Purification of omega-3 polyunsaturated fatty acids from fish oil using silver-thiolate chromatographic material and high performance liquid chromatography

• Isocratic separation of fatty acid ethyl esters (FAEEs) from fish oil.
• Baseline resolution between ω-3 PUFAs (EPA/DHA) within 5–10 min.
• Fundamental properties of the silver(I)-mercaptopropyl stationary phase examined.
• Eicosapentaenoic acid purity (EPA, >95%) and docosahexaenoic acid (DHA, >99%).

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have become increasingly popular in dietary supplements worldwide and global demand for higher purity ω-3 PUFAs in clinical applications has been rising rapidly in the recent years. Traditional methods for isolating ω-3 PUFAs however, generally require multiple, cumbersome steps to obtain high purity (>95%) products. In this study, we report an efficient liquid chromatography method for purifying individual omega-3 fatty acid ethyl esters (FAEEs), eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6), at >95% purity using a silver(I)-mercaptopropyl stationary phase, otherwise known as silver-thiolate chromatographic material (AgTCM). We demonstrate the key variables controlling separation of FAEEs with a silver(I)-thiolate stationary phase and examine important similarities and differences between silver-thiolate and silver-ion chromatography in regards to the separation mechanism taking place. We demonstrate the separation behavior of FAEEs under various conditions such as mobile phase composition, flow rate and sample injection amount. Purification of EPA and DHA was carried out on an analytical and semi-preparative scale and the resulting purity was determined by GC-FID. We report high attainable purity for EPA (>95%) and DHA (>99%) from a single isocratic separation of fish oil FAEEs within 5–10 min using a mobile phase of heptane/acetone (95:5, % v/v).