666 ENANTIOMER SEPARATIONS 14.7.2 Thermodynamics of Direct Chromatographic Enantiomer Separation, 716 14.7.3 Site-Selective Thermodynamics, 717 14.1 INTRODUCTION Previous chapters have described HPLC procedures of wide applicability; that is, separations that can be used for many different kinds of samples. Usually method development can be started with any of various columns or conditions, and resolution is then systematically improved by varying separation conditions. Consequently there are often many different ways of successfully separating a particular sample. Such an approach cannot be used for enantiomers, however, which require highly specialized techniques and separation materials. Furthermore, in most cases, the selection of the column is the critical step; unless a suitable column is selected, subsequent changes in other conditions are unlikely to be successful. Nevertheless, enantiomer separations are today performed routinely in many research and routine laboratories, and are of great importance in the pharmaceutical industries. Various technologies and tools have been developed that provide a rich toolbox to separate virtually any sample. Among the available tools, direct separation with a chiral stationary phase (CSP; an enantioselective or ‘‘chiral’’ column) has become the pre- dominant and most accepted procedure (but not the only procedure). However, the identification of the most suitable CSP/mobile-phase combination for a particular analyte can be challenging. Due to the specificity of molecular-recognition for each combination of CSP (chiral selector) and analyte—and its sensitivity to minor struc- tural variations in either the solute or CSP—reliable predictions of an appropriate column from analyte molecular structure are as yet hardly possible. Databases such as ChirBase (http://chirbase.u-3mrs.fr) can provide help in the selection of column and starting mobile phase, based on analyte structure [1, 2]. A database approach will be of greatest value when the enantiomers of interest are included in the database. This approach can also be useful for enantiomers of related structures—where presumably similar separation conditions will be successful, but it must fail for completely new structures. Alternatively, automated screening procedures are used in large pharmaceutical companies to solve this problem efficiently [3, 4]. This way the most promising CSP can be found quickly, followed by optimization of the mobile phase. Overall, direct HPLC enantiomer separation based on CSPs has become an extraordinarily powerful technology, and this will be the primary focus of the present chapter. 14.2 BACKGROUND AND DEFINITIONS Non-enantiomeric separations can often be developed from only a general descrip- tion of the sample components, for example, acidic, basic, or neutral solutes. In 14.2 BACKGROUND AND DEFINITIONS 667 some cases the class of compounds within a sample (e.g., peptides, proteins, car- bohydrates) suggest a range of suitable conditions, including column type (but not requiring a specific column). Consequently further details of solute molecular structure are unnecessary for non-enantioselective method development. This is not the case for enantiomeric separations, where solute molecular structure and related physicochemical properties play a critical role in method development. For this reason a basic understanding of the behavior of enantiomers is essential for their efficient separation. 14.2.1 Isomerism and Chirality Isomers are molecules that possess identical atomic composition, yet are not superimposible upon each other [5, 6]. A classification of isomeric structures is given in Figure 14.1 [6]. As distinguished from the case of molecules which are actually identical (‘‘homomers’’), a structural isomer can be defined in various ways. Compounds whose atom-to-atom connections are different (e.g., 1-butanol, 2-butanol) are defined as constitutional isomers. Stereoisomers, by contrast, have identical atom-to-atom connections, but distinct orientation of atoms or groups in three-dimensional space. The latter can be further divided into enantiomers and diastereomers (or ‘‘diastereoisomers’’). While the former are always chiral, the latter may also include nonchiral stereoisomers such as cis/trans isomers (e.g., cis/trans-1,2-dichloroethylene). Chirality, the synonym for ‘‘handedness’’ (from the Greek word for hand), refers to the geometric property of an object which is nonsuperimposable on its mirror image (e.g., the left and right hand). Such an object has no symmetry elements of the second kind, such as a plane of symmetry, a center of inversion, or a rotation-reflection axis. Chirality may arise from various distinct chiral elements (Fig. 14.2): centers of chirality (stereogenic centers; Fig. 14.2a), chiral axes (axial Constitutional isomers Isomers (structural isomers) Homomers (identical) Molecules with identical atomic composition yes no superimposible yes no Same constitution Enantiomers (R S) (1:1 mixture = racemate) Diastereomers (SR SS) Stereoisomers Criteria for distinction: Property Non-enantiomers Enantiomers Symmetry No mirror images Non-superimposible mirror images Energy Distinct Identical Figure 14.1 Classification of isomeric structures. Adapted from [6]. 668 ENANTIOMER SEPARATIONS chirality; Fig. 14.2b), chiral planes (planar chirality; Fig. 14.2c), chiral helices (helical chirality; Fig. 14.2d), and topologically chiral elements (topological chirality). The most well known example is a center of chirality, where typically a carbon atom within the molecule is substituted by four different entities (see the example of carbons 1 and 2 in Figure 14.3a. Enantiomers and diastereomers can be differentiated by either of two proper- ties: symmetry or energy content (i.e., the free energy of the molecule). Enantiomers are molecules that are nonsuperimposable mirror images of each other having identical energy content (because of exactly identical atomic distances, angles, and torsions, as well as interatomic interactions). They are indistinguishable in an achiral environment and therefore cannot be separated by achiral chromato- graphic methods such as conventional reversed-phase chromatography (RPC). For example, (1S,2R)-ephedrine and (1R,2S)-ephedrine (Fig. 14.3a) are enantiomers. Moreover (1R,2R)- and (1S,2S)-pseudoephedrines are also enantiomeric to each other (Fig. 14.3b). Note that the corresponding enantiomers always exhibit opposite configurations at the two stereogenic centers (as in Fig. 14.3). An exactly equimolar mixture of enantiomers is called a racemate. All other mixtures of enantiomers with a composition deviating from 1:1 are defined as nonracemic mixtures. Enantiomers are provided with stereochemical descriptors (e.g., R and S,or L and D,or+ and −) that distinguish them (with the R and S system being (a) Centers of chirality X = C, Si, Ge, Sn, N + , P + , As + a X b c d a X b c d tetra-coordinate X = N, S + , P tri-coordinate X c b a X c b a (b) Axial chirality CCC a b c d c d a b c d a b a b d c N a b c d CCC H HOOC COOH H CCC H COOH HOOC H CH 3 COOH H 3 C HOOC H 3 C HOOC CH 3 COOH Figure 14.2 Structural features that contribute to chirality. 14.2 BACKGROUND AND DEFINITIONS 669 (c) Planar chirality CH 2 CH 2 H 2 C H 2 C a CH 2 CH 2 H 2 C H 2 C COOH H 2 C H 2 C CH 2 CH 2 HOOC P (plus) M (minus) (d ) Helical chirality Figure 14.2 (Continued). highly preferred). R and S refer to configurations in which the substituents at the stereogenic center are in clockwise and counterclockwise arrangement regarding their Cahn–Ingold–Prelog priorities when the substituent with the lowest priority is oriented away from the observer. + and − refer to the optical rotation properties of a chiral molecule; that is, its ability to rotate the plane of linear polarized light to the right or left (dextrorotatory and levorotatory). L and D refer to the Fisher designations for amino acids and sugars that specify relative configurations chemically derived from D-(+)-glyceraldehyde [6]. Diastereomers are not mirror images of each other, are characterized by distinct physical and chemical properties, and can be separated by achiral chromatography. In the example of Figure 14.3, each stereoisomer of Figure 14.3a is a diastereomer of any of the stereoisomers in Figure 14.3b because they differ solely in the configuration of one stereogenic center rather than both (as for enantiomers). Thus ephedrines and pseudoephedrines are diastereomers because they are not mirror images; they can also be termed epimers, since only one of several stereogenic centers is inverted. This distinction between enantiomers and diastereomers is the fundamental basis for all chiral differentiation processes including all enantiomer separation concepts. 