(ii) practical investigations to analyse biological solutions using paper…

The idea

A practical chromatography investigation starts with a prepared sample: a small, concentrated spot of the biological solution is applied to the origin line, drawn in pencil, above the level of the solvent. The solvent is allowed to travel up the paper or TLC plate by capillary action. When the solvent front nears the top, the chromatogram is removed and the front is marked immediately in pencil. Spots may be colourless, so they are located using a locating agent such as ninhydrin for amino acids, or viewed under ultraviolet light. Each spot's distance from the origin is measured to its centre, and Rf is calculated as spot distance ÷ solvent-front distance. The sample is identified by comparing its Rf values with those of known standards run on the same chromatogram under identical conditions.

What earns the marks

• Apply a small, concentrated spot of the sample on a pencil origin line, keeping the spot above the solvent level so the sample does not dissolve into the reservoir.
• Allow the solvent to move up the stationary phase by capillary action and mark the solvent front in pencil as soon as the run finishes.
• Locate colourless spots using a locating agent such as ninhydrin for amino acids, or by viewing under ultraviolet light.
• Measure from the origin line to the centre of each spot and to the solvent front, then calculate Rf = spot distance ÷ solvent-front distance.
• Identify components by comparing sample Rf values with those of known standards run on the same chromatogram under the same conditions.
• Use the results to analyse the biological solution, for example identifying which amino acids or sugars are present in a mixture.

Common mistakes and how to put them right

• Spotting the sample below the solvent level.

  Put it right: The sample must sit above the solvent so it is carried up the stationary phase rather than dissolving into the reservoir.

• Using a pen to mark the origin or solvent front.

  Put it right: Pen ink runs in the solvent, so pencil must be used.

• Measuring to the edge of a spot rather than its centre.

  Put it right: Measurement is to the centre of the spot for a reliable Rf.

• Failing to run known standards alongside the sample.

  Put it right: Without standards under identical conditions, Rf values cannot reliably identify components.

Exam tips

• Describe the practical sequence in order: spot, run, mark the front, locate spots, measure, calculate Rf, compare with standards.
• Name a suitable locating agent and say why it is needed, linking it to colourless spots such as amino acids.
• Explain why the same solvent and conditions are needed for the sample and standards when identifying substances.
• State that Rf is calculated from the origin to the centre of the spot divided by the origin to the solvent front.