How do you do the chromatography required practical?

What does the chromatography practical investigate?

How paper chromatography separates a mixture of coloured substances, such as food colourings or inks, and how Rf values help you tell substances apart. It is a required practical on both GCSE Chemistry and Combined Science: Trilogy.

The paper is the stationary phase and the solvent (usually water) is the mobile phase. Each substance is shared between the two, and one that spends more time dissolved in the moving solvent travels further up the paper. So a mixture spreads out into separate spots, while a pure substance gives a single spot.

What equipment do you need?

You need chromatography paper, a pencil and ruler, fine capillary tubes for spotting, and a beaker with a shallow layer of water as the solvent. The samples are usually a few known food colourings and one unknown mixture to identify.

• A rectangle of chromatography paper
• A pencil and a ruler
• Glass capillary tubes, one for each sample
• Known food colourings and an unknown mixture
• A 250 cm³ beaker, and water as the solvent
• A wooden spill and a paper clip, to hang the paper in the beaker

What is the method?

Spot each sample on a pencil start line, hang the paper in a shallow layer of solvent and let the solvent rise most of the way up. Then mark how far it got and measure.

• Draw a pencil line 2 cm from the bottom of the paper. This is the start line.
• Mark evenly spaced dots on the line in pencil, keeping at least 0.5 cm from each edge.
• Use a capillary tube to put a small spot, 2 to 3 mm across, of each known colouring on its own dot, with a clean tube for each. Spot the unknown mixture on the last dot and label every spot in pencil.
• Pour water into the beaker to a depth of no more than 1 cm.
• Clip the top of the paper to the spill and rest the spill across the beaker, so the bottom edge dips into the water but the start line stays above it. The paper must not touch the sides.
• Leave it undisturbed until the water has risen about three quarters of the way up the paper.
• Take the paper out and mark the solvent front (the highest point the water reached) in pencil straight away.
• Hang the paper up to dry, then measure from the start line to the solvent front, and from the start line to the centre of each spot.

What are the variables?

You are comparing substances rather than changing one thing to see its effect, so there is no true independent variable. What matters is running every sample under the same conditions, so their Rf values can be compared fairly.

How do you work out and present the results?

Calculate an Rf value for each spot: Rf = distance moved by the substance ÷ distance moved by the solvent. Measure both from the start line, one to the centre of the spot and the other to the solvent front.

Example: the solvent front is 8.0 cm above the start line, and the centre of colouring B's spot is 4.0 cm above it. Rf = 4.0 ÷ 8.0 = 0.50. Rf has no units and is always less than 1, because no spot can travel further than the solvent.

Record the distances and Rf values in a table like the one below, then read the chromatogram. Here the unknown mixture U gives two spots. One has the same Rf as colouring A, so U probably contains A. The other matches none of the known colourings, so U contains something else too. A sample that gives a single spot is probably pure: to be sure, it should give one spot in other solvents as well.

How do you make it accurate and safe?

Draw the start line in pencil. Graphite does not dissolve, but ink would run up the paper with your samples. Keep the start line above the solvent, or the spots dissolve straight into the beaker.

Small, concentrated spots separate cleanly, while big ones smear into each other. Do not let the paper touch the sides of the beaker, because the solvent creeps up unevenly where it does. Leave the beaker alone while the solvent rises, and mark the solvent front as soon as the paper comes out, since it is hard to see once the paper dries. If your solvent evaporates easily, put a lid on the beaker.

With water as the solvent, this is one of the lowest-risk practicals. If your teacher uses a flammable solvent such as ethanol, keep it well away from flames. Take care with the glass capillary tubes, which break easily.

What do exam questions ask about it?

Expect to calculate an Rf value from a diagram or from measurements, and to say which substances are in a mixture by comparing spots. Method questions ask why the start line is drawn in pencil and kept above the solvent, and what it means when a spot stays on the start line (that substance did not dissolve in the solvent). For example:

"On a chromatogram, the solvent front is 7.5 cm above the start line. A spot from ink X has moved 3.0 cm. Calculate the Rf value of the spot. Ink Y has an Rf value of 0.40 in the same solvent. What does this suggest about inks X and Y? (3 marks)"

The Rf of X is 3.0 ÷ 7.5 = 0.40, the same as Y, which suggests X and Y could be the same substance. Running them in a second solvent would be a stronger test.

Common mistakes

Each one has a simple fix:

• Measuring to the top of a spot. Measure to its centre.
• Measuring the solvent distance from the bottom of the paper. Both distances start at the pencil line.
• Drawing the start line in ink. It dissolves and runs up the paper with the samples, so use pencil.
• Putting the start line below the solvent level. The spots wash off into the beaker, so keep the line above the surface.
• Giving Rf a unit, or a value above 1. It is one distance divided by another, so it has no unit and cannot be more than 1.