Comparative study of capillary electroendosmotic chromatography and electrically assisted gradient nano-liquid chromatography for the separation of peptides

Capillary electroendoendosmotic chromatography (CEC), being a hybrid of hig h-performance liquid chromatography (HPLC) and capillary electrophoresis, o ffers considerable changes to enhance column efficiency, speed of analysis and additional selectivity as compared to the parent methods. The analytes are driven by the electroendosmotic flow (EOF) and separated by surface-sol ute interactions as well as by differences in electromigration. In this pap er on the separation of peptides on C-18 reversed-phase and mixed-mode (sul phonic acid-n-alkyl) packings in CEC and electrically assisted reversed-pha se gradient nano-LC are investigated. It is shown that mixed mode packings generate a higher EOF than reversed-phase packings that is scarcely depende nt on the pH of the eluent. Applying a potential in gradient elution revers ed-phase nano-LC of peptides shortens the analysis time as compared to sepa rations without a potential. Electrically assisted reversed-phase gradient elution nano-LC is a powerful separation tool for analysis of tryptic diges ts. Peptides can be successfully resolved in acidic organic mobile phase at pH 2-3 with and without trifluoroacid as ion pairing reagent under isocrat ic conditions. It is demonstrated that CEC with mixed mode packing and an e luent of pH 2.3 with varying acetonitrile content can be applied to monitor impurities in a synthetic peptide. (C) 2000 Elsevier Science B.V. All righ ts reserved.