Comparison of a three-peptide separation by capillary electrochromatography, voltage-assisted liquid chromatography and nano-high-performance liquid chromatography

A mixture of three peptides was separated by capillary electrochromatography (CEC), nano-HPLC and voltage-assisted LC. In the latter case the charged analytes migrate through a neutral stationary phase driven by electrophoresis while their interaction with the stationary phase provides the basis for a chromatographic separation. The stationary phases used were poly(glycidyl methacrylate-co-ethylene dimethacrylate)-based monoliths that could be used directly as neutral “C1”-type columns for voltage-assisted LC and nano-HPLC, while their application in CEC became possible after derivatization of the epoxy groups with ionogenic N-ethylbutylamine functions. The separation of the peptide mixture was possible in all three modes. Highest plate numbers and resolutions were obtained under voltage-assisted conditions. The elution order showed dependencies on the charge density but also on the hydrophobicity of the peptides and was different in the three investigated chromatographic modes. The effect of changes in the ionic strength and the organic solvent content of the mobile phase on the resolution and the migration behavior of the peptides was investigated and showed the expected behavior. Voltage-assisted LC is suggested as an alternative to CEC for the separation of charged analytes by electrochromatography.