14.2.2 Chiral Recognition and Enantiomer Separation Enantiomers have identical physicochemical properties, so their separation requires their conversion to either (1) diastereomers (the indirect method) or (2) diastere- omeric complexes (the direct method) [7, 8]. Today the use of the indirect approach 670 ENANTIOMER SEPARATIONS (1S, 2R)−(+) (1R, 2S)−(−) erythro ephedrine (1R, 2R)−(−)(1S, 2S)−(+) threo p seudo-e p hedrine C 6 H 5 C C CH 3 OHH NHCH 3 H C 6 H 5 C C CH 3 HO H H 3 CHN H 1 2 C 6 H 5 C C CH 3 HHO NHCH 3 H C 6 H 5 C C CH 3 HOH H 3 CHN H (a) (b) Figure 14.3 Structures of ephedrines and pseudoephedrines. is decreasing because of certain problems discussed below [8]. Nevertheless, the indirect method is the procedure of choice for some applications, and its discussion in following Section 14.3 will cover issues that are also relevant to the later treatment of the direct method in Section 14.4. When planning an enantioselective separation by the direct method, different chromatographic modes can be used, as in the case of achiral chromatography. In this connection we will distinguish enantioselective separations by designating them as reversed phase (RP) or normal phase (NP); this contrasts with the previously used abbreviations RPC and NPC for achiral separations. 14.3 INDIRECT METHOD The indirect method involves the formation of diastereomers by reaction of an analyte (X)intheR or S configuration with an enantiomerically pure compound (hereafter with R-configuration), which we will refer to as a chiral derivatizing reagent (CDR): (R)-X + (R)-CDR → (R, R)-X-CDR (14.1) 14.3 INDIRECT METHOD 671 and (S)-X + (R)-CDR → (S, R)-X-CDR (14.1a) The reactions above must go to completion, and the diastereomeric products must be stable (chemically and configurationally). The diastereomers (R, R)-X-CDR and (S, R)-X-CDR can then be separated by achiral chromatography (usually RPC). One advantage of the indirect method is its use of conventional HPLC columns, which offer higher plate numbers compared to the enantioselective (‘‘chiral’’) columns of Section 14.4. A higher column efficiency can be especially important for the measurement of impurities at the <0.1% level in complex mixtures, as required in pharmaceutial products. Even more important is the possible use of CDRs for enhanced detection by UV, fluorescent, electrochemical, or mass spectromet- ric means, for example, fluorescent tags for the sensitive detection of otherwise difficult-to-detect amino acids. The indirect method is also relatively economical, in contrast to the direct method with its requirement of a battery of different (and generally expensive) enantioselective columns. A large number of CDRs have been developed that provide adequate diastereo- selectivity and in some cases enhanced detection [8]. Chiral derivatizing reagents must be both chemically and stereochemically stable, and should be commercially available in both enantiomeric forms (e.g., R and S). The choice of R or S CDRs Table 14.1 Commonly Employed Chiral Derivatizing Reagents (CDR) and Their Application CDR Analyte Class Reference 1. (R)or(S)−α-methoxy- α-trifluoromethyl phenylacetic acid and corresponding acid chloride (Mosher’s reagent) Alcohols, amines [9] 2. O,O -dibenzoyl tartaric acid anhydride (DBTAAN) Primary and secondary amines, alcohols, aminoalcohols [10] 3. (R)- or (S )−1-(9-fluorenyl)ethyl chloroformate (FLEC) Primary and secondary amines, amino acids [11] 4. ortho-phthaldialdehyde (OPA) in combination with chiral thiols such as (S)-or (R)-enantiomers of N-acetyl-cysteine, N-t-Boc-cysteine, N-acetyl-penicillamine, 1-thio-β-glucose Primary amines, primary amino acids [12, 13] 5. 1-fluoro-2,4-dinitrophenyl- 5-(S)-alanine amide (FDAA) (Marfey’s reagent) Primary and secondary amines, amino acids, thiols [14, 15] 6. 2,3,4,6-tetra-O-acetyl- β- D-glucopyranosyl isothiocyanate (GITC) Primary and secondary amines, amino acids, thiols [16] 672 ENANTIOMER SEPARATIONS enables a reversal of elution order for two enantiomers, which can be used to position a minor peak in front of a major peak, when one enantiomer is in considerable excess (see Section 2.4.2 and compare Fig. 2.17e, f ). Some popular examples for CDRs are given in Table 14.1. A still popular, indirect method for the analysis of the enantiomer composition of amino acids is the use of the o-phthaldialdehyde (OPA) reagent with a chiral thiol such as N-acetyl-cysteine (CDR 4 in Table 14.1). The reaction scheme is shown in Figure 14.4. OPA derivatization is fast and amenable to automation, so derivative instability can be overcome by reacting just before injection (an excess of the non-fluorescent reagent will not interfere with detection). The chromatogram