Effects of electroosmotic flow on zone mobilization in capillary isoelectric focusing

The electroosmotic mobilization of focused protein zones in a fused-silica capillary is investigated using a mixture of model proteins, including alpha-chymotrypsinogen A (bovine pancreas), myoglobin (horse heart) and carbonic anhydrase II (bovine erythrocytes). The presence of carrier ampholytes in the entire capillary and the adsorption of carrier ampholytes onto the capillary wall almost eliminate the electroosmotic flow in the fused-silica capillary, obviating the need for polymer additives such as methylcellulose and hydroxypropylmethylcellulose. In fact, the electroosmotic displacement of focused protein zones can only be achieved by injecting a mixture of proteins and ampholytes as a plug at the inlet of a capillary that has been pre-filled with the catholyte. Various approaches for protein mobilization in the uncoated capillary completely filled with carrier ampholytes are studied. The addition of methylcellulose to the sample mixture of carrier ampholytes and protein analytes serves as an anticonvective medium during the gravity mobilization step and contributes to the reduction of protein adsorption onto the capillary wall.