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Separation of photosynthetic pigments by paper chromatography.

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Separation of photosynthetic pigments by paper chromatography Introduction Chlorophyll is in fact only one pigment in a group of closely related pigments commonly found in photosynthesising plants called photosynthetic pigments. This can be demonstrated by extracting the pigments from leaves with acetone and separating them by means of paper chromatography. With a bit of luck five pigments can be identified: chlorophyll a (blue-green), chlorophyll b (yellow-green), xanthophylls (yellow), carotene (orange) and phaeophytin (grey, it is a breakdown product of chlorophyll). Absorptive paper with a concentrated spot of leaf extract is used in this experiment. When dipping in a suitable solvent, the pigments ascend the absorptive paper at different rates because they have different solubilities in the solvent. In this way they become separated from one another and can be identified by their different colours and positions. Requirements: * Large test tube (24 * 150 mm); * Stopper to fit test tube; * Pin; * A small glass tube to transfer pigment solution; * Chromatography paper or filter paper; * Rack of test tube; * Pigment solution; * Solvent (5 cm3). ...read more.


When the solvent is about 20 mm from the top of the paper remove the strip, rule a pencil line to mark the solvent front and dry the paper; * Detectable pigments can be identified by their colours and the Bf values; * Measure the distance from the pencil line to the leading edge of each clear pigment and work out the Rf value for each one using this formulae: Rf = a / b Where a = distance moved by substance from its original position; b = distance moved by solvent from the same position. Name Colour Rf Carotene Yellow 0.95 Phaeophytin Yellow-grey 0.83 Xanthophyll Yellow-brown 0.71 Chlorophyll a Blue-green 0.65 Chlorophyll b Green 0.45 Table 1 Colours and Rf values of the pigments found in a typical leaf (Rf values for propane/ ether mixture). Result and calculation: b = 8.4 cm For the first pigment: a = 8.1 cm Rf = 8.1 / 8.4 = 0.965 For the second pigment: a = 7.2 cm Rf = 7.2 / 8.4 = 0.857 For the third pigment: a = 5.9 cm Rf = 5.9 / 8.4 = 0.70 For ...read more.


Light could have affected the nature of chlorophyll thus led to inaccurate results. I would recommend not taking out the pigment until the last minute when it is needed in order to minimise the effect of light; * An alternative technique (thin layer chromatography) mentioned in the preparation paper can be employed: instead of absorptive paper, a glass of plastic slide (or aluminium sheet) coated with silica gel is used. The gel is spotted and then the slide or aluminium sheet is placed vertically in a beaker or Coplin jar containing solvent at the bottom; * Further experiment can employ pigments from different plants. Chlorophyll a is of universal occurrence in all photosynthesising plants but we can compare the different pigments in different plants. Because different pigments absorb light of different wavelengths, by comparing the occurrence of different pigments we can deduce the plants' habitat; * We can do further experiment to determine the absorption spectrum of each pigment. We can make separate solution of each pigment and use a colorimeter to find out what wavelength of light each one absorbs. Then we can find out what wavelength of light a plant uses most to carry out photosynthesises. ...read more.

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