A biphasic system based on guanidinium ionic liquid: Preparative separation of eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester by countercurrent chromatography.

Abstract

eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are high nutritional components. Evidence for unique effects of them is increasing. Further understanding of their independent biological functions urgently needs more efficient separation techniques. Nowadays, most of the commercially available fish oil products are the mixture of eicosapentaenoic acid ethyl ester (EPAEE) and docosahexaenoic acid ethyl ester (DHAEE). It will be convenient to directly separate esterified epa and DHA without saponification pretreatment. However, it is of great challenge to separate EPAEE and DHAEE because of their extremely fat-soluble nature and the equivalent chain length rule. In this research, the suitability of green guanidinium ionic liquid (IL) in countercurrent chromatography (CCC) solvent system for the separation of them was evaluated for the first time. Compared with imidazolium IL and phosphonium IL, guanidinium IL based non-aqueous biphasic system showed more outstanding separation performance. The separation mechanism was elucidated in depth through quantum mechanical calculations. It was found that guanidinium IL acted a crucial role in the CCC separation, which resulted in difference of partition behavior of EPAEE and DHAEE via different hydrogen-bonding affinity. EPAEE and DHAEE were successfully separated by solvent system (n-heptane/methanol/propylguanidinium chloride ([C(3)Gun]Cl, 1:1:5%, v/v/m)) with high purity (>95%) in one step, which was not achieved beforehand. Moreover, an easy recycling procedure of IL had also been devised, which significantly reduced waste generated. It opens up a new way for reasonable design water-free two-phase solvent system for efficient separation of very non-polar lipid compounds.