Technical Note: Rapid Normal-phase Separation of Phytoplankton Lipids by Ultra-High Performance Supercritical Fluid Chromatography (UHPSFC)

Abstract. Lipid metabolism is one of the cornerstones of biochemistry, and these chemically diverse biomolecules play key roles in molecular physiology and mediate interactions between microbes and their environment that play out on cell ular to ecosystem scales. Marine phytoplankton fix in the range of 1 billion tonnes of carbon as lipid biomass each year, which goes on to fuel higher trophic levels or ends up in the marine dissolved organic matter pool. Yet despite the importance of the vast marine lipidome for global biogeochemistry, surprisingly little is known about its diverse inventory of molecular structures, or the influence that dynamic environmental conditions exert on microbial lipid synthesis, remodelling and turnover.

To aid in this research, a high-throughput platform for comprehensive analysis of phytoplankton lipids was developed using Ultra-High Performance Supercritical Fluid Chromatography (UHPSFC). This recently developed technology combines a primary supercritical fluid (CO₂) mobile phase with an organic co-solvent of choice. Using a simple 10 minute gradient and a sub-2 μm particle column, UHPSFC efficiently separates all of the major neutral and polar lipid classes encountered in phytoplankton in a single analysis. These can then be measured by tandem mass spectrometry using established precursor and neutral mass loss scans.

To demonstrate the analytical power of this novel platform the lipid compositions of a diverse range of phytoplankton species grown in culture, as well as phytoplankton community samples from the Western Antarctic Peninsula, were analysed. With higher chromatographic resolution and a much shorter analysis time than current liquid chromatography methods, the application of U HPSFC has considerable potential to benefit large-scale lipidomic studies, including in the field of environmental microbiology.