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

Abstract

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 (<20 mM). An acid modifier of formic acid or acetic acid exhibited a similar effect on the separation, and a low acid concentration (<0.5%) led to peak broadening by additional ion exchange with a silanol group at high pH. With respect to the composition of organic solvents, aprotic solvents such as tetrahydrofuran or acetonitrile afforded appropriate retention. However, protic solvents led to the considerably reduced retention except positively charged amino acids. Novel, practical information for the optimization or development of applications with a CEX/HILIC column for polar analytes such as amino acids was obtained.

Assessment of Highly Polar Chemicals in Dutch and Flemish Drinking Water and Its Sources: Presence and Potential Risks

“…Limited research has been performed on the analysis and monitoring of highly polar chemicals in water. − Analytical separation techniques like supercritical fluid chromatography, 7 , − chromatography using a bi- or trifunctional mixed-mode column combining RP, anion, and cation exchange, chromatography with a core–shell biphenyl stationary phase, and hydrophilic interaction liquid chromatography (HILIC), , often in combination with high-resolution mass spectrometry (HRMS) have been applied to study highly polar compounds in water samples. HRMS has been shown to be particularly useful in the identification of emerging substances. − In particular for organic acids and bases, new analytical methods need to be developed to enable the monitoring of their levels in drinking water sources.…”

Quantitation of non-derivatized free amino acids for detecting inborn errors of metabolism by incorporating mixed-mode chromatography with tandem mass spectrometry

“…The important parameters for peak shape, retention time, and separation using HILIC/CEX MM are defined by the salt concentration, acid modifier, and choice and composition of organic solvent(s) used in the mobile phases [16] . The priority during development was the separation of all 3 leucine isomers and separation of remaining amino acids for accurate quantitation (i.e., minimum of 12 data points across each chromatographic peak).…”

“…Recently, mixed-mode (MM) chromatography has been reported as an alternative strategy for amino acid analysis [15] , [16] , [17] , [18] , [19] , [20] . However, most MM amino acid methods published to date either do not effectively separate the leucine isomers or have smaller panel sizes.…”

Overview of chromatographic and electrophoretic methods for the determination of branched‐chain amino acids

“…Comparatively, as an alternative to conventional RPLC and IEC, mixed‐mode (MM) RPLC, which uses both RPLC and IEC, maintains the hydrophobic effect of RPLC and takes full advantage of ion exchange. It has been demonstrated to be capable of retaining and analyzing highly polar compounds that are not retained in RPLC [138]. Hydrophobic interactions occur between the hydrophobic side chains of AAs and hydrophobic functional groups on the stationary phase, and ion exchange interactions occur between charged side chains of AAs and ion exchange functional groups on the stationary phase.…”

“…A detailed comparison of the influence of the following factors on chromatographic separation is presented in reference [138]. (1) Buffer salt type and concentration: In the analysis of AAs, ammonium formate (AMF) separates better than ammonium acetate (AMAc).…”