Describe paper chromatography, interpret a chromatogram to determine the number and identity of components, and calculate and use the Rf value

What this dot point is asking

SEAB wants you to describe how paper chromatography separates a mixture of soluble (usually coloured) substances, read a finished chromatogram to find how many components a mixture contains and to identify them, and calculate the $R_f$ value as a number that characterises each substance. The technique is a staple of qualitative analysis and appears in both the written and practical papers.

The answer

How chromatography separates a mixture

A small spot of the mixture is placed on a start (pencil) line near the bottom of a strip of chromatography paper. The paper is stood in a shallow layer of solvent so the solvent level is below the start line. The solvent rises up the paper by capillary action, dissolves the substances in the spot, and carries them up at different speeds.

Each substance is in a balance between two pulls: how strongly it is attracted to the paper and how well it dissolves in the moving solvent. A substance that dissolves well and is weakly held by the paper travels far; one that is strongly held by the paper travels only a little. Because different substances have different balances, they end up at different heights and are separated.

Reading a chromatogram

A finished chromatogram tells you two things directly:

• Number of components. Each separate spot from one mixture is one substance, so three spots means three components. A single spot means a pure substance.
• Identity of components. Running the mixture alongside known reference substances lets you match spots: a spot in the mixture that rises to the same height as a known reference is the same substance.

The Rf value

To give each substance a fixed number, measure how far the spot moved and how far the solvent front moved, both from the start line, and divide:

The $R_f$ value has no units and always lies between $0$ and $1$. For a given paper and solvent it is constant for a substance, so it can be compared with reference values to identify it. A small $R_f$ means the substance barely moved (strongly held by the paper); an $R_f$ close to $1$ means it travelled almost as far as the solvent (weakly held).

Locating colourless substances

If the substances are colourless (such as amino acids or sugars), the separated spots are invisible. They are revealed by spraying the paper with a locating agent that reacts to give a colour, or by viewing under ultraviolet light if the spots fluoresce.

Examples in context

Example 1. Checking a food dye. A manufacturer runs a food colouring next to permitted reference dyes. If the colouring shows a spot that does not match any permitted reference, an unapproved dye is present. The technique gives a quick visual check of both how many dyes are present and whether they are allowed.

Example 2. Identifying amino acids. In analysing the products of protein breakdown, the colourless amino acids are separated on paper and then sprayed with ninhydrin, a locating agent that turns each spot purple. Their $R_f$ values are compared with known amino acids to identify which were present.

Try this

Q1. A mixture produces four spots on a chromatogram. State how many substances it contains. [1 mark]

• Cue. Four spots means four different substances in the mixture.

Q2. A spot moves $3.6\ \text{cm}$ while the solvent front moves $9.0\ \text{cm}$. Calculate its $R_f$ value. [2 marks]

• Cue. $R_f = \dfrac{3.6}{9.0} = 0.40$ (no units).

Q3. Explain why the start line on a chromatogram is drawn in pencil and not in ink. [2 marks]

• Cue. Pencil (graphite) is insoluble and stays put; ink would dissolve in the solvent and travel up the paper, adding its own spots and ruining the result.