Factors affecting selectivity and resolution in micellar electrokinetic chromatography and capillary electrochromatography

Abstract

This work involves the theoretical models of separation selectivity for charged analytes in micellar electrokinetic chromatography (MEKC), and comparison of selectivity of neutral monoliths for peptides and proteins separation in capillary electrochromatography (CEC). Equations and theoretical models for MEKC separation selectivity (αMEKC) were established, in which αMEKC is related to dimensionless values of mobility selectivity (αCZE) in capillary zone electrophoresis (CZE) and retention selectivity (αk) in MEKC, and where αCZE and αk are defined as the ratio of electrophoretic mobility in CZE and the ratio of retention factor in MEKC for two charged analytes, respectively. For example, MEKC separation of two charged analytes can enhance αMEKC when the selectivity ratio (ρ) is greater than 1.0 (ρ = αk/αCZE), while lower αMEKC is obtained with ρ ≤1.0. Using alkylparabens as test analytes, excellent agreement was found between the observed αMEKC and prediction according to the theoretical models of αMEKC. In addition, neutral monolithic CEC columns with C8-, C12- and C16- alkyl moieties were prepared by the copolymerization of the functional monomers C8-methacrylate, C12-acrylate and C16-methacrylate, respectively with the crosslinker pentaerythritol triacrylate. The C16-monolith provided better separation efficiency (N) for alkylbenzenes (N of 200,000, 162,000, 150,000 plates/m for C16-, C12- and C8-monoliths, respectively), and yielded better separation for tryptic peptide mapping. However, the C8-monolith provided better separation efficiency for proteins (N of 332,000, 236,000, 225,000 plates/m for C8-, C12- and C16-monoliths, respectively).