Polyfunctional low-capacity cation-exchange packing material for the separation of underivatized amino acids

A polyfunctional low-capacity cation-exchange column-packing material was developed for the simultaneous separation and determination of underivatized 20 amino acids including 16 proteinogenic ones using a binary gradient HPLC. The new packing material was prepared by sulfoacylating a highly crosslinked macroreticular poly(styrene-divinylbenzene) copolymer of 3 μm in diameter commercially available for size-exclusion chromatography. The polyfunctionality could derive from unintentional carboxy groups innately present in the base polymers probably came from polymerization initiators in addition to the intentionally introduced sulfopropionyl groups, which was certified and evaluated by measuring dynamic capacity curves of the cation exchanger. Sixteen underivatized proteinogenic amino acids were separated in 23 min using a binary high-pressure gradient elution system with two liquids of 1 mmol L−1 H3PO4 and 20 mmol L−1 NaH2PO4/30% (v/v) CH3CN, which led to the cycle time of 25 min. In the case of separation of 20 amino acids, the cycle time was 27 min. The developed low-capacity cation-exchange chromatography with post-column fluorescence detection was sufficiently quantitative, providing linear calibration lines ranged through almost three digit for all analytes. The detection limits were calculated as nmol L−1 order of magnitude with 20-μL injection. The method was applicable to the direct analysis of urinary amino acids of diagnostic markers for inborn errors of metabolism.