Chlorophyll and chlorophyll catabolite analysis by HPLC

Abstract

The most obvious event of leaf senescence is the loss of chlorophyll. Chlorophyll degradation proceeds in a well-characterized pathway that, although being common to higher plants, yields a species-specific set of chlorophyll catabolites, termed phyllobilins. Analysis of chlorophyll degradation and phyllobilin accumulation by high-performance liquid chromatography (HPLC) is a valuable tool to investigate senescence processes in plants. In this chapter, methods for the extraction, separation, and quantification of chlorophyll and its degradation products are described. Because of their different physicochemical properties, chlorin-type pigments (chlorophylls and magnesium-free pheo-pigments) and phyllobilins (linear tetrapyrroles) are analyzed separately. Specific spectral properties and polarity differences allow the identification of the different classes of known chlorins and phyllobilins. The methods provided facilitate the analysis of chlorophyll degradation and the identification of chlorophyll catabolites in a wide range of plant species, in different tissues, and under a variety of physiological conditions that involve loss of chlorophyll.