in Figure 14.5 shows the analysis of amino acids in a bacitracin sample, after its hydrolysis and oxidation of Cys to cysteic acid (Cya) by the OPA method [13]. This example illustrates the ability of the indirect approach to resolve several enantiomer pairs in a single separation, which is much less likely with a direct method (unless MS detection is used, which introduces additional chemoselectivity so that co-elution of species with distinct MS properties does not matter). Nevertheless, despite its apparent simplicity, the development of indirect enantiomer separation methods is far from a trivial task [8]. A crucial requirement of the indirect method is an analyte that possesses a selectively derivatizable functional group such as hydroxyl, amino, carboxylic, carbonyl, or thiol. Another important requirement is a derivatizing reagent that is chemically and (especially) enantiomerically pure, that is, an enantiomeric excess > 99.9%. If the CDR contains a significant amount of enantiomeric impurity, erroneous quantification data will result. For example, the (S)-enantiomer impurity in (R)-CDR gives upon derivatization, besides the main diastereomeric pair of products, the formation of a second pair of diastereomers yielding all four stereoisomers (see Fig. 14.6; note that the impurity and its diastereomeric products are distinguished CHO CHO + analyte H 2 N R 1 R 2 HS COOH N H CH 3 O R OPA N S R 1 R 2 COOH NH CH 3 O R R + N S R 1 R 2 COOH NH CH 3 O R S fluorescent derivatives frequently employed reagents: • OPA / N-acetyl-cysteine • OPA / Boc-cysteine • OPA / Isobutyryl-cysteine • OPA / N-acetyl-O-penicillamine • OPA / 1-thio-β-glucose • OPA / 1-thio-β-mannose Figure 14.4 OPA-chiral thiol derivatization for the stereoselective analysis of primary amines and amino acids (indirect enantiomer separation). 14.3 INDIRECT METHOD 673 Cya Asp Glu L-His L-Val D-Val L-Phe L-Ile D-Phe L-Leu D-Ile D-Orn L-Lys D-Lys 10 20 300 40506070 D-His L D L D D L ( min ) Figure 14.5 Enantiomeric separation of amino acids obtained from the hydrolysis of baci- tracin A, followed by OPA-chiral thiol derivatization (indirect separation with fluorescence detection; Cya = cysteic acid). Adapted from [13]. pair of enantiomeric analytes (R)-X k R k S chiral derivatizing agent (bold, R) with enantiomeric impurity (plain, S) two pairs of diastereomers (4 stereoisomers, two pairs of enantiomers) (S)-X + (R)-CDR + (S)-CDR k′ R k′ S (R, R)-X-CDR (S, R)-X-CDR (R, S)-X-CDR (S, S)-X-CDR ee d d d d d diastereomeric to each othe r e enantiomeric to each other Figure 14.6 Reaction scheme for indirect HPLC enantiomer separation (in the presence of an S-CDR impurity in the chiral derivatization reagent R-CDR). All four stereoisomers are formed and two pairs of enantiomers, respectively (d , diastereomeric to each other; e, enan- tiomeric to each other). by unbolded type). Achiral chromatography (e.g., RPC) will be unable to resolve the products that are enantiomeric to each other. Hence the stereoisomers arising from the enantiomeric contamination of the CDR will co-elute with the peaks of the opposite enantiomers (i.e., opposite configurations), and only two peaks will be observed. Needless to say, these co-elutions will prohibit accurate quantitation. Corrections are possible for CDR contamination, if the enantiomeric impurity level in the CDR is known; however, this adds considerable complexity to both method development and validation. 674 ENANTIOMER SEPARATIONS k R > k S Time (h) 0510 % Conversion 100 50 0 R S Peak area ratio: (R)/(S) = 55:45 Kinetic resolution Peak area ratio: (R)/(S) = 50:50 No kinetic resolution Figure 14.7 Illustrative kinetic profiles for the reaction of a sample with a chiral derivatizing reagent. The two enantiomers are assumed to have different rate constants and an identical detector response for the resulting diastereomers. Two additional complications exist for the indirect method. First,fora given derivatization procedure, stereochemical integrity must be fully preserved; no racemization is allowed of the derivatizing reagent, analytes, or diastereomeric products [8]. The absence of racemization can be easily checked by derivatizing and analyzing a sample of known enantiomer composition. Second,itmustbeverified that the derivatization has reached completion, in order to avoid potential kinetic resolution problems (Fig. 14.7) [8]. Specifically, the reaction rates for derivatization of the R-andS-enantiomers of the analyte may differ; if the