Zeta Potential Of Protein And Its Correlation To Separation Behavior Of Ion Exchange Chromatography

Chromatography is an efficent method to separate and purify biomacromolecules, which can provide high resolution. Among different chromatographic technology, ion exchange chromatography (IEC) is used most widelyl. The essence of ion exchange chromatography is the electrostatic interactions between target protein and functional ligand in the adsorbent. Zeta potential is a characteristic parameter indicating the electrostatic property of charged particles in solutions, and used to describe the electrostatic interations between charged particles. In the present work, the concept of zeta potential was introduced to the study of protein separation with ion exchange chromatography. Firstly, the measurement methods of zeta potential of protein and adsorbents were established, and some important factors, such as pH, and ionic type and ion concentration, were investigated. Then, the capacity factors were measured under varying fluid phase conditions, and used to characterize the protein separation during the chromatograpy process. Finaly, the relation between zeta potential and capacity factor was explored to bridge the microscopic electrastatic interactions and macroscopic protein separation behavior, which could certainly be used for chromatography process design.The thesis is divided into five sections. Section I is the review of zeta potential and ion exchange chromatography, including the history, principles and operation. Furthermore, the idea of research is put forward: aiming at the predicting difficulty of separation behavior of proteins in ion exchange chromatography, zeta potential would be introduced as an intermediate parameter to correlate the capacity factor in ion exchange chromatography with surface charge properties of protein.Section II is the analysis on zeta potein of protein. Bovine serum albumin (BSA) and lysozyme were used as model protein, and the influences of pH, ionic concentration and ionic types, surfactants on the zeta potential of proteins were investigated. The results indicated that zeta potential is useful and feasible parameter to characterize the surface charge properties of protein.Section III is the analysis on zeta potein of ion exchangers. Some typical ion exchange absorbents, such as DEAE Sepharose FF, Q Sepharose FF, CM Sepharose FF and SP Sepharose FF were used as model absorbents in the present work. The influences of pH, ionic concentration and ionic types, surfactants on the zeta potential of absorbents were investigated.Section IV is on the protein retention behavior during IEC. The capacity factor was chosen as the parameter to describle the protein separation behavior during chromatography process. Three chromatographic systems was investigated, including BSA/ DEAE Sepharose FF, BSA/Q Sepharose FF and lysozyme/CM Sepharose FF. The infuences of pH and salt concentration of fluid phase on the capacity factor were investigated.Section V is the relation between zeta potential and capacity factor. Considering the impacts of both protein (ï¿¡1 )and adsorbent ( ï¿¡2 ), the zeta potential parameter (-ï¿¡1*^2) was chosen as the intermediate parameter. It was found that there is a good linear relation between the parameter (-C,i*Qi) and the logarithm of retention factor under different pH and ionic concentration tested. The relationship obtained in the present work could be used to predict the separation behavior of protein in ion exchange chromatography with zeta potential measurement, and which would certainly improve the optimizing design of chromatography process.