The Determination and Quantification of Photosynthetic Pigments by Reverse Phase High-Performance Liquid Chromatography, Thin-Layer Chromatography, and Spectrophotometry

Chorophylls and carotenoids are functionally important pigment molecules in photosynthetic organisms. Methods for the determination of chlorophylls
a
and
b
, β-carotene, neoxanthin, and the pigments that are involved in photoprotective cycles such as the xanthophylls are discussed. These cycles involve the reversible de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin, as well as the reversible de-epoxidation of lutein-5,6-epoxide into lutein. This chapter describes pigment extraction procedures from higher plants and green algae. Methods for the determination and quantification using high-performance liquid chromatograpy (HPLC) are described as well as methods for the separation and purification of pigments for use as standards using thin-layer chromatography (TLC). In addition, several spectrophotometric methods for the quantification of chlorophylls
a
and
b
are described.