The separation was achieved in 65 min at
52 °C using a gradient between two solvents: 70 mM sodium acetate
at pH 6.55 containing 2.5% of acetonitrile (solvent A) and water–
acetonitrile–methanol, 40:45:15 (solvent B), as described by Flores,
Aristoy, Spanier, and Toldrá (1997). Separated amino acids were
detected at 254 nm.