Cyanogen bromide activation and coupling of ligands to diol-containing silica for high-performance affinity chromatography: Optimization of conditions

To obtain silica supports for high-performance affinity chromatography, a method of preparing CNBr-activated diol–silica under anhydrous conditions was developed. Activation of the silane-derived hydroxyls with cyanogen bromide and triethylamine was optimized and demonstrated to efficiently couple several amino ligands (tryptophan, 6-aminohexyl-Cibacron Blue, and DNA). Sonication and vacuum degassing, a procedure used to remove air from the silica beads, increased activation. Coupling of an amino ligand under slightly basic conditions (pH 8.0) gave the highest yield. The linkage between the immobilized ligands and silica was stable from pH 2–10. Anhydrous acetone was the most effective solvent for activation but dimethylformamide and 2-propanol were also good choices. The high-performance affinity chromatography columns obtained by coupling sequence-specific DNA binding sequences for Lac repressor-β-galactosidase fusion protein were compared to affinity columns obtained by coupling the same DNA element to Sepharose beads; 5 μm silica gave the best performance.