Combinatorial effects of the configuration of the cationic and the anionic chiral subunits of four zwitterionic chiral stationary phases leading to reversal of elution order of cyclic β3-amino acid enantiomers as ampholytic model compounds

•Enantiomers of cyclic β3-amino acids were separated on zwitterionic chiral stationary phases.•N-methylation has a marked effect on the elution order.•Elution order can be tuned by changing the configuration of the chiral subunits of the selector.

In a systematic way enantioseparations of non-methylated and the corresponding N-monomethylated ampholytic cyclic ß3-amino acids were carried out on four zwitterionic chiral stationary phases (CSPs; ZWIX(+)™, ZWIX(−)™, ZWIX(+A), ZWIX(-A)). CSPs were based on the combinations of quinine and quinidine as the cationic and of (R,R)- and (S,S)-aminocyclohexane sulfonic acid as the anionic sites. In polar-ionic mobile phase systems, the effects of the composition of the bulk solvents, the additives, the concentration of the co- and counter-ions, the temperature, and the structures of the ampholytic analytes were investigated. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the studied temperature range (5–40 °C). Unusual temperature behavior was observed on the ZWIX(−)™ column: decreased retention times were accompanied by increased separation factors with increasing temperature, and separation was entropically-driven. For the other three CSPs, enthalpically-driven enantioseparations were observed. Via the consequent determination of the elution order of the resolved enantiomers, the effects of the absolute configuration of the chiral anionic and cationic subunits of the zwitterionic CSPs could be elucidated. N-methylation of the amino acids led unexpectedly to a reversal of the elution sequence, which can be interpreted by a subtle shift of the hierarchical order of the sterically most important driving interaction sites from the cationic to the anionic units, and vice versa.