ChromatographyEdexcel A-Level Chemistry: Revision notes

Chromatography separates the components of a mixture between a mobile phase and a stationary phase.

• The mobile phase moves through the system carrying the mixture (a liquid solvent or a gas)
• The stationary phase does not move (a solid, or a liquid held on a solid)

Each component has a different affinity for the two phases, through its solubility in the mobile phase and its adsorption on or attraction to the stationary phase. A component more strongly attracted to the stationary phase moves more slowly, and one more soluble in the mobile phase moves faster. This is why the components separate.

In TLC the stationary phase is a thin layer of silica (or alumina) on a plate. In paper chromatography it is water held in the paper. A pencil baseline is drawn, the sample is spotted on it, and the plate is placed in solvent below the baseline. The solvent rises, carrying components different distances.

Colourless spots are made visible with a locating agent (such as ninhydrin for amino acids) or ultraviolet light. These are one-way chromatograms, developed in a single direction.

The Rf value for a spot is:

$R_f = \dfrac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}}$

Both distances are measured from the baseline, to the centre of the spot. Rf has no units and is always between 0 and 1.

Worked example: a solvent front moves 12.5 cm and a spot moves 4.5 cm.

$R_f = \dfrac{4.5}{12.5} = 0.36$

Under identical conditions (the same solvent, stationary phase and temperature) a given compound has the same Rf, so a spot can be identified by comparing its Rf with reference values.

In column chromatography the stationary phase is held in a column and the sample passes through it. Components leave the column at different times, called retention times, and a detector records each as a peak.

Retention time is the time from injection of the sample to the detection of a component. A component more strongly attracted to the stationary phase has a longer retention time.

Gas chromatography (GC): the mobile phase is an inert gas (such as helium). The stationary phase is a liquid held on a solid support. The sample must be vaporised, so GC suits volatile, thermally stable compounds.

HPLC: the mobile phase is a liquid pumped at high pressure through a column packed with a solid stationary phase. The sample is not vaporised, so HPLC suits compounds that are non-volatile or that decompose on heating.

Retention time on its own cannot prove identity, because different compounds can have the same retention time. A mass spectrometer connected to the end of the column (GC–MS or HPLC–MS) gives the relative molecular mass and fragmentation pattern of each separated component. These are compared with a database to identify the compound.

Applications:

• Forensics: identifying accelerants in fire debris (GC–MS) or drugs and poisons in samples
• Drug testing in sport: detecting banned substances in an athlete's urine or blood (commonly HPLC–MS or GC–MS)

Choose GC for volatile, stable compounds and HPLC for non-volatile or thermally unstable ones.