Reduction in the sample complexity enables more thorough intact protein analysis using MS-based proteomics. A capillary electrophoresis method, namely the velocity gap mode of capillary electrophoresis (VGCE), is proposed to separate protein mixtures with high resolution. Although the separation mechanism of VGCE is also based on the difference of the mass-to-charge ratios of the proteins, it fractionates the sample zone into small pieces of subunits. In this way, the resolution can be dramatically improved due to less longitudinal dispersion of the sample. The effect of the new approach is evaluated by separation of three groups of reference protein mixtures, i.e. a mixture of lysozyme and BSA; a mixture of lysozyme, β-lactoglobulin, and ribonuclease A; and a mixture of cytochrome C, lysozyme, BSA, β-lactoglobulin, ribonuclease A, conalbumin, carbonic anhydrase, and hemoglobin. The results indicate that the new approach shows great potential to couple with MS for top-down analysis of complex mixtures.

•The ee% measurements in the case of extreme ratios can be performed.•The dispersion effect on the enantioseparation is reduced.•Fully resolving enantiomers in the case of insufficient resolution.Capillary electrophoresis (CE) has been applied to determine the percentage of enantiomeric excess (ee%) of chiral compounds. In such assays, the quality of chiral selectors (CSs) plays vital roles in resolving the enantiomers for accurate determination of the ee%. Selecting an efficient CS is usually by trial and error, and is, if ever possible, time-consuming and costly. Here we propose a new approach by using the velocity gap mode of CE (VGCE) method, to simplify the method development process for ee% determination. With VGCE, it is still possible to measure ee% even when the CS has a weak resolving power. This is especially important at the extreme cases where one of the enantiomers is significantly higher than the other one. The key point of VGCE in this case is to fractionate the small part of the mixture containing both enantiomers from the major component of the enantiomer, which is already enantiopure. Baseline separations can be achieved between the two enantiomers for the small mixture due to less longitudinal dispersion, making it possible to determine the ee%. The feasibility of this VGCE approach was confirmed by the ee% measurements of amlodipine and ofloxacin, respectively. And the practical application of VGCE was tested by analyzing levamlodipine besylate tablet.