In flow activation of diol–silica with cyanogen bromide and triethylamine for preparing high-performance affinity chromatographic columns

A new coupling strategy using pre-packed diol–silica supports to obtain affinity columns for high-performance affinity chromatography (HPAC) is described. These columns were prepared by “in flow” activation in which solutions containing anhydrous solutions of CNBr and triethylamine are separately pumped to a mixer and then onto a pre-packed diol–silica column. Recycling the amino ligand to be coupled several times over the activated silica diol columns results in ligand immobilization. DNA (the Op1 lac operator), 6-aminohexyl-Cibacron and a peptide (melittin) were all successfully “in flow” coupled to freshly activated columns. Methods for CNBr activation of pre-packed diol–silica column were developed for one, two or three pump HPLC systems. The supports were successfully used for the HPAC purification of a Lac repressor-β-galactosidase fusion protein, alcohol dehydrogenase, and calmodulin. Columns prepared by in flow activation/coupling procedures were shown to be stable for at least 14 months. Also, in flow activated silica columns could be stored in anhydrous acetone for at least 3 months prior to coupling. Our experiments with these affinity ligand columns (DNA–silica, aminohexyl-Cibacron F3GA–silica, and melittin–silica), suggests that this is a very successful coupling protocol for producing a variety of HPAC columns.