Object:
To separate the chlorophyll pigments by Thin Layer Chromatography (T.L.C.).
Requirements:
Spinach leaves, slides, staining tube, acetone, benzene, mortar, pastel, Kieselgel G, drier, pencil, sand, distilled water and oven.
Preparation of T.L.C. Slides:
1. Prepare a slurry of Kieselgel by suspending about 2 gm. of Kieselgel in 10 ml. of distilled water.
2. Take 4 clean microscopic slides and place them in a horizontal position.
3. Spread the suspension over these slides very quickly m such a manner that a homogenous layer of Kieselgel is formed. Wait for 10 to 15 minutes.
4. Keep these slides in an oven for about 30 minutes at 120°C.
The slides become dry and are now ready for the further treatment.
Method:
1. Take a TLC slide and draw a small circle at a distance of about 1.5 cm from one edge.
2. Put a spot of chlorophyll extract in the circle and make it dry with a drier. Make the spot concentrated by applying the extract 2-3 times.
3. Take a beaker having the benzene-acetone (8.5:1.5) solvent and put the lower edge of the TLC slide (having the chlorophyll spot) in the solvent.
4. Cover the beaker with a petri-dish and keep it in an undisturbed condition for about 20-30 minutes.
Observations and Results:
Different pigments separate at different levels on T.L.C. slide.
The sequence of pigments from top to bottom is as under:
Carotene — Orange yellow
Xanthophylls — Yellow bands
Chlorophyll a — Bluish green
Chlorophyll b — Yellowish green
Calculate the Rf values with the following formula:
