Chromatography: how it works and Rf values

1. Draw a start line in pencil about 1 to 2 cm from the bottom of the chromatography paper.
2. Put small spots of each sample on the line, spaced apart.
3. Stand the paper in a beaker of solvent, with the solvent level below the start line.
4. Cover the beaker and wait while the solvent moves up the paper.
5. Remove the paper before the solvent reaches the top, mark the solvent front in pencil, and let it dry.

Pencil is used because it's insoluble: ink would dissolve and run with the samples. The start line is above the solvent so the samples don't dissolve into the beaker.

Every type of chromatography has two phases:

• Stationary phase: the part that doesn't move (the paper).
• Mobile phase: the solvent that moves through it (often water or ethanol).

Each substance is distributed between the two phases. A substance that is more soluble in the solvent and less attracted to the paper spends more time in the mobile phase, so it travels further. Different substances travel different distances, which separates them.

Measure both distances from the start line: to the centre of the spot, and to the solvent front.

Example: a spot moves 3.2 cm and the solvent front moves 8.0 cm. $R_f = 3.2 \div 8.0 = 0.40$.

Rf values have no units and are always less than 1, because a substance can't travel further than the solvent. Check your answers with our Rf value calculator.

A substance has the same Rf value every time in the same solvent and conditions. To identify the substances in a mixture, run known reference substances on the same paper. If a spot from the mixture matches a reference spot (same colour and same height, so the same Rf value), it's likely to be the same substance. A substance can have different Rf values in different solvents.

• A pure substance gives one spot in any solvent.
• A mixture gives two or more spots.
• A spot that stays on the start line is insoluble in that solvent; try a different solvent.

In chemistry, a pure substance contains only one element or compound, not the everyday meaning of "natural" or "with nothing added".

Chromatography is used to check the purity of medicines, to identify food colourings and additives, in forensic science (for example, comparing inks), and to test for drugs in blood or urine. More advanced kinds, such as gas chromatography, work on the same principle of a stationary and a mobile phase.

• Measuring from the bottom of the paper instead of the start line.
• Drawing the start line in pen.
• Putting the start line below the solvent level.
• Getting the Rf fraction upside down: it's always less than 1.

1. The solvent moves 10.0 cm and a dye moves 6.5 cm. Calculate the Rf value.
2. An ink gives three spots. Is it pure?
3. Why should the beaker be covered during the experiment?

Answers: 1. $6.5 \div 10.0 = 0.65$. 2. No, it's a mixture of at least three substances. 3. To stop the solvent evaporating, so it moves up the paper steadily.

More practice in our Rf value questions.