The Synthesis Of Task-specific Ionic Liquids And Its Application In Protein Extraction Separation

Proteome was a protein of a genome or all a cell expression. Proteomics is aboutquantitatively, dynamically, to study biology in protein level. As part of the organism,proteins play an important role in life activities, such as metabolism, gene expression,signal transduction etc. Most of proteins can be used for treatment or diagnosisapplication. It is necessary to preparation of purey protein samples which does notcontain any pollutants. Therefore, separation and purification of protein is one of themajor areas of biological technology in the process of modernization.As a “green solvent”, room temperature ionic liquids have received great attentionbecause of many unique physical and chemical properties. Task-specific ionic liquidswas a new kind of ionic liquids. It has particular physical and chemical properties bychanging its chemical structure. Task-specific ionic liquids have been used in organicsynthesis, electrochemical and analytical chemistry et al. In analytical chemistry, itwas mainly used for CO2adsorption and extraction of metal ions, and relatively fewreports in the extraction separation of biological macromolecules.In this paper, Task-specific ionic liquids which we have synthesized were used forextract solvent. Modern sample pretreatment techniques （the aqueous two-phasesystem extraction） were serve as the extraction means to extract protein. In this study,we researched various factors on extraction process, and determined the optimalconditions of extraction method. The paper was carried out mainly as the followingaspects:1. Amine functionalized ionic liquids-aqueous two-phase system extractionof ovalbuminIn this work, a new green solvent ionic liquid （[NH₂-pmim]Cl） was used to extractthe target ingredient of ovalbumin by the aqueous two-phase system extraction. Fourkinds of ionic liquids including [bmim]Br,[bmim]Cl,[bmim]BF4and [NH₂-pmim]Clfor extracting ovalbumin were compared in this work. Extraction conditions of[NH₂-pmim]Cl/K2HPO4system were optimized. The mechanism of extractionmethods was also studied. And the extraction conditions of ILMAE including theconcentration of ionic liquid aqueous solution, salt concentration, extractingtemperature and extracting time were mainly investigated for extracting ovalbumin.And the obtained optimal conditions of ionic liquid-based aqueous two-phase system extraction were as follows:0.6g/mL of [NH₂-pmim]Cl solution concentration,0.8g/mL of salt concentration,20min of extraction time,30℃of extraction temperature.Qualitative and quantitative analysis of ovalbumin was determined by UV at235nm.The linear range from0.1to1.0mg/ml of ovalbumin solution was good, and the linearregression equation were Y=0.6502X+0.00589（R2=0.9995）. Repeatability andintermediate precision were both satisfactory. Furthermore, the mechanism of ionicliquid-based aqueous two-phase system extraction was preliminarily studied by meansof Ultraviolet spectrum, Infrared spectrum, Dynamic laser scattering and Transmissionelectron microscope techniques.2. Choline-like Ionic liquid-based aqueous two-phase extraction of proteinIn this work, we designed and synthesized a Choline-like Ionic liquid which havetwo hydroxys.[OH-choline]Cl-based Aqueous Two-Phase （ATP） System technique hasbeen developed for the effective extraction of BSA. Compared with the effects of fourdifferent ionic liquids used in aqueous two-phase system, Choline-like Ionic liquid wasselected as the suitable solution for Bovine serum albumin. Based on the single-factorexperiment, serial significant operating parameters of ILs-ATP were respectivelyoptimized by ultraviolet spectroscopy （UV）. Experimental results show that theobtained optimal conditions were as follows:0.6g/mL of [OH-choline]Cl solutionconcentration,0.7g/mL of salt concentration,25min of extraction time,25℃ofextraction temperature. The experiment indicated that the linear range from0.2to2.5mg/ml of BSA solution was good, and the linear regression equation wereY=0.61093X+0.0113（R2=0.9992）. Furthermore, the mechanism of ionic liquid-basedaqueous two-phase system extraction was preliminarily studied by means ofUltraviolet spectrum, Fluorescence spectra, Infrared spectrum, Dynamic laserscattering and Transmission electron microscope techniques. Under the optimalconditions, the ovalbumin, trypsin and lysozyme were extracted by the method. Theextraction rate was96.85%and103.6%（n=3）.All these results showed that this studycould provide a new approach for efficient separation of protein.3、Synthesis of Magnetic Molecularly Imprinted Polymer and its applicationin separation and analysis of rutin from Chinese medicinal plantsIn this work, we designed and synthesized a hyamine-like Ionic liquid whichhave epoxy functional group. The cost of [（Bu3N）-PO]Cl was cheap.[（Bu3N）-PO]Cl-based Aqueous Two-Phase （ATP） System technique has been developed for theeffective extraction of trypsin. Compared with the effects of four different ionic liquidsused in aqueous two-phase system,[（Bu3N）-PO]Cl was selected as the suitable solution for trypsin. Experimental results show that the obtained optimal conditionswere as follows:0.6g/mL of [OH-choline]Cl solution concentration,0.5g/mL of saltconcentration,15min of extraction time,30℃of extraction temperature. Under theoptimal conditions, the extraction of trypsin was92.56%。The linear range from0.1to1.0mg/ml of ovalbumin solution was good, and the linear regression equation wereY=0.12409X+0.00353（R2=0.9996）. Repeatability and intermediate precision wereboth satisfactory. Furthermore, the mechanism of ionic liquid-based aqueoustwo-phase system extraction was preliminarily studied by means of Ultravioletspectrum, Fluorescence spectra, Infrared spectrum. All these results showed that thisstudy could provide a new approach for efficient separation of protein.