Development and Optimization of Organic Based Monoliths for Use in Affinity Chromatography

Affinity chromatography is an important and useful tool for studying biological interactions, such as the binding of an antibody with an antigen. Monolithic supports offer many advantages over traditional packed bed supports in affinity chromatography, including their ease of preparation, low back pressures and good mass transfer properties. Monoliths can be broken down into two basic categories: organic (polymer) and inorganic (silica) monoliths. There are many varieties of polymer based monoliths; however, a large focus has been on co-polymers of glycidyl methacrylate (a functional monomer) and ethylene dimethacrylate (a cross-linking agent). The solvents of choice for making this type of monolith are typically 1-dodecanol and cyclohexanol. The combination of monolith supports with biological ligands of interest in affinity chromatography has given rise to a technique known as affinity monolith chromatography (AMC). In order to study the conditions needed for preparing affinity monolithic supports, a combinatorial library was prepared in which the polymerization temperature and relative ratio of cyclohexanol to1-dodecanol was varied to determine the effects on the total protein content that could be achieved with such materials. In the first of this work, glycidyl methacrylate was used along with a cross linking agent that was either ethylene dimethacrylate or trimethylolpropane trimethacrylate. It was found that changing the ratio of these agents could be used to obtain a high protein content for monoliths containing immobilized human serum albumin (HSA). It was also found that these materials could be used for the separation of chiral substances such as (R/S)-warfarin and (D/L)-tryptophan. The second study utilized a monolith comprised of a co-polymer of glycidyl methacrylate and ethylene dimethacrylate to examine the effectiveness of this material to entrap carbon-based nanomaterials for eventual use in characterizing such materials or using them in separations based on biologically-relevant proteins or ligands. ACKNOWLEDGEMENTS I am profoundly indebted to several people for their support and encouragement during this experience. First and foremost, I would like to thank my advisor Dr. David S. Hage for his patience, advice and guidance. His encouragement and support has aided in my development as both a scientist and a researcher. I would also like to thank my supervisory committee members, Dr. their assistance and guidance in my academic career. I would also like to thank the members of the Hage lab both past and present. I am very thankful and fortunate to have met a terrific co-worker and lab mate: Jeanethe Anguizola.. You have …