Experiment10-Pigment - Experiment 10(Lab Period 11...

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Experiment 10 (Lab Period 11) Separation of Photosynthetic Pigments The chloroplasts of spinach, as of most plants, look green because the major photosynthetic pigments, the chlorophylls, look green. However, there are many other pigments present in the chloroplast, primarily the xanthophylls and the carotenoids, which are masked by the chlorophylls. Non-green pigments must be separated from the chlorophylls to be detectable. In this experiment, a solvent-partitioning technique will be used to attempt to separate four main pigment types in spinach chloroplasts: chlorophyll a, chlorophyll b, carotenoids (like ? -carotene) and xanthophylls (such as lutein). The procedure, outlined below, is designed to yield four solutions, each containing one of these pigment types due to differences with respect to solubility in organic solvents and susceptibility to alkaline hydrolysis. By measuring the absorption spectrum of each of the final four solutions, you will also determine whether the pigments can be distinguished by their abilities to absorb different wavelengths of visible light. The absorbance of each of the solutions will also provide a rough estimate of the relative abundance of the pigments in the extracts. The following procedure can be diagrammed as: Spinach Acetone Pet Ether Methanol Pet Ether Methanol Ethyl Ether Ethyl Ether Methanol Discard MeOH KOH Ethyl Ether Solution C MeOH KOH Solution D MeOH KOH MeOH KOH Solution B Pet Ether Solution A
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The following steps will be performed in the separation: Acetone should extract lipids. Petroleum ether should remove lipids from the acetone, which mixes with water. Methanol , with a small proportion of water, should remove slightly more hydrophilic lipids from relatively hydrophobic lipids, which remain in the petroleum ether layer. Additional water should drive the slightly hydrophilic lipids into the ethyl ether from the methanol, which (like acetone) mixes with water. Methanolic KOH should hydrolyze the lipid (phytyl) side-chains of chlorophylls, freeing the water- soluble chlorophyllides. This step should leave any lipid pigments (e.g., carotenes and xanthophylls) in the ether layers, and the light-absorbing portion of the chlorophyll molecules (the chlorophyllides) should move into the lower, aqueous layers. After studying the procedure and looking at the structures of the different pigments, try to
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Experiment10-Pigment - Experiment 10(Lab Period 11...

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