Development and evaluation of N-hydroxysuccinimide-activated silica for immobilizing human serum albumin in liquid chromatography columns

A method was developed for preparing N-hydroxysuccinimide (NHS)-activated silica for use in immobilizing human serum albumin (HSA) within HPLC columns. This support was made by reacting aminopropyl silica with disuccinimidyl suberate (DSS). Solid state 13C NMR was used to examine the steps in this synthesis. The number of NHS groups on the silicaʹs surface was controlled by varying the amount of DSS or density of surface amine groups used in the preparing this material. Items considered in the use of this material for the immobilization of HSA included the amount of protein added to the support, the reaction time, and the pH of the coupling buffer. These supports were then evaluated in terms of their ability to perform chiral separations for R/S-warfarin and d/l-tryptophan. Advantages of this method compared to current immobilization techniques for HSA included its better long-term stability and the fact that it did not require the use of any reducing agents. The approaches developed in this work are not limited to HSA but can be used with other proteins or amine-containing ligands.