HPLC Separation of Geometric Carotene Isomers Using a Calcium Hydroxide Stationary Phase

HPLC methodology employing a calcium hydroxide stationary phase selective for separations of phytofluene, zeta-carotene, gamma-carotene, alpha-carotene, and beta-carotene geometric isomer sets was developed. The retention behavior of certain members of these isomer sets and the resulting impact of modifier selection and concentration was demonstrated. Pertinent modifications for specific analytical situations are recommended. Individual purified carotenoids were isomerized by iodine catalysis. Resolution of phytofluene isomers was achieved using hexane, that of alpha-carotenes using 0.1% p-methylanisole in hexane, that of beta-carotenes using 2.0% p-methylanisole in hexane, that of zeta-carotenes using 1.4% benzyl ether in hexane, and that of gamma-carotenes using 2.45% benzyl ether in hexane. Retention characteristics on calcium hydroxide and electronic absorption spectra allowed assignment of geometric configurations to some of the resolved peaks. These methods may be used, with modifications where necessary, to analyze isomeric sets of carotenes and to isolate individual isomers.