Research And Application Of Hydroxy Functional Ionic Liquids For The Separation Of Proteins

As the material basis of life and the various forms of life activities, Protein participates in the metabolism of organisms, gene expression, signal conversion and other biological phenomena. As a “green solvent”, ionic liquids have drawn much attention because of the many unique physical and chemical properties. Aqueous two-phase extraction technology is a new separation method for the extraction and purification of the biologically active substance. Functional ionic ionic liquid aqueous two-phase system?FIL-ATPs? is effectively combining the advantages of ionic liquids and aqueous two-phase extraction, which gets more and more attention due to the characteristics of nontoxic, safe, convenient and fast. Functionalized ionic liquids combine the magnetic solid phase extraction technique?FIL-MSPE? by modificated the functionalized ionic liquids on the surface of the magnetic nanoparticles, thus forming a particular function or application of a new type of solid phase extraction agent.In recent years, with the rise of the concept of "green chemistry", building a fast,accurate and high efficiency of protein separation, extraction and detection method is particularly urgent. In this work, hydroxy functional ionic liquid has been choosen as the extractant, with modern new sample pretreatment technique: FIL-ATPs and FIL-MSPE which is applied successfully for protein extraction of separation. The main research content is as follows:1. Extraction of proteins in hydroxyl ammonium ILs based ATPsHydroxyl ammonium ionic liquids have been designed and synthesized. An ionic liquid aqueous two-phase system based on hydroxyl ammonium ionic liquid was studied for the extraction of Bovine serum albumin?BSA?. After extraction, the concentration of BSA was measured by UV-vis Spectrophotometer at 278 nm. Based on the single-factor experiment and the orthogonal array experiments, the optimum conditions of ILs-ATPs were set as follows: the mass of ILs and K₂HPO₄ was 1.6mg and 2.2 mg, the concentrations of BSA was 5 mg/m L and 2 m L, the extraction temperature and time was carried out at 25 ? for 25 min. Under the optimal conditions the extraction efficiency could reach up to 99.5%. The RSD of extraction efficiencies in precision experiment, repeatability experiment and stability experiment were 0.27%,1.1% and 1.5%, respectively. The possible mechanism for the extraction of protein was investigated by UV-vis spectra, Fourier transform infrared spectroscopy?FTIR?,dynamic light scattering?DLS? and transmission electron microscopy?TEM?. The circular dichroism spectral experiment analysis indicated that the secondary structures of the protein were unchanged after extract.2. Extraction of proteins in hydroxy functional ILs based MSPEHydroxy functional ionic liquids have been designed and modied on the surface of silica-coated magnetic Fe3O4 nanoparticles?MNPs? for the investigation of the extraction performance of proteins by magnetic magnetic solid-phase extraction?MSPE? and characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, vibrating sample magnetometer?VSM?, X-raydiraction?XRD?and thermogravimetric analysis?TGA?. After extraction, the concentration of BSA was measured by UV-vis Spectrophotometer at 278 nm. Based on the single-factor experiment, the optimum conditions of ILs-MSPE were set as follows: the mass of[Simam][Cl]-MNPs was 60 mg, the concentrations of BSA was 0.5 mg/m L and 2 m L,the p H of the solution was 6, the extraction time and temperature was carried out for20 min under 30 ?. Under the optimal conditions the extraction efficiency could reach up to 86.9%. The RSD of extraction efficiencies in the precision, repeatability and stability experiments were 0.55%, 1.5% and 1.3%, respectively.The desorption of proteins was up to 94.9% when the concentrations of Na Cl were greater than1.1mol/L.3. Extraction of proteins in dual hydroxyl functional ionic liquid based multiwall carbon nanotubes MSPEA novel adsorbent based on silica-coated magnetic multiwall carbon nanotubes?MWCNTs? surface modied by dual hydroxyl functional ionic liquid?FIL? has been designed and used for the purication of lysozyme?Lys? by magnetic solid-phase extraction. Fourier transforms infrared spectroscopy, a vibrating sample magnetometer, transmission electron microscopy, X-raydiraction and thermogravimetric analysis were employed to. After extraction, the concentration of Lys was determined by a UV-Vis spectrophotometer at 278 nm. Based on the single-factor experiment, the optimum conditions of FIL-MSPE were set as follows:the mass of [OH]-FIL-m-MWCNTs@Si O2 MNPs was 10 mg, the concentrations of BSA was 0.5 mg/m L and 1m L, the p H of the solution was 6, the extraction time and temperature was carried out for 20 min under 30 ?. Under the optimal conditions the extraction amount could reach up to 94.6 mg/g. The RSD of the precision, the repeatability and the stability experiments were 0.37%, 0.47% and 0.52%,respectively. Comparison of different types of solid phase extraction agent for Lys extraction and [OH]-FIL-m-MWCNTs@Si O2 for different types protein extraction.The circular dichroism spectral experiment analysis indicated that the secondary structure of Lys was unchanged after extraction.