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Relationship between Retention and Effective Selector Concentration in Affinity Capillary Electrophoresis and High-Performance Liquid Chromatography

Many chiral selectors are used in both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). One would expect their behavior in both techniques to be similar, since the underlying molecular interactions should be the same, and to a great extent this is the case. However, our work with human serum albumin as a buffer additive in CE has shown that compounds separated by this technique cannot always be separated by HPLC with a human serum albumin chiral stationary phase, and vice versa. In this paper, results are presented which suggest that these discrepancies can be understood if one considers the concentration of protein present in the column in both separation systems. Human serum albumin is usually used at tens of micromolar concentrations in CE, while the effective concentration of the immobilized protein in a HPLC column may approach millimolar levels. This disparity in protein concentration is shown to be reflected in the capacity factors for a test solute (benzoin) in a v...