Preparation Of Enzyme Reactor Based On Thiol-ene Click Reaction And Double-doped Monolithic Column

Capillary monolithic column is a new generation of chromatographic stationary phase,which can be made by introducing many functional groups through changing the monomer of reaction.It has the advantages of easy preparation,easy surface improvement and good permeability.To adjust the reagents and conditions of reaction,changing the performance of the monolithic column is always the front topic of sample preparation and chromatographic technology for the growing requirements of complex system separation.In the first part of thesis,a monolithic enzyme reactor based on a strategy of green synthesis was successfully prepared in a capillary with trypsin immobilized by " thiol-ene " click reaction.A polymer of poly(butyl methacrylate-co-α-methacrylic acid-co-ethylene glycol dimethacrylate)was prepared in a mixture of 1-butyl-3-methylimidazolium tetrafluoroborate and choline chloride/ethylene glycol as the support of enzyme reactor.After “thiol-ene” reaction was used for enzyme immobilization,the Michaelis constants and maximum reaction rate of the resulting immobilized enzyme reactors(IMER)were determined by capillary electrophoresis to be 2.1 m M and 0.028 μmol/min,respectively.The enzymatic hydrolysis of the enzyme reactor under different experimental conditions were investigated.A on-line digestion of bovine serum albumin(BSA)on the new IMER can be achieved within 50 s,up to 864 times faster than in-solution digestion(12 h).BSA can be well digested and the numbers of identified peptides were 73 with the coverage rates of 82.7%.Further,a mixture of four proteins,consisted of Cyt C,My O,BHb and BSA,was passed through the enzyme reactor.The coverage of BSA,Cyt-C,BHb and My O was 36.08%,77.14%,80.69% and 85.06%,respectively.The IMER was further used for the analysis of protein extracts from rat liver,and 1034 protein groups were identified.All these results demonstrated that such a click reaction based IMER would be of great prospect in the high throughput analysis for proteome with high confidence.In the second part of the thesis,we successfully doped two nanomaterials,metal organic frameworks(MOFs)and carbon nanotubes(CNTs)into the monolithic column,and successfully used the green solvent ionic liquid as a porogen.A MOF(Nd)-SWCNT hybrid monolithic column was prepared.Metal organic frameworks(MOFs)were a chain or cluster crystal porous framework material formed by the coordination of metal ions and organic ligands through coordination bonds.They have a large surface area and adjustable surface chemistry features.Carbon nanotubes(CNTs)were divided into single-walled carbon nanotubes(SWCNTs)and multi-walled carbon nanotubes(MWCNTs)according to the number of layers of graphene sheets.The narrow diameter range of SWCNTs was conducive to the retention of small molecules in the stationary phase,thereby improved the separation between molecular.The separation ability of MOF(Nd)-SWCNT hybrid monolithic column was demonstrated by separating five groups of small molecular substances(phenol analogues,alkylbenzenes,polycyclic aromatic hydrocarbon,naphthalene substitutes,and aniline)in CEC mode.The synthesis conditions and chromatographic separation conditions of the monolithic column were investigated to improve the column efficiency and separation effect.The hybrid monolithic column separated alkylbenzene analogs with a maximum column efficiency of 246,000 plates/m,which was about twice that of a blank monolithic column.Therefore,the prepared MOF(Nd)-SWCNT hybrid monolithic column has better separation performance and can be used for the separation and analysis of many substances.