Photosynthetic pigments can be separated and identified by chromatography, using paper or a thin-layer chromatography (TLC) plate.Method:
1. Grind a leaf with a little sand and a solvent such as propanone to extract the pigments.
2. Draw an origin line in pencil about 2 cm from the bottom of the paper or plate (ink would dissolve and run).
3. Using a capillary tube, add a small, concentrated spot of extract to the origin, letting it dry between applications.
4. Stand the paper in a container of running solvent with the solvent level below the origin line, and cover it so the air stays saturated with solvent vapour.
5. Remove it when the solvent is near the top, and mark the solvent front immediately.The pigments separate because they differ in solubility in the solvent and in how strongly they are attracted to the paper or plate. The most soluble, least attracted pigments travel furthest.
Rf = distance moved by pigment ÷ distance moved by solvent front Distances are measured from the origin to the centre of each spot, and Rf values are compared with known values for the same solvent. Carotene usually has the highest Rf, running close to the solvent front.Worked example: a spot moves 3.6 cm and the solvent front moves 8.0 cm, so Rf = 3.6 ÷ 8.0 = 0.45.The solvents are flammable and harmful, so work in a well-ventilated room away from flames.
1. Grind a leaf with a little sand and a solvent such as propanone to extract the pigments.
2. Draw an origin line in pencil about 2 cm from the bottom of the paper or plate (ink would dissolve and run).
3. Using a capillary tube, add a small, concentrated spot of extract to the origin, letting it dry between applications.
4. Stand the paper in a container of running solvent with the solvent level below the origin line, and cover it so the air stays saturated with solvent vapour.
5. Remove it when the solvent is near the top, and mark the solvent front immediately.The pigments separate because they differ in solubility in the solvent and in how strongly they are attracted to the paper or plate. The most soluble, least attracted pigments travel furthest.