluminum oxide).

c- Magnesium Silicate (florisil) for Lipids.

2-Partition: Cellulose.

3-Ion Exchange TLC: Cellulose phosphate

4-Reversed – phase partition: a) C-18 silica gel b) C-8 silica gel c) C-4 silica gel

5- Polyamides: E-poly caprolactam &Poly acrylonitrite

6- Gel chromatography (Size exclusion):

Sephadex G25, Sephadex G50 , Sephadex G75, Sephadex G100, Sephadex LH20


Thin layer chromatography Cont…

Binders:These are materials used to hold the thin layer of

the coating material into the surface of the

supporting plates.

Types of binders:a- CaSO4 (Plaster of Paris) Gypsum (10-15%)

b- Silicon dioxide

c- Starch (1-3 %)

d- Organic polymers e.g. polyvinyl alcohol.

Indicators:These are materials mixed

with the coating material and binder to

help locating the spots on the TLC. The

most common used indicator is the

fluorescent materials (silica gel 60 F254).


Thin layer chromatography Cont…Developing system:

(Mobile phase – developing solvent)

Using a single solvent (very rare) or mixture of solvents to allow the separation. The type of adsorbent used will affect the choice of the developing system.

Adsorption: Usually mixture of non polar organic solvents are used.

Partition: More polar organic solvents such as butanol- acetic acid –water are used. Buffer solution are also used in partition chromatography.

Ion Exchange: Acidic or basic solutions are used.(HCl, NaOH, NaCl, LiCl)

Reversed phase: Methanol- acetonitril- water- acetone-acetic acid are used as mixtures.

Polyamide: Mixtures of Water – ethanol- acetone can be used.

Gel: Buffer solutions and aqueous acidic or basic solutions can be used


Thin layer chromatography Cont…Types of developments:

1- Single development: The solvent system is allowed to

move through the stationary phase one time only against

gravity.

2- Repeated developments:a- Multiple developments: The plated are developed more

than one time using the same solvent system. The plates must

be completely dried after each development.

b- Stepwise developments: The plated are developed more

than one time using different solvent systems.

3- Two-dimensional developmentIs used to verify if a given spot on TLC using the above

methods of development (one Dimensional) is one pure

compound or mixture of two closely related compounds. The

spots are applied to one corner and the plate developed as

usual. The plate is then rotated 90 ˚C and then developed

again. This method allow better separation of related

compounds.

A- Ascending:


Thin layer chromatography Cont…Types of developments:

This method of

development require the use of

Chromatotron. Simply it is

composed of motor rotate in high

speed (about 1000 rpm) to

accelerate the speed of the mobile

phase. Circular plates are used and

the mixture is applied to the center

of the plate. Mobile phase is also

allowed to flow from the plate

center to the edges. The separated

materials will appear as concentric

zones. Chromatotron is used only

for preparative work.

B- Centrifugal (chromatotron):


Preparing the Chamber

To a jar with a tight-fitting lid add enough of the appropriate developing liquid so that it is 0.5 to 1 cm deep in the bottom of the jar.

Close the jar tightly, and let it stand for about 30 minutes so that the atmosphere in the jar becomes saturated with solvent.

Thin layer chromatography Cont.…



Preparing the Plates for Development

With a pencil, etch two small notches into the adsorbent about 2 cm from the bottom of the plate.

The notches should be on the edges of the plate, and each notch should be the same distance up from the bottom of the plate.

The notches must be farther from the bottom of the plate than the depth of the solvent in the jar.

Using a drawn-out capillary tube, spot the samples on the plate so that they line up with the notches you etched.



Developing the Plates

After preparing the development chamber and spotting the samples, the plates are ready for development.

Be careful to handle the plates only by their edges, and try to leave the development chamber uncovered for as little time as possible.

When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the plate) with a pencil.



Identifying the Spots (visualization)

If the spots can be seen, outline them with a pencil.

If no spots are obvious, the most common visualization technique is to hold the plate under a UV lamp.

