Study On Ligand Selection And Application Of IEC/HIC Dual-function Stationary Phase

Mixed mode chromatography(MMC)is a new chromatographic technique which can improve separation ability,and make selectivity more unique and convenient in the separation process.In recent years,researchers have paid more and more attention for preparation,separation mechanism and the application of MMC.However,the selection of ligand is very critical in the synthesis of a dual-function stationary phase.A series of silica stationary phases based on silica gel for mixed-mode interaction chromatography have been synthesized and applied in the research of adsorption,retention mechanism and the separation of proteins.Based on the hydrophobic binding constant,the effect of the hydrophobic ligand of the dual-functional stationary phase on the separation of protein was discussed in detail.Some of the dual-functional stationary phases were applied in active protein purification from actual biological samples.The dissertation includes the following four chapters:1.Literature ReviewThis chapter introduced the advantages of the mixed mode chromatography over traditional mode chromatography.The comments focused on the preparation and application of the combined different types of ligands of mixed-mode stationary phase.2.Retention mechanism and adsorption properties of the dual-function stationary phaseThe mixed retention mechanism of proteins on two kinds of the dual-function stationary phases,such as SAX/HIC and SCX/HIC synthetized by our group,was investigated in detail based on the stiociometric displacement theory(SDT).The "U-shape" elution curves of protein on the dual-fuction stationary phase indicated that the dual-function column displayed IEC and HIC characters.By comparing the "U-shape" curves,it can be found if the column has good separation capacity under both IEC and HIC modes,the "U-shape" curves have good symmetry.Meanwhile,adsorption capacity of proteins was measured under different modes:in IEC mode,qm of consecutive and non-consecutive frontal chromatography were 112.63mg/mL and 87.71mg/mL,KL were 7.42 and 9.50,respectively;in HIC mode,qm of consecutive frontal chromatography was 270.27mg/mL and KL was 2.64.The effect of salt concentration on the adsorption capacity was also discussed.The result showed that with the increase of salt concentration,the adsorption capacity was presented as "U-shape".The experimental data was matched using Langmuir and Freundlich isotherm and it was found that Langmuir isotherm was available.3.The Effects of the hydrophobicity of the ligands of the dual-functional stationary phase on protein separationA series of SAX/HIC silica stationary phases were prepared using different alkyl homologous series,such as methyl,ethyl,propyl,butyl,heptyl,decyl,myristyl and octadecyl bromosilane as the ligands.We tested the separation performance to proteins of these stationary phases in IEC mode and HIC mode,respectively.The results indicated that the retention time of protein gradually increased with the increasing of the hydrophobicity of ligand,and the peak shape was gradually widen.In addition,the selectivity of stationary phase was influenced by the hydrophobicity of ligand at the same time.Under two modes,using benzyl as hydrophobic ligand of dual-function stationary phase,the dual-function stationary phase can display the best resolution and peak shape.The results indicated that the hydrophobicity of ligand to match the electrostatic interaction is the key to have good reolution under IEC and HIC modes.Combining hydrophobic binding constant,the theoretical guidance on the selection of ligand on dual-function stationary phase was offered.In addition,the mass recovery and active recovery of the eight kinds of dual-function stationary phases were determined.The results indicated that the stronger hydrophobicity was,the smaller mass recovery and active recovery were.Meanwhile,the retention mechanism of protein on the five dual-function stationary phase was discussed and illustrated by SDT theory.By comparing the "U-shape" curves,it indicated that the higher the critical point was,the stronger hydrophobicity of ligand becomes and the stronger the retention of proteins was.If the range of elution curve was wide,the peak shape was narrow,vice versa.4.The purification of the active proteins from egg white and milk using dual-function columnThree kinds of SAX/HIC dual-functional stationary phases modified with methyl,butyl,and myristyl,were selected for isolation and purification of ovalbumin and ovotransferrin from the egg white using on-line two dimensional liquid chromatography with a single column(2DLC-1C).The first dimension was SAX mode,and the second dimension was HIC mode.After off-line or on-line two-dimensional chromatography,the best purifaciton can be obtained using the dual-function stationary phase with butyl as ligand.The purity of ovalbumin was 96%,and the purity of ovotransferrin was 98%.The dual-function stationary phase with butyl as ligand also was used for the separation and purification active protein from milk.In SAX mode,only a-lactalbumin was purified and the purity was 97%;In HIC mode,?-lactoglobulin and a-lactalbumin were purified,the purity was 94%and 96%,respectively.