Losses of chlorophylls and carotenoids in aqueous acetone and methanol extracts prepared for RPHPLC analysis of pigments

Summary

RPHPLC methods for analysis of photosynthetic pigments (chlorophylls and carotenoids) usually require addition of water to methanol or acetone extracts to prevent distortion of early-eluting peaks corresponding to the more polar compounds. In this work we have investigated the short-(<2 min) and long-term (up to 48 h) effect of adding water to acetone and methanol extracts from two marine phytoplankton species,Emiliania hyxleyi andDunaliella tertiolecta. Solvent extracts were prepared and separated into fractions that were subsequently diluted with water to 90%, 80%, 70%, 60%, 50%, and 40% for methanol, and the same range extended to 30% and 20% for acetone. Changes in pigment concentration with time were followed spectrophotometrically and chromatographically. Losses of pigments as a result of precipitation were clearly observed immediately after dilution of acetone extracts to 60% or less and methanol extracts to 80% or less. For chlorophyll a the most substantial losses were recorded for 50% acetone (up to 27% decrease) and for 70% methanol (31% decrease). This effect increased considerably with time. Only for 90% and 80% acetone were the initial concentrations of all the pigments unchanged after 24h, and even up to 48 h. In contrast, more than 60% and 57% of the initial amounts of chlorophyll a were lost after 24 h in 50% acetone and 70% methanol extracts, respectively. These losses increased to 83% and 60% after 48 h. There was a clear correlation between the polarity of a pigment and the polarity of the solvent at which maximum precipitation occurred. Losses of pigment from pure acetone and methanol extracts with time were also observed, although we attribute these to pigment degradation rather than precipitation. Some of the losses occurring with time can be avoided by use of autosamplers in which the sample can be mixed with water immediately before injection.