Object:
To separate leaf pigments by column chromatography.
or
To separate the chloroplast pigments by solvent method.
Requirements:
Pigments extract, magnesium oxide, filter glass disc, glass wool, flask, suction pump, chromatographic tube, petroleum ether, benzene.
Method:
1. Take a chromatographic tube having a filter glass disc and glass wool at its base.
2. Fill the chromatographic tube with magnesium oxide.
3. Fix the chromatographic tube on the mouth of the filter flask and attach the side arm of the flask with a suction pump (Fig. 42).
4. Fill petroleum ether solution of leaf pigments on the top of magnesium oxide in the tube.
5. Wait for some time and observe that each pigment forms a distinct colour band.
6. After passing whole of the pigment solution into the magnesium oxide, develop the chromatogram by adding about 50 ml of equal proportion of petroleum ether and benzene.
Observations:
Different bands of yellowish-green, blue, yellow and orange colours separate in the column from the top towards the bottom.
Results:
Yellow-green band present at the top of the column represents chlorophyll b; dark-blue colour band present next to yellowish-green represents chlorophyll a, and the yellow and orange-coloured bands represent xanthophyll and carotene in the column, respectively.