High-performance anion-exchange chromatography of proteins using aza-ether bonded silica-based phases.
The use of wide-pore silica-based hydrophilic aza-ether bonded phases for the chromatographic separation of proteins under anion-exchange conditions was studied. Polyether silanes containing terminal morpholine or piperazine derivatives are synthesized for attachment to the silica surface and provide a flexible approach to bonded phase design. In one instance, a quaternized amine support may be prepared by further derivatization of the methylpiperazine bonded phase. The supports provide high-performance anion-exchange chromatographic separations of proteins using gradients of increasing salt content, e.g., to 1.0 M sodium acetate at pH 7.0. The salt type and concentration can be varied to control protein retention while the buffer system used at pH 7.0 exerts a minimal influence on the separation. The anion exchangers may be reproducibly prepared and exhibit chromatographic retention stability at pH 7.5 for at least 2 months of operation. Acceptable capacity for protein on the bonded phase is demonstrated with high recovery of solute mass. The flexibility in anion exchanger design provides a probe of bonded ligand hydrophobic effects which can contribute in an undefined and deleterious manner to the desired ion-exchange separation. Taken together, these results provide a greater insight into the operating characteristics of anion exchangers, especially with regard to competing retention mechanisms.