Preparation Of Immobilized Metal Ion Affinity Silica Hybrid Monolith And Its Application To The Separation Of Phosphorylated Polypeptide

Immobilized metal affinity monolithic column has the advantages of outstanding bio-compatibility and high affinity selectivity.This paper is based on the advantages of simple preparation process,high mechanical strength,acid and alkali resistance of hybrid monolithic silica column,using "one-pot" method and post-column modification to prepare the immobilized metal affinity hybrid monolithic silica column.Then,the chromatographic properties of the hybrid monolithic silica column and the separation of phosphorylated peptide in the column were studied.The main contents are as follows:Chapter 1 is a review.The classification of capillary column was summarized.The classification of capillary monolithic column and the research progress of hybrid monolithic silica column are introduced in detail.Then,the thesis selection basis and research program of this thesis were put forward.Chapter 2 is the preparation and evaluation of the immobilized metal ion affinity organic silica hybrid monolithic silica column,Based on "one-pot method",ethylene glycol phosphate methacrylate(EGMP)was used as functional monomer and polyethylene glycol(PEG-10000)and urea as crosslinking agents,and tetramethoxysilane(TMOS)and vinyl trimethoxy silane(VTMS)as silane agents,the poly(EGMP-co-TOMS-VTMS)hybrid silica monolithic column with surface bound phosphate ion was prepared by sol-gel one-step polymerization.Then,the morphology,structure,mechanical stability,reproducibility and hydrophilic retention mechanism of the hybrid column were investigated.The content of functional monomers in the hybrid column is analyzed by energy spectrum,According to the separation between benzoic acids and nucleoside base substances to explore the separation performance of the hybrid column on charged materials.Chapter 3 is the preparation of the poly(Zn2+-EGMP-co-TMOS-VTMS)monolithic column based on post-column modification,by bonding Zn2+ on surface of the poly(EGMP-co-TMOS-VTMS)monolithic column thought passing ZnSO4 salt solution into the poly(EGMP-co-TMOS-VTMS)monolithic column.Then,the mechanical stability and reproducibility of poly(Zn2+-EGMP-co-TMOS-VTMS)column were investigated.The effect of post-column modification was explored by comparing the changes of element content and electroosmotic flow of poly(EGMP-co-TMOS-VTMS)monolithic column before and after post column modification.Results indicate that mechanical stability of the poly(Zn2+-EGMP-co-TMOS-VTMS)monolithic column and its electroosmotic flow are enhanced.Therefore,the hybrid monolithic column has excellent performance in the enrichment and elution of tyrosine phosphorylated peptide.Chapter 4 is the preparation of the poly(ZnA-co-TMOS-VTMS)monolithic column by one-step polymerization,which based on "one pot method",in order to improving properties of the hybrid monolithic column in Chapter 3 through using Zn-Acrylate(ZnA)as monomer.Then,the optimization for the formula of monolithic column polymerization was carried out by analyzing the effect of the morphology and elemental analysis of monolithic column on phosphorylated polypeptide by skeleton.The prepared poly(ZnA-co-TMOS-VTMS)hybrid column has uniform structure and excellent mechanical stability after the optimization.And the content of zinc is high in the poly(ZnA-co-TMOS-VTMS)monolithic column.After that,the retention of phenylphosphonic acids on poly(ZnA-co-TMOS-VTMS)hybrid column was studied.Results show that the separation performance between benzoic acid and nucleoside bases with different charge is excellent.Finally,the results of on-line enrichment and elution by adopting Liquid Chromatography-Mass Spectrometry proved that the poly(ZnA-co-TMOS-VTMS)monolithic column is effective on the enrichment and elution of tyrosine phosphorylated polypeptide.