Critical conditions for separating the microheterogeneous components of glycoproteins by capillary electrophoresis

To separate the microheterogeneous components of glycoproteins, capillary electrophoresis (CE) conditions were systematically investigated with ovalbumin from chicken egg white as the main testing sample. In addition to the well-known adsorption effect, a reversible capillary wall partitioning effect was found to contribute greatly to the separation and the favorable tubing wall was that coated with polyacrylamide. The separation media, especially the buffer composition, its concentration and pH, were also found to be critical. Buffers composed of boric acid and alcohol amines were demonstrated to be more effective media than gels and other solutions, but the selection of the buffer concentrations and pH was dependent on samples. By using free-solution CE with polyacrylamide-coated capillaries and boric acid-alcohol amine (BA) buffers, ovalbumin of agarose electrophoresis purity can be split into more than twenty peaks at pH 8.0+/-0.3 (0.2-0.4 mol/l BA). In contrast, free-solution CE with uncoated capillaries and gel electrophoresis with polyacrylamide-filled capillaries yielded only five or less peaks.