Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. IV. Mobile phase and surface-mediated effects on recovery of native proteins in gradient elution on non-porous, monodisperse 1.5-microns reversed-phase silicas.

The reversed-phase chromatography of proteins by gradient elution with acidic, low-ionic-strength aqueous-organic eluents is often associated with losses of the biological activity of the protein. In this study, the enzymatic activities of catalase, horseradish peroxidase and pepsin were examined under static and dynamic column conditions on non-porous, monodisperse 1.5-microns reversed-phase silicas with various n-alkyl ligands. Catalase readily lost its enzymatic activity under the influence of the acidic aqueous-organic eluents in the absence of the reversed-phase packing, whereas peroxidase was partially deactivated as a result of combined mobile phase and stationary phase effects but regained its activity on storage after elution. The enzymatic activity of pepsin was found to be very dependent on the column residence time and on the type of bonded n-alkyl ligand employed.