Preparation Of Boronate Affinity Monolithic Capillary Columns And Its Application In Two-Dimensional Liquid Chromatography

Boronate affinity chromatography(BAC)is widely used in separation,enrichment and purification of cis-diol-containing compounds relied on the reversible formation between boronate ligands and cis-diol-containing compounds.Monolithic capillary columns have attracted great attention due to its simple preparation,low backpressure,good permeability and so on.Combined the merits of BAC and monolithic capillary columns,the boronate affinity monolithic capillary columns is playing a significant role in glycomics and proteomics.Moreover,an online two-dimensional liquid chromatography without interface can be estabilished based on both boronate affinity interaction and other secondary interactions on boronate affinity monoliths.Also,it can be further coupled with micro mass spectrum to seperate and qualitatively analyze biosamples.This paper is divided into six chapters.The first chapter introduced micro liquid chromatography(?LC)and pressurized capillary electrochromatography(pCEC).And it then described the principles,preparation methods,development and current status on both monolithic capillary column and boronate affinity monolithic column in details.Also,the background of two-dimensional liquid chromatography was summarized.The second chapter included the preparation,optimization,SEM characterization and separation mechanism of poly(AAPBA-co-PETA)monolithic capillary column.With optimized preparation condition(AAPBA 7 mg,PETA 5 mg,cyclohexanol 35 mg,ethylene glycol 40 mg,AIBN 1 mg),the poly(AAPBA-co-PETA)monoliths was synthesized and the results showed that the monoliths had a porous and high cross-linking polymer structure,and persisted both boronate affinity mechanism and reversed phase retention mechanism.It could be used for separation of cisdiol-containing small molecules such as caffeic acid in ?LC,also for separation of alkylbenzenes and peptides in pCEC.The third chapter introduced the preparation,optimization,SEM and FT-IR characterization of the poly(VPBA-co-TMPTMA)monolithic capillary columns,also its reproducibility was investigated.This monoliths is synthesized under the optimized condition(VPBA 19 mg,TMPTMA 16 mg,ethylene glycol 37.5 mg,cyclohexanol 32.5 mg,AIBN 1mg).The results showed that the monoliths had good permeability and exposed boronate ligands and TMPTMA.Relatively good repeatability was obtained with RSD of retention time of 5 alkylbenzenes less than 1.5% for run-to-run(n = 6)and less than 9.7% for column-to-column(n = 3).Furthermore,the monoliths persisted mechanism of boronate affinity chromatography(BAC)for separating of cis-diol-containing small molecules such as caffeic acid and glycoproteins such as HRP in ?LC as well as reversed phase chromatography(RPC)for separating of alkylbenzenes,polycyclic aromatic hydrocarbons,phenols and peptides in pCEC.Based on the results,the poly(VPBA-co-TMPTMA)monoliths demonstrated better separation than poly(AAPBA-co-PETA)monolithis.In the fourth chapter,due on the dual retention mechanism of the poly(VPBA-co-TMPTMA)monoliths,an on-line BAC/RPC two-dimensional micro liquid chromatography with a single column(BAC/RPC 2D ?LC-1C)without interface was established.In view of analysis of principle and operation,opitimization of pH,ACN content and concentration of buffer,BAC/RPC 2D ?LC-1C could be successfully utilized to separate eight compounds.The fifth chapter studied on the enrichment and separation of norepinephrine and dopamine in Portulaca oleracea L.on poly(VPBA-coTMPTMA)monoliths.Firstly,based on the two-dimensional liquid chromatography platform which was estabilished on poly(VPBA-coTMPTMA)monoliths,the cis-diol-containing compounds including norepinephrine and dopamine in Portulaca oleracea L.were enriched and separated.In the meantime,the enriched compounds were confirmed by capillary reversed-phase liquid chromatography and micro liquid chromatography-micro mass spectrometry(?LC-?MS).As a result,the norepinephrine and dopamine in Portulaca oleracea L.were indeed specifically captured by the monolithic column with the imformation of retention time and mass-to-charge ratio.Chapter Six summed up this thesis,and the prospect of the further direction was speculated.