Protein In The Weak Cation Exchange Column Dual Retention Mechanism Studies And Applications

In the multi-step purification of proteins in biotechnology, two-dimensional and multi-dimensional liquid chromatography in analytical chemistry has become a powerful tool for separation and analysis of complex system. In this research, several columns with mixed weak cation-exchange (WCX) and hydrophobic interaction (HIC) mechanism were selected to study chromatographic behavior of proteins on them. By selecting a suitable mobile phase, some proteins usually required to separate with two dimensional chromatography can now be accomplished by only using the single column.The full text of study includes following four parts:Literature Review. This chapter summarizes liquid chromatographic method and research status of separation of biological macromolecules. It focuses on the research progress and significance of mixed-mode column in two-dimensional liquid chromatography (2D-LC). At the same time, the basic theory of liquid chromatography—stoichiometric displacement model for retention(SDT), short column theory and on-line buffer exchange has been briefly described.Experimental methods. The experimental instuments, reagents, experimental methods are explained in details.Optimization of the retention behavior of proteins on two-dimensional TSKgel CM-5PW column. It was found that all proteins have the character of both ion exchange and hydrophobic interaction chromatographic modes on this column with high resolution. We studied the retention role of the transfer process of proteins in the column using both kinetic and thermodynamic theory. Three linear equations from SDT were tested and found to fit well. All these studies of seperation characters and optimization of chromatographic separation conditions of proteins provide a possible selection that to employ the column for protein separation by 2D-LC.Effect of hydrophobic interaction to protein retention on WCX column and applications. The retention behavior of protein separation was investigated by using four commercial columns which have been popularly used in WCX. It was found that all proteins have the character of HIC mode on these WCX columns, but have different extents. The obtained elution curve exhibits "U" shape when the retention time was polted vs. the changes in the salt concentration in the employed mobile phase. This factor indicates that the existence of the hydrophobic interactions contributes to the protein retention and may change the eluted order of protein separation on these WCX columns. This phenomenon was qualitatively explained from the standpoint of molecular mechanism. In addition, two linear equations in the SDT were employed to test the character of interaction forces between the proteins and the stationary phase. One of the columns PolyCAT A^TM was used for two-dimensional rapid separation and purification of insulins in the cow pancreas .