REPORT
Optical Isomer Separation by Liquid Chromatography Daniel W. Armstrong Department of Chemistry and Biochemistry Texas Tech University Lubbock, Tex. 79409
The resolution of enantiomers (nonsuperimposable, mirror image isomers) traditionally has been considered one of the more difficult problems in sepa ration science. In an isotropic liquid environment enantiomers have identi cal physical and chemical properties, with the exception that they rotate plane-polarized light in opposite direc tions. Substances capable of rotating plane-polarized light are said to be op tically active. The French physicist Biot first reported the existence of op tical activity in α-quartz in the early 1800s (1). Pasteur was well aware of the find ings of Biot and others when he began his classic work with tartrate salts in the mid-1800s. In 1848 Pasteur report ed the first deliberate separation of en antiomers from a racemic mixture (2). This separation was possible because at temperatures below 27 °C, racemic sodium ammonium tartrate forms two types of crystals, each of distinct shape and containing but one optical isomer. The morphologically distinct crystals were then separated by hand. Equally important was Pasteur's conclusion that molecular dissymmetry (the nonsuperimposability of an object and its mirror image) is the necessary condi tion for optical activity in small mole cules and crystals. Pasteur, however, never determined exactly what made a molecule dissymmetric or chiral.
This problem was successfully ad dressed by Van't Hoff and Le Bel in 1874 when they independently pro posed the "asymmetric carbon atom" (3, 4). Van't Hoff has historically re ceived a greater share of the credit, largely because he correctly favored a tetrahedral arrangement for the substit u e n t s about the carbon whereas Le Bel favored a square pyramid geom etry. Van't Hoff also proposed a "sec ond case of optical activity" that did not require the presence of an asym metric carbon. Examples of this are tetrasubstituted aliènes. Although it is not the purpose of this REPORT to thoroughly discuss the history and ramifications of stereochemical theory, analytical chemists should be familiar with both. The literature contains assorted mistakes, misunderstandings, and controversies about stereochemical matters of factual, conceptual, and semantic origin. Efforts to systematize concepts and nomenclature continue to the present, as evidenced by the recent work of Mislow and Siegel (5).
ent chemical and physical properties and thus may be separated by conventional techniques. For example, racemic substances containing carboxylic acid functional groups form diastereomeric salts with a number of enantiomerically pure alkaloids. Separation of t h e d i a s t e r e o m e r s can often be achieved by fractional recrystallization, and a single enantiomer of the acid can then be retrieved from each diastereomer. Enantiomeric excesses have also been obtained via microbiological or enzymatic digestion. In this case, the enzyme must preferentially catalyze the reaction of one enantiomer relative to the other. Crystallization followed by mechanical separation, à la
Evolution of enantiomeric separations Enantiomeric separations are important in many scientific disciplines. Typical examples include chiral synthesis, mechanistic studies, catalysis, kinetics, geochronology, biology and b i o c h e m i s t r y , pharmacology, and medicine. Several nonchromatographic methods classically have been used to isolate optically pure compounds from racemic mixtures. The most generally useful of these involves conversion of the racemic mixture to a pair of diastereomers that have differ-
84 A · ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 15, 1987
Support
Figure 1. Schematic of the ternary complex formed at the surface of a proline bonded phase-ligand exchange medium This technique effectively separates a number of racemic amino acids
0003-2700/87/0359-84A$01.50/0 S) 1987 American Chemical Society
Figure 2. Computer projection of the X-ray crystal structure of a segment of triacetyl cellulose
Figure 3. Computer projection of the structure of W-(3,5-dinitrobenzoyl)phenylglycine This compound was used in the first commercially successful and widely applicable liquid chromatographic CSP
Pasteur, are useful for the few compounds that segregate into morphologically distinct crystals. Another type of crystallization technique involves seeding a supersaturated racemic solution with a small optically pure crystal. The resulting crystals often contain an enantiomeric excess. Although all of these techniques are successful in specific instances, none can be considered generally useful, and all are relatively time-consuming and tedious. In addition, these methods often fail to accomplish total separation of enantiomers. An alternative approach uses the differential association of enantiomers with a chiral substance, as in certain chromatographic media. In essence, short-lived diastereomeric adsorbates that may differ in stability are formed, and the enantiomer forming the more stable adsorbate is preferentially retained. Although this possibility was first recognized at the turn of the century (6), there were only a few successful applications over the next six decades. In the mid-1960s researchers began to use a greater number of chiral stationary phases in a variety of chromatographic techniques, including conventional column liquid chromatography (LC), planar chromatography, and gas chromatography (GC). GC, in particular, looked like a promising method for separating enantiomers. At least four factors limited its usefulness, however. The first problem (common to all of GC) is that the solute must be volatile. Second, the relatively high column temperatures, for entropie reasons, cause the stability differences between the diastereomeric adsorbates
