Chromatography and Separation Chemistry - American Chemical

6 Chromatographic Separation of Enantiomers on Rationally Designed Chiral Stationary Phases

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: January 27, 1986 | doi: 10.1021/bk-1986-0297.ch006

William H . Pirkle School of Chemical Sciences, University of Illinois, Urbana, IL 61801

The separation of enantiomers by liquid chromato graphy is now feasible on both analytical and preparative scales. Recent developments in chiral stationary phase (CSP) design have extended the scope of enantiomer separations to a point unimagined a few years ago. The enantiomers of l i t e r erally tens of thousands of compounds can now be separated chromatographically, often with consid erable understanding of the mechanism of the sep aration process. Such understanding enhances one's ability to design improved CSPs and to assign ab solute configurations from observed elution orders. It has long been understood that chromatography of racemates on c h i r a l adsorbents might r e s u l t i n enantiomer separation. Initial e f f o r t s to use convenient c h i r a l adsorbents ( c e l l u l o s e , starch, wool) usually met with scant success. With the advent of more sophisticated techniques, i t has become possible to separate the enantiomers of a large number, l i t e r a l l y tens of thousands, of compounds by l i q u i d chromatography. Because of the close t i e s between l i f e and c h i r a l i t y , the a b i l i t y to separate enantiomers by HPLC has an eager audience of potential users. Since enantiomer separation requires the intervention of some c h i r a l agent, one may u t i l i z e either c h i r a l mobile phase additives (CMPA) or c h i r a l stationary phases (CSPs). While the requirement that one add a c h i r a l substance to the mobile phase has obvious l i m i t a t i o n s for preparative separations, i t i s not a serious problem for a n a l y t i c a l separations. Indeed, for some types of compounds (e.g. amino acids) t h i s approach may be preferred. Quite an extensive l i t e r a t u r e exists for the use of mobile phases containing c h i r a l bidentate ligands and copper ions for the "ligand exchange" resolution of underivatized amino acids (1,2) and for N-dansyl derivatives of amino acids ( 3 , ^ 0 . T a r t a r i c acid derivatives have also been used as CMPAs ( 5 ) . When one uses CMPAs, there i s always as question as to why enantiomer separation occurs ( i f i t does). The same i s true for those CSAs that we may term "biopolymers". This constitutes a 0097-6156/ 86/ 0297-0101 $06.00/ 0

© 1986 American Chemical Society

In Chromatography and Separation Chemistry; Ahuja, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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C H R O M A T O G R A P H Y A N D SEPARATION CHEMISTRY

problem of sorts although one needn't understand why a separation occurs in order to use the separation. Once enantiomers are caused to give r i s e to separate chromatographic peaks, area measurement affords the enantiomeric purity of the sample. However, i f one doesn't understand why a separation should occur, then one i s reduced to a " t r i a l and error" search for a system capable of producing the desired r e s u l t . A great many interesting and useful enantiomer separations have been performed on "biopolymer" CSPs, several of which w i l l be mentioned for they are commercially available. Columns containing swollen t r i a c e t y l a t e d microcrystalline c e l l u l o s e , available from Merck, have been extensively investigated by Mannschrek (6^,7) and used in both a n a l y t i c a l and preparative modes. Although there i s l i t t l e detailed understanding as to how and why this CSP works, i t is thought that the laminar nature of the swollen crystals offers c h i r a l c a v i t i e s into which enantiomers must intercalate. The recent cyclodextrin CSPs, available from Advanced Separations Technology, c l e a r l y require i n t e r c a l a t i o n into the hole in the c h i r a l cyclodextrin (8). Beyond t h i s , understanding i s dim. S t i l l less well defined i s the mode of action of various c e l l u l o s e derivatives coated onto diphenyl-silanized s i l i c a ( 9 , 1 0 ) . Daicel markets columns made from actylated, benzoylated, cinnamoylated, phenyl carbamoylated and benzylated c e l l u l o s e s . While these columns afford a number of interesting enantiomer separations, there i s no clear pattern as to what w i l l resolve on which column nor i n what order the enantiomers w i l l elute. Consequently, the design of these CSPs i s approached empirically and their use i s on a t r i a l and error basis. A variety of polysaccharides has been so investigated. Again, no clear pattern of performance i s evident ( 1 0 ) . Columns packed with silica-bound proteins have been devised; bovine serum albumin-derived columns ( 1 J _ ) are available from Macherey Nagel. These CSPs show a f a i r l y extensive scope of action, although the nature of the c h i r a l recognition processes employed i s s t i l l vague. Owing to the low concentration of active s i t e s , these columns require quite small samples ( 0 . 5 - 5 nmol). Mobile phase variation i s possible (over a limited range) and a l t e r s the chromatographic behavior of the enantiomers markedly. The columns must be treated c a r e f u l l y so as to not destroy the protein CSP. The biopolymer CSPs are a t t r a c t i v e owing to the ready a v a i l a b i l i t y of the c h i r a l precursors. Offsetting this advantage is an innate complexity that more or less baffles one*s a b i l i t y to deduce the d e t a i l s of the operative c h i r a l recognition processes. Moreover, one cannot easily a l t e r or " f i n e tune" the structure of the CSP to enhance s e l e c t i v e l y . F i n a l l y , there may well be l i m i t a t i o n s as to the mobile phases which can be used owing to possible swelling, shrinking, denaturation, or dissolution. A great deal of the complexity of polymeric CSPs stems from the analyte perceiving the CSP as a c h i r a l array of subunits (monomers) which may themselves be c h i r a l . Unless one knows the structure of the array, one w i l l have d i f f i c u l t y i n specifying a possible c h i r a l recognition mechanism. Even then, an abundance of closely spaced potential interaction s i t e s can preclude mechanistic understanding. This i s also true for the synthetic



