Commercial applications of chromatography

Chromatography is an analytical technique chemists use to separate the components of a mixture.

It separates the compounds in a mixture using their relative affinities for a mobile phase and a stationary phase. The greater a compound’s affinity for the mobile phase, the further it travels. The greater its affinity for the stationary phase, the shorter the distance it travels.

There are different types of chromatography, including paper chromatography, thin-layer chromatography (TLC), column chromatography, high-performance liquid chromatography (HPLC) and gas chromatography (GC).

Paper chromatography

The simplest type of chromatography, which chemists use to identify the dyes in mixtures such as ink or food colouring. The stationary phase is a sheet of chromatography paper and the mobile phase is a solvent, usually water, that soaks up through the paper by capillary action.

Thin-layer chromatography

Also known as TLC, scientists use it to quickly identify organic compounds present in a mixture.

TLC starts with a baseline drawn in pencil on the TLC plate near to one end. The scientist dissolves the mixture they plan to analyse in a suitable solvent. They place a spot of this solution on the line. They then place the plate vertically in a tank containing just enough of the mobile phase to keep the liquid level below the baseline.

As the mobile phase moves up the TLC plate, the components in the mixture move different distances depending on how attracted they are to the stationary phase and how soluble they are in the mobile phase. You can vary the relative distances by changing the polarity of the mobile phase.

The components of an organic mixture are often colourless and not visible on the TLC plate, so scientists use specific visualisation techniques:

• Fluorescence – most TLC plates contain an additional substance which fluoresces in the visible region when placed in UV light. If the compound you’re analysing absorbs the UV light, it will not fluoresce and the substance shows up as a dark spot.
• Chemical stains – you can make compounds containing certain functional groups visible by reacting them with chemicals to produce a coloured compound. For example, amino acids and amines can be visualised using ninhydrin, which produces blue spots.

We call the distance a substance moves compared to the mobile phase its retention factor, or R_f value. If the stationary and mobile phases are known, we can use this to identify the compound by comparison to a database of known R_f values.

Column chromatography

We can use column chromatography to separate and isolate organic compounds from a mixture.

Scientists add the sample of the mixture to analyse to the top of the column and the components move through it at different speeds. 

They can combine any fractions containing the same compound after they’ve collected them in test tubes and isolate the pure compound by gently evaporating off the mobile phase.

High-performance liquid chromatography

Also known as HPLC, we use this analytical technique to separate, identify and quantify soluble compounds in a mixture. The stationary phase is silica or resin packed inside a long narrow tube coiled within the machine. The mobile phase is water or a non-polar organic solvent that the machine pumps through the tube under pressure. 

By forcing the mobile phase under high pressure through a coiled narrow column containing the stationary phase, chemists make the process much faster and it gives better separation due to the high surface area of the small solid particles. Scientists can use a variety of materials for the stationary phase, including ion-exchange resins that separate components based on their charge, size exclusion resins that separate components based on their size and chiral resins, which can separate optical isomers.

A detector identifies the compounds as they leave the column. The detector the chemists use depends on the properties of the compounds in the mixture. The output from the detector shows as a series of peaks.

We call the time a substance takes to pass through the column its retention time and we can compare with that of known compounds separated under the same conditions (mobile phase, stationary phase, pressure and temperature).

We can use the area under each peak to determine the relative quantities of compounds in a mixture, as long as they all respond similarly to the detector.

Gas chromatography

Chemists use gas chromatography (GC) to separate, identify and quantify volatile compounds present in trace amounts in a mixture. We can use the retention time to identify the compounds.

Gas chromatography–mass spectrometry (GC–MS) has the advantage of being able to detect very small quantities of compounds. Its uses include detecting banned substances in urine samples from athletes, detecting traces of pesticide residues in food and in environmental monitoring to detect pollutants in the air, soil or water.