THE SEPARATION MECHANISM OF AMINO ACID ENANTIOMERS RESOLVED BY REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING A METAL ION AND CHIRAL LIGAND IN THE MOBILE PHASE (HPLC)

A quantitative model is proposed for the separation of valine enantiomers eluted through a reversed phase HPLC column. The mobile phase for this separation consisted of divalent copper and the chiral ligand aspartame (L-aspartyl-L-phenylalanine methyl ester). The model accounts for the dependence of the observed valine enantiomer retention time data on the total mobile phase concentrations of copper and aspartame.;The equilibrium distribution of species in the mobile phase is calculated using the measured constants. The diastereomeric mixed ligand complexes show no stereoselectivity. Thus, the separation does not occur through the mobile phase formation of these complexes. The protonated species of aspartame is seen to inhibit the retention of the enantiomers, while the copper-aspartame complexes enhance retention. The dependence of the adsorbed copper and aspartame on the concentrations of the mobile phase species is determined through the use of a competitive Langmuir adsorption model. The influence of each adsorbed adduct on the retention of the enantiomers is quantified using an equation based on the formation of diastereomeric mixed ligand complexes on the stationary phase. The differences in stability of the adsorbed mixed ligand complexes accounts for the separation of the valine enantiomers.;The influence of the mobile phase concentrations on the separation is studied by measuring the formation constants for the mobile phase adducts. Direct evidence for the participation of adsorbed copper and aspartame in the separation process is provided by measuring their adsorption isotherms.