Changes in Electrolyte pH Due to Electrolysis during Capillary Zone Electrophoresis

The positioning of the capillary relative to the electrode in the electrolyte vial and buffering of the background electrolyte (BGE) in capillary zone electrophoresis have been found to have a crucial influence on the magnitude of pH changes of the BGE in the separation capillary. On-capillary measurement of the pH changes was accomplished by adding a suitable pH indicator dye to the BGE and employing absorbance detection in the visible region. This method has been demonstrated using xylenol blue in a chromate BGE commonly used for anion analysis (pH 8.0) and bromocresol green in an acetate BGE (pH 4.8). Using the chromate BGE, high pH changes of more than 2.5 pH units were observed when the capillary end was positioned close to the electrode surface in the inlet electrolyte vial, but changes of less than ± 0.3 pH unit resulted if the capillary end was displaced at least 1 mm laterally from the electrode and/or at least 1 mm vertically below the electrode end. These effects were explained by direct observations of pH changes around a platinum electrode placed in a photometric cell serving as a BGE reservoir. These observations showed a zone of altered pH being generated close to the electrode and spreading over time through the surrounding BGE by thermal convection. Further, a simple way of buffering the chromate electrolyte with a suitable cation (Tris) is presented, with the buffered electrolyte showing pH fluctuations in the capillary of less than ± 0.2 pH unit. Practical implications of these findings on the practice of capillary zone electrophoresis are discussed.