Optimization of dynamic pH barrage junction focusing for weakly alkaline or zwitterionic analytes in capillary electrophoresis

Dynamic pH junction focusing prior to electrophoretic separation has been widely used for online pre-concentration of biologically important analytes, which are mostly weakly alkaline/acidic or zwitterionic species such as neurotransmitters, peptides, and proteins. A pH junction is formed when background electrolytes with different pH values are injected sequentially into the separation column of a capillary electrophoresis (CE) system. Unlike the traditional dynamic pH junction configuration with analyte molecules located in a different chemical environment to the separation background electrolyte (BGE), the pH barrage junction has a separate high pH (or low pH) region containing no analyte. Based on Simul 5 Complex simulations and experimental verification with three series of electrolyte combinations, four basic principles for pH barrage junction focusing were identified for its optimization. First, the peak shape after focusing is slightly asymmetric, but this has negligible influence on the analysis result. Second, longer length of the barrage segment is needed for complete focusing with lower concentration of the buffering species. Third, this technique is more advantageous for analytes with relatively high electrophoretic mobility in a capillary without electroosmotic flow. Fourth, provided the analyte region and pH junction buffering species are separated, this quantitative technique is compatible with both optical and mass spectrometric detection.