HPLC - PDA ANALYSIS OF CAROTENOIDS AND CHLOROPHYLLS FROM CUCURBITA PEPO L.CONVAR.GIROMONTINA FRUITS

Abstract

A reversed-phase high-performance liquid chromatographic (HPLC) procedure, which operates with gradient elution and detection by a photodiode-array detector, has been developed to analyze both carotenoids and chlorophylls from the fruits of Cucurbita pepo L.convar.giromontina. HPLC analysis was performed on a system consisting of a 322 Kontron Instruments pumping system, a Rheodyne 7125 injection valve with 20 l loop, a Waters 990 photodiode array detector and a computer running Waters 990 software for data analysis. HPLC separations were achieved on a Nucleosil 120 - 5 C18 column, using the following mobile phases: A - acetonitrile : water (9 : 1) and B - ethyl acetate. The flow rate was 1 ml/ min. and the solvent gradient was as follows: initial conditions - 90% A, 10%B/ from 0 to 15 min. - 30%A, 70% B/ from 16 to 22 min. - 90%A, 10%B. Separations were monitored at 450 nm; peak identities were established by comparing their HPLC retention times and their on-line recorded VIS spectra with those of known reference compounds. Eleven carotenoids were identified: the major ones are -carotene and lutein, these being followed by smaller amounts of neoxanthin, violaxanthin, lactucaxanthin, -cryptoxanthin, -cryptoxanthin, -carotene 5,6-epoxide, -carotene, 9Z--carotene and 15Z--carotene. Besides carotenoids, the proposed procedure separates also chlorophylls a, a’, b, b’, pheophytins a, a’, b and piropheophytins a and b which were identified in unsaponified extracts based on their VIS spectra.