Analyte zone sharpening in pressurized capillary electrochromatography based on electrophoretic migration under a heterogeneous field

Significant peak width reductions, or peak height enhancements, of angiotensins were observed when a high voltage was applied to hydrophilic interaction pressurized capillary electrochromatography using gradient elution with mobile phases containing perchloric acid. The investigation using a contactless conductivity detector revealed that perchloric acid was adsorbed on the surface of the stationary phase, when the acetonitrile content in the mobile phase was high, and released from the stationary phase by increasing the water content during a gradient procedure. The released perchloric acid formed a highly concentrated zone moving from the column inlet to the outlet. The electrochromatographic behavior of the analytes, primarily electrophoretic migration, was changed in this zone. As a consequence of the significant variation in migration velocity of the analytes, the sample band width was reduced similar fashion to on-capillary concentration in capillary electrophoresis. Using this result, the reduction of band width and enhancement in separation efficiency was demonstrated in reversed-phase pressurized electrochromatography, in which the conductivity of the mobile phase was significantly altered using a step gradient. The resolution between benzoic acid and 1-naphthalene sulfonic acid was successfully improved from 2.7 to 4.3 by using the band width reduction method based on field-amplified stacking.