Solving Multicomponent Chiral Separation Challenges Using a New SFC Tandem Column Screening Tool CHRISTOPHER J. WELCH, 1 * MIRLINDA BIBA, 1 * JOSEPH R. GOUKER, 1 GARY KATH, 1 PAUL AUGUSTINE, 1 AND PAUL HOSEK 2 1 Department of Process Research, Separation and Analysis Technologies, Merck Research Laboratories, RY 801-C101, Rahway, New Jersey 2 Mettler-Toledo AutoChem, Inc. Newark, Delaware ABSTRACT A tool for improved tandem column chiral supercritical fluid chromatog- raphy (SFC) method development screening was prepared by modification of a commer- cial analytical SFC instrument with two different software-controllable, six position high- pressure column selection valves, each controlling a bank of five different columns and a pass through line. The resulting instrument, which has the ability to screen 10 differ- ent individual columns and 25 different tandem column arrangements, is a useful tool for facilitating the screening of tandem column SFC arrangements for separation of complex mixtures of stereoisomers or other multicomponent mixtures. Strategies for optimal use of the instrument are discussed, and several examples of the use of the instrument in developing tandem SFC methods for resolution of multicomponent mixtures are presented. Chirality 19:184–189, 2007. V V C 2006 Wiley-Liss, Inc. KEY WORDS: tandem chiral SFC; multicomponent chiral FSC; SFC screening; coupled columns INTRODUCTION In its simplest form, chiral chromatography involves the separation of only two components-the two enantiomers being studied. 1 However, ‘real world’ separation challenges in pharmaceutical process research and other fields often require chromatographic methods that allow for resolution of other components in addition to the enantiomers of in- terest, for example, residual starting materials, byproducts or other closely related stereoisomers. The use of mass spectrometry (MS) or other detection strategies can some- times be used to deconvolute overlapping peaks, 2 but the technique is far from universal, and is often plagued by in- terference problems. Recent advances in multidimensional chromatography offer promise for improved separation of multicomponent mixtures 3 through the use of mixed media 4 or mixed-bed approaches, 5 or through the use of dual column strategies. Column switching is often used in bioanalysis of chiral drugs, where separation of matrix- related proteins using an achiral column is carried out prior to enantiopurity analysis using a chiral column. 6 Analysis of complex mixtures of closely related stereoisomers has long relied on the use of fixed tandem column arrange- ments, a typical approach being the pairing of an achiral column (to separate diastereomers) with a chiral column (to separate enantiomers). 7–9 A certain inconvenience is involved in the setup and use of tandem column arrangements, as mechanical plumbing of the assembly is required, and the two columns used in the study become unavailable for conventional single column use while in the tandem arrangement. To over- come these limitations and to create a tool for systematic exploration of tandem column approaches for solving mul- ticomponent chiral separation problems, we have devel- oped a modification of the Berger analytical supercritical fluid chromatography (SFC) system that allows for soft- ware-controlled selection of 25 different tandem column arrangements and 10 different single column arrange- ments, thereby affording a convenient tool for developing and utilizing tandem SFC separation methods. We herein describe our general approach, the key instrument modi- fications, and several examples of the utilization of this new tandem chiral SFC analysis tool. EXPERIMENTAL SECTION Chemicals Methanol, HPLC-grade solvent, was purchased from EMD Chemicals. (Gibbstown, NJ). Carbon dioxide (bone dry) was purchased from Airgas. (Radnor, PA). The mix- ture of stereoisomers illustrated in Figure 4 was prepared by coupling of racemic ibuprofen obtained from Aldrich (Milwaukee, WI) and racemic 1-phenylethylamine ob- tained from Aldrich (Milwaukee, WI) using the peptide coupling reagent, 1-Ethyl-3-(3-dimehtylaminopropyl)-carbo- diimide (EDC), obtained from Aldrich (Milwaukee, WI), in dichloromethane. Stationary Phases Chiral stationary phases evaluated in the study include Chiralpak AD-H, AS-H and IA, Chiralcel OD-H, OJ-H, OF *Correspondence to: Christopher J. Welch or Mirlinda Biba, Department of Process Research, Separation and Analysis Technologies, Merck Research Laboratories, RY 801-C101, Rahway, New Jersey 07065, USA. E-mail: christopher_welch@merck.com or mirlinda_biba@merck.com Received for publication 10 August 2005; Accepted 20 October 2006 DOI: 10.1002/chir.20357 Published online 27 December 2006 in Wiley InterScience (www.interscience.wiley.com). CHIRALITY 19:184–189 (2007) V V C 2006 Wiley-Liss, Inc.