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

Abstract

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.

Introduction

Since the end of the 1970s the interest in analysing marine lipids has increased. The interest was originally focused on their role as energy reserve in marine organisms and they became useful as biomarkers in studies of marine foodchains. Today the analysis of marine lipids is more relevant than ever, mainly because of their potential role as carriers of environmental toxins. For example, it has been shown that the distribution of mainly particulate bound PCDD/F's and PAH's in the Baltic Sea is dependent on the lipid concentration in the particulate material (Broman et al., 1991). In a study of the accumulation of 40 different PCBs in phytoplankton it was established that the lipid composition had an important role in the accumulation process (Stange and Swackhamer, 1994). In later years, the toxical effects that appear in bloomings of certain species of algae have also been shown to relate to lipids (Yasumoto et al., 1990). The toxicity is associated with the presence of specific fatty acids, both in free form and esterified in glycolipids. This has motivated extensive studies of the lipid class composition of specific algae species (Parrish et al., 1994).

Lipids are divided into classes according to their structure and polarity, from nonpolar (hydrocarbons, wax- and sterol esters) via neutral (triglycerides, sterols) to polar (glyco- and phospholipids). Each class is further divided into molecular species according to their length of carbon-chains and degree of saturation. The lipid class composition in marine particulate material can be very complex. For example, in a review, Parrish (1988)described as many as 16 individual classes. The complexity of the lipid pool is explained by the large number of contributing species from the variety of microbial life forms in seawater. The pool can contain lipids from particles of highly varying size, e.g., bacteria, algae and protozoa, all having different membrane structures and metabolisms.

Most studies of marine lipid classes are based on different thin-layer chromatography (TLC) methods (Parrish, 1987; Olsen and Henderson, 1989; Volkman et al., 1989; Parrish et al., 1992) and solid phase extraction (SPE) procedures (Yongmanitchai and Ward, 1992; Ikawa et al., 1994). Eventhough these techniques are well developed and partly automated, they still have the disadvantages of being time-consuming and limited in terms of separation capacity. The use of HPLC instead of TLC offers the possibility of full automation and higher separation capacity. Another benefit of HPLC is that fractions containing single lipid classes can easily be collected for further identification and analysis of molecular species.

Evaporative light-scattering detectors (ELSDs) have greatly simplified the development of HPLC methods for lipid class separations (Christie, 1992). For example, this type of detector has been used with a ternary gradient system to separate all the simple and complex lipids from animal tissues on a silica gel column in a single chromatographic run (Christie, 1985, Christie, 1986).

Compared to animal lipids, the separation of plant lipids requires an improved selectivity to separate pigments and different glycolipids that are frequently occurring in vegetable tissues (and algae). Today, there are several alternative polar stationary phases to silica, such as diol, cyanopropyl and polyvinyl alcohol. Such chemically bonded stationary phases have been used successfully to separate lipids from potato tubers, rich in glycolipids (Christie and Urwin, 1995).

This paper describes an HPLC-based method for separation and quantification of lipid classes in marine particulate material, using a polyvinyl alcohol-bonded stationary phase and evaporative light-scattering detection.