Preparation Of Phenylboronate Capillary Monolithic Columns And Its Application In Separating Small Molecules And Proteins

Monolithic column, also called rod column or fritless column, is acontinuous piece of material prepared by in situ polymerization. It hasdrawn considerable attention owing to its significant advantages such aslow back pressure, high loading capacity, versatile surface modificationsand simplicity of fabrication.4-vinylphenylboronic acid (VPBA),possessing a hydrophobic benzene ring and an ionizable boronic acidgroup, is a versatile compound. It has a unique chemistry feature that canreversibly form complexation with cis-diols to generate five-orsix-membered cyclic esters in a basic aqueous media while disassociate atacidic condition. Such a property of VPBA was exploited as an affinityligand to capture cis-diol-containing compounds. In addition, VPBA,served as a weak acid, can become cation-exchangers after being titratedto boronates at a pH above its pKa.In this work, we prepared a novel poly (VPBA-co-PEGDA)monolithic column. The resulting monolith demonstrated mixed-mode separation performances: boronate affinity, reversed-phase retention andcation-exchange interactions towards different analytes. Then, thereversed-phase and cation-exchange mechanisms of the monolith wereutilized simultaneously to separate four kinds of proteins successfully. Atlast, the monolithic column was applied to isolate basic proteins fromprotein mixtures and purify lysozyme from egg whites.This thesis contains five chapters as follows:The first chapter introduced the background of this project, includingthe theory of pCEC, the descriptions and merits of monolithic column,and the mechanisms and applications of boronate affinity andion-exchange interactions.Chapter two specified the preparation, optimizing process andcharacterization of the poly (VPBA-co-PEGDA) monolith. Wesynthesized the monolithic column using4-vinylphenylboronic acid(VPBA) and poly (ethylene glycol) diacrylate (PEGDA) in a binaryporogen system comprising diethylene glycol and ethylene glycol. Thefabricated monolith was characterized by morphology, pore property andbreak-through capacity. The prepared monolith exhibited uniform openchannel network and the monolithic beds were well linked to the innerwall of the capillary.In chapter three, we discussed the boronate affinity andreversed-phase mechanisms of the poly (VPBA-co-PEGDA) monolith in μHPLC and pCEC. Two pairs of compounds: Quinol and catechol,benzoic acid and caffeic acid were separated on the column as testsamples, and catechol and caffeic acid, which both contain cis-diolgroups, were specifically captured. The result demonstrated a goodaffinity performance for small molecules with cis-diols groups, while thecolumn showed typical reversed-phase retention when separatingbenzenes homologous series and heterocyclic compounds.In the forth chapter, we analyzed four kinds of proteins includingBSA, RNase, Cyt C and Lyz according to the cation-exchangemechanism of poly (VPBA-co-PEGDA) monolith. We found thatnon-basic proteins cannot be retained on the column while the basicproteins were adsorbed; as a result, the non-basic and basic proteins werespecifically separated. And then, we innovatively utilized thereversed-phase and cation-exchange mechanisms of the monolithsimultaneously to separate four kinds of proteins successfully.Chapter five confirmed the the ability of capturing basic proteins ofthe poly (VPBA-co-PEGDA) monolith. We tried to isolate Cyt C and Lyzfrom protein mixtures, and then the column was applied to enrich Lyzfrom egg whites and a satisfied outcome was obtained.