Enantiomer separation of hydrophobic amino compounds by high-performance liquid chromatography using crown ether dynamically coated chiral stationary phase

CROWNPAK CR(+) column, which is powerful for the separation of amino acid enantiomers, must be used at a column temperature below 50°C and a mobile phase containing less than 15% methanol, because the chiral crown ether moiety of the stationary phase is dynamically coated on an ODS matrix. The second peak of the enantiomers of alanine-β-naphthylamide (Ala-β-NA) appeared at 204 min (k₂=148) by using ordinary mobile phase, that is, a mixture of 10 mM perchloric acid and 15% methanol. In this study, enantiomer separations of Ala-β-NA and 1-(1-naphthyl)ethylamine (1-NEA), both of which are hydrophobic amino compounds, were investigated through the modification of the mobile phase. Addition of crown ether, cyclodextrins (CDs), cations, etc., affected the stability of the complex between an analyte and the chiral moiety, leading to fast separation. The second peak of the enantiomers of Ala-β-NA appeared at 68 min (k₂=49) through the addition of 10 mM β-CD, or at 61 min (k₂=44) using potassium dihydrophosphate as a buffer component. This method was applied for the optical purity testing of -Ala-β-NA, which is used as one of the chiral derivatization reagents for carboxylic compounds. Validations such as reproducibility and linearity were also demonstrated and this method was found to be sufficient as a quality control method for the optical purity testing of -Ala-β-NA. As little as 0.05% -form in -Ala-β-NA could be determined.