13.1.6—Pigment chromatography

Chromatography separates a leaf-pigment mixture because its components move through a stationary phase at different speeds. The separated bands provide evidence for different pigments and their relative movement under the chosen solvent conditions.

1. Extract and apply: dissolve pigments from leaf material in a suitable solvent, then apply a concentrated sample to the stationary phase. Keep the starting spot small so separated bands can be distinguished.
2. Develop the chromatogram: allow the solvent mobile phase to travel through the stationary material. Different pigment components move at different rates because their interactions with the two phases differ.
3. Observe separation: record the number, colour and relative positions of the bands. Separate bands are evidence that the extract contained more than one pigment; colour supports description but is not, by itself, a complete identification.
4. Compare movement: measure from the origin to a pigment band and from the origin to the solvent front. Where required, calculate the retardation factor: Rf = distance travelled by component ÷ distance travelled by solvent front.
5. Interpret cautiously: compare bands or Rf values with a reference run made using the same solvent and conditions. A larger Rf means farther movement in that setup; it does not provide a universal value independent of solvent.

Use the same solvent, stationary material, sample preparation and development conditions when comparing extracts. Repeat or run a reference where possible, and report that Rf values and band positions depend on the solvent and experimental conditions. Chromatography separates pigments; it does not measure their absorption spectrum or demonstrate the full light-dependent pathway.

Do not invent exact Rf values, treat a band colour alone as conclusive identification, or confuse chromatographic movement with wavelength absorption or photosynthesis rate.