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THIN LAYER CHROMATOGRAPHY LAB REPORT.

Introduction:

Chromatography is defined as the separation of a mixture of two or more different compounds by distribution between two phases, one of which is stationary and the other moving. Chromatography depends on the difference solubility and mobility speed of the substances to be separated

Procedures:

· Isolation of Pigments: Weigh about 0.1 g of fresh spinach leaves. Place the small pieces of spinach leaves in a mortal along with 1.0 ml of anhydrous magnesium sulphate and add 2.0gm of sand. Grind the mixture until a light green powder is obtained. Transfer the mixture into a centrifuge tube and add 2.0ml of acetone, rinse the mortal with another 2.0ml of acetone and transfer the mixture in the centrifuge tube. Cap and shake the mixture and then allow it to stand for 10 minutes. Carefully pour the liquid to a vial and prepare the solvent(70:30 hexane: aceton)

· TLC of spinach extract: On the TLC slide make a spot using a pencil approximately 1cm from the end of the slide. Using a clean capillary tube transfer the solution to the TLC plate. Let the first spot dry then touch it two or three more times to ensure adequate sample.

· Let the spot dry, then carefully place the TLC in the developing jar containing ½ cm of the solvent at the bottom.

· When the solvent have reached ¾ of the way up the slide, remove it from the chamber and quickly mark the solvent front. Let the slide dry and then measure the distance from the original sporting to each spot and to the solvent front. Calculate the Rf Values.

CALCULATION

Rf Value = Distance spot moved

Distance Solvent moved

Rf( compound 1)=22/65=0.34

Rf( compound 2)=50/65=0.77

DISCUSSION

The colored pigments from a plant fall into 2 categories, Chlorophylls and Carotenoids.

Caroteniods are yellow pigments that are involved in the photosynthesis process.

Xanthophylls are oxygen-containing carotenes (OH and C=O) The carotenes are shown

below. The Green pigments are the Chlorophylls that act as the principal photoreceptor

molecules of plants.

On the TLC place you will see in the following in order of decreasing Rf value Carotenes (yellow), Pheophytin a (grayish), Pheophytine b (grey, may

not be visible), Chlorophyll a (blue-green), Chlorophyll b (green)and Xanthophylls .

Conclusion

The Rf( compounds 1) which are carotenoids moved further than the Rf compound 2)( which were xanthophylls.