Separation of Photosynthetic Pigments by Paper Chromatography Experiment

By using paper chromatography experiment we can determine more than one type of pigments are involved in the photosynthesis. Photosynthetic pigments carry out photosynthesis. Pigments (organic molecules) are separated by dissolving them in organic solvent and are separated by paper chromatography. In paper chromatography separation of the molecules is governed by the following points.

1. Electrostatic forces of pigment molecules.
2. Molecular weight of pigment of molecules.
3. Adsorption capacity of the pigment molecules.

Apparatus/Chemicals for Paper Chromatography Experiment

Fresh and healthy green leaves, mortar, acetone, petroleum ether, separating funnel, Watmann paper, pipette, distilled water.

Procedure for Paper Chromatography Experiment

Crush the leaves thoroughly with the help of mortar and pestal. To aid the crushing add little sand and continued the crush till a pulpy material is formed. Add acetone to it and Filter the contents to get a clear filtrate. With medicine dropper apply 2-3 drops of filtrate (extracted chlorophyll) at a particular point in the center of Watmann paper and let the spot dry.

Mix 100 parts of petroleum ether with 12 parts of acetone to formulate the solvent with the help of dropper or pipette and apply the solvent in the. center of the dried pigment spot. Preferably apply 1ml solvent in a single stroke. The solvent will move out wards taking pigment molecules along with it. The different pigments are differently absorbed and hence are carried through different distances on the paper. This allows the observer to see different pigments involved in photosynthesis.

Dry the paper and label the zones formed by the pigments. Note the reading from the center outward.

Chlorophyll b               Olive green

Chlorophyll a                Blue green

      Xanthophyll                 Yellow-brown

Carotene                         Yellow

Other Method for Paper Chromatography Experiment

You can also use chromatogram paper strip in case whatmann paper is not available. On paper strip also different pigments are differently absorbed and carried through different distances on the paper strip.

Also study absorption spectra of the chlorophyll extract by using spectrophotometer.

Precautions for Paper Chromatography Experiment

1. Use fresh extract for preparing chromatogram.
2. Avoid the formation of emulsion in extract.
3. Solvent should be applied once and in sufficient quantity so that it bath all the pigment in one stroke.