Paper chromatography

Paper chromatography is another method that can be used to test if a substance is pure or impure. It can also be used to separate mixtures of soluble substances in order to find out what substances are in the mixture. These are often coloured substances such as food colourings, inks, dyes or plant pigments.

A pencil line is drawn across a sheet of chromatography paper and spots of ink or plant dye are placed along it. The paper is held abovea basin containing solvent.

1. Spots of ink or plant dye are placed on a pencil line

Paper chromatography can be used to analyse colourless substances. A locating agent reacts with these substances to form coloured products, or products that glow under ultraviolet light. For example, iodine vapour reacts with fats and oils, turning them brown.


Chromatography relies on two different ‘phases’:

The different dissolved substances in a mixture are attracted to the two phases in different proportions. This causes them to move at different rates through the paper.

Water is often used as a solvent but if the substances being tested do not dissolve in an aqueous solvent then an alternative non-aqueous solvent must be used such as alcohol.

Interpreting a chromatogram

Separation by chromatography produces a chromatogram. A paper chromatogram can be used to distinguish between pure and impure substances:

  • a pure substance produces one spot on the chromatogram
  • an impure substance produces two or more spots

A paper chromatogram can also be used to identify substances by comparing them with known substances. Two substances are likely to be the same if:

  • they produce the same number of spots, and these match in colour
  • the spots travel the same distance up the paper compared to reference spots (have the same Rf value)
Results of a chromatogram of three pure substances and brown inkInterpreting the chromatogram for a brown ink

In this chromatogram, the brown ink is made of a mixture of the red, blue and yellow inks. This is because the spots in the brown ink have travelled the same distance (and have the same Rf value) as the reference inks.

Rf values

This distance a spot travels is partly dependent upon the time it is left in the solvent. This distance travelled compared with the distance travelled by the solvent is the same for a particular substance.

The Rf value of a spot is calculated using:

Rf = \frac{distance~travelled~by~substance}{distance~travelled~by~solvent}

The Rf value is always the same for a particular substance using the same stationary phase and mobile phase. Rf values can be used to identify unknown chemicals if they can be compared to a range of reference substances.

Rf values vary from 0 (the substance is not attracted to the mobile phase) to 1 (the substance is not attracted to the stationary phase).

Chromatography paper next to a measurement scale, showing the distance travelled by the solvent, and a coloured dot showing the distance travelled by the substanceMeasurements needed to calculate an Rf value