Study On Purification Of Palmitoleic Acid From Anchovy Oil By Chromatographic Techniques

Palmitoleic acid(POA)is an omega-7 monounsaturated fatty acid with a variety of physiological benefits such as decreasing blood sugar,reducing blood lipids,combating inflammation,and regulating metabolism.The main natural sources of POA are macadamia nut oil and sea buckthorn fruit oil,followed by deep sea fish oil,with different fish oils containing between 5% and 15% POA.As of now,most fatty acid enrichment researches are concentrated on polyunsaturated fatty acids,and monounsaturated fatty acids enrichment technology,particularly the enrichment of POA using fish oil as a raw material,is still immature.In this study,ethyl anchovy oil was used as raw material.The crude oil was first been processed using composite solvent-low temperature crystallization method,and then was further purified utilizing high-speed countercurrent chromatography(HSCCC)and preparative high-performance chromatography(Prep-HPLC).This study provided theoretical justification for monounsaturated fatty acid enrichment as well as an experimental support for large-scale commercial preparation.The specific research contents and results were as follows:(1)The composite solvent-low temperature crystallization method was used as a pretreatment for the anchovy oil.The effects of eight single solvents on the content and recovery of POA were investigated: ethanol,acetonitrile,n-hexane,methanol,acetone,n-heptane,trichloromethane,and petroleum ether.Finally,acetone was selected to mix with ethanol,acetonitrile,n-hexane and methanol.The acetone-acetonitrile group of the four combined solvents performed better than the other three groups.A single-factor test was carried out to optimize the acetone-acetonitrile compound ratio,oil solubility ratio,crystallization temperature,and crystallization time,as well as to raise the sample size of fish oil to 7 and 17 times,respectively,and the POA recoveries were increased to 49.73±3.91%and 53.66±5.99%.The response surface method was used to further optimize the fish oil to solvent ratio,crystallization temperature,and crystallization time.The main saturated fatty acids in crude oil,palmitic acid(PA)and myristic acid(MA),were considerably fewer present in the oil,while the content and recovery of POA increased to 29.82±0.24% and 58.93±0.36%respectively.(2)The pretreated samples were further purified by HSCCC.The sample was extracted numerous times and for a lengthy period of time(> 24 h)with the low polar solvent n-heptane in order to prevent the influence of highly polar fat-soluble contaminants on the analysis and detection of liquid chromatography.After extraction,the sample’s color became lighter and the mass spectra revealed a material loss in the m/z 250～700 region.Two solvent systems were created: n-hexane-methanol-water and n-heptane-ethanol-acetonitrile.Based on the partition coefficient and the retention rate of the stationary phase,three successful solvent systems for HSCCC separation were chosen: n-hexane-methanol-water(14:7:0.06,v/v/v),n-heptane-ethanol-acetonitrile(20:7:13,v/v/v),and n-heptane-ethanol-acetonitrile(20:8:12,v/v/v).The POA content increased to 70.57%,49.62%,and 42.16% with the same rotational speed and mobile phase flow rate,respectively.The sample separated by the n-hexane-methanol-water(14:7:0.06,v/v/v)system contained POA and oleic acid(OA),which was chosen for further purification.(3)To separate POA and OA,high performance liquid chromatography technology was employed,and the chromatographic conditions were amplified to the Prep-HPLC through an amplification formula to realize large-scale POA preparation.The optimization of chromatographic conditions was carried out on Anal-HPLC.Taking the degree of separation and separation efficiency of POA and OA as indications,methanol-water was chosen as the mobile phase,and the methanol-to-water volume ratio,mobile phase flow rate,sample concentration,and injection volume were all optimized in turn.The degree of separation,separation efficiency,and elution time were within the acceptable range when using methanol-water(94:6,v/v),flow rate of 0.8 m L/min,sample concentration of 80 mg/m L,and injection volume of 15 μL.This condition was amplified to the preparation column,the flow rate was adjusted to 425 m L/min,the injection volume was 1275 mg,the final POA purity was94.89±0.68%,and the recovery was 65.66±0.45%.