Write a lab discussion on photosynthesis with the object of identifying the colour pigments responsible for photosynthesis in coleus leaves.

Also use paper chromatography to separate and identify various pigments present in spinach leaves and determine which pigments are polar and which are nonpolar.

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Photosynthesis is a series of chemical reactions by which living plants utilize carbon dioxide and water in presence of sunlight to form carbohydrates. These carbohydrates are the food that they prepare.

6 CO2 + 12 H2O  ----light-------->     C6H12O6 + 6 H2O + 6 O2

Speciallized colored pigments like chlorophyll act as a light harvesting complex that receives sunlight and uses its radiant energy to power up the process of photosynthesis.

The main photosynthetic pigments are chlorophyll a and b. There are accessory pigments like carotene and xanthophyll which transmit absorbed light energy to chlorophyll a.

Inorder to identify the pigments that are present in leaves, they need to be separated and characterized. Separation of the pigments can be done by paper chromatography. In paper chromatography, a paper strip has its tip dipped in a non-polar solvent that contains the pigments to be separated. The water that is bound to the cellulose of the paper acts as the polar medium that attracts polar molecules. When the solvent moves through the paper, more polar pigments are bound easily to the water in the cellulose of the paper and stops moving. They form a band near to the bottom. Less polar or non-polar pigments have more affinity to the solvent rather than the paper. Thus they move farther and form their bands later. The distance of the bands are measured to calculate the Rf value (Ratio of fronts).

Rf = Distance moved by pigment/ Distance from pigment origin to solvent front.

Inorder to separate the pigments in coleus leaves and spinach leaves, grind them separately in acetone medium. In a chromatography paper, draw a pencil mark at around 15 - 20 mm from the tip of the paper. With a capillary tube or rod, place a spot of the pigment on the pencil mark. ie., of coleus in one paper and of spinach in the other paper. Let the pigments dry. Now place each paper in separate arrangements of chromatography solvents and keep each closed. Let the solvent front rise to around 2 - 3 cm and then remove the papers. Allow them to dry. Once dried, measure the distances of the pigments moved and then calculate the Rf values.

(Those pigments that moved the farthest are non-polar while those that stayed near are polar in nature.)

In case of the spinach leaf extract, the chromatogram showed a first band of chlorophyll, then chlorophyll a, then xanthophyll and finally carotene at the top. (Please see the attached image)

This shows the polarity of the pigments. We can see that chlorophylls are more polar than xanthophyll or carotene.

Coleus leaves are variegated in nature. Some have white patches and some have red patches. Those with white patches have no color pigments in those areas. Red patches have pigments called anthocyanins. But anthocyanins are not involved in photosynthesis. Only chlorophylls and carotenoids (xanthophyll, carotene etc) are involved in photosynthesis. Paper chromatogram of coleus thus shows bands of pigments like anthocyanins which are not involved in photosynthesis.


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