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Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis.

A method for determining the accurate effective mobility value of an analyte in the presence of a charged interacting agent, such as a charged cyclodextrin, a micellar agent, a protein, or a DNA fragment that binds the traditional electroosmotic flow markers, is presented. Part of the capillary is filled with the charged interacting agent-containing background electrolyte; the other part is filled with the charged interacting agent-free background electrolyte. The analyte band is placed in the charged interacting agent-containing background electrolyte zone, while a neutral marker (electroosmotic flow marker) is placed in the adjacent charged interacting agent-free background electrolyte zone. The initial, preelectrophoresis distance between the analyte band and the neutral marker band is determined by pressure mobilizing the bands past the detector and recording the detector trace. Subsequently, by applying reverse pressure, the bands are moved back into the first portion of the capillary and a brief electrophoretic separation is carried out. Then, the bands are pressure mobilized again past the detector to obtain their final, postelectrophoresis distance. If (i) the neutral marker does not come into contact with the charged interacting agent and (ii) the analyte does not migrate out of the homogeneous portion of the charged interacting agent zone, the accurate effective electrophoretic migration distance of the analyte, corrected for bulk flow transport, can be determined. The actual electric field strengths in the different zones of the heterogeneously filled capillary can be calculated from the integral of the electrophoretic current and the conductivity of the charged interacting agent-containing background electrolyte measured in a separate experiment. Once the effective mobility of an analyte in the charged resolving agent-containing background electrolyte is determined by this method, the analyte becomes a mobility reference probe for that background electrolyte and can be used to calculate the bulk flow mobility in subsequent conventional CE separations utilizing the same charged interacting agent. The new method can also be used to probe the interactions of the charged interacting agents and the wall of the capillary.