Separation of lipid classes from marine particulate material by HPLC on a polyvinyl alcohol-bonded stationary phase using dual-channel evaporative light-scattering detection

Using gradient HPLC and evaporative light-scattering (ELS) detection, a variety of lipid classes from marine particulate material have been separated on a polyvinyl alcohol bonded stationary phase in a single run. These classes, which cover the entire polarity range, include a nonpolar group (e.g., hydrocarbons, sterol esters, wax esters), sterols, triacylglycerols, chlorophyll a, monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols, phosphatidylglycerols and phosphatidylcholine. An extract from a spring bloom sample in combination with separate lipid standards were used to optimize the chromatographic parameters. Nine classes could be identified and quantified in this extract, the major classes being triacylglycerols and nonpolar lipids. Detection limits attainable were in the range 16 ng for cholesterol to 820 ng for sulphoquinovosyldiacylglycerols. In an extract of the cyanobacteria Aphanizomenon sp. the dominating classes were found to be chlorophyll a, sulphoquinovosyldiacylglycerols, monogalactosyldiacylglycerols and nonpolar lipids. In a separate study, significant differences in lipid composition and concentrations were found between samples from various depths in the water column. The accuracy of the method was tested by comparing with gravimetric determinations. It was found to be acceptable provided that the main lipid classes can be identified and quantified. The ELS detector was modified electronically in such a way that two chromatograms with different gain factors could be obtained from the same run, thus halving the time for analysis for the majority of samples since evaluation of various lipid concentration levels often require that a sample is run twice with different gain factors. The time between injections, including column equilibration, was 55 min.