Phytoplankton pigment biomarkers: HPLC separation using a pentafluorophenyloctadecyl silica column

The use of pigment data to map microalgal populations in natural waters has become an established and convenient way of studying phytoplankton. However, the chromatographic analysis of algal pigments is a major challenge due to the diversity of molecules spanning a wide range of polarities, but also including many that have closely similar chemical structures. Among these sets of compounds of similar structure, the separation of mono and divinylic pairs of chlorophylls is of particular importance due to their relevance as chemotaxonomical biomarkers. In this work, we have taken advantage of the special type of solute-stationary phase interactions provided by pentafluorophenyl phases together with the high retention values resulting from an octadecyl spacer in the column to develop a method for the joined analysis of chlorophylls and carotenoids. The mobile phase contains organic solvents of low toxicity, methanol and ethanol and ammonium acetate buffer, in a simple binary elution gradient. More than seventy photosynthetic pigments (chlorophylls, carotenes and xanthophylls) can be determined in the same chromatographic analysis employing the method here presented. The complete resolution of mono and divinylic forms of chlorophylls a, b and c is achieved in less than 42min. The same analysis allows the separation of most chemotaxonomically important carotenoids, including positional isomers. The method can successfully be applied to the characterization of the pigment composition of members of different classes in the main chloroplast lineages (red and green) of the evolution of photosynthetic eukaryotes, in the study of biosynthetic processes of chlorophylls and especially in the description of plankton populations in natural waters. It is particularly suited for the simultaneous detection of green algae and the cyanobacteria of outstanding global importance Prochlorococcus marinus.