The Performance Investigation of Bimodal Cation Exchange/Hydrophilic Interaction Liquid Chromatography–Electrospray Ionization Mass Spectrometry by Modifying Mobile Phase Composition in Amino Acid Separation

Owing to its unique selectivity, mixed‐mode chromatography (MMC) has been extended to the analysis of biological fluids. Among various MMC applications, ion exchange/hydrophilic interaction liquid chromatography (IEX/HILIC) is particularly beneficial for the analysis of polar compounds without derivatization or ion pairing. Nevertheless, no practical information about the retention behavior is discussed. In this study, the chromatographic behavior of amino acids in a CEX/hydrophilic interaction mixed‐mode column via the modification of the mobile phase was investigated. A low buffer salt concentration led to increased retention; particularly, positively charged amino acids were not eluted by excessive interactions with the negatively charged stationary phase at a moderate salt concentration (