6. P I R K L E

Chromatographic Separation of Enantiomers

polymeric CSPs which have been devised. The more noteworthy of these i s that of Blaske, derived from the acrylamide of ot-phenylethylamine (^2) and the c h i r a l polymer of Okamoto, derived from the triphenylmethyl ester of methacrylic acid ( 1_3). The l a t t e r i s available from D a i c e l . Both phases perform useful separations and are thought to work by i n c l u s i o n - l i k e processes. An alternative approach (and one we have favored) i s to consider c h i r a l recognition as i t might occur between small c h i r a l molecules i n s o l u t i o n . C h i r a l recognition cannot occur unless there are at least three simultaneous interactions, at least one of which i s stereochemically dependent, between a c h i r a l "recognizer" and one of the enantiomers whose configuration i s to be "recognized." The interactions so employed are the usual intermolecular interactions which occur in s o l u t i o n . One simply engineers a c h i r a l molecule that contains functional groups capable of undergoing these interactions, anchors i t to a support, packs a column, and chromatographs a racemate which contains appropriate f u n c t i o n a l i t y . Complimentary f u n c t i o n a l i t y must be present i f the analyte i s to undergo the required multiple simultaneous i n t e r a c t i o n s . This simple rationale has great value and far-reaching implications. It provides an a p r i o r i basis for CSP design and i t provides a means of understanding the subsequently observed chromatographic behavior of assorted racemates on that CSP. It provides a means for r a t i o n a l l y improving the performance of a given CSP. F i n a l l y and importantly, the CSP i s synthetic and i t s structure can be altered and controlled at w i l l . For example, our f i r s t CSP was 9-anthryl trifluoromethyl c a r b i n o l , linked at the 10-position to s i l i c a through a six atom connecting arm. This CSP was intended to u t i l i z e π-π interactions, hydrogen bonding interactions, and s t e r i c interactions to separate the enantiomers of compounds containing π-acidic and basic s i t e s (14). Among the analytes which are part of this c l i e n t e l e are N-(3,5-dinitrobenzoyl) derivatives of amines and amino a c i d s . Chiral recognition i s reciprocal i n that i f a CSP derived from (+)-A s e l e c t i v e l y retains (+)-B, then a CSP derived from (+)-B should s e l e c t i v e l y r e t a i n (+)-A. On t h i s basis, CSPs derived from N-(3,5-dinitrobenzoyl) amino acids were prepared and evaluated (147l 5). The N-(3,5-dinitrobenzoyl) amino acid-derived CSPs show extraordinary scope and follow readily understandable patterns of behavior. These CSPs have been commercialized; a n a l y t i c a l and preparative columns and packings are available from the Regis, J . T. Baker, and Sumitomo companies. The s t r u c t u r a l requirements these CSPs exact from resolvable analytes are s t i p u l a t a b l e , a l b e i t s t i l l in imprecise terms. For example, the N-(3,5-dinitrobenzoyl) amino acid columns u t i l i z e combinations of π-ττ; hydrogen bonding, d i p o l e - d i p o l e , and s t e r i c interactions to achieve c h i r a l recognition. Thus, the analyte must contain a combination of ir-basic, basic or a c i d i c " s i t e s , " possibly have a strong dipole, or perhaps contain a bulky s t e r i c interaction s i t e . These interaction s i t e s must be arranged so as to act i n concert. Generalized (16,1_7) and s p e c i f i c c h i r a l recognition models have been presented (1_8) for a variety of analyte types.


