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Study Notes: What’s This Thing Called ‘Chromatography’?

Chromatography is the science of separation
Separating the species of interest (the analyte) from other material in the sample and especially ‘interferences’ (substances which may interfere with the subsequent analysis) is often a vital first step before further analytical processes are instituted. For instance, isolating a protein of interest (analyte) from the cellular materials to which it is bound may be the first step in the further study of the protein. If the cellular material contains interferences (for instance proteases that may degrade the protein of interest) these also need to be removed.

Chromatography is an extremely useful tool for performing such separation. Chromatography may also be used as an analytical tool to identify and measure the relative amounts of species of interest in a complex sample.

View an animation of Thin Layer Chromatography (a technique very similar to paper chromatography). The components of the substance separate into bands or spots as the substance and solvent move up the length of the plate. The green coloured sample separates into 2 components, one blue and one yellow.

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Paper chromatography is a simple but effective method for separating species in a mixture. Try this experiment at home. Put some spots (6 mm diameter) of different coloured marking pens on a 6 cm wide piece of photocopy paper in a line about 3 cm from the bottom of the sheet. Place the sheet carefully into 1.5 cm of methylated spirits in a jar. Be careful that you do not splash any of the liquid onto the spots. Allow the liquid to migrate up the paper for about an hour. What you see is chromatography in action!

 

A mixture containing three components is passed through a chromatography column. Two components separate into bands of red, blue and yellow. The first band (red) is shown eluting off the column.

 

 

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Chromatography in chemical analysis works in the same way. Put very simply, a mobile phase (the liquid methylated spirits in this case but can be gas) carries a sample (the marker ink) past or through a stationary phase (paper) and in the process separates out the different components in the sample mixture.

Mikhail Tswett invented chromatography about 100 years ago. This Russian botanist separated various plant pigments by passing solutions of the pigments through a glass tube (column) packed with fine calcium carbonate. The species present in the samples appeared as coloured bands on the column. The name he chose for this method was chromatography from the Greek, chroma meaning ‘colour’ and graphein meaning ‘to write’.

Chromatography comes in many formats

Image shows Paper Chromatography, separating a solution into colours.

Paper Chromatography

Image shows Thin Layer Chromatography.

Thin Layer Chromatography (TLC)

Image of a Liquid Column Chromatograph.

Liquid Column Chromatography (LC)


Photograph that shows some detail of the HPLC equipment.

High Performance Liquid Chromatography (HPLC)

A photograph of a gas chromatograph instrument commonly found in an analytical laboratory.

Gas Chromatography (GC)

In all these techniques the sample is dissolved in the mobile phase (may be liquid or gas). The mobile phase is then forced through an immiscible stationary phase that is fixed in a column or on a solid surface.

If a component is strongly retained by the stationary phase it will move slowly with the mobile phase. In comparison, components that are weakly held by the stationary phase travel rapidly and are first off the column. Movement of analyte off the column is called ‘elution’. These differences in mobility allow the components in the sample to separate into discrete bands that can be analysed both qualitatively (presence or absence of components) and quantitatively (a measure of the amount of component present).

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