Ilaria D’Acquarica 1 Francesco Gasparrini 1 Marco Pierini 1 Claudio Villani 1 Giovanni Zappia 2 1 Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Università degli Studi di Roma “La Sapienza”, Rome, Italy 2 Institute of Chemical Sciences and Institute of Pharmaceutical Chemistry, University of Urbino, Urbino, Italy Review Dynamic HPLC on chiral stationary phases: A powerful tool for the investigation of stereomutation processes Dynamic HPLC on enantioselective stationary phases has become a well-established technique to investigate chiral molecules with internal motions that result in ste- reoinversion and occur on the time scale of the separation process. Kinetic param- eters for the on-column interconversion phenomena can be extracted from experi- mental peak profiles by computer simulation or by direct calculation methods. The technique has been used in a wide range of temperatures and is complementary in scope to dynamic NMR spectroscopy. Keywords: Dynamic HPLC / Chiral stationary phases / Stereomutation, enantiomerization / Com- puter simulation / Received: March 22, 2006; revised: April 21, 2006; accepted: April 23, 2006 DOI 10.1002/jssc.200600129 1 Introduction The direct HPLC separation of enantiomers on chiral sta- tionary phases is nowadays one of the preferred modes to quantitatively characterize chiral compounds in terms of enantiomeric composition, and to obtain discrete amounts of pure, single enantiomers on large scales. Enantiomer separation is realized through non-covalent, reversible formation of diastereomers as the two enantio- mers of the analyte interact with the chiral, enantiomer- enriched selector present in the system. Reversible inter- actions of the individual enantiomers with the station- ary phase are usually referred to as primary equilibria [1 – 5]. The enantioselectivity has a thermodynamic origin and is simply related to a difference in the stability of the dia- stereomeric species formed by the two enantiomers and the chiral selector. A simple equation (Eq. 1) relates chro- matographically derived enantioselectivity a with this energy difference. This value may approach the true value of the thermodynamic enantioselectivity of the selector-analyte-solvent system when the association with the chiral selector governs the retention of the enantiomers in the chromatographic system and other, non-selective types of interactions are negligible. DDG8 2,1 = DG8 2 – DG8 1 = –RTln(k9 2 /k9 1 ) = –RTln a 2,1 (1) where dDG8 2,1 is the difference in the interaction Gibbs energies between the two enantiomers and the system selector, R is the gas constant, T is the absolute tempera- ture and a 2,1 is the ratio of the retention factors k9 2 and k9 1 of the two enantiomers. Both enantioselective and unspecific association of the enantiomers with the stationary phase may have a retarding or activating effect on the internal molecular motions of stereolabile chiral species during their pas- sage through the chromatographic column. 2 Theory of dynamic HPLC of chiral stereolabile compounds In recent years enantioselective HPLC has been extended to the investigation of stereolabile compounds. Dynamic HPLC (DHPLC) in the form of variable temperature or variable flow chromatography can be employed to deter- mine the enantiomerization (the reversible isomeriza- tion of one enantiomer into the other) barrier of chiral stereolabile species when the interconversion takes place at the time scale of the separation process [6 – 12]. This technique has been recently used for the study of internal molecular dynamics of a range of chiral, stereo- labile compounds, and for the determination of the per- tinent kinetic parameters. Attractive features of this Correspondence: Professor Claudio Villani, Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente At- tive, Università degli Studi di Roma ”La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy E-mail : claudio.villani@uniroma1.it Fax: +39-06-4991-2780 Abbreviations: CSP, chiral stationary phase ; DHPLC, dynamic HPLC i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 1508 I. D’Acquarica et al. J. Sep. Sci. 2006, 29, 1508 – 1516