Chromatography and Rf Values

Chromatography separates the substances in a mixture and can help identify what they are. It works because of two phases: a stationary phase that stays put and a mobile phase that moves through it.

In paper chromatography, the stationary phase is the chromatography paper and the mobile phase is the solvent. When the solvent soaks up the paper, it carries the dissolved substances with it. Each substance is separated according to how it is shared, or distributed, between the two phases.

A substance strongly attracted to the mobile phase (solvent) and weakly held by the stationary phase (paper) travels far up the paper. A substance strongly held by the paper travels only a short distance.

Because different substances in a mixture are attracted to the two phases by different amounts, they move at different speeds and end up at different heights. That spreads the mixture out into separate spots, which is how the components are told apart.

The finished, dried piece of paper is called a chromatogram. The number of spots tells you how many substances are present, and their positions let you compare them.

The rules for reading a chromatogram are:

• A pure substance produces a single spot in every solvent it is tested with.
• A mixture separates into two or more spots, one for each substance that has moved a different distance.
• Two spots that travel the same distance in the same solvent are likely to be the same substance.

Observation on the chromatogram	What it tells you
One spot only, in all solvents	The sample is a pure substance
Two or more spots	The sample is a mixture
Spots at the same height as a known sample	Likely to be that same substance

Comparing the spots from an unknown mixture against reference substances run on the same paper is how chromatography helps to identify what is in the mixture, not just separate it.

To describe how far a substance has travelled in a way that can be compared, you calculate its Rf value.

$R_f=\frac{\text{distance moved by the substance}}{\text{distance moved by the solvent}}$

Both distances are measured from the pencil start line. The distance moved by the substance is measured to the centre of its spot; the distance moved by the solvent is measured to the solvent front (the furthest point the solvent reached).

Rf is a ratio of two lengths, so it has no units and is always between 0 and 1. A substance can never travel further than the solvent that carries it.

Worked example. A spot moves 4.5 cm from the start line while the solvent front moves 6.0 cm.

$R_f=\frac{4.5}{6.0}=0.75$

The Rf value is 0.75 (2 significant figures). Giving the answer to a sensible number of significant figures matters: the spec expects an appropriate number of significant figures.

Different compounds have different Rf values, and the same compound gives a different Rf in a different solvent. That is why Rf is quoted together with the solvent used.

Worked example. On the same chromatogram, three substances are separated. The solvent front travelled 8.0 cm from the start line. The three spots travelled 2.4 cm, 5.6 cm and 7.2 cm.

Spot	Distance moved by substance (cm)	Rf calculation	Rf value
A	2.4	2.4 ÷ 8.0	0.30
B	5.6	5.6 ÷ 8.0	0.70
C	7.2	7.2 ÷ 8.0	0.90

Checking one: $R_f=\frac{5.6}{8.0}=0.70$.

To identify an unknown, compare its Rf value in a given solvent against a table of reference Rf values measured in the same solvent. A match in Rf points to the same substance.

This required practical investigates how paper chromatography separates and distinguishes coloured substances, and how to calculate their Rf values. A common version separates the dyes in food colourings or in ink.

Method:

1. Draw a horizontal start line in pencil near the bottom of the chromatography paper.
2. Add a small spot of each sample onto the line, letting each dry, so the spots are concentrated.
3. Pour solvent into a beaker to a depth below the start line.
4. Hang the paper so the bottom edge dips into the solvent but the start line stays above the solvent surface.
5. Cover the beaker with a lid or watch glass and leave it undisturbed until the solvent has risen most of the way up.
6. Remove the paper, immediately mark the solvent front in pencil, and leave it to dry.
7. Measure the distances and calculate the Rf value of each spot.

Variable	In this practical
Independent variable	The substance/dye being tested
Dependent variable	How far each substance travels (its Rf)
Control variables	Same solvent, same paper, same start-line height, same distance solvent is allowed to rise

Examiners reward understanding of why the method works, not just the steps. Each instruction fixes a specific problem.

Pencil start line, not pen. Pencil is carbon (graphite), which is insoluble in the solvent, so the line will not run up the paper and contaminate the results. An ink line would dissolve and separate into its own spots.

• Start line above the solvent surface so the samples are not simply washed off the paper into the solvent before separation can happen.
• Cover the beaker to stop the solvent evaporating, which would change the solvent level and give unreliable, uneven results.
• Mark the solvent front straight away because once the paper dries you can no longer see how far the solvent reached, and you need that distance for every Rf calculation.
• Small, dry, concentrated spots so each substance stays a tight spot rather than spreading into a smear that is hard to measure.

Get any of these wrong and the Rf values become inaccurate or impossible to work out.

1. Drawing the start line in pen

The start line must be in pencil. Ink dissolves in the solvent, runs up the paper and separates into its own spots, ruining the chromatogram.

2. Starting the paper below the solvent level

If the start line sits below the solvent surface, the samples dissolve straight into the solvent and wash away before they can separate. The solvent must start below the spots.

3. Measuring Rf to the wrong point

Measure to the centre of the spot for the substance distance and to the solvent front for the solvent distance, both from the start line. Measuring to the top or bottom edge of a spot loses accuracy.

4. Giving Rf a unit or a value above 1

Rf is a ratio of two distances, so it has no units and is always between 0 and 1. An answer greater than 1 means you have divided the wrong way round.

5. Assuming spots at the same height are definitely identical

Spots that travel the same distance in the same solvent are likely the same substance, but confirming it may need testing in a second solvent, since two different substances can share an Rf value in one solvent.