Synthesis And Characterization Of Temperature-responsive Stationary Phase And Their Application In Protein Separation

Temperature-responsive chromatography is a novel chromatographic method. Only adjusting the temperature of the separation process, can we change the separation efficiency of solutes. It has been gotten a lot of attention in the separation of small molecules. This paper has synthesized a series of temperature-responsive polymer with different low critical solution temperature (LCST), and further explore the relationship between salt concentration and polymers' LCST. The temperature-responsive polymer was bonded to the porous silica particles made into a thermo-sensitive hydrophobic stationary phase. We compared this new thermo-sensitive hydrophobic packing material with regular hydrophobic packings from the point of column efficiency, and disucssed the relationship between the temperature-responsive stationary phase and several factors, such as salt concentration, temperature and pH. Finally, this novel chromatographic method was applied to purification of the lysozyme from egg-white. This article consisted of three parts:1.Literature Review:This part briefly described the temperature-responsive polymers and the development and significance of the temperature-responsive chromatography. We detailedly discussed the principle of different types of temperature sensitive chromatography. Finally, the applications of the temperature-responsive chromatography was reviewed.2.Synthesis and characterization the temperature-responsive polymers:Through the copolymerization of N-isopropylacrylamide (NIPAm) and butyl methacrylate (BMA), we synthesized a series of temperature-responsive polymers with different LCSTs, the changes of the LCST of the temperature-responsive polymers was studied in different concentrations of ammonium((NH4)2SO4) and sodium chloride(NaCl) solution. It was concluded that the temperature-responsive polymers' LCST reduced, when the ionic strength in polymers' solution increased. 3.Synthesis, characterization and application of the thermo-sensitive hydrophobic stationary phase:We successfully synthesized a series of temperature-responsive stationary phase with different hydrophobicity. Calculation the dynamic adsorption of the thermo-sensitive chromatographic column was carried out by the frontal chromatography method, we found thermo-sensitive chromatographic column had good column capacity. We applied the thermo-sensitive chromatographic column to separate the intact proteins, and compared its separation efficiency with conventional hydrophobic stationary phase (PEG 600 and phenyl). From the research of the effects of the temperature,ammonium sulfate concentration, pH of mobile phase, we found that the temperature-responsive hydrophobic chromatographic stationary phase was more sensitive to temperature at a lower ammonium sulfate concentration. In a certain temperature range, the higher of the temperature, the better of the resolution of the temperature-responsive hydrophobic stationary phase. Thermo-sensitive stationary phase is more sensitive to pH of mobile phase compared with the conventional hydrophobic stationary phase. Finally the thermo-sensitive chromatographic stationary phase was used to the separation and purification of Lysozyme from egg-white, we found that it had a good resolution when temperature on a higher degree.