Study On The Preparation And Application Of Chromatographic Stationary Phase Using Betaine As Ligand

The development of mixed-mode chromatography(MMC)can remedy the native defect of single chromatography in the separation of complicated samples.Among them,the selection of ligand for MMC stationary phase has become a hot area of research.Zwitterionic compounds are a kind of material with bionic structure,which attracted researchers’ interest in separation,analysis,purification and refolding of proteins.Because of its unique structure,the stationary phase prepared with this kind of material as ligand can provide a variety of forces and show obvious advantages.In the previous work,many research groups include us had used phosphorylcholine(PC)groups with natural structure and its derivatives 2-methacryloyloxy ethylphosphorylcholine(MPC)as chromatographic ligand to prepared zwitterionic exchange chromatography(ZIC),and hydrophilic interaction chromatography(HILIC),and HILIC/weak cation exchange chromatography(WCX)mode stationary phases respectively.The results prove that this kind of stationary phase could separate proteins as well as small molecules and showed good separation performance.In addition,there are obvious advantages for the refolding and simultaneous purification of inclusion bodies.In this work,in order to systematically study zwitterionic materials,we chose3-N,N-dimethyl-N-methacryloyloxyethyl ammonium(SBMA)and carboxybetaine(CBMA)as ligand to prepare the MMC stationary phase with ion exchange chromatography(IEC)and HILIC as well as other modes.The chromatographic properties of these two stationary phases were evaluated,and application of separation of small molecules and proteins,as well as refolding and simultaneous purification of inclusion bodies,and their separation mechanism and application value were preliminarily discussed respectively.These dissertations include four sections:1.Literature reviewIn recent years,MMC has been applied to many fields such as the detection,separation andanalysis of biomedicine,preparation of proteins,etc.The selection of ligand has become the key to the development of MMC stationary phase.Therefore,this chapter mainly reviews some of the MMC materials as well as their applications,and briefly discusses the mechanism.2.Preparation,characterization and performance evaluation of mixed mode chromatography stationary phase with sulphobetaine ligandAs the representative of sulfoammonium zwitterionic compounds,sulfobetaine has quaternary ammonium ion group and negatively charged sulfonic acid group.The surface of the sulfobetaine exhibits electronegativity over a wide p H range and can provide hydrophilic interactions,as well as ionic exchange.When the former was used as ligand,the separation of small molecules(melamine,benzene and homologues)can be achieved in HILIC mode,but the reports of latter are rare for the ionic exchange force of sulfonic acid groups is weak.In this experiment,we used sulfobetaine as ligand to prepare a silica-based MMC phase,abbreviated as SBMA-silica.Element analysis and thermogravimetry as well as Fourier infrared and XPS were used to characterized SBMA-silica and the result show SBMA was successfully bonded with a optimal ligand density of 1.03 μmol/m2 and dynamic adsorption of49.6 mg/g.The separation performance of SBMA-silica was evaluated by separating nucleosides,base mixtures,and standard proteins,respectively.The water content and p H of the mobile phase were examined to confirm that the stationary phase conformed to the retention rules of HILIC and SCX mode.3.Preparation,characterization and performance evaluation of mixed mode chromatography stationary phase with carboxylbetaine ligandIn order to study the effects of betaine ionic groups and hydrophobic interactions on protein separation simultaneously,the zwitterionic compound carboxybetaine was selected as ligand.This molecule has similar chemical structure to glycine with good biocompatibility.The distance between the anions and cations can be adjusted by the length of the alkyl chain.Compared with sulfobetaine,it contains cation group and hydrophobic interaction group.Firstly,the 1HNMR was used to characterize prepared(6-carboxypentyl)-3-acrylamidopropyld-imethylammonium(5-CBMA).Then the MMC stationary phase with HILIC,IEC,HIC and RPLC mode modified by the 5-CBMA was prepared and characterized by the element analysis,Fourier infrared and XPS.The result shown 5-CBMA was successfully bonded with the ligand density of 1.18 μmol/m2 and dynamic adsorption of 42.1 mg/g.The proteins and nucleosides were used to evaluate separation performance and the results confirmed that this stationary phase can achieve baseline separation of five nucleosides in HILIC mode,but the separation of proteins in IEC,HIC and RPLC mode are poor,which should be deeply investigated.4.The refolding and simultaneous purification of inclusion body by mixed mode chromatography stationary phase with betaine ligandProtein folding liquid chromatography(PFLC)is an ideal,efficient,and scalable method for protein refolding and simultaneous purification.However,the denatured proteins,especially E.coli-expressed inclusion bodies,are inactive and insoluble expect in strong denaturants(such as 8M urea or 6-7M guanidine hydrochloride).The hydrophobicity of inciusion body is strong and easy to re-aggregate that there were no retention or the retention is weak in many stationary phases,resulting in very low renaturation.Some studies,including our previous experiments,have found that multiple groups of the MMC stationary phase favor refolding and simultaneous purification of denatured proteins.The 5-CBMA-silica and SBMA-silica columns were used to purification and simultaneous refolding of the recombinant human Notch ligand delta-like 1(rh Dll1)inclusion bodies expressed in Escherichia coli(E.coil),respectively.The results showed that both of the columns can refold and purify the rh Dll1 inclusion bodies,but mass recovery of 5-CBMA-silica(34.6%)column was significant lower than that of SBMA-silica column(56.4%),whose mass recovery could reach up to 60.3% with urea concentration of mobile phase is 4mol / L with the purity of 98%.Fourthermore,the results of the circular dichroism spectra show that after one-step refolding and purification of the rh Dll1,the conformation conforms to the secondary structure(α-helix)characteristics.