Zwitter-ion Modified Silica-A Novel Multi-interaction Stationary Phase For Protein Separation

Zwitterionic stationary phase contains both positive and negative charges and carries zero net charge.Zwitterionic stationary phase has excellent chromatographic performance and has been widely used for chromatographic analysis of the inorganic ions,organic ions,the polarity of small molecules,and peptide but zwitterionic stationary phase in reversed-phase mode are still rare,especially for the analysis of biological macromolecules.In this paper,we synthesized a series of zwitterionic stationary phase modified with histidine cysteine and reduced glutathione.The chromatographic performance on RPLC/IEC mode was tested for proteins separation.We carried out further research on mixed-mode chromatograph for retention mechanism of proteins using stoichiometric displacement theory(SDT)and were applied in protein separation from actual biological sample.The dissertation includes the following four chapters:1.Literature ReviewThis chapter introduced the advantages of the mixed-mode chromatography and different types of ligands over traditional mode chromatography.Then the preparation,properties and application of zwitter-ion-based stationary phase were reviewed as well.2.The preparation and chromatographic performance of mix-mode chromatography stationary phase modified with histidine cysteine and reduced glutathione.Three kinds of mix-mode chromatography(MMC)stationary phase based on silica gel was prepared using histidine cysteine and reduced glutathione as the ligand.These were used for the separation of biological macromolecules,results indicate that the three kinds of stationary phase has good selectivity,At pH = 8.0,three kinds of zwitterionic stationary phase has good selectively for bsaic protein,acid proteins retained on the stationary phase by the change of mobile phase pH,At pH = 6.0 cysteine column can separate the seven kinds of acid and basic protein and at pH = 6.0 glutathione column can separate the six kinds of acid and basic protein.These show special selectivity and better resolution for basic proteins as compared to commonly used commercial ODS column.In addition,the mass recovery of the three kinds of stationary phases were high.The influence of acetonitrile concentration,salt concentration and pH value was experimentally investigated.The results showed that with the acetonitrile concentration decreasing,the retention of proteins increases by hydrophobic interaction,and with the sodium perchlorate concentration increasing,the retention of proteins reduces due to the electrostatic interaction.Hydrophobic and electrostatic interaction effect the retention of proteins as varying the pH value and the selectivity of zwitterionic stationary is strongly effected by pH.3.The Effects of the hydrophobicity and electrostatic interactions of the ligands of the zwitterionic stationary phase on protein separationCysteine stationary phase were further modified using different alkyl homologous series,such as ethyl,heptyl,and octadecyl as the ligands.We tested performance of these stationary phases in RPLC/IEC mode for the separation of protiens.This demonstrate that three kinds of quaternary amine cysteine modified stationary phase at pH = 8.0 show specific retention for basic protein,while retention of acid protein can be achieved at low pH.Proteins retention time was studied by changing the organic solvent concentration,salt concentration and pH of the mobile phase.Then structure of ligands,hydrophobic binding constant and proteins retention time was investigated the hydrophobic and electrostatic interactions in RPLC/IEC mode.Therefore theoretical guidance was offered for the selection of ligand on mixed-mode stationary phase.Meanwhile,the retention mechanism of protein on the ethyl quaternary amine cysteine modified stationary phase was discussed and illustrated by SDT theory.Two different modes were set for the retention behavior of proteins.In the RPLC mode,the retention followed by SDT that show the chromatographic performance of the stationary phase in reverse phase.In the IEC mode,the retention satisfied the first set of linear equation.The results agreed with this idea-the electrostatic interaction is a type of selective force.As the R values were greater than 0.99 its better to use SDT to explain the retention mechanism of proteins in zwitterionic stationary phase.4.The separation of proteins from egg white?pig heart crude extract and milk on RPLC/IEC modeZwitterionic stationary phase were applied in protein separation from actual biological sample.The mixed mode stationary phase with ethyl at pH 8.0 and at pH 4.0 was used for the separation of Cytochrome C from pig heart crude extract and a-lactalbumin from milke,with purity 93.2%and 94.3%respectively.Due to specific pH synthetic stationary phase only retain and separate the basic proteins,therefore we build the offline two-dimensional chromatography technology with a single column by changing the mobile phase pH and successfully applied for separation of acidic and basic protein from egg white.First,at pH=8.0 two basic protein such as avidin and lysozyme from egg white were separated with the purity of 93.5%and 98.3%respectively,then acid protein like ovalbumin were separated at pH=4.0 with purity of 87.5%.In summary the synthesized stationary phase is suitable for the separation of proteins and can be applied for proteomic analysis.