Owing to their light-harvesting and protective functions', the chlorophylls and carotenoids of the photosynthetic membrane are of key interest in studies on thylakoid architecture2, chloroplast development3 and senescence4, adaptation to environmental conditions5 and effects of pollution-induced stres6. In higher plants, the pattern of plastid pigments is highly conserved. In addition to the porphyrin-derived pigments chlorophyll a, chlorophyll b and pheophytin u7, the chloroplasts generally contain p-carotene, lutein, violaxanthin and neoxanthin as major and lutein epoxide and antheraxanthin as minor carotenoidss. Zeaxanthin, formed from violaxanthin via antheraxanthin under light'," may be present at different levels depending on the light exposure of the plant. Only a few deviations from this general pattern have been observed; spruce needles, for example, further contain a-carotene" and Lactucu species and some closely related genera synthesize lactucaxanthin'2. Thus, the plastid pigments include groups of isomeric carotenoids with either B-/S, or #?-E-or E-Eend-groups (i.e., /3-or a-carotene, zeaxanthin or lutein or lactucaxanthin, antheraxanthin or lutein epoxide) and cover a wide range of polarity.
In recent years, high-performance liquid chromatographic (HPLC) techniques have facilitated the separation and quantification of plastid pigments, but fast and efficient one-step procedures for higher plants have not yet been fully developed. Clearly, separation procedures developed for carotenoids of similar polarity'33'4 are of little use for mixtures of plastid pigments. The HPLC systems in refs. 15-21 allowed the major groups of plastid pigments to be separated but were either not successful in or not tested for the separation of carotenoid isomers. With other procedures22*23 carotenoid isomers could be separated, but the pigment extracts examined contained no chlorophylls. In one studyz4 more than 40 chlorophyll and carotenoid pigments from phytoplankton species were separated. This procedure covered an extended polarity range and even resolved the highly polar algal pigments chlorophyll c, peridinin and fucoxanthin. Lutein and zeaxanthin were partially resolved, but the resolution of a-and p-carotene was not satisfactory.
In this paper a fast one-step separation of chlorophylls and carotenoids from higher plant chloroplast using HPLC is described. By modifying the HPLC system of