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Large columns of the N-(3,5-dinitrobenzoyl) amino acid CSPs have been prepared and multigram quantities of racemates have been separated automatically (19.). F i f t y grams of racemic methyl N-(2-naphthyl) alaninate have been resolved per pass on a 6" X 1' column containing 13 kg of c h i r a l packing. Among the analyte enantiomers separable on the N - 3 , 5 - d i n i t r o benzoylphenylglycine CSPs are N-acylated a-arylalkylamines (20,21_). After a study of the r e l a t i o n between analyte structure, chromatographic s e p a r a b i l i t y , and operative c h i r a l recognition mechanism(s), several reciprocal α-arylalkylamine-derived CSPs were designed and constructed (22). These CSPs afford excellent s e l e c t i v i t y for the N-3,5-dinitrobenzoyl derivatives of amines, α-amino esters, amino alcohols and α-aminophosphonates. From systematic study of such analytes, a deeper understanding of c h i r a l recognition processes has been attained and employed to further improve the design of the a^arylalkylamine-derived CSPs. As a consequence, unusually large s e p a r a b i l i t y factors are noted; values ranging between 2 and 8 are considered t y p i c a l (16,17). Large s e p a r a b i l i t y factors f a c i l i t a t e preparative separations. These CSPs are not yet commercially a v a i l a b l e . Somewhat similar but less optimized (hence, less selective) CSPs are available from Sumitomo. These CSPs stem from the work of Ôi et a l and have many p r a c t i c a l applications ( 2 3 ) . They are derived from N-acylated 1-(a-naphthyl)ethylamine, commercially available from several sources. Similar CSPs derived from l i n k i n g a-arylalkylamines to s i l i c a through urea functionality have been prepared by Ôi (23,25) and by ourselves (26). These CSPs are workable but usually i n f e r i o r to the corresponding amide-linked CSPs. A commercial urea-linked α-phenylethylamine CSP column i s available from Supelco but w i l l almost always show considerably less s e l e c t i v i t y and narrower scope than the more sophisticated amide-linked CSPs derived from more "optimized" a-arylalkylamines. One important aspect of the α-arylalkylamine CSPs, both amide and u r e a - l i n k e d , i s that they often have more than one c h i r a l recognition process available to them. It was recently shown that these CSPs u t i l i z e two competing processes of opposite e n a n t i o s e l e c t i v i t i e s (16,1_7). Optimization e n t a i l s not only a l t e r i n g the structure of the c h i r a l entity so as to maximize the strengths of essential interactions, i t also e n t a i l s the manner in which the c h i r a l entity i s connected to the s i l i c a support and the spacing between adjacent strands of bonded phase (27). In simple terms, one process i s more i n t e r c a l a t i v e than the other. Densely packed strands disfavor the i n t e r c a l a t i v e process, thereby enhancing the contribution of the nonintercalative process. S i m i l a r l y , the orientation of the c h i r a l entity with respect to the s i l i c a surface determines whether, using a given combination of analyte-CSP interactions, a portion of the analyte i s intercalated between adjacent strands. In organic mobile phases, i n t e r c a l a t i o n can lead to s t e r i c repulsion. Thus, different orientations of the c h i r a l entity with respect to the s i l i c a w i l l a l t e r the r e l a t i v e contributions of the two competing processes. By largely suppressing one process, rather high s e l e c t i v i t y may be obtained from the other. In aqueous mobile phases, l i p o p h i l i c interaction begins to compensate for the s t e r i c repulsion which



6.

