Study On Preparation And Chromatographic Behaviors Of Mixed-mode Monolithic Columns For Capillary Liquid Chromatography

Monolithic columns are defined as "continuous stationary phases that form as a homogeneous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures". Due to the advantages of ease of preparation, low back pressure and convective mass transfer, versatile surface chemistry for ligand attachment and ease of miniaturization in channels and capillaries, great attention has been paid on monolithic columns in recent decades. Mixed-mode chromatography is emerging as a powerful tool in separation of various molecules. Mixed-mode is defined as liquid chromatography in which at least two modes of interactions exist simultaneously, both controllable by mobile phase selection. Modern mixed-mode columns offer high selectivity and high separation efficiency for separation of a wide range of compounds. In this work, we developed two kinds of mixed-mode monolithic columns for capillary liquid chromatography. The separation mechanisms and retention behaviors of the two monoliths were also discussed in detail.The thesis consists of three chapters as following.In chapter 1, the general introduction, which included monolithic columns for capillary liquid chromatography (cLC) and their classifications, as well as the applications in biomolecule separation, was described in detail. Furthermore, the aim and significance of this thesis were also briefly presented.In chapter 2, a mixed-mode monolithic stationary phase was prepared for cLC by in situ copolymerization of SPE and pentaerythritol triacrylate (PETA) in a binary porogenic solvent. PETA was introduced as a hydrophilic crosslinker. The resultant monoliths with different column properties (e.g. morphology, permeability and selectivity) were optimized by adjusting the ratio of SPE to PETA and the composition of porogenic solvent. A series of alkylbenzenes, amides, and acidic analytes were used to evaluate the column performance in terms of hydrophobic, hydrophilic and cation-exchange interactions. The poly(SPE-co-PETA) monolithic column was applied to the separation of phenols, nucleobases, and peptides and tryptic digest, respectively. These successful applications demonstrate the purposed monoliths are promising for cLC separation of small molecules and complex sample analysis.In chapter 3, a novel glutathione (GSH)-silica hybrid monolithic column synthesized via a combination of thiol-ene click reaction and one-pot process was described, where thiol-end GSH organic monomer and 2,2-azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethyloxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) and then introduced into a fused-silica capillary for simultaneous polycondensation and "thiol-ene" click reaction to form the GSH-silica hybrid monolith. The effects of the molar ratio of TMOS/γ-MAPS, the amount of GSH, and the volume of porogen on the morphology, permeability and pore properties of the prepared GSH-silica hybrid monoliths were studied in detail. A uniform monolithic network with high porosity was obtained. A series of test compounds including alkylbenzenes, amides, and anilines were used to evaluate the retention behaviors of the GSH-silica hybrid monolithic column. The result demonstrated that the prepared GSH-silica hybrid monolith exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange interaction. In addition, the GSH-silica hybrid monolith was applied to the separation of nucleotides, peptides and tryptic digest, respectively. The successful applications suggested the potential of the GSH-silica hybrid monolith in complex sample analysis.