When acetate buffer was used in chiral separation of D,L-serine derivatives using a gamma-cyclodextrin (CD) column, both retention factor and resolution were high below the pKa of acetic acid and decreased sharply as the pH approached the pKa. A similar result was obtained by increasing the buffer concentration at a fixed pH. These observations suggest that hydrogen bonding interaction between the carboxylate group of the amino acid and the secondary hydroxyl groups at the CD rim plays an important role in chiral separation and is disrupted by the buffer anion.