Locating Agents and Rf Values in Chromatography

Chromatography separates a mixture into its parts. Sometimes the separated spots are colourless, so we need a way to see them. We also measure how far they move to help identify them.

Locating agents: making invisible spots visible

After the solvent has travelled up the paper or TLC plate, some substances leave no colour. Locating agents reveal these hidden spots:

• Iodine vapour: put the dry chromatogram in a closed jar with iodine crystals. Brown iodine sticks to many organic spots, making them brown.
• Ninhydrin spray: spray then gently warm. Amino acids turn purple.
• UV light: TLC plates often glow under UV. Spots appear as dark patches because they block the glow.

Always draw the start line and the solvent front in pencil. Ink can run and spoil results.

Rf values: measuring how far spots travel

An Rf value tells how strongly a substance moves with the solvent. It helps compare unknowns with known substances (under the same conditions).

Measure from the pencil start line to the centre of a spot, and from the start line to the solvent front. Then use:

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

• No units; values are between 0 and 1.
• Rf depends on the solvent and the stationary phase, so compare only if these are the same.

Using Rf values

• Match an unknown spot’s Rf with a known standard run in the same solvent on the same paper/plate.
• Two different substances can share similar Rf values; use locating agents or another solvent to confirm.

Common mistakes

• Using pen for the start line or labels.
• Forgetting to dry the chromatogram before using a locating agent.
• Measuring from the bottom of the paper instead of from the start line.