Abstract

Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride (AETAC) and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion exchanger and quaternary amine as a strong anion exchanger, respectively. An alternative weak anion exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. The dynamic binding capacities of the three monoliths were comparable or superior to values that have been reported for various other monoliths. Chromatographic performances were also similar to those provided by a modified poly(GMA-co-ethylene glycol dimethacrylate) monolith. Separations of standard proteins were achieved under gradient elution conditions using these monolithic columns. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, laborious surface modifications were avoided and the PEGDA crosslinker improved the biocompatibility of the monolithic backbone. Protein separations by capillary size exclusion chromatography (SEC) require a monolith that is biocompatible, has sufficient pore volume, has the appropriate pore size distribution, and is rigid. Most polymer monoliths have not possessed a biomodal pore-size distribution, i.e., especially with one distribution in the macropore region and the other in the mesopore region. Furthermore, non-specific adsorption of proteins in these stationary phases has persisted as a major unresolved problem. To overcome these difficulties, a porous poly[polyethylene glycol methyl ether acrylate (PEGMEA)-co-PEGDA] monolith which can resist adsorption of both acidic and basic proteins when using an aqueous buffer without any organic solvent additives was developed. Based on this biocompatible monolith, surfactants were introduced as porogens with the hope of significantly increasing the mesopore volume within the polymer. Two types of surfactants were studied, including poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO and Brij. Pore size distributions were examined using a well-defined molecular weight range series of proteins and peptides by inverse size exclusion chromatography, which indicated relatively large volume percentages of mesopores and micropores. The two new monoliths demonstrated different SEC behaviors, low nonspecific adsorption of proteins, and high mechanical rigidity. High density lipoprotein (HDL) is a heterogeneous class of lipoprotein particles with subspecies that differ in apolipoprotein and lipid composition, size, density, and charge. In this work, I developed a new capillary SEC method for size separation of native HDL particles from plasma using a capillary packed with BioSep-SEC-4000 particles, Three major sizes of HDL particles were separated. Additionally, capillary SEC and capillary strong anion-exchange chromatography of non-delipidated HDL were accomplished using poly(PEGMEA-co-PEGDA) and poly(AETAC-co-PEGDA) monoliths. These new LC methods using packed and monolithic stationary phases provided rapid separation of HDLs and excellent reproducibility.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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