Mixed carotenoids are obtained by solvent extraction of alfalfa, removal of chlorophylls through saponification and subsequent purification of the carotenoids by solvent extraction. The main colouring principle consists of carotenoids, of which lutein accounts for the major part. Variable amounts of neoxanthin, violaxanthin and b-carotene are present.
Acetonitrite, dichloromethane, ethyl acetate, acetone (all HPLC grade), butylated hydroxytoluene (BHT), potassium hydroxide, methanol, sodium sulfate (all analytical reagents of better grade). Add 1 g/L of BHT to all solvents.
Grind dried alfalfa to pass through 1-mm mesh screen and thoroughly mix. Accurately weigh 1-2 g sample in a glass stoppered boiling tube. Add 30 mL dichloromethane:acetone (2:1) and shake. Let it stand overnight. Add 20 mL of dichloromethane, 2 mL of 40% (w/v) methanolic potassium hydroxide, shake and let it stand for 60 minutes. Add 30 mL of 10% (w/v) aqueous sodium sulfate, shake and let it stand for 60 minutes. Remove an aliquot of the lower layer and centrifuge for 10 minutes at 2,000 rpm. Remove a 3-mL aliquot of the lower yellow layer, mix with 3 mL acetonitrile and use this solution for analysis.
10. ORGANIC ACIDS, LIPIDS AND RELATED COMPOUNDS
Aqueous extracts of plant tissues contain large amounts of organic acids. Citric, malic, tartaric, succinic and oxalic acids are usually the most abundant and the wide distribution of these particular acids is well recognized. These acids accumulate in plant cell vacuoles, sometimes in considerable amount, such as in Crassulaceae, where high quantities of malic, citric and isocitric acids are accumulated in the vacuoles during the day (Kluge and Ting, 1978). Plant organic acids can contain a variable number of carboxylic groups. Other chemical functions, such as hydroxyl and keto, can be present. Some widespread organic acids are present in small quantities in plant tissues, but have important plant functions. Oxalic, ascorbic, shikimic, quinic and monofluroacetic (inhibitor of the tricarboxylic acid cycle) acids are some of them. Among the dicarboxylic acids, malonic and succinic acids are worth mentioning. Plants also contain unsaturated and cyclic acids.
Determination of organic acids from plant tissues is difficult due to the diversity of acidic substances found in plants. Furthermore, the organic acids must be estimated in the presence of more or less mineral acids, a circumstance that places restrictions on the use of ordinary techniques.
Organic acids are water-soluble, colourless liquids with relatively low melting points. In general, they are non-volatile compounds. They are chemically stable, with the exception of a-ketoacids, which are easily descarboxylated. Their solutions in water are acidic and their presence is detected by the change in colour of acid-base indicators.
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