Fast enantiomeric separation of amino acids using liquid chromatography/mass spectrometry on a chiral crown ether stationary phase.

Fast enantiomeric separation of amino acids was studied by liquid chromatography/mass spectrometry (LC/MS) on a chiral crown ether stationary phase. A chiral crown ether bonded silica column (3 mm internal diameter (i.d.), 5 cm long) packed with 3 μm particles was employed instead of a 15 cm column packed with 5 μm particles used in our previous study. In addition, the extra-column variance, becoming more serious for smaller columns, was reduced by replacing 0.127 mm i.d. post-column tubes with shorter, smaller-diameter (0.0635 mm i.d.) tubes. The results demonstrated the benefits of using shorter columns packed with smaller particles and the reduction of the extra-column band broadening for fast enantiomeric separation. Finally, the enantiomeric separation of 18 pairs of proteinogenic amino acids was achieved within 2 min with a resolution (Rs) > 1.5 for each pair using an isocratic mobile phase of acetonitrile/water/trifluoroacetic acid (ACN/W/TFA) = 96/4/0.5, and a flow rate 1.2 mL/min at 30°C. This is the highest throughput method for simultaneous chiral separation of all proteinogenic amino acids except proline to date.