Effects of extraction on HPLC quantification of major pigments from benthic microalgae

The first crucial step in the chromatographic analysis of pigments by HPLC is the extraction of a sample. In this study, the extraction efficiencies of major pigments from a benthic microalgae community of an intertidal sediment by acetone and methanol are compared. The influence of extraction time and the use of sonication on pigment recovery is also examined. Extraction with acetone underestimated chlorophyll-c(2) and pheophorbide-a, while methanol extraction underestimated pheophythins-a and beta-carotene. In methanol, long extraction times (6-24 h) generally yielded lower pigment concentrations, possibly due to isomerization and/or pigment degradation. Acetone is a better option if long extraction times are used. For extraction times of 15 min, higher pigment concentrations were usually found in sonicated extracts. No significant effect of sonication was observed for longer extraction times (2, 6 and 24 h). There is no ideal method for pigment extraction and results are probably highly dependent on several factors such as the sediment type, the microphytobenthic community present, the solvent gradient and the HPLC column. Nevertheless, in view of the strong underestimation of chlorophyll-c(2) in acetone extracts, we recommend the use of 15 min extraction in methanol after 30 s of sonication for HPLC sedimentary pigment analysis.