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Aim : to separate and identify of the pigments in leaves by paper chromatography
Principle of design:
In this experiment, a strip of filter paper containing a small spot of concentrated chlorophyll pigment is dipped in a mixture of developing solvent, the pigments was carried up the strip as the solvents move up. Since the solvents moved at different rates and pigments had different solubilities in them, the pigments gradually become separated. When the solvent was near the top of the paper, the paper was removed. After that, Rf (Relative front) for that substance were calculated. In this experiment, Rf values were used to identify the different substances present in the leaf extract mixture.
1. 3 leaves of spinach were ground in pestle and mortar by 2 cm3 of extraction solvent
2. the developing solvent was added into the boiling tube and it just touched the lower edge of the paper strip.
3. The tube was kept stopper to allow it to saturate with the solvent vapour
4. Both sides of paper strip were trimmed to make sure that the sides of paper strip didn’t touch the sides of the boiling tube
5. A light pencil line was ruled 20mm from the lower edge of paper strip
6. A capillary tube was dipped into the extract and a small drop of the extract was put in the middle of the starting line. When it was dried, another drop was added and adding was continued until the spot was quite dark. The spot was kept as small as possible.
7. One end of the strip was folded at 90 so that when the paper strip was suspended with a pin from the stopper, the opposite end would just dip into the surface of the solvent.
8. The chromatogram was allowed to develop until the solvent front is 2 cm from the upper edge of paper strip.
9. The paper strip was taken out and a light pencil line was ruled to mark the solvent front.
10. The pigment patches were circled immediately with a pencil.
11. The Rf values for the different pigments in the leaf extract were calculated.
The distance that the solvent travels = 9.3cm
Rf of the top yellow spot = 8.9/ 9.3 = 0.96cm
Rf of the middle orange spot = 6.8/ 9.3 = 0.73cm
Rf of the bottom bright green spot = 5.7/9.3 = 0.61cm
Rf of the lowest green spot = 4.4/9.3 = 0.47cm
Therefore the top yellow spot is carotene pigment, the middle orange spot is xanthophyll pigment, the bottom bright green spot is chlorophyll a pigment, and the lowest green spot is chlorophyll b pigment.
Chromatography is an effective method of separating the solutes in a solution. Paper chromatography is the process we used to separate various chemicals found in the pigment, in the case is chlorophyll. The four pigments that being separated were chlorophyll a, chlorophyll b, xanthophyll II, and carotene. These pigments were each separated at a different point on the paper. This is because of few different factors.
First, each pigments has a different mass, and therefore are heavier and cannot travel up the paper as far, or as quickly. In this experiment, the heaviest pigment is chlorophyll b as it is the lowest, and the lightest pigment is carotene as it is the top of all pigments.
The second factor was the solubility of the pigment. The least soluble pigment should travel up the shortest distance, and the most soluble pigment should travel up the longest distance. In this experiment, the least soluble pigment was chlorophyll b, and the most soluble one was carotene.
The third factor was adsorption, that is the ability to attract other substances strongly and hold on their surface. The pigment with the greatest adsorption ability traveled up the shortest distance, and the pigment with the weakest adsorption ability traveled up the longest distance. In this experiment, carotene had the weakest adsorption ability, and chlorophyll b had the greatest adsorption ability.
The strip of filter paper should be freely suspended in the test tube, this is because the movement of the pigment on the paper in contact or near the glass would cause the particles to be attracted to the side such that net charge of the force would be towards the side.
The test tube must be stoppered. This is to produce a saturated atmosphere in the test tube. Also, it was to prevent the evaporation of the solvent, which is toxic.
There were four pigments being separated from chlorophyll, they were chlorophyll b, which is the least soluble, heaviest and have the greatest ability of adsorption, chlorophyll a, xanthophyll II and carotene, which is the most soluble, lightest and have the weakest ability of adsorption.