Total marine lipid profiling by short column gas chromatography

Kehoe, Jonathan Joseph (2003) Total marine lipid profiling by short column gas chromatography. Masters thesis, Memorial University of Newfoundland.

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Abstract

Analysis of lipids is a very informative way of determining the physiological state of a marine ecosystem. The information derived from analysis of these hydrophobic, carbon rich compounds is very important to researchers in fields such as aquaculture and biological research. -- Thin layer chromatography with flame ionization detection (TLC-FID) has been a common method for identification and quantitation of the lipid classes of samples derived from marine sources such as sediments, plants and animals. However, TLC suffers from some analytical problems such as low sensitivity, non-linear calibration curves, long analysis times and use of copious amounts of hazardous solvent. -- Gas chromatography (GC) is an ideal choice for lipid analysis as it allows for automation, high sensitivity, short analysis times and low cost. Marine lipid samples often contain high proportions of polar lipid classes such as the phospholipids (PL) and the acetone mobile polar lipids (AMPL). While neutral lipid classes are readily analyzed by short column GC, AMPL retain on the GC column rendering the column useless after time. Thus GC determination of polar lipids requires enzymatic treatment and derivatization prior to chromatographic analysis. -- This project optimized a short column GC method for marine lipid class profiling by incorporating Kuksis' GC profiling strategy (1984) that used the enzyme phospholipase C to hydrolyze PL to diacylglycerols, with the optimized short column GC method for marine neutral lipids developed by Yang (1996). Combination of enzymatic hydrolysis and short column GC makes a near complete lipid profile of marine lipid samples possible. Hydrogenation of samples allows for compounds to be separated according to their carbon numbers and functional groups. -- In this project, the dephosphorylation procedure was optimized for marine samples, which were 50 units of phospholipase C for every milligram of phospholipid present in the sample. Comparison of percent lipid data obtained by short-column GC with Iatroscan TLC-FID data showed that equally accurate and sensitive data could be obtained. Hydrogenation of samples prior to analysis allows for excellent peak resolution and sensitivity to individual compounds within each lipid class. Achieving this information is not possible with TLC-FID when performing total lipid profiles. The pretreatment of samples resulted in 63.7 ± 3.7% recovery of samples, however the overall analytical precision was 1.7% error between replicate samples.

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