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Dept.

of Chemistry, University of the West Indies Mona


C.A.P.E. CHEMISTRY WORKSHOP

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.

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.

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)

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

What is the stationary phase?


This is the immovable phase and may either be a solid support: 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

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)

PARTITIONING AND ADSORPTION


Adsorption chromatography occurs when the components of 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.

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.

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 those least soluble.

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 is sealed

The Process of Paper Chromatography

https://defra.jot.com/System/TmpImageUpload/Chromatogr aphy.jpg

Methods of Analysis and Detection, Cambridge University Press

As the solvent moves up the paper, the different components are partitioned between the stationary and mobile phase.

Least polar molecules move ahead of the more polar molecules.

Can be used to separate components of black pen ink

http://www.columbia.edu/cu/biology/courses/c2005/handouts/chrom2.4.gif

Separation of black ink using Paper Chromatography

http://wps.prenhall.com/wps/media/objects/165/169061/GIFS/AAAVBCN0.JPG

Retention factor (also called retardation factor) : Rf

The movement of a solute relative to the movement of the solvent front

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 this utilises adsorption and not partition.

Useful for separating colourless components which can be detected by use of appropriate chemicals or techniques (visualising agents)

Thin layer chromatography used to separate the components of chlorophyll

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

Visualising agents 1. Iodine crystals may be added to the solvent and as 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

1. Spraying amino acids with ninhydrin causes them to appear lilac in colour

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

1. Visualising colourless solute using UV light

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)

Application of TLC
Wide range of applications
1. 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)

COLUMN CHROMATOGRAPHY
Stationary phase is silica (SiO2) or alumina (Al2O3) placed in a column. (note, this is a polar phase)

The mixture is placed above the stationary phase and eluted 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

The aim is to achieve good separation by carefully choosing solvent systems which allow one component to move through the column faster than the other components.

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 successively polar components

Useful for purification purposes as mixtures can be separated into individual components and be collected in fractions.
http://www.chemguide.co.uk/analysis/chromatography/column.html

Application of Column Chromatography


1. Purification of natural products
2. Purification of pharmaceuticals 3. Purification of chemical reactions 4. Separation of dyes and pigments

GAS-LIQUID CHROMATOGRAPHY
Used to separate and identify small quantities of gases, liquids and 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

Diagram of a typical Gas-Liquid Chromatograph

The components leave the column after a definite time period.

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)

Application of GLC
1. Pesticide analysis
2. Analysis of crude oil 3. Environmental analysis: detection of pollutants 4. Determination of the components of natural oils and flavours

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. longer B. 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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