Chromatography is a method used to separate and identify the components of a mixture of soluble substances. We can also use it to carry out purity tests. One of the most well-known uses of chromatography is in the separation of different pigments in ink, such as colourings or plant pigments like chlorophyll.

The process of chromatography involves two phases: the mobile phase and the stationary phase.

Paper Chromatography

A specific type of chromatography is paper chromatography. In paper chromatography:

  • The stationary phase is the non-moving phase, which doesn’t move through the chromatography paper when the solvent carries the sample.
  • The mobile phase is the solvent in which the sample can move. Solvent (which is usually a liquid) moves along the chromatography paper, and the size of the molecules determines the rate at which they move.

The mobile phase moves through the stationary phase, picking up compounds. Meanwhile, the stationary phase remains fixed in place.

You can see the chromatography apparatus below.

The image illustrates the process of paper chromatography. On the left side, there's a vertical strip of paper with a sample spot near its bottom, labelled "Sample at origin". The paper strip is placed inside a container partially filled with a blue solvent. As time progresses, shown by the representation on the right, the solvent travels upwards through the paper, labelled as "Solvent flow". As it does so, it carries the components of the sample with it, separating them into distinct coloured bands, which are referred to as "Separated components". The leading edge of the solvent as it travels up the paper is marked as the "Solvent front". The entire setup is labelled "Paper Chromatography" at the top.


To perform paper chromatography, follow these steps:

1. Start by drawing a baseline on the chromatography paper using a pencil. The baseline is insoluble, so it won’t move with the solvent.

2. Place a small spot of the sample mixture on the baseline.

3. Pour the solvent into a beaker. Place a glass rod over the beaker, and tape or clip the paper to it, ensuring that the paper’s base just touches the solvent without being submerged.

  • It’s important to note that the solvent does not have to be water. For example, it could be ethanol.

4. Allow the solvent to slowly travel upwards through the paper, taking with it a few soluble pigments from the sample mixture. This will create different spots along the paper.

5. Remove the paper from the beaker before the solvent reaches the top. You now have a chromatogram.

6. Analyse the chromatogram to identify the different components of the sample mixture. Each spot on the chromatogram represents a different component of the mixture.

8. Finally, mark the distance travelled by the solvent.

  • This will be useful for calculating the Rf value

Interpreting a Chromatogram

After carrying out paper chromatography, we can interpret the results to distinguish between pure and impure substances.

  • A pure substance will only produce one spot on the chromatogram
  • An impure substance will separate into two or more spots

Substances that are identical will produce identical chromatograms. This means that they will produce the same number of spots, and those spots will travel the same distances up the paper, with matching colours.

For example, when analysing brown ink using chromatography, we can see that it is a mixture of red, blue, and yellow inks.

The image depicts the separation of brown ink into its constituent colours through chromatography. At the top, three distinct coloured dots – blue, yellow, and red – are shown, representing the pure colours extracted from the brown ink. Below them, a rectangular representation displays a line marked as "Brown ink" from which the colours red, blue, and yellow emanate. These are enclosed in a larger box labelled "Pure substances". There's an arrow pointing to a distinct line within the rectangle and this is labelled "Pencil line".

These colours separate out, with each ink travelling the same distance up the paper as its respective reference ink.

For example, the red spot from the brown ink travelled the same distance up the paper as the red ink, which we used as a reference. This allows us to identify the different components of the mixture.

Rf Value

The Rf value is the ratio between the distance travelled by the dissolved substance (solute) and the distance travelled by the solvent. It is used to identify the components of a mixture.

To calculate the Rf value, we use the equation:

Formula for Rf value, shown as 'Rf equals distance travelled by substance divided by distance travelled by solvent'

Therefore, it is a number between 0 and 1. A particular substance will always give you the same Rf value, as long as the solvent is the same. However, changing the solvent will change the Rf value.

We can compare the Rf value of unknown substances with the Rf values of known substances under the same conditions. These are called reference values.

To find the distance travelled by substance, measure from the baseline to the centre of the spot.

The image illustrates a chromatography paper with measurements on the left side, ranging from 0 to 6. On the paper, a line labelled "Solvent front" is drawn near the top. Two red dots on the paper represent separated substances. A green arrow pointing upward from the bottom of the paper to the solvent front is labelled "Distance travelled by solvent". Another green arrow, shorter in length, pointing upward from the bottom of the paper to one of the red dots, is labelled "Distance travelled by substance". The depiction aims to explain how to measure the movement of substances and solvent during chromatography.

The Rf value is determined by the solubility of the sample in the chosen solvent. In other words, the efficiency with which the sample dissolves in the solvent will influence the Rf value.