Effect of the flow profile on separation efficiency in pressure‐assisted reversed‐polarity capillary zone electrophoresis of anions: Simulation and experimental evaluation

Abstract

Capillary electrophoresis connected to electrospray ionization mass spectrometry is a promising combination to analyze complex biological samples. The use of sheathless electrospray ionization interfaces, such as a porous nanoelectrospray capillary emitter, requires the application of forward flow (either by pressure or electroosmosis) to maintain the electrospray process. The analysis of solute molecules with strong negative charges (e.g., aminopyrenetrisulfonate labeled glycans) necessitates a reversed-polarity capillary electrophoresis separation mode, in which case the electroosmotic flow is counter current, thus pressure assistance is necessary. In this study, we compared the effect of forced convection with and without counter electroosmotic flow on the resulting separation efficiency in capillary electrophoresis based on flow profile simulations by computational fluid dynamics technique and by actual experiments. The efficiencies of the detected peaks were calculated from the resulting electropherograms and found approximately 950 000 plates/m for electrophoresis with counter electroosmotic flow, 20 000 plates/m with pressure only (such as would be in open tubular liquid chromatography), and 480 000 plates/m for electrophoresis with simultaneous counter electroosmotic flow and forward pressure assistance, which validates the simulation data.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Below flow rates of 50 nL/min, ion suppression was reduced, demonstrating the benefits of low flow rates. In another article, Javas et al evaluated the performance of separation systems for analysis of strongly negative charged analytes in reversed polarity mode with counter EOF, which is especially of interest for the use of sheathless interfaces [15]. Since a flow toward the ion source of the MS is necessary for sheathless systems, an inlet pressure is needed to maintain electrospray.…”

Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis–Top-Down Mass Spectrometry

“…Under different parameters (evaluated considering separation performance; Discussion S1), migration times and peak widths increase noticeably when decreasing the supplemental pressure from 5.0 to 2.2 psi and electrophoretic resolution is significantly improved from R = 0.37 to 1.09 ( n = 2 each, Figure b), which becomes even more evident by comparing the mass spectra in Figure c–f. The influence of superimposed pressure on CZE flow profiles was described in detail previously . The S/N also improved by ∼3-fold, likely due to reduced ion suppression from overlapping peaks and a higher number of scans averaged.…”

“…The influence of superimposed pressure on CZE flow profiles was described in detail previously. 46 The S/N also improved by ∼3-fold, likely due to reduced ion suppression from overlapping peaks and a higher number of scans averaged. In short, the final "high-resolution" CZE method is based on 40 mM AmAc (pH = 6.8) as BGE and a separation voltage of +18 kV with 2.2 psi of supplemental pressure, resulting in a total run time of about 135 min.…”

Database search assisted N-glycan structure identification

“…The EOF is the motion of the background electrolyte toward the cathode because of the formation of the electric double layer on the inner surface of the separation capillary. The effect of the EOF on the convective transport can be neglected at the pH under typical glycan separation conditions, especially when polymeric matrices are used [20]. Electrophoresis, on the other hand, is the motion of charged analytes under the influence of an applied electric field.…”