COLORIMETRIC DETERMINATION OF CAROTENE IN PLANT TISSUE

The discovery by Borodin (2) in 1883 that the carotenoid pigments could be separated into alcohol soluble and petroleum ether soluble groups has been the basis for all the procedures that have been described for the determination of carotene and other carotenoid pigments. Arnaud (1), in 1887, extracted dry plant tissue with petroleum ether and used a colorimetric method for the estimation of the amount of pigment present, a carotene solution being employed as a standard. No attempt was made to separate carotene from other yellow pigments and the purity of the carotene standard was not given. In 1913, Monteverde and Lubimenko (9) reported a spectro-colorimetric method for the estimation of the pigments of green leaves, and in the same year Willstatter and Stoll (17) presented a method for the determination of carotene and xanthophyll which has served as the starting point for all subsequent modifications. The latter method consists essentially of acetone extraction of plant tissue, saponification of chlorophyll, separation of the carotenoids by means of petroleum ether and aqueous methyl alcohol, and the colorimetric estimation of the pigments. A petroleum ether solution of carotene or an aqueous solution of potassium dichromate served as a colorimetric standard. Coward (3), in 1926, modified the procedure by making the first step the decomposition of chlorophyll, which was followed by extraction with petroleum ether and the separation of carotene from xanthophyll by aqueous methyl alcohol. The use of diethyl ether in addition to acetone in the extraction of plant tissue was introduced by Schertz (12) in 1928. In 1923 he described a method (13) for the spectrophotometric estimation of carotene. Sprague and Shive (15), 1929, employed the method as modified by Schertz (12), except that they used petroleum ether rather than diethyl ether as a solvent for the carotenoids. These investigators (15) and Sprague and Troxler (16) developed a color standard of dye solutions for use in colorimetric readings. Pyridine has been employed by Smith and Smith (14) for the extraction of small quantities of fresh fruit, the pigments being transferred to petroleum ether. Recently, Kuhn and Brockmann (6) have described the use of petroleum ether and methyl alcohol in the extraction of plant tissue and the subsequent separation into 1 Journal Series paper of New Jersey Agricultural Experiment Station, Department of Agricultural Biochemistry.