Journal of Chromatography A, 1289 (2013) 94–104 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Strong cation exchange-type chiral stationary phase for enantioseparation of chiral amines in subcritical fluid chromatography Denise Wolrab a , Michal Kohout a,∗ , Mario Boras b , Wolfgang Lindner a a Department of Analytical Chemistry, University of Vienna, Währinger Strasse 38, A-1090 Vienna, Austria b Waters Ges.m.b.H, Hietzinger Hauptstrasse 145, A-1130 Vienna, Austria a r t i c l e i n f o Article history: Received 13 January 2013 Received in revised form 9 March 2013 Accepted 11 March 2013 Available online 17 March 2013 Keywords: Enantiomer separation Ion exchange chromatography Subcritical fluid chromatography Transient buffer species Beta-blockers a b s t r a c t A new strong cation exchange type chiral stationary phase (SCX CSP) based on a syringic acid amide derivative of trans-(R, R)-2-aminocyclohexanesulfonic acid was applied to subcritical fluid chromatogra- phy (SFC) for separation of various chiral basic drugs and their analogues. Mobile phase systems consisting of aliphatic alcohols as polar modifiers and a broad range of amines with different substitution patterns and lipophilicity were employed to evaluate the impact on the SFC retention and selectivity charac- teristics. The observed results point to the existence of carbonic and carbamic acid salts formed as a consequence of reactions occurring between carbon dioxide, the alcoholic modifiers and the amine species present in the sub/supercritical fluid medium, respectively. Evidence is provided that these species are essential for affecting ion exchange between the strongly acidic chiral selector units and the basic analytes, following the well-established stoichiometric displacement mechanisms. Specific trends were observed when different types of amines were used as basic additives. While ammonia gave rise to the formation of the most strongly eluting carbonic and carbamic salt species, simple tertiary amines con- sistently provided superior levels of enantioselectivity. Furthermore, trends in the chiral SFC separation characteristics were investigated by the systematic variation of the modifier content and temperature. Different effects of additives are interpreted in terms of changes in the relative concentration of the tran- sient ionic species contributing to analyte elution, with ammonia-derived carbamic salts being depleted at elevated temperatures by decomposition. Additionally, in an effort to optimize SFC enantiomer sepa- ration conditions for selected analytes, the impact of the type of the organic modifier, temperature, flow rate and active back pressure were also investigated. © 2013 Elsevier B.V. All rights reserved. 1. Introduction In the last decades, chromatographic separation of chiral com- pounds has developed into the preferred technology for analytical assessment of stereochemical purity and preparative separation of enantiomers [1]. In context with these applications, HPLC and in particular SFC have gained considerable popularity, and are rou- tinely employed to address separation challenges in academic and industrial settings [2,3]. Supercritical fluid was first applied as a mobile phase for chromatographic separation in early 1960s [4]; however, the method was in the following decades outperformed ∗ Corresponding author. Current address: Department of Organic Chemistry, Insti- tute of Chemical Technology, Technická 5, Prague CZ-166 28, Czech Republic. Tel.: +420 220445012; fax: +420 220444288. E-mail addresses: michal.kohout@univie.ac.at, Michal.Kohout@vscht.cz (M. Kohout). by HPLC. For quite some time SFC was considered as a niche sep- aration technique. The current rapid development of SFC can be mainly ascribed to the development of a new generation of reliable and compact instruments, which enable to take the main advan- tage of SFC over HPLC – its environmental friendliness (less organic solvents and short analysis times) and high separation efficiency. Generally, supercritical fluids have lower viscosities and better dif- fusion properties than usual liquids, thus considerably reducing the elution time [5]. Currently, carbon dioxide (CO 2 ) is the most popular medium employed in SFC, due to its conveniently accessible critical parame- ters (31 ◦ C, 74 bar). As a serious limitation, it usually lacks sufficient solvent strength to elute medium to highly polar compounds. Thus the addition of polar organic modifiers is usually required to achieve adequate elution strength. The presence of these co- solvents, however, shifts the inherent critical parameters to higher pressure/temperature figures. Therefore, the majority of today’s separations in SFC mode are actually performed under subcritical 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.03.018