Many organic compounds can be seen using this technique, and many commercially made platesoften contain a substance which aids in the visualization of compounds.

http://chemscape.santafe.cc.fl.us/chemscape/catofp/chromato/tlc/fluoresc.htm




In these methods the materials can be recovered.

• Day light for colour compounds.

• UV light for fluorescentcompounds (conjugated doublebonds).

• I2 vapour for any compoundscontain at least one double bond

• Spray with water where organiccompounds appear as whiteopaque spots.

1- Physical methods (Non –

Destructive methods)




The use of color-forming reactions is another

valuable technique for visualizing spots.

A very specific procedure involves spraying the

plate with a reagent that will react with a

compound on the plate to form a colored

product. For example, an acid-base indicator,

like phenolphthalein, can be used to identify

acidic or basic compounds.

Other specialized sprays contain ferric chloride (to

identify phenols), 2,4-dinitrophenylhydrazine,

DNP (to identify aldehydes and ketones),

ninhydrin (to identify amino acids), and

dragendorff’s reagent (to identify alkaloids).

1-Chemical methods Destructive methods

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Application of TLC

1- Qualitative:

Identification through comparison of the Rf value with that of Reference material.

Determination of Complexity of mixtures. That will be indicated from number of spots.

Determination the purity of materials.

Monitoring the progress of Chemical reactions.

Monitoring of column chromatography.

Development of finger print TLC for extracts, volatile oils or pharmaceutical preparation for future identification and comparison.

In this application plates 5×5, 5×10 cm with thin film of coating material are usually used.


Thin layer chromatography Cont.…Application of TLC

2- Quantitative:In this case an accurate volume of samples are

applied using syringes. The dimensions of plates

range from 5x10 to 20x20 according to the

number pf spots used.

The plates are developed as usual in the

chromatographic tanks. After development the

concentration of material can be determined by:

Spot area measurement: Which is directly

proportional to the conc. of materials.

Photodensitometry: Measure transmittance,

reflection or fluorescence of spots.

Radioactivity: For radioactive material.

These measurements are done using TLC Scanner

connected to computer that perform all

calculations.


Thin layer chromatography Cont.…Application of TLC

3- Preparative TLC:

In preparative application

20×20 plates with thick layer of

adsorbent 0,25m are used. The mixture

is apply as bands and a pilot or guide

spots may be used in one side of the

plate to enable the detection of the spots

location.


Thin layer chromatography Cont.…Interpreting the Data

The Rf (retention factor) value for each spot should be calculated.

It is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates.

Hence, known Rf values can be compared to those of unknown substances to aid in their identifications.

Rf = distance moved by substance

distance moved by solvent front

For substances that are very soluble in the

liquid Rf will be close to … 1

For substances that are rather insoluble in

the liquid Rf will be close to…. 0

• The distance that the spot of a particular compound moves up the plate relative to the distance moved by the solvent front is called the retention factor, or Rf value.



Rf values often depend on the temperature and the solvent used in the TLC experiment.

the most effective way to identify a compound is to spot known substances – authentic - next to unknown substances on the same plate.)

In addition, the purity of a sample may be estimated from the chromatogram.

An impure sample will often develop as two or more spots, while a pure sample will show only one spot

Important note

http://chemscape.santafe.cc.fl.us/chemscape/catofp/chromato/tlc/purity.htm

In some cases instead of getting roundspots a Tailed or comet like spots areobtained leading to overlapping of the spotsand poor resolution.



Reasons and solution for tailing problem:

1-Ionic characters of acids and bases when they

are chromatographed under neutral conditions.

Solution: add acids or bases to the developing

system.

2-Application of large amounts of material.

Solution: decrease conc. of material.

3-Unproper choice of solvent system.

Solution: change the solvent system.

Tailing in TLC:

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pharmacognosy- 1 phg 222 - psau · mikhail tswett, russian, 1872-1919 botanist in 1906 tswett used...

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