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to separate leaf pigments by chromatography

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Name: Kern Pemberton Date: 13th October, 2008 Lab#: 2 Aim: To separate leaf pigments by chromatography Theory: All biological pigments selectively absorb certain wavelengths of light while reflecting others. The light that is absorbed may be used by the plant to power chemical reactions, while the reflected wavelengths of light determine the color the pigment will appear to the eye. Pigments also serve to attract pollinators. These pigments in plant can be separated in the laboratory via chromatography. This works on a basic principle which is the partition of the components of a mixture between a stationary phase and a mobile phase. The different components of a mixture experience slightly different absorption forces with the stationary phase (cellulose paper) and have different solubilities in the mobile phase (solvent) therefore resulting in different components having different Rf values which is the distance travelled by each component divided by the total distance travelled by the solvent. ...read more.


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. ...read more.


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. ...read more.

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