Benedetto Natalini Roccaldo Sardella Antonio Macchiarulo Roberto Pellicciari Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di Perugia, Perugia, Italy Original Paper S-Trityl-(R)-cysteine, a powerful chiral selector for the analytical and preparative ligand-exchange chromatography of amino acids S-Trityl-(R)-cysteine [(R)-STC] is the new selector of a dynamically coated, chiral ligand-exchange stationary phase which proved to be highly effective in both analyt- ical and preparative-scale separation of enantiomers of some natural and unnatural underivatized amino acids, with good separation and resolution factors. With the aim of identifying the best chromatographic conditions suitable for the preparative- scale separations, some parameters controlling retention, separation and resolution factors (such as the type and amount of cupric salt and the eluent pH) were investi- gated. The relatively easy removal of the Cu(II) ions renders this technique suitable for obtaining small amounts of enantiomerically pure samples for preliminary bio- logical evaluations. Keywords: Chiral coated phase / Chiral ligand-exchange chromatography / Enantiomer separa- tion / High performance liquid chromatography / Preparative separation / Received: October 17, 2007; revised: December 5, 2007; accepted: December 5, 2007 DOI 10.1002/jssc.200700511 1 Introduction One of the most elegant approaches in the resolution of enantiomers through the formation of diastereomers is the in situ conversion of the racemic mixture. In this frame, chiral ligand-exchange chromatography (CLEC) fulfils such a requirement by means of an optically active reagent as a component of either the mobile phase or the stationary phase [1 – 3]. In order to achieve the best chro- matographic performances with this technique, the chi- ral discriminating agent has been often selected with particular attention to its hydrophobic/hydrophilic bal- ance, using longer alkyl/aralkyl substituents either in the side chain or attached to the amino nitrogen. Gener- ally, in a CLEC system, a combination of the complexa- tion process and hydrophobic interactions occurs, with the former resulting as the dominant factor [4]. As a confirmation, a higher alkyl or aralkyl chain length and hydrophobicity of the selector does not very often parallel an analogous increase in the retention, and a controversial trend in the enantioselectivity values and elution order has also been observed. However, this is not surprising since retention and enantioselectivity are governed by different factors which can be fine-tuned through proper changes in the eluent composition [4, 5]. Consequently, an increase in the lipophilic character of the chiral selector is not necessarily followed by a con- comitant enhancement in the retention of a specific ana- lyte. Moreover, a parallelism between retention and selectivity is anything but expected. Various examples enlighten for this situation. Karger and coworkers [3] observed a linear increase in retention by employing L-2- alkyl substituted 4-octyldiethylenetriamine-metal com- plex as the chiral selector added to the mobile phase. A different trend was observed for the selectivity, with the intermediate isopropyl being the best performing group: additionally, in some examples an unexplained inver- sion in the elution order was also obtained. Weinstein [6] observed an intriguing result, in terms of retention, selectivity and elution order, by eluting a series of race- mic amino acids with different N,N-dialkyl-substituted L- alanine and L-valine selectors added to the mobile phase: an increase in the bulkyness of the nitrogen alkyl sub- stituent did not correlate with a linear response both in retention and selectivity. Similar results have been shown by Davankov and coworkers [7]: a lack of linearity Correspondence: Professor Benedetto Natalini, Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di Pe- rugia, Via del Liceo 1, 06123 Perugia, Italy E-mail: natalini@chimfarm.unipg.it Fax: +39-075-5855161 Abbreviations: AIDA, 1-aminoindane-1,5-dicarboxylic acid; CIP- A, 3a,5,6,6a-tetrahydro-4H-pyrrolo[3,4-d]isoxazole-3,4-dicarbox- ylic acid; CIP-B, 3a,5,6,6a-tetrahydro-4H-pyrrolo[3,4-d]isoxazole- 3,6-dicarboxylic acid; CSP, chiral stationary phase; CLEC, chiral ligand-exchange chromatography; (R)-SBC, S-benzyl-(R)-cysteine; (R)-STC, S-trityl-(R)-cysteine i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 696 B. Natalini et al. J. Sep. Sci. 2008, 31, 696 – 704