| Ionic liquids(ILs) are a group of green solvents with unique extraction properties,while counter-current chromatography(CCC) is an efficient liquid-liquid partition chromatography which has been widely used in bio-separation during recent years.However, the application of ILs in CCC sepration is still seldomly reported. In this paper,ILs and CCC were combined to separation non-polar tocopherol homologues and high polar anthocyanin in eggplant peel, the modification effect of ILs and their partition in traditional CCC solvent systems were explored. The main contents include:(1) Separation of tocopherol mixtures and plant oil extract using ILs-CCC solvent system.First of all, according to the polarity of tocopherols, a basic non-polar solvent system composed of hexane-methanol(5:5, v/v) was selected to separate a mixture of α, β,γ and δ tocopherol homologues. Then different ILs were added into the system to test their modification effects on the separation of tocopherols. The results showed that the suitable ionic liquid is 1-butyl-3-methylimidazolium chloride([C4MIM][Cl]), and using the ILs-CCC system n-hexane-methanol-[C4MIM][Cl](5:5:0.5, v/v) tocopherols can be well separated into α, β+γ, and δ three fractions at the sample amount lower than10.0mg/mL.When the amount of ILs increase to n-hexane-methanol-[C4MIM][Cl](5:5:3, v/v), the sample amount can be increase to 40.0mg/mL. Secondly, it is found that almost all of the ILs distribute between the lower phase of the solvent system and with the increase of[C4MIM][Cl] volume, the miscibility of two phases reduced, because the high cohesive energy of ILs. Finally, soybean oil was selected as a real sample to extract and separate tocopherols due to its higher amount of tocopherols among the tested vegetable oil samples.Three tocopherol fractions with HPLC purity higher than 95% can be obtained through the ILs-CCC system n-hexane-methanol-[C4MIM][Cl](5:5:0.5, v/v) under the 130mg/mL sample loading amount.(2) Separation of anthocyanin in eggplant peels using ILs-CCC solvent systems and removal of ILs from target.Eggplant peel pigment obtained by ultrasonic extraction was analyzed by HPLC and HPLC-MS, and an anthocyanin identified as delphinidin-3- rutinoside was locked as the target compound. Through the determination of of partition coefficient K value in different systems, a proper system composed of methyl tert-butyl ether(MTBE)-butanolacetonitrile-1%trifluoroacetic acid(TFA) water(2:4:1:5, v/v) was selected as the basic system. The effect of different ILs and the amount on the separation of target anthocyaninwere then investigated. The results indicated that the suitable ILs is 1-butyl-3-methylimidazolium hexafluorophosphate([C4MIM][PF6]), the optimum volume ratio is0.2. Compared with the basic solvent system, better separation can be achieved by the ILs-CCC system MTBE-butanol-acetonitrile-1%TFA water-[C4MIM][PF6](2:4:1:5:0.2,v/v), where 95.79%(HPLC 280nm) target can be obtained, while only 55.85% be obtained by the basic system. The distribution of ILs between the two phases of the basic system revealed that different ILs partitioned in both of two phases to different extent, the ILs containing [PF6]-are more preferably to distribute into the upper phase, and the longer cationic chain, the more hydrophobic, and the more distribution. Finally, the removal method of ILs were explored including organic solvent extraction, adsorption by inorganic absorbants and macroporous resin, only the macroporous resinwas effective to remove ionic liquids.In this paper, the advantages of the ILs extraction with mutiple selectivity and the CCC separation with high mass transfer efficiency were combined to form novel ILs-CCC separation systems for the separation of non-polar and polar compounds, which provide an alternative way for the research and development of bioactive compounds. |
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