Development of monolithic supports and improved immobilization methods for high-performance affinity chromatography and free drug analysis

This work combines five projects. In the first project, affinity monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were developed for ultrafast immunoextractions. Rabbit immunoglobulin G (IgG) and anti-FITC antibodies were used as model ligands for this work. The antibody content of the monoliths was optimized by varying both the polymerization and immobilization conditions for preparing such supports. When a 4.5 mm i.d. x 0.95 mm monolith disk containing anti-FITC antibodies was used, 95% extraction of fluorescein was achieved in 100 ms.; In the second project, several immobilization methods were explored for the preparation of high-performance affinity monolithic columns containing human serum albumin (HSA). These monoliths were based on a copolymer of GMA and EDMA. Each HSA monolith was evaluated in terms of its total protein content and its retention of (R/S)-warfarin and D/L-tryptophan.; In the third project, affinity monoliths based on silica and containing immobilized HSA or alpha1-acid glycoprotein (AGP) were developed and evaluated in terms of their binding, efficiency and selectivity in chiral separations. It was found that the amount of immobilized protein per unit volume, retention, efficiency and resolving power (for chiral drugs) of silica monoliths containing HSA or AGP were better than columns containing silica particles or GMA/EDMA monoliths.; The fourth project introduced two techniques using maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method) for the immobilization of HSA and other ligands to silica through sulfhydryl groups. A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods.; The fifth project introduced a new ultrafast extraction technique for free drug analysis and determination of association equilibrium constants for drug-protein binding. Some advantages of this method include its speed (approximately 5 min per run) and its ability to use the same affinity column for more than one type of drug.