Stationary phase optimization for miniaturized chromatography and electrophoresis columns

The objective of this work was to develop synthetic methods to apply stationary phases to miniaturized columns using biopolymers and organic polymers to achieve high efficiency and fast separation. The enzyme alkaline phosphatase (ALP) was immobilized in capillaries as a narrow zone through riboflavin photoinitiated polymerization. Subsequent to surface silylation with methacryl silane, a localized coating was applied by polymerization of N-hydroxysuccinimide (NHS) derivatized acrylic acid with a directed light source. Primary amine groups of ALP were reacted with NHS activated polymer. The success of the coating was confirmed by enzyme assays. Neither nonspecific binding of ALP on the capillary nor diffusion during photopolymerization was observed. A new synthetic route was also developed for application of activated coatings to microchannel devices using NHS activated polyacrylic acid of well-defined molecular weight. The NHS activated polymer was applied to aminopropyl silane supports and surfaces. Subsequent to this bonding step, this activated polymer was further derivatized with different functionalities to form so-called arboreal coatings. The electroosmotic flow (EOF) of derivatized capillaries with a neutral surface was reduced 80% relative to uncoated fused silica capillaries. The EOF of derivatized capillaries with a charged surface was almost independent of pH. Successful electrophoretic separations of proteins and pyridine derivatives were achieved in two types of derivatized capillaries. Arboreal polyacrylate phases with sulfonic acid groups, carboxylic acid groups, and the affinity ligand concanavalin A (con A) were used in separations. Comparisons of polyacrylate coatings prepared by this new process were made with the established cerium initiated polyacrylate coatings. Coatings were characterized on silanol rich surfaces of high surface area that were amenable to characterization