Separation of Chloroplast Pigments Using Reverse Phase Chromatography

Abstract

SOLVENT extraction and separation of chlorophylls and carotenoids by paper, thin layer chromatography (TLC), or normal phase column chromatography are common components of many high school biology programs and most general botany and plant physiology courses. Protocols for these experiments can be found in teaching journals and most laboratory manuals designed for these classes (Heim 1970; Dobbins 1972; Moore 1973; Storey 1980; Witham et al. 1986; Dean & Schuhmacher 1987; Reiss 1994; Fail 1995). The established methods are reliable and, accompanied by examination of fluorescence and absorption of the pigments, provide valuable learning experiences for students. The primary limitation of the established protocols is their reliance on toxic and highly flammable compounds such as acetone, carbon tetrachloride, chloroform, diethyl ether, hexane, and petroleum ether (Dobbins 1972; Moore 1973; Witham et al. 1986; Dean & Schuhmacher 1987; Reiss 1994; Fail 1995). Ethanol, which is less toxic and has a lower flash point than most of these solvents, has been used successfully for extraction of plant photosynthetic pigments (Storey 1980). The protocol described be-