Development of monolithic polymers for the use in capillary-based separations
2017-02-09T03:08:30Z (GMT) by
The identification of biomolecules, particularly of proteins, is one of the recognized toughest challenges in analytical biotechnology due to the sample complexity. With the availability of modern instrumentation, a variety of analytical techniques are currently employed for this quest, typically including chromatography and electrophoresis. However, most current techniques have environmental and economic challenges since they consume a large amount of hazardous and expensive highly purified organic solvents, and sometimes, use large amount of expensive compounds. Therefore, the study of a greener analytical technique for the identification of biomolecules in biotechnology is crucial to reduce the environmental and economic impact. Miniaturized separation techniques could be one of the greener options to address these issues. They offer a number of advantages, for example, reduced chemical and sample consumption, separation improvement, and better sensitivity. Capillary electrochromatography (CEC), one of the most rapidly developing miniaturization modes of separation science in recent years, combines high performance liquid chromatography with capillary electrophoresis. Initial efforts have been devoted to the use of HPLC stationary phases in CEC, taking advantage of the presence of free silanol groups to generate electroosmosis as the driving force. However, the traditional column packing technology for HPLC was unusable in micro-scale columns due to inherent packing difficulties. Although various alternative approaches have been proposed to overcome the problems, the utilization of monolithic (continuous rod) polymers as capillary column sorbents is considered a promising approach. This thesis project aims at the development of novel monolithic polymer materials for the use in capillary-based techniques for the separation of amino acids. Compared to the traditional particle-based chromatographic polymer sorbents, monoliths provide higher rates of mass transfer, faster separation and easier permeability for biomolecules. In this thesis, targeted monolithic polymers have been synthesized and characterized. Two synthetic methods have been employed for the monolithic polymer preparation, namely, molecularly imprinting technology synthesis and post-modification polymerization. Through systemic investigations of various alternatives in the preparation and thorough characterization of numerous monolithic polymers, a better understanding of the influencing factors in the preparation methods and operating conditions on their morphology and electrochromatographic properties have been obtained. Evaluation of the electrochromatographic / chromatographic behaviors of the selected analytes by electrically-driven and pressure-driven modes under various conditions in terms of pH, temperature, buffer composition and other operational parameters have delivered some fundamental insights into their retention / separation mechanisms. Moreover, new practical methods for the production of partially filled monolithic capillaries have been established. The results provide an efficient and greener approach for the preparation and characterization of novel monolithic columns containing highly selective functional moieties for the separation of biomolecules in the field of pharmaceutical technology, medical diagnostics, proteomics and metabolomics.