Effect of Column Temperature on Improvement of Resolution in Separating Triglyceride Molecular Species Containing Highly Unsaturated Fatty Acids by Reverse Phase High Performance Liquid Chromatography

摘要

By taking advantage of the low melting point of fish oil, chilled-column reverse-phase high-performance liquid chromatography (HPLC) was examined in order to obtain good resolution of triglycerioe (TG) molecular species. TGs containing high proportions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were synthesized by using 90% pure EPA and DHA with a bacterial lipase. Four TG molecular species thus obtained were subjected to HPLC analysis, from which the difference in retention time, theoretical plate number, and resolution of the adjacent peaks were discussed in relation to the column temperature of the system. The lower the column temperature, the better the theoretical plate number and resolution. However, they reached a maximum at around -15°C. It was concluded that low temperature, wherever it is practicable not to exceed the melting point, is the best temperature for fish oil TG molecular species analysis and fractionation