Isothermal calorimetric titrations (ITC), circular dichroism (CD), and UV spectroscopy have been employed to investigate and quantify binding of the enantiomers of N-3,5-dinitrobenzoyl-leucine (DNB-Leu) and nonchiral N-3,5-dinitrobenzoyl-glycine (DNB-Gly), denoted as selectands (SAs) to the following chiral selectors (SOs): quinine (QN), quinidine (QD) and their derivatives O9-tert-butylcarbamoyl quinine (t-BuCQN) and O9-tert-butylcarbamoyl quinidine (t-BuCQD). The results reveal that DNB-Leu binds to all SOs in a 1:1 association mode. Although DNB-Leu exhibits higher affinity for QN and QD than for t-BuCQN and t-BuCQD, no preferential binding of any of the two DNB-Leu enantiomers to QN or QD was observed. By contrast, t-BuCQN binds (S)-DNB-Leu with high enantioselectivity (K-b,K-S/K-b,K-R approximate to 10), whereas the t-BuCQD derivative shows similarly high selectivity for the (R)-DNB-Leu enantiomer (K-b,K-R/K-b,K-S approximate to 10). The results of optical (CD, UV) titrations of t-BuCQN with (S)-DNB-Leu and t-BuCQD with (R)-DNB-Leu are fully consistent with those obtained from the corresponding calorimetric titrations. The induced CD spectra of (S)-DNB-Leu-t-BuCQN and (R)-DNB-Leu-t-BuCQD ionic complexes display bands of opposite sign indicating that binding of DNB-Leu enantiomers within the SOs molecules occurs at well-defined domains in a "pseudo-enantiomeric fashion" (Lammerhofer and Lindner, J. Chromatog. 1996, 741, 33). The relative binding constants derived from ITC, UV, and CD titrations are in good agreement with the enantioseparation factors observed with the corresponding immobilized SO versions under HPLC conditions in prior studies. The thermodynamic analysis shows that the ion-pair formation between cinchona alkaloid type SOs and DNB-leucine is a strongly enthalpy-driven process (DeltaH(b)(o) up to -38 KJ/mol), accompanied by unfavorable entropic contributions ( T DeltaS(b)(o)T DeltaS(b)(o) up to -15 kJ/mol). The observed highly exothermic DeltaH(b)(o) values result most likely from the attractive noncovalent intermolecular interactions, such as van der Waals interactions, hydrogen bonding and pi-pi interactions, whereas the negative entropy contributions apparently reflect the generation of highly ordered bimolecular ionic associates.