Copyright © 2005 John Wiley & Sons, Ltd. Biomed. Chromatogr. 19: 434–438 (2005) ORIGINAL RESEARCH 434 C. Roussel et al. ORIGINAL RESEARCH Copyright © 2005 John Wiley & Sons, Ltd. BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 19: 434–438 (2005) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bmc.502 Enantiorecognition on solid chiral selectors using microbatch technology: an example of limitation in case of strong association in the racemate Christian Roussel,* Esfandiar Rafii, Alberto Del Rio and Nicolas Vanthuyne Laboratoire Stéréochimie Dynamique et Chiralité, UMR CNRS 6180, ‘Chirotechnologies: catalyse et biocatalyse’, Université Paul Cézanne-Aix- Marseille III, 13397 Marseille Cedex 20, France Received 30 December 2004; accepted 20 January 2005 ABSTRACT: When they were independently tested, the enantiomers of N,N′-bis(salicylidene)-trans-1,2-cyclohexanediamine showed a large difference in adsorption on new chiral selectors using microbatch technology. Surprisingly, when these enantiomers were applied on the same supports as a racemic mixture, no discrimination was observed even though suitable adsorption existed. When a mixture enriched in one enantiomer (scalemic mixture) was applied, the resulting supernatant con- tained the racemic form and the enantiomer in excess was adsorbed on the support together with a part of racemate. This behavior, which militates in favor of a strong heterochiral dimer formation in the racemate, was revealed using microbatch tech- nology but remained hidden on classical column chromatography on chiral support. Molecular dynamics calculations corroborate this hypothesis, showing a favorite binding mode of the heterochiral dimer, which is stabilized by various inter- and intramolecular interactions. Our findings may be considered as a new limitation of microbatch technology, but they may have some inference in case of chiral amplification using the N,N′-bis(salicylidene)-trans-1,2-cyclohexanediamine enantiomers as chiral ligands. Copyright © 2005 John Wiley & Sons, Ltd. KEYWORDS: chiral amplification; microbatch; enantiorecognition; heterochiral dimer; chiral selector *Correspondence to: C. Roussel, UMR ‘Chirotechnologies: catalyse et biocatalyse’, case A-62, Université Paul Cézanne-Aix-Marseille III, 13397 Marseille Cedex 20, France. E-mail: christian.roussel@univ.u-3mrs.fr Abbreviations used: CSP, chiral stationary phase; TTB, 1,3,5-tri- tertiobutylbenzene. Contract/grant sponsor: CHIRBASE, Université Paul Cézanne-Aix- Marseille III. INTRODUCTION In the last few years, we and others have developed microbatch methodologies to evaluate the application field of new chiral selectors or to select commercially available chiral stationary phases (CSPs) (Wolf et al., 1993; Nakano et al., 1998, 2001; Welch et al., 1999, 2001, 2002; Roussel et al., 2000, 2001; Bluhm et al., 2000; Tobler et al., 2000; Wang et al., 2000; Xu et al., 2001). The so-called microbatch method for the evaluation of solid chiral selectors as potential CSP involves the enantioselective partition of a racemate between the solid chiral support and an achiral solvent. Chiral HPLC analysis of the supernatant allows the deter- mination of the ratio of enantiomers left in the liquid phase. We have already applied microbatch technology to evaluate some new polysaccharide CSPs developed in our group. We revised the microbatch method by adding an internal standard, a compound which is not adsorbed on the chiral selector and which allows a more accurate evaluation of the absolute enantio- selective adsorptions (Roussel et al., 2001). Some limitations have been put forward in the use of microbatch methodology, dealing with the poor sensitivity of the method, which will be dedicated to separation involving large enantioselectivity α values for preparative applications (Welch et al., 1998). To the best of our knowledge, no example has been pro- duced reporting a limitation which results from strong heterochiral dimer formation between the enantiomers in the liquid phase. The purpose of our report is to pro- vide such an example. EXPERIMENTAL n-Hexane and 2-PrOH were HPLC-grade, obtained from SDS (Peypin, France), the solvents for chromatography experi- ments were degassed and filtered on Millipore membrane (0.45 μm) before use. Cellulose Tris(3,5-dimethylphenylcar- bamate) chiral stationary phases, Chiralcel OD-H (250 × 4.6 mm, 5 μm) and Chiralcel OD (250 × 10 mm, 10 μm) were obtained from Chiral Technologies Europe (Illkirch, France), Sumichiral OA-2500 (250 × 4.6 mm, 5 μm) was obtained from Sumitomo Chemicals (Osaka, Japan). Chiral HPLC experiments were performed with a Merck-Hitachi LiChrograph L-6000 pump, a Merck-Hitachi L-4000 UV de- tector and a Merck D-7000 system manager. The retention times Rt i in minutes and retention factors k i = (Rt i - Rt 0 )/Rt 0