Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

The effect of the mobile phase parameters flow rate, temperature, pH and ionic strength, as well as the addition of various organic modifiers on the enantiomer separation of various aromatic α-amino acids was investigated using two antibody-based chiral stationary phases that have opposing stereoselectivity. On both columns, a decrease in flow rate or temperature resulted in increased interaction with the retained enantiomer. It was found that the retention factor k2 depends on the affinity between the analyte and the immobilized antibody and is not independent of the flow rate. Optimum separations of all amino acids investigated were obtained at pH 7.4 on both columns. While increased k2 values were obtained at low ionic strength on the anti-d-amino acid antibody column, no such effect was observed on the anti-l-amino acid antibody column. The addition of organic modifiers did not improve separations. In all studies, the unretained enantiomer eluted with the void volume.