cape chromatography 1 (1)

7/27/2019 CAPE Chromatography 1 (1)

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Dept. of Chemistry, University of the

West Indies MonaC.A.P.E. CHEMISTRY WORKSHOP


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What is Chromatography?

Recall the first time you were introduced to colour in art

class and you learnt of the three primary colours and the

resulting secondary colours.


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Chromatography is concerned with being able to separate

mixtures into their individual components.

This can be useful for detection purposes, but may also be

used to quantify the different components.


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Types of chromatography

There are many types, however, you need to be familiar

with the following

1. Paper

2. Thin layer (tlc)

3. Column

4. Gas-liquid (glc)


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Principles of Chromatography

All types of chromatography have the following principles

in common.

1. There are two phases: the stationary phase and the

mobile phase

2. The separation of the mixture is due to differing

interaction of the components with the stationary phase

3. The two mechanisms which can occur are partitioning

and adsorption


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What is the stationary phase?

This is the immovable phase and may either be a solidsupport:

Cellulose in the paper

Silica or alumina

or it may be a non-volatile, viscous liquid coated unto a

solid surface

A long-chain alkane of high boiling point on a SiO2

support


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What is the mobile phase?

This is the solvent which moves through the column or

over the surface of the stationary phase and can be a

gas or a liquid.

There are different types of solvents, ranging from polar

(such as water) to non-polar (such as alkanes)

The mobile phase can be a mixture of solvents, in which

case the ratio is quoted

e.g. methanol:water (2:1)


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PARTITIONING AND ADSORPTION

Adsorption chromatography occurs when the componentsof the mixture (solute molecules) are bound to the surface

of the stationary phase.

The stationary phase is a polar solid (such as silica or

alumina) and the polar solute molecules are bound to this

surface. The more polar the solute, the more strongly

bound it is to the stationary phase

As the mobile phase passes over this, the solute

molecules are eluted, from least polar to most polar in

turn.


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Partitioning occurs between liquids.

If colourless chloroform is added to aqueous iodine (a

brown solution,) two layers develop, with the chloroform

layer below the aqueous layer and having a purple colour.

The iodine moves between the two layers until it reaches

an equilibrium, at which point it is in a definite ratio. This is

partitioning.

If both the stationary and the mobile phases are fluids, then

the solute will be partitioned between the two.


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Solute in the mobile phase will move along with it and

elution will take place starting with those compounds

more soluble in the mobile phase and ending with thoseleast soluble.


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PAPER CHROMATOGRAPHY

Filter paper is used to aid in separation

Cellulose fibres contain water which acts as the stationary

phase.

A small dot of mixture is placed on the paper, which is

placed in a jar containing a shallow layer of solvent and issealed


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The Process of Paper

Chromatography

https://defra.jot.com/System/TmpImageUpload/Chromatography.jpg


16/39Methods of Analysis and Detection, Cambridge University Press


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Separation of black ink using Paper Chromatography


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Retention factor (also called

retardation factor) : Rf

The movement of a soluterelative to the movement of

the solvent front


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THIN LAYER CHROMATOGRAPHY

Stationary phase is a thin layer of silica (SiO2) or alumina

(Al2O3) coated unto a plastic or glass support.

Setup is similar to that of paper chromatography, but thisutilises adsorption and not partition.

Useful for separating colourless components which can bedetected by use of appropriate chemicals or techniques

(visualising agents)


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Thin layer chromatography used to

separate the components of chlorophyll

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


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Visualising agents

1. Iodine crystals may be

added to the solvent andas iodine vapour

evolves, this is

accumulated on the

spots of the separated

solute.

Result is dark brown

spots on a yellow

background

http://www.chem.ucsb.edu/~kalju/chem110L/public/TLC_2004.png


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1. Spraying amino acids with ninhydrin causes them to

appear lilac in colour

http://www.chemguide.co.uk/analysis/chromatography/thinlayer.html


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1. Visualising colourless solute using UV light


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Advantages of TLC over Paper Chromatography

1. TLC is faster (approximately three times as fast)2. TLC works well with very small samples

3. The thin layer can be made from different solids, so a

wide range of mixtures can be separated by carefully

selecting the mobile and stationary phases

4. TLC can be used to quickly select the best conditions for

larger scale separation (such as by column

chromatography)


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Application of TLC

Wide range of applications1. Pesticide analysis: separation of chlorinated insecticides

2. Chemical Forensics: separation and detection of

alkaloids (e.g. morphine and opium) and cannibis also

used as a screen for explosives

3. Drug testing: Detection of steroids

4. Pharmaceutical: Determination of the purity of new drugs

(quality control purposes)


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COLUMN CHROMATOGRAPHY

Stationary phase is silica (SiO2) or alumina (Al2O3) placedin a column. (note, this is a polar phase)

The mixture is placed above the stationary phase andeluted with the mobile phase under gravity or under

pressure (flash chromatography.)

Can be used to separate quantities ranging from

micrograms to kilograms and is based on adsorption

mechanism


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The aim is to achieve good separation by carefully

choosing solvent systems which allow one component to

move through the column faster than the othercomponents.

Stationary phase is polar and will bind strongest to the

most polar component of the mixture

Mobile phase ranges from non-polar through to polar, with

a different solvent mixture being used to elute successivelypolar components


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http://www.chemguide.co.uk/analysis/chromatography/column.html

Useful for purification purposes as mixtures can be separated into individual

components and be collected in fractions.


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Application of Column Chromatography

1. Purification of natural products2. Purification of pharmaceuticals

3. Purification of chemical reactions

4. Separation of dyes and pigments


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GAS-LIQUID CHROMATOGRAPHY

Used to separate and identify small quantities of gases, liquidsand volatile solids

Vapourised sample is carried by a mobile phase (inert gas)over a stationary phase (non-volatile liquid on a solid support)

Partitioning of the components of the sample occurs and they

move through the column based on

their volatility

their relative solubility in the mobile and stationary phases


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Diagram of a typical Gas-Liquid Chromatograph


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The components leave the column after a definite time

period.


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Retention time: time between the injection of the mixture

and the centre of the peak corresponding to a component

Area under a component peak is proportional to the

amount of that component in the mixture

Useful for detection, determination and quantification of

compounds and is used in conjunction with mass

spectrometry for confirmation of the components eluted.

(GC-MS)


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Application of GLC

1. Pesticide analysis2. Analysis of crude oil

3. Environmental analysis: detection of pollutants

4. Determination of the components of natural oils andflavours


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1. A particular component with a low affinity for the stationary phase will

generally move a ________ distance along the stationary phase than a

component with a high affinity for the stationary phase in a

chromatography experiment.

A. longerB. shorter

2. Let Ds be the distance the solvent travels, and let D1 be the distance

component 1 travels on the chromatogram. The retention factor for

component 1 is defined to be:

A. D1 x Ds

B. D1/Ds

C. 1/( D1 x Ds)

3. If a beaker is used as a "developing jar" in an experiment. When the

TLC plate is set in this beaker, the solvent in the beaker must be:

A. above the pencil line used to guide the spotting of samples

B. deep enough to cover the entire TLC plate

C. deep enough to come about halfway up the TLC plate

D. below the pencil line used to guide the spotting of samples

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