Apparatus:
- Boiling tube
- Cellulose Paper
- Pencil
- Test tube racks
- Dropper
- Green leaf pigment
- Stopper
- Dropper
- Solvent
Method:
- A line 2.5 cm away from the end of the chromatography paper or cellulose paper was drawn using a graphite pencil.
- A dropper was dipped into the green pigment provided and touched on the centre of the line drawn in pencil on the chromatography paper. This was repeated until a small, dark green spot was obtained on the chromatography paper.
- A small amount of the solvent provided was then placed into a boiling tube.
- The paper was then carefully immersed into the test tube, not allowing the green spot or the pencil line to come into contact with the solvent. Therefore just enough solvent was placed in the test tube so that it would not exceed the line drawn in pencil.
- After 20 minutes the chromatography paper was removed then dried.
- The position of the pigments and solvent were then marked off and recorded.
Diagram:
Diagram 1.0 shows setup of apparatus for chromatography.
Results:
Table 1.0 shows the results obtained from the chromatography paper.
Discussion: Green plants have five closely related photosynthetic pigments which are; Carotene, Phaeophytin, Xantophyll, Chorophyll a and Chlorophyll b, in order of increasing polarity. Chlorophyll a is the most present in every plant that performs photosynthesis as it is the primary pigment whereas the other are considered primary pigments.
The pigments mentioned above can be found in the light harvesting cluster in the thylakoids of the chloroplast. All the pigments except for chlorophyll a are considered accessory pigments. Their purpose in the light harvesting cluster is to absorb energy from light and channel it the primary pigment (chlorophyll a). The combined pigments absorb at different wavelengths in different light harvesting clusters thus there is photosystem I (750nm) and photosystem II (650nm).
In photosystem I, the accessory pigments trap energy from the sunlight and funnel it to the primary pigment or chlorophyll a. This then becomes excited and releases an electron which is accepted by an electron acceptor. The electron then moves through a series of electron carriers arranged in order of their redox potentials. Meanwhile, the photosystem is said to be unstable as a result of chlorophyll a losing an electron. The electron then moves back down the series of electron carriers to return to stability producing energy thus A.T.P.
Photosystem II differs in that the process is non cyclic (i.e. the electron emitted from chlorophyll a does not return to chlorophyll a), it produces NADPH and O2 as a by-product in addition to A.T.P. and photosystem I is in fact a component of photosystem II. In this case the electron emitted from chlorophyll a travels through a series of electron carriers and then is accepted by a hydrogen ion (which is then picked up by NADP+) and this upsets the equilibrium for the splitting of water, causing more water to split. However, the electrons harvested from the splitting of water goes to photosystem II(which would have been energized to release an electron simultaneously with photosystem I), which would have been unstable. Also, the electron that left chlorophyll a from photosystem I would have passed through a series of electron carriers, arranged in redox potentials, to stabilize photosystem I producing A.T.P.
The extract provided in the experiment in the experiment contains a mixture of the pigments involved in the above processes. The process above then explains why chlorophyll a is needed in all photosynthesizing plants as it is the primary pigment. However, chromatography was used to separate and identify all the mixtures of the pigments. As mentioned in the theory, this is due to the absorption of the pigments to the stationary phase or cellulose paper and its solubility in the solvent therefore providing Rf values. Earlier in the discussion, the different pigments were mentioned in order of increasing polarity which was also the order of decreasing Rf values. This then explains that the solvent used was non-polar and carotene had the greatest attraction for it, thus being separated at the longest distance on the paper and chlorophyll b had the greatest attraction for the cellulose paper.
Precautions:
- The test tube was stoppered to prevent interaction of the chromatograph paper with the air thus preventing any evaporation.
- A graphite pencil was used to draw the base line to insure there were no impurities or foreign pigments being picked up by the solvent.
Limitations:
- There could have been presence of impurities in the extract provided.
Sources of error:
- There may have been some absorption of light by the pigments while the experiment was taking place.
Improvements:
- The tube could have been placed in dim light to ensure that no absorption was taking place.
