HPLC separation of cyanobacterial and algal photosynthetic pigments

Abstract

Photosynthetic pigments analysis has become a standard tool in ecological and physiological studies of photosynthetic organisms. With numerous methods previously published, there is no one ideal protocol that could satisfy all the research needs. Therefore, regarding the purpose of HPLC pigment analyses, a suitable method should be chosen. In this study, two C18 columns (i.e., LiChrospher 100 RP18e and Spherisorb ODS2) and three sets of eluents (based on acetone, acetonitrile, methanol, and water) were used to develop three separation protocols. They were then examined with respect to their resolution and sensitivity, by analyzing pigment extracts obtained from 10 cyanobacterial and algal cultures and two types of environmental samples, i.e. phytoplankton and microphytobenthos. All the protocols provided highly repeatable results and allowed for the separation of all taxonomically most relevant chlorophylls and carotenoids. They had similar resolution and sensitivity. The Syst1 method was the shortest, while Syst2 had better resolution of pigments in the middle part of the protocol when most of the diagnostic pigments are separated (i.e., alloxanthin, diatoxanthin, lutein, chlorophyll b). Syst3, on the other hand, enabled distinguishing the highest number of pigments, including their derivatives and degradation products. The results showed that all protocols may be used for routine analysis of cyanobacterial and algal pigments in various sample types.