Isoelectric focusing sample injection for capillary electrophoresis of proteins

Carrier ampholyte-free isoelectric focusing (IEF) sample injection (concentration) for capillary electrophoresis (CE) is realized in a single capillary. A short section of porous capillary wall was made near the injection end of a capillary by HF etching. In the etching process, an electric voltage was applied across the etching capillary wall and electric current was monitored. When an electric current through the etching capillary was observed, the capillary wall became porous. The etched part was fixed in a vial, where NaOH solution with a certain concentration was added during the sample injection. The whole capillary was filled with pH 3.0 running buffer. The inlet end vial was filled with protein sample dissolved in the running buffer. An electric voltage was applied across the inlet end vial and etched porous wall. A neutralization reaction occurs at the boundary (interface) of the fronts of H+ and OH-. A pH step or sharp pH gradient exists across the boundary. When positive protein ions electromigrate to the boundary from the sample vial, they are isoelectricelly focused at points corresponding to their pH. After a certain period of concentration, a high voltage is applied across the whole capillary and a conventional CE is followed. An over 100-fold concentration factor has been easily obtained for three model proteins (bovine serum albumin, lysozyme, ribonuclease A). Furthermore, the IEF sample concentration and its dynamics have been visually observed with the whole-column imaging technique. Its merits and remaining problem have been discussed, too.