• Draw a start line in pencil about 1.5 cm from the bottom (ink would run).
• Put small spots of the unknown and of known substances on the line.
• Stand the paper in a little solvent: the solvent must be below the line. Put a lid on.
• Take the paper out before the solvent reaches the top, mark the solvent front and let it dry.

Colourless substances such as amino acids need a locating agent sprayed on the dry paper to make the spots show.

The stationary phase is the paper; the mobile phase is the solvent moving up it. Each substance is shared between them. One that dissolves well in the solvent spends more time in the mobile phase and moves further. A pure substance gives one spot; a mixture gives a spot for each substance it contains.

Rf = distance moved by substance ÷ distance moved by solvent

AQA: recall. Both distances are measured from the start line.

1. Start line 1.5 cm up the paper; spot centre at 5.7 cm; solvent front at 10.5 cm
2. spot moved 5.7 − 1.5 = 4.2 cm; solvent moved 10.5 − 1.5 = 9.0 cm
3. Rf = 4.2 ÷ 9.0 = 0.47 (2 d.p.)

The same substance has the same Rf in the same solvent. Run the unknown next to known substances on one sheet: spots level with each other are the same substance. Repeat the run, leave out anomalies and take a mean distance before working out Rf.

If the unknown has a spot that matches nothing, it contains a substance you did not test for.