Thin-layer chromatography

Thin-layer chromatography (TLC) separates the substances in a mixture on a plate coated with a thin layer of solid, usually silica gel. A solvent moves up the plate and carries each substance a different distance, depending on how strongly it sticks to the solid. You identify substances by their R_f values, or by comparing their spots with known standards run on the same plate.

How thin-layer chromatography works

Every kind of chromatography has two phases:

• The stationary phase stays put. In TLC it is a thin layer of a solid such as silica gel or aluminium oxide on a glass, plastic or aluminium plate.
• The mobile phase moves. In TLC it is a solvent, which soaks up the plate.

AQA's A-level specification sums up why substances separate: it depends on the balance between solubility in the moving phase and retention by the stationary phase. A substance that dissolves well in the solvent and is held only weakly by the solid travels a long way. One that is held strongly by the solid stays near the start.

Silica gel is very polar. So polar substances, which are attracted strongly to it, move slowly and have low R_f values, while less polar substances travel further. In a Royal Society of Chemistry practical on painkillers, for example, paracetamol is more polar than aspirin and ends up lower on the plate.

TLC method

Separating species by thin-layer chromatography is AQA A-level Chemistry required practical 12. A typical method:

1. Wearing gloves, hold the plate by its edges. Draw a faint pencil line about 1 cm from the bottom: the baseline.
2. Use a fine capillary tube to put a small spot of each sample, and of each known standard, on the baseline, well apart. Let them dry.
3. Pour solvent into a beaker or tank to a depth below the baseline, and put the plate in. Cover it with a lid so the air inside fills with solvent vapour.
4. Leave it until the solvent has almost reached the top, then take the plate out and immediately mark the solvent front in pencil.
5. Let the plate dry, then locate the spots. Many plates contain a fluorescent dye, so colourless spots show up as dark patches under a UV lamp; iodine vapour is another way to show them. Draw round each spot in pencil.

In the figure, the sample S has spots level with reference A and reference B, so S contains both. Its middle spot matches neither, so it is a third substance.

How to calculate Rf values

Measure both distances from the baseline, to the centre of the spot and to the solvent front. R_f has no units and is always between 0 and 1.

Example: identifying spots

In the figure, the solvent front is 7.0 cm above the baseline. Spot A has moved 5.6 cm and spot B 2.1 cm.

• R_f of A = 5.6 ÷ 7.0 = 0.80
• R_f of B = 2.1 ÷ 7.0 = 0.30
• The middle spot of S has moved 3.4 cm, so its R_f = 3.4 ÷ 7.0 = 0.49 (2 d.p.).

An R_f value only identifies a substance if the conditions are the same: the same solvent, the same type of plate and the same temperature. That is why AQA asks you to compare R_f values with standards, and why running the standards on the same plate as the unknown is the most reliable way to do it.

TLC vs paper, column and gas chromatography

Compared with paper chromatography, TLC is usually quicker and gives sharper, better-separated spots. Column chromatography uses the same idea on a larger scale to purify a product. In gas chromatography, AQA notes that mass spectrometry can be used to analyse the separated components.

Uses of TLC

• Identifying substances, such as the active ingredients in painkillers, by comparison with standards.
• Checking purity: a pure substance gives a single spot; extra spots mean impurities.
• Following a reaction: spot the reaction mixture every few minutes; the starting material's spot fades as a new product spot appears.
• Identifying amino acids or metal ions, using a locating agent to make colourless spots visible.

Common mistakes

• Drawing the baseline in ink. Ink would dissolve and separate too. Use pencil.
• Putting the solvent above the baseline. The spots would dissolve into the solvent in the tank instead of moving up the plate.
• Forgetting to mark the solvent front. It evaporates quickly, and without it you can't calculate R_f.
• Measuring from the bottom of the plate. Measure both distances from the baseline.
• Comparing R_f values from different solvents. R_f depends on the conditions, so only compare like with like.

Paper chromatography is the GCSE version of the same idea. To practise analysis questions in a short timed paper, try today's A-level Chemistry: Year 2 or GCSE Chemistry Higher paper.

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