to be quite small and separation of the enantiomers to be minimal. Moreover, the high column temperature often causes racemization of the chiral stationary phase, resulting in a loss of enantioselectivity with time. Finally, large-scale preparative separations are not generally feasible in GC. There was also some concern over the racemization of the analyte during analysis. Although the extension of chiral separations to LC began in the 1970s, the decade of the 1980s is proving to be a major turning point in the field for the following reasons. First, a tremendous number of new and improved chiral stationary phases and additives have been introduced, accompanied by a corresponding increase in publications in this area. Second, the first widespread commercialization of chiral stationary phases occurred during this time. Third, a number of extensive theoretical and mechanistic studies involving chiral recognition are beginning worldwide. This will lead to greater understanding and optimization of these techniques. Finally, and perhaps most significant, the psychological barrier—the belief that enantiomeric separations are very difficult and to be avoided if possible—is breaking down. Indeed, we are not far from the point when the majority of enantiomeric separations will be considered routine. This, in addition to advances in the synthesis of chiral compounds (see inset, p. 90A), will greatly increase the availability of optical isomers. There are at least two general approaches for the direct LC separation of enantiomers. The first involves the
use of chiral stationary phases (CSPs); the second involves the use of chiral mobile-phase additives in conjunction with achiral stationary phases. Chiral stationary phases
The present boom in chiral separations was foreshadowed by reports on classic column chromatographic separations (7, 8). Davankov's studies on ligand exchange chromatography were particularly significant. A polystyrene-p-divinylbenzene cross-linked copolymer was used as a support. An amino acid such as L-proline was attached to the resin. The mobile phase contained copper(II) ions and the amino acid anions to be separated. A ternary complex was formed with the stationary phase, copper ion, and the free amino acid as shown in Figure 1. A variety of studies have been done on this method, analyzing the effect of resin type, metal type, immobilized amino acid, and so on. Today commercial varieties of this media are available for modern LC (Table I). This technique (or variations of it) remains one of the most effective means of separating underivatized amino acid racemates. Subsequently, Cram synthesized several chiral, binaphthylbased crown ethers and found that the crown-6 derivative made an effective bonded phase (9). This was because 18crown-6 complexes ammonium ions. Therefore, chiral compounds containing primary amines could be complexed and often resolved by these substrates. Researchers continued to adapt and improve some of the more classical CSPs for HPLC. This generally result-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 15, 1987 · 85 A
Table I. Examples of commercially available chiral stationary phases'1 Typical mobile phase used
Representative compounds separated
Regis, Baker, Alltech
Normal phase (hexane-isopropanol)
(S)-/V-(3,5-dinitrobenzoyl)leucine; ionically or covalently bonded
Regis, Baker
Normal phase (hexane-isopropanol)
(R)- or (S)-/v-(2-naphthyl)alanine
Regis
Normal phase (hexane-isopropanol)
(S)-1-(a-naphthyl)ethylamine
Sumitomo
Normal phase (hexane-isopropanol)
Aromatic sulfoxides; 3,5-dinitrobenzoyl derivatives of amines, alcohols, thiols, amino acids, amino alcohols, and hydroxy acids; aryl-substituted lactams, succinimides, hydantoins, hydroxylphosphonates; oxazolidones; binaphthols; drug derivatives Aromatic sulfoxides; 3,5-dinitrobenzoyl derivatives of amines, alcohols, thiols, amino acids, amino alcohols, and hydroxy acids; aryl-substituted lactams, succinimides, hydantoins, hydroxylphosphonates; oxazolidones; binaphthols; drug derivatives Very high selectivities for a variety of dinitrobenzoyl derivatized compounds 3,5-dinitrobenzoyl derivatives of amino acids; 3,5-dinitroanilide derivatives of carboxylic acids
(fl)-, (R)- or (Sy, (S)-a-naphthylethylaminocarbonylvaline. Several other analogous CSPs based on different amino acid derivatives are offered by this company
Sumitomo
Normal phase (hexane-isopropanol)
3,5-dinitrobenzoyl derivatives of amines, amides, amino acid esters, and fungicides; 3,5dinitroanilide derivatives of carboxylic acids; other deriva tives of the above compounds
Advanced Separation Technologies
Reversed phase0 (aqueous buffersacetonitrile or meth anol)
Dansyl and naphthyl amino ac ids, several aromatic drugs, steroids, alkaloids, metallocenes, binaphthyl crown ethers, aromatic acids, aro matic amines, aromatic sulf oxides
Manufacturer''
Chiral phase type
•π-complex-hydrogen bond (fi)- or (S)-W-(3,5 dinitrobenzoyl)phenylglycine; available ionically or covalently bonded and as the racemate
Cyclodextrin (3-Cyclodextrin
ed in more widely applicable media. One example of this is the work of Lindner and Mannschreck (10) on triacetyl cellulose (Figure 2). This sta tionary phase and recent commercial variations of it were shown to separate a number of racemic thioamides, sulf oxides, organophosphorus compounds, drugs, and amino acid derivatives. As previously mentioned, the hall mark of the 1980s is the rapid evolution of CSPs accompanied by commercial ization and widespread public accep tance and use. The first commercially successful (and widely applicable) CSP was developed by William Pirkle, an organic chemist with experience in enantioselective synthesis and chiral in teractions (11). The CSP consists of
(/?)-iV-(3,5-dinitrobenzoyl)phenylglycine attached ionically or covalently to γ-aminopropylsilanized silica gel (Fig ure 3). Pirkle's rationale was to develop a normal-phase medium in which the chiral-bonded molecule could interact at three points with chiral solutes (for example, there might be a jr-ir interac tion, a hydrogen bond, and a dipolar interaction). If the two diastereomeric adsorbates are of different energy, sep aration can be achieved. The success of this approach is evident from the large number and variety of separations achieved. Although the covalently bonded version of these CSPs can be used with relatively polar mobile phases, the enantioselectivity and sta bility are much greater when using nor
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mal-phase eluents. A number of addi tional 7r-complex hydrogen bond sta tionary phases have been synthesized by Pirkle and by others (12, 13). Being synthetic, these CSPs are typically available in either absolute configura tion. Changing the configuration of the CSP changes the elution order of the enantiomers. Enantioselectivities (as) exceeding 100 have recently been re ported on a commercial version of this new generation of CSPs (14). Stable cyclodextrin-bonded phases developed in our laboratory (Figure 4) were the first commercial CSPs intend ed to be used in a traditional reversedphase mode (15). Retention and resolu tion are controlled by altering the amount of organic modifiers (e.g.,
. ν ,
••-•*
••'
