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Friday, April 1, 2011

SEPARATION OF PLANT PIGMENTS BY THIN LAYER CHROMATOGRAPHY


SEPARATION OF PLANT PIGMENTS BY THIN LAYER CHROMATOGRAPHY
Aim:
To separate the constituents of plant pigments (leaf chlorophyll) by TLC.
Principle:
In TLC the compound under examination moves along the surface of the adsorbent. The moving substance is attracted by the polar sites on the surface of the adsorbent by electrostatic forces and this binding is reversible. The solvent also interacts with the adsorbent and the compound interacts with the solvent. This three fold competitive interaction among solute, solvent and adsorbent establishes the relative rates at which the solvent front and solute ascend the layer of adsorbent on the glass plate. A more polar solute is attracted to the adsorbent more strongly than a less polar solute. This is reflected in the faster movements of the less polar compounds.
The experiment is based on the principle of adsorption. The chlorophyll extract of plant leaves get adsorbed by the solvent molecules. The extent of adsorption of plant pigment decides partition and separation. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried and separated components of the mixture are visualized. If the components are colored, visualization is straight forward. Usually the components are not colored so a UV lamp to visualize the plate.
Chemicals and equipments required:
1.      Acetone
2.      Petroleum ether
3.      Mortar and Pestle
4.      Developing Chamber
5.      Glass plate
6.      Distilled Water
Procedure:
1.      Green leaves were crushed and or squashed using a mortar and pestle until a green pulp was obtained.
2.      Mixture of acetone, petroleum ether (7.3), was added to dissolve the chlorophyll. The green extract was centrifuged and chlorophyll extract was obtained as a supernatant.
3.      The slurry (stationary phase + water) is prepared and poured on to a glass plate which is maintained on a leveled surface. The slurry is spread uniformly on the surface of the glass plate. After setting, the plates are dried in an oven.
4.       Add a sufficient mixture of solvent system (acetone, petroleum ether (7:3)) and cover the bottom of the tank to a depth of 0.5cm. Allow it to stand for 15minutes.
5.      Select a TLC plate, draw a very fine line with a pencil above 1cm from one edge of the plate and apply mixtures as spot on to the plate.
6.      Place the plate into the tank and allow the solvent to develop up to 15 cm.
7.      Remove the plate from the tank, and allow dry it.
8.      Dry the chromatogram in an oven at 60.c for 10-15 minutes. Spots will be visualized.
Result:



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