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High Performance Liquid Chromatographic Analysis of Phytoplankton Pigments Using a C16-Amide Column
JAYARAMAN, S., M. L. KNUTH, M. G. CANTWELL, AND A. SANTOS. High Performance Liquid Chromatographic Analysis of Phytoplankton Pigments Using a C16-Amide Column. JOURNAL OF CHROMATOGRAPHY A. Elsevier Science Ltd, New York, NY, 1218(22):3432-3438, (2011).
Analysis of phytoplankton pigments using high performance liquid chromatography has been increasingly used to determine composition and biomass of phytoplankton. In this study, a reverse-phase HPLC method was developed to analyze most polar and non-polar chlorophylls and carotenoids present in phytoplankton. The method was able to resolve more than sixty pigments, ranging from very polar acidic chlorophylls to the non-polar carotenes in less than thirty-six minutes. The ability of this method to resolve important chlorophyll marker pigments is particularly important for the quantification and identification of prochlorophytes in oceanic waters. The described protocol is a significant improvement to existing methodology and the will help to better assess the distribution and dynamics of major phytoplankton groups in marine and freshwater ecosystems.
A reverse-phase high performance liquid chromatographic (RP-HPLC) method was developed to analyze in a single run, most polar and non-polar chlorophylls and carotenoids from marine phytoplankton. The method is based on a RP-C16-Amide column and a ternary gradient system consisting of methanol, acetonitrile and ammonium acetate. Under the gradient conditions employed, the C16-Amide column provides excellent peak resolution of most taxonomically important pigments and an elution profile similar to RP-C18 phases. Analysis of mixed pigment standards, extracts of phytoplankton monocultures and field samples show that this method is able to resolve more than fifty pigments, ranging from the very polar acidic chlorophylls to the non-polar hydrocarbon carotenes, in less than thirty six minutes. This includes chlorophylls c1, c2 and c3, and the carotenoids lutein and zeaxantin. The ability to completely separate divinyl chl b from monovinyl chl b and quantifiably resolve divinyl chl a from monovinyl chl a is unique to this method. The described protocol is rapid and reproducible and could be used to assess the distribution and dynamics of major phytoplankton groups in the ocean.