Effects of mobile phase ratios on the separation of plant carotenoids by HPLC with a C30 column

Plant products are dietary sources of lutein and zeaxanthin. Lutein and zeaxanthin have been implicated in the protection of age related macular degeneration (AMD) and in cardiovascular diseases. However, xanthophylls and unidentified components (lambda(max) = 423 and 468 nm) in plant products are often not separated well, and affect an accurate quantitative determination of lutein and zeaxanthin. A high performance liquid chromatography (HPLC) system equipped with a Bischoff C30 column and a mobile phase of methanol, metkyl-tert-butyl ether (MTBE) and water was used to separate lutein, zeaxanthin and other unidentified components in plant products. Mobile phase A containing methanol, MTBE and water with a ratio of 60:33:7 by volume (1.5% ammonium acetate, NH4Ac), combined with mobile phase B with a ratio of 8:90:2 by volume (1.0% NH4Ac) is optimal for the separation. This method was successfully applied to the quantitative determination of lutein and zeaxanthin in extracts of plant products, such as chlorella, spirulina, celery and mallow.