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

Highlights

• A novel biphasic system based on green guanidinium ionic liquid was developed.

• Successful separation was achieved with high loading capacity, yield and purity.

• Performance of ionic liquid was interpreted with help of σ-profile and σ-surface.

• Preparative separation was efficient without resolution loss and time extension.

• A simple method was proposed for recovery of targets and recycling of ionic liquid.

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 ([C₃Gun]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.

Introduction

Polyunsaturated fatty acids (PUFAs) of the omega-3 [(ω -3)] have obtained growing attention, owing to their beneficial impacts on human health, for example enhancing cognition [1], improving immune profile [2], optimizing neuromuscular function [3], and regulating blood lipid [4]. Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are the most researched ω -3 fatty acids which can be found in numerous dietary supplements, algae and oily fishes. They are necessary substances for the production of anti-inflammatory and inflammation resolving mediators [5]. However, increasing studies reported that EPA and DHA had largely different biological activity. Compared to EPA, evidence to date suggested that DHA significantly had more efficiency in the decrease of heart rate, platelet aggregation and blood pressure [6]. EPA but not DHA promoted protein synthesis and inhibited protein breakdown [7]. DHA has been shown to have indispensable and unique role in the neuronal membrane [8]. There are different anti-adipogenic activities of EPA and DHA in obesity [9]. A greater understanding of their individual roles in molecular mechanism and further exploring vast possible applications of different ω -3 PUFA in industry are not trivial. More individual EPA (DHA) is urgent to obtain. Several technologies have been used for enrichment and purification of fatty acid methyl esters such as solid phase extraction, membrane technology, solvents washing, decantation, and filtration. Most of them have proven to be inefficient, time and energy consumptive, and less cost-effective [10].

Countercurrent chromatography (CCC) is a kind of liquid-liquid partition chromatographic technology, which utilizes two non-miscible liquid phases to separate solutes based on different partition coefficient [11]. The diverse choices of two-phase solvent system avoid permanent adsorption of targets by eliminating the use of solid stationary phase. It has made great advancement in preparative separation and purification of active components. Moreover, CCC does not require expensive solid phase column, and it can use cheap solvents and gentle operating condition. These make it as a good choice for industrial production [12]. Separation of EPA and DHA has been developed by ordinary CCC using conventional solvent system, which achieved in two steps with unsatisfactory resolution [13]. pH-zone-refining CCC were also used to separate EPA and DHA, which is a technique for separating basic and acidic substances according to the differences of hydrophobicity and pKa values [14]. However, pretreatment of raw materials including saponification, acidification and extraction is obligatory before the separation of free EPA and DHA. 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 more convenient to directly separate esterified EPA and DHA without saponification pretreatment. Nevertheless, the almost same 1-octanol/water partition coefficients (logP) of EPAEE and DHAEE (7.642 ± 0.362 and 8.154 ± 0.375, respectively, SciFinder predicted property) and the effect of equivalent chain length (ECL) rule make their separation a great challenge [14].

Ionic liquids (ILs) are salts consisting of ions, which remain liquid at room temperature or near room temperature. In recent years, they have attracted extensive scientific and commercial interest because of their unique features, which are viewed as green alternative to traditional reagents [15], [16]. Their characters can be effectively tuned by random combination of abundant ions. Thus, ILs are labeled as ‘‘designer solvents’’, due to they can be tuned to tackle specific problems, for instance producing multiphasic solvent system to improve separation performance and allow simplified separations [17], [18]. Imidazolium IL or phosphonium IL is the preferred choice for many researchers working on separation approaches [19], [20], [21], [22], [23], [24]. But their poor biodegradability and photodegradability are still controversial issues [25]. It is necessary to comprehensively investigate more biocompatible and benign ILs with combination maximized task-specific superior properties and minimized environmental impacts [26]. Besides, it is of high importance to develop/explore procedures for both the targets recovery and IL recycling [27]. These are all essential for industrial and commercial utilization. Guanidinium IL is kind of more biocompatible and biodegradable material compared with universal used IL, which exhibits distinct molecular recognition, strong coordination property and outstanding catalyst efficiency [28], [29], [30]. Accordingly, it may be served as a more effective and green constituent in CCC solvent system for EPAEE and DHAEE separation.

Herein, a new biphasic system based on guanidinium IL was firstly developed for the preparative separation of EPAEE and DHAEE from commercially fish oil. The basic solvent systems, type of IL, alkyl chain length, IL content, sample loading capacity, preparative separation and IL recycling were investigated and optimized. The reagents used in the final solvent system of CCC were desirable and environmentally friendly solvents. High pure EPAEE and DHAEE can be simultaneously yielded in one cycle process.