Optimization of an isocratic high-performance liquid chromatographic separation of carotenoids

Abstract

Using a polymeric C 18 high-performance liquid chromatographic (HPLC) column, which demonstrated excellent separation selectivity toward carotenoid compounds in an earlier column evaluation, the effects of mobile phase modifier, modifier concentration, and column temperature were investigated. A seven-component carotenoid mixture was used to monitor changes in separation selectivity in response to variations in HPLC conditions. Both acetonitrile and tetrahydrofuran (THF) improved the resolution of echinenone and α-carotene; THF was selected for use as a modifier due to its solvating properties. At concentrations greater than 6% THF, the resolution of lutein and zeaxanthin deteriorated significantly. Temperature was varied from 15 to 35°C in 5°C increments. Resolution of lutein/zeaxanthin and β-cartone/lycopene were better at lower temperatures while echinenone/α-carotene separation improved as temperature increased. An acceptable separation of all seven carotenoids was achieved at 20°C using 5% THF as a mobile phase modifier. Method applicability is demonstrated for serum and food carotenoids.