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

RPHPLC methods for analysis of photosynthetic pigments (chlorophylls and ca rotenoids) usually require addition of water to methanol or acetone extract s to prevent distortion of early-eluting peaks corresponding to the more po lar compounds. In this work we have investigated the short-(< 2 min) and lo ng-term (up to 48 h) effect of adding water to acetone and methanol extract s from two marine phytoplankton species, Emiliania huxleyi and Dunaliella t ertiolecta. Solvent extracts were prepared and separated into fractions tha t 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, Chang es in pigment concentration with lime were followed spectrophotometrically and chromatographically. Losses of pigments as a result of precipitation we re clearly observed immediately after dilution of acetone extracts to 60% o r less and methanal extracts to 80% or less. For chlorophyll a the most sub stantial 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 pig ments unchanged after 24 h, 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 t o 83% and 60% after 48 h. There was a clear correlation between the polarit y of a pigment and the polarity of the solvent ai which maximum precipitati on occurred. Losses of pigment from pure acetone and methanol extracts with time were also observed, although we attribute these to pigment degradatio n 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.