HPLC Chiral Optimization of a Unique β-Amino Acid and Its Ester

Enantiomeric optimization of a unique ß-amino acid (ß-amino-3-pyridylpropionic acid) and its ethyl ester was achieved with a Daicel Crownpak CR (+) column requiring no derivatization. Using this column, the pH of the mobile phase was adjusted with perchloric acid and studied to determine the optimum pH. A plot of the capacity factor as a function of pH for the amines showed the critical pH required for chiral recognition. From this data, the retention of the amines shift from a hydrophobic non-enantioselective retention mechanism (pH > 2) to complex formation between the crown ether and the amines causing enantioselectivity (pH ≤ 2). At pH 1, lowering the column temperature from ambient to sub-ambient temperatures caused increased retention and enantiomeric separation of the amines. At pH 1 and a column temperature of ∼7°C, concurrent enantiomeric baseline resolution (Rs > 1.5) of the ß-amino acid and ester was achieved. The addition of methanol under these conditions caused a decrease in retention and enantiomeric separation of the amines, especially for the ß-amino acid enantiomers.