Biology Lab report
Title : Paper chromatography
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.
Instructions:
- 3 leaves of spinach were ground in pestle and mortar by 2 cm3 of extraction solvent
- the developing solvent was added into the boiling tube and it just touched the lower edge of the paper strip.
- The tube was kept stopper to allow it to saturate with the solvent vapour
- 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
- A light pencil line was ruled 20mm from the lower edge of paper strip
- 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.
- 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.
- The chromatogram was allowed to develop until the solvent front is 2 cm from the upper edge of paper strip.
- The paper strip was taken out and a light pencil line was ruled to mark the solvent front.
- The pigment patches were circled immediately with a pencil.
- The Rf values for the different pigments in the leaf extract were calculated.
