High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-l-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas

Nonporous, microparticulate, monodisperse silicas with particle diameters between 0.7 and 2.1 μm are introduced as stationary phases in high-performance affinity chromatography. The immobilization of m-aminophenylboronic acid, p-aminobenzamidine, tri-l-alanine, and concanavalin A onto these silicas was successfully achieved using 3-isothiocyanatopropyltriethoxysilane as an activation reagent. Immobilized phenylboronic acid was applied to the isolation of nucleosides, nucleotides, and glycoprotein hormones such as bovine follicotropin and human chorionic gonadotropin, while immobilized benzamidine was employed for the isolation of the serine proteases thrombin and trypsin, immobilized tri-l-alanine for the separation of pig pancreatic elastase and human leukocyte elastase, and immobilized concanavalin A for the isolation of horseradish peroxidase. In all affinity chromatographic systems studied, the nonporous monodisperse silicas showed improved chromatographic performance compared to results obtained with porous silica supports using identical activation and immobilization procedures. Furthermore, frontal analysis was used as a method to evaluate the influence of experimental parameters on biological activity and accessible ligand densities. Only minor changes in bioactivity were found with the nonporous affinity supports, where accessibilities were typically higher than ca. 60%. The immobilization of affinity ligands onto porous supports as used in this and associated papers thus represents a successful general procedure for the preparation of stable matrices with fast kinetics for use in high-performance affinity chromatography.