5.7—Chloroplast pigment chromatography

Paper chromatography separates photosynthetic pigments because they differ in solubility in the solvent and attraction to the paper. Each band can be compared using its Rf value: distance travelled by pigment divided by distance travelled by the solvent front.

Keep the origin above the solvent, mark the solvent front quickly, and compare Rf values only when solvent, paper and conditions are comparable. Different bands indicate different pigments or pigment mixtures.

If a pigment travels 3.0 cm while the solvent front travels 6.0 cm, its Rf is 0.50. A second sample with the same Rf under the same conditions may contain the same pigment, but the result is comparative rather than absolute identification.

Rf has no units and should lie between 0 and 1. A changed solvent or a smeared band prevents direct comparison; Rf alone is not proof of molecular identity.