Study On The Synthesis And Applications Of High Performance Liquid Chromatography Stationary Phase Modified With 3-phenyl-propylamine

As a common method for rapid analysis and separation,High performance liquid chromatography(HPLC)has been widely used in different samples,such as small organic molecules,proteins and other biological samples.Studies on the synthesis and applications about mixed mode chromatography(MMC)stationary phase are the most important part for complex sample analysis and inclusion body(IBs)refolding.So synthesizing some new MMC stationary phase and studying the retention behavior of proteins is the key to understand MMC mechanism.In our previous work,we found that high performance hydrophobic interaction chromatography(HPHIC)stationary phase with phenethylamine ligand,which had phenyl as the hydrophobic group and amino as the charged groups,could be used for denatured lysozyme and recombinant human FLt3 IBs refolding.The denaturation efficiency had been improved significantly than the traditional HPHIC stationary phase.Further study on the MMC stationary phase with indole,carboxyl,and amino group showed a great refolding efficiency on recombinant human-Dll1-RGD(RGD-rhDlll)in HPHIC mode.The mass recovery was 68.6%,which compared with 36.4%when using traditional HPHIC stationary phase at the same time.In the present work,we synthesized a new HPHIC stationary phase with 3-phenyl-propylamine as the ligand.It could also provide weak anion exchanger.The stationary phase was used to separate protein and refold RGD-rhDlll inclusion body by HPHIC mode.Then,the obtained results were compared with stationary phase with phenylethylamine ligand(1)and stationary phase with tryptophan ligand(2),in order to figure out how could electrostatic force influence protein retention and protein refolding in HPHIC mode.This article includes four sections as following:1.Literature review Stationary phase is the crucial part for HPLC development.Traditional HPLC stationary phase can only provide a single mechanism for sample's separation,and it can not meet the need for complex sample analysis.Literatures found that the combination of multiple chromatographic separation modes can get better results.Thus,it is important to develop new MMC stationary phase and make its mechanism clear.This chapter focuses on some new stationary phases which based on HIC and IEC and their separation mechanism.2.The synthesis and separation performance of HPLC stationary phase modified with 3-phenyl-propylamineA new HPLC stationary phase based on silica was prepared with 3-phenylpropylamine as its ligand(3).It was characterized by elemental analysis and UV-Vis spectrophotometry to confirm that the ligand has been successfully,and applied for standard protein separation in HIC and WAX mode respectively.Six kinds of proteins(Cyt-C,RNase A,Lys,?-chyA,?-Amy,Ins)could be baseline separated by the new stationary phase(3)in HIC mode.But proteins can not be catched in IEC mode.The performance and retentions of same proteins were also observed by columns with stationary phase(1),(2).We found that pH could only affect the retention of protein with strong or weak hydrophobicity.3.The separation and purification of protein samplesThe stationary phase with 3-phenyl-propylamine ligand was applied for egg white,IgG sample and human umbilical cord mesenchymal stem cell sample.The purification result showed that OVA were obtained with high purity from egg white.The purity and mass recovery could reach 99.5%and 99.7%,respectively.And the purity of target protein in IgG samples could be raised from 50.1%to 96.3%.From human umbilical cord mesenchymal stem cell sample,we obtained a protein with the molecular weight of 65 kDa,which was F(ab')? fragments.4.Refolding with simultaneous purification of recombinant human Noth ligand Delta-like 1 by HPLC stationary phase with different ligandIn this study,we compared the refolding efficiency of RGD-hDll1 by three different kinds of stationary phase,and optimized the urea concentration,pH and ammonium sulfate concentration in the mobile phase.The results showed that on every column,the best mass recoveries could be obtained with 3.0 mol/L urea added in mobile phase.For(1),(3),pH 7.0 and 3.0 mol/L ammonium sulfate concentration were the best condition,when pH 7.5 and 2.5 mol/L ammonium sulfate concentration for(2)meanwhile.Under the optimal chromatographic conditions,the RGD-rhDll1 purity were:3-phenyl-propylamine(98.6%)>phenethylamine(98.3%)>tryptophan(97.9%),and mass recoveries were:tryptophan(69.3%)>3-phenyl-propylamine(62.4%)>phenethylamine(57.2%).Conformational changes were measured by circular dichroism spectroscopy analysis before and after refolding.