Separation of lipid classes from marine particulate material by HPLC on a polyvinyl alcohol-bonded stationary phase using dual-channel evaporative light-scattering detection
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.
Section snippets
Materials
All solvents used in the mobile phases were HPLC or Analar grade, supplied by Merck (Kebo Lab, Sweden). N-ethyl morpholine (approx. 99%) was supplied by Sigma (Labkemi, Sweden). The chloroform was of high purity grade from Burdick and Jackson™, distributed by Fluka Chemie (Switzerland). Galactolipid, chlorophyll a and phospholipid standards were purchased from Sigma, whereas hydrocarbon, wax ester, sterol ester, sterol, triglyceride and sulphoquinovosyldiacylglycerol standards were from Larodan
Optimization of chromatographic parameters
Preliminary experiments to optimize separations were carried out on a diol phase (using marine particulate lipids) and a cyanopropyl phase (using potato lipids). The diol phase has shown a good performance for separation of phospholipids by others (Herslöf et al., 1990). We found it limited for our purposes since it required long conditioning periods between injections for adequate separation of nonpolar and neutral lipids, even 1 h was not enough to achieve reproducible results (unpublished
Acknowledgements
This research was supported by the Swedish Environmental Protection Agency, project number 20509, and in part by the Scottish Office Agriculture, Environment and Fisheries Dept. A grant from the foundation `In memory of Bengt Lundqvist' is gratefully acknowledged.
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