Determination of chlorophylls by reversed-phase high-performance liquid chromatography with isocratic elution and the column-switching technique

Abstract

A reversed-phase high-performance liquid chromatographic (HPLC) system was designed for the separation and determination of chlorophylls and their degradation products. Two separation columns packed with octadecylbonded silica gel (ODS) and octadecyl-bonded vinyl alcohol copolymer (ODP) were employed with the aid of a column-switching technique. The ODS column (1 cm) functioned for the seperation of very hydrophobic compounds, such as chlorphyll a, chlorophyll b, pheo phytin a and pheophytin b, and the ODP column (15 cm) for separating the compounds of low hydrophobicity, such as chlorophyll c 1, chlorophyll c 2, pheoporphyrin c 1, pheoporphyrin c 2, pheophorbide a and pheophorbide b. The HPLC system could cope with a variety of hydrophobicities of chlorophyll pigments by means of isocratic elution with methanol-phosphate buffer (pH 3) (92:8, v/v). Application to several seaweed samples was demonstrated.