reaction is stopped before completion, this can give rise to a stereoisomer ratio that deviates from the actual ratio of enantiomers in the sample. For example, if the sample is a racemate and k R > k S (with k being the rate constants of the derivatization reaction), the analyzed enantiomer composition after 2 minutes in Figure 14.7 would significantly deviate from the 1:1 ratio that is expected for a racemate. Systematic error due to kinetic resolution can be easily avoided by driving the derivatization reaction to completion. This is generally achieved by increasing the reaction temperature and time, and by the use of a large excess of the corresponding CDR (the CDR is typically employed in 10-fold molar excess relative to the enantiomers). Another drawback of the indirect method is that enantiomeric ratios cannot be directly calculated from peak-area ratios measured in the final separation (as is possible with the direct method), since the two diastereomeric products can differ considerably in their detector response [8]. This holds true for most detection modes, including UV, fluorescence, and mass spectrometry, with response factors varying typically by a factor of 1.1 to 1.5. Consequently a correction for this difference in response factors will be necessary. External calibration with individual standards of 14.4 DIRECT METHOD 675 the diastereomers circumvents this problem, but individual standards are not always available. It might appear that these limitations make indirect methods questionable, which is certainly not the case. However, users should carefully check for all of the complications above, most important, the enantiomeric purity of the CDR [17] (it is unacceptable to rely only on specifications given by the supplier). Moreover the numerous method-validation issues that need to be addressed during the development of an indirect assay render this approach time-consuming, labor intensive, and more prone to systematic errors. Nevertheless, some enantioselective analysis assays are still carried out by indirect methods. 14.4 DIRECT METHOD The direct approach relies on the reversible formation of (transient) diastereomeric complexes between the two enantiomers [(R) − X and (S) − X] and a chiral com- plexing agent termed chiral selector (CS). As in the case of the indirect method, the selector must be enantiomerically pure, but this requirement is less stringent for the direct method. If the difference in complex stabilities is sufficiently large for the diastereomeric associates, a less-pure selector can still be used in a direct method: (R)-X + (R)-CS ⇔ (R)-X (R)-CS (14.2) and (S)-X + (R)-CS ⇔ (S)-X (R)-CS (14.2a) The direct method circumvents the laborious derivatization with a CDR. Two distinct experimental modes for direct enantiomer separation exist: The chiral mobile-phase-additive (CMPA) mode (or simply, ‘‘additive mode’’) and the chiral stationary-phase (CSP) mode. 14.4.1 Chiral Mobile-Phase-Additive Mode (CMPA) The additive mode makes use of an achiral stationary phase (e.g., a reversed-phase or normal-phase column) with a mobile phase that contains the chiral mobile- phase-additive (CMPA) at an appropriate concentration. The CMPA (selector, CS) may be present in the mobile phase and/or retained by the stationary phase as described by its distribution coefficient K d,CS . Upon injection of the sample, various equilibria will be established that involve both the analyte X and the selector CS (Fig. 14.8; note that subscripts m and s refer to species in the mobile and stationary phases, respectively, and the R-form of the selector is assumed). These equilibria include: • complex formation between selector (R)-CS and enantiomers (R)-X and (S)-X in the mobile phase, with association constants K a,(R)-X and K a,(S)-X • distribution of (R)-X and (S)-X between the mobile and stationary phases with distribution constants K d,(R)-X and K d,(S)-X . Compounds whose atom -to- atom connections are different (e.g., 1-butanol, 2-butanol) are defined as constitutional isomers. Stereoisomers, by contrast, have identical atom -to- atom connections,. and − refer to the optical rotation properties of a chiral molecule; that is, its ability to rotate the plane of linear polarized light to the right or left (dextrorotatory and levorotatory). L. diastereomeric to each other; e, enan- tiomeric to each other). by unbolded type). Achiral chromatography (e.g., RPC) will be unable to resolve the products that are enantiomeric to each other.