PIRKLE

Chromatographic Separation of Enantiomers

attends i n t e r c a l a t i o n . Although m e c h a n i s t i c a l l y r a t h e r i n t e r e s t i n g , the p r a c t i c a l consequence o f use o f aqueous m o b i l e phases i s u s u a l l y a r e d u c t i o n i n s e l e c t i v i t y . However, s e l e c t i v i t y i s s t i l l g r e a t enough f o r h i g h q u a l i t y a n a l y s i s of enantiomeric p u r i t y . I n the a r e a o f enantiomer s e p a r a t i o n , t h e number o f a v a i l a b l e CSPs i s r a p i d l y p r o l i f e r a t i n g . W h i l e t h i s may be c o n f u s i n g , i t does o f f e r the r e s e a r c h e r an o p p o r t u n i t y t o p e r f o r m a number o f " t r i a l and e r r o r " experiments t o e f f e c t the d e s i r e d r e s o l u t i o n . Among t h e s e p r o l i f e r a t i n g CSPs a r e some whose mode o f i n t e r a c t i o n i s r e l a t i v e l y w e l l u n d e r s t o o d . T h i s o f f e r s t h e r e s e a r c h e r a way to r a t i o n a l l y match a CSP t o h i s a n a l y t e so as t o maximize the p r o b a b i l i t y of success.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Davankov, V. Α.; Kurganov, Α. Α.; Bochkov, A. S.; in "Advances in Chromatography, Vol. 22"; Giddings, J. C.; Grushka, E; Cazes, J . and Brown, P. R. Ed.; Marcel Dekker, New York, 1984; p 71-116. Weinstein, S.; Engel, M. H.; Hare, P. E. Anal.Biochem. 1982, 121, 370-377. Weinstein, S. Tetrahedron Lett. 1984, 985-9. Feibush, B.; Cohen, M. J.; Karger, B. L. J . Chromatogr. 1983, 282, 3-26 Dobashi, Α.; Hara, S. Anal Chem. 1983, 55, 1805-6. Koller, H.; Rimbock, K. H.; Mannschreck, A. J . Chromatogr. 1983, 282, 89-94. Mannschreck, A; Koller, H.; Wernicke, R. Merck Kontakte H1 1984, 2-14. Hinze, W. L.; Riehl, T. E.; Armstrong, D. Α.; DeMond, W.; Alak, Α.; Ward, T. Anal. Chem. 1985, 57, 237-242. Okamoto, Y; Kawashima, M; Hatada, K. Chem Lett. 1984, 739. Okamoto, Y.; Kawashima; Hatada, K. J . Am. Chem. Soc. 1984, 106, 5357-9. Allenmark, S. LC 1985, 3, 348-353. Schwanghart, Α.; Backmann, W.; Blaschke, G. Chem. Ber. 1977, 110, 778. Yuki, H; Okamoto, Y.; Okamoto, I. J . Am. Chem. Soc. 1980, 102, 6356. Pirkle, W. H.; House, D. W.; Finn, J . M. J . Chromatogr. 1980, 192, 143-158. Pirkle, W. H.; Finn, J . M.; Schreiner, J . L.; Hamper, B. C. J . Am. Chem. Soc. 1981, 103, 3964-6. Pirkle, W. H.; Hyun, M. H.; Bank, B. J . Chromatogr. 1984, 316, 585-604. Pirkle, W. H.; Hyun, M. H.; Tsipouras, Α.; Hamper, B. C.; Bank, B. J . Pharm.Biomed. Anal. 1984, 2, 173-181. Pirkle, W. H.; Finn, J . M.; Hamper, B. C.; Schreiner, J.; Pribish, J . R. in "Asymmetric Reactions and Processes in Chemistry, No 185"; Eliel, E. and Otsuka, S. Ed.; American Chemical Society: Washington, D.C., 1982. Pirkle, W. H.; Finn, J . M. J . Org. Chem. 1982, 47, 4037-4040. Pirkle, W. H.; Welch, C. J . J . Org. Chem. 1984, 49, 138-140.


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21. Pirkle, W. H.; Welch, C. J.; Hyun, M. H. J . Org. Chem. 1983, 48, 5022-5026. 22. Pirkle, W. H.; Hyun, M. H. J . Org. Chem. 1984, 49, 3043-3046. 23. Ōi, Ν.; Nagase, M.; Doi, T. J . Chromatogr. 1983, 257, 111-117. 24. Ōi, N.; Kitahara, H.; Doi, T; Yamamoto, S. Bunseki Kagaka 1983, 32, 345. 25. Ōi, Ν.; Kitahara, Η.; "Abstracts of the 49th Biannual Meeting of the Chemical Society of Japan, I", 1984, 386. 26. Pirkle, W. H.; Hyun, M. H. J . Chromatogr. 1985, 322, 295-307. 27.

Pirkle, W. H.; Hyun, M. H. J . Chromatogr. 1985, in press.

RECEIVED May 6, 1985


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