Preparation Of L-proline And Tetrazole-based Stationary Phase And Their Chromatographic Properties

The separation of solute in liquid chromatography was achieved within the chromatographic column, and the separation efficiency of solute was closely related to the property of stationary phase and mobile phase. Consequently, the preparation of stationary phase and the interaction mechanism between the solute, stationary phase and the mobile phase have being the hot research topics in the field of chromatography. Based on the previous research on ligand exchange chromatography and affinity chromatography, this dissertation attempts to give a new explanation on the chiral separation mechanism in ligand exchange chromatography, as well as synthesize a new tetrazole-based stationary phase. The whole work can be divided into the following three parts.1. The separation ofα-amino acid enatiomer on L-proline ligand exchange chromatographic stationary phase and the influences of pH, concentration of metal ion and salt in mobile phase, and temperature of column on the retention and resolution of solute were investigated. The contribution of salt in mobile phase to the retention of solute in the ligand exchange chromatography was quantitatively explained with stoichiometric displacement model for retention.2. Five tetrazole analogues were synthesized with Fmoc protected leucine, valine, alanine, isoleucine and phenylalanine as starting material. One novel tetrazole-based stationary phase was prepared by bonding the deprotected phenylpropyl tetrazole onto silica, and then chelating zinc(II). The separation property of protein on the prepared column and the effects of pH on protein retention were investigated preliminarily. The results indicated that the stationary phase had some separation ability to the mixture of proteins.3. One novel route to prepare tetrazole-based chromatographic stationary phases was founded by forming tetrazole ring on silica support. Compared to the first route that is to synthesize tetrazole analogues first and then bond it to silica, this method was simple and timesaving. The chromatographic property of proteins on this tetrazole-based column and the effects of pH on protein retention were investigated. The results showed that four proteins were separated well on both naked column as well as chelated zinc(II) column. The mass recovery of proteins on the column was more than 93.41%. In the two cases, the separation models of proteins belonged to immobilized metal ion affinity chromatography and affinity chromatography respectively. In addition, the retention times of proteins became longer on chelated zinc(II) column and the selectivity of protein on chelated zinc(II) column was different from that on the naked column obviously. It was suggested that the stationary phase can not only be used for the separation and purification of biological macromolecules but also can achieve the selective separation to proteins by chelating metal ion on the same column.