The Research Of Extraction And Its Mechanism Analysis Of Biological Active Material By Ionic Liquids Aqueous Two Phase

Aqueous two phase extraction （ATP） is a new separation technology of extractionand purification of biologically active substances. The mild operation condition, easilyamplification and continuous operation are the advantages of the aqueous two phaseextraction. Ionic liquid aqueous two phase separation system （ILs ATPS） is anefficient mildly extraction which was found after polymer aqueous two phase. It isdifferent from the traditional ATP technology. The aqueous solution of hydrophilicionic liquids and inorganic salt of high concentration are mixed, and then the twoimmiscible phases could form. The advantage of ionic liquids （ILs） and the edge ofaqueous two phase were emerged effectively. It not only has the characteristics ofnontoxic, safe, convenient, fast, but also could accommodate the pH and solubility,uneasily emulsificated, get a more clearly interface. Owing to maintain the advantageof the biological activity and the stable conformation, the technique arouses more andmore attention.As the research object, the hyperin and isoquercitrin of Apocynum venetum ofsmall molecule substances and macromolecules protein were applied in the ILs ATPS,and its mechanism was also studied. The specific contents are as follows:1ILs-based microwave-assisted extraction （ILs-MAE） coupled with aqueoustwo-phase for the extraction of hyperin and isoquercitrin from Chinese medicineILs MAE of medicinal or useful compounds from Apocynum venetum was investigated as an alternative to conventional organic solvent extractions. The extraction and the preconcentration of ATP systems have been integrated. Variousoperating parameters were systematically considered by single factor and L9（34）orthogonal array experiments.0.2g/mL1Butyl3methylimidazolium tetrafluoroborate （[bmim][BF₄]） has been selected to extract Apocynum venetum. As the solid liquid was set at0.02g/mL, the Apocynum venetum was extracted in60oC during10min. And then, the extract was converted to the top phase by [bmim][BF4]/NaH2PO4system which was suitable for the preconcentration. Reversed phase high performance liquid chromatography （RP HPLC） with ultraviolet detectionwas employed for the analysis of hyperin and isoquercitrin in Apocynum venetum. The optimal experiment approach could provide higher detection limit of hyperin and isoquercitrin which were3.82g/L and3.00g/L in Apocynum venetu m. The recoveries of hyperin and isoquercitrin were64%and62%. Methodological study results indicate that: were0.75%and0.59%; the RSD of the reproducibility were3.2%and2.9%; the RSD of the stability were0.49%and0.96%.Moreover, the extraction mechanism research results show that the extraction rate of ILs MAE mainly depends on the microstructure damage by microwave, overlong extraction time is too long to extract hyperoside and isoquercitrin.2Extraction and separation of protein by ILs-ATPSA satisfactory protocol of extraction and ultraviolet detection has been establishedbased on ionic liquid aqueous two phase system applied in the analysis of Bovineserum albumin （BSA）, Hemoglobin （Hb） and Lysozyme （Lys）. The results show thatthe BSA of277nm, Hb of404nm and Lys of280nm were determined as theultraviolet detection wavelength. The extraction effect of imidazolium ionic liquid wasbetter than that of guanidine and morpholinium ionic liquid, and the longer alkyl chainwas better for protein extraction. Octyl3methylimidazolium Bromide （[omim][Br]）was choosed as the suitable ionic liquid in the present work. This experiment selected3.0mmol ionic liquid aqueous solution,55%K₂HPO₄solution, the sample amount isdetermined as10mg/mL, the pH value was fixed at the optimal condition, extractioncan be extracted in10min at45oC. The recovery of BSA was96.90%（RSD=2.8%）,the recovery of Hb was97.83%（RSD=2.9%）, the recovery of Lys was97.14%（RSD=2.4%）. The recovery and reproducibility of this method were get the satisfactoryresults, and it was suitable for detection in the mixed samples of BSA and Hb. Theseparation of protein method has exhibited potential value.3The research of mechanism extraction and separation of protein byILs-ATPSUltraviolet spectrum structure was used to analysis the structure of BSA, Hb, Lysand trypsin （Try） before and after extraction. The result was that protein has notchanged significantly. The Try was stored in2h and the storage temperature was set at45oC. The higher the temperature, the ATPS on the top phase was become darker.Therefore, the high storage temperature could affect the structure of protein. Thenormal temperature is more benefit to preservation the [omim][Br]/K₂HPO₄systemafter extraction. In order to further study on the mechanism of protein extraction, theconductivity of the aqueous solution of ionic liquid [omim][Br] were determined. TheCAC was determined at about0.07g/ml. By dynamic light scattering （DLS） andtransmission electron microscopy （TEM）, compared the cluster phenomenon of the topphase in the before and after ILs ATPS. The results show that the intensity of aggregates （300600nm） was larger than the cluster of the ionic liquid. Protein andionic liquid could form aggregates, which are involved in the partitioning of proteinbetween the ILs rich phase and phosphate phase.