Anal. Chem. 1997, 69, 2610-2612
Correspondence
Direct Chromatographic Separation of Racemates on the Basis of Isotopic Chirality Kazuhiro Kimata,† Ken Hosoya,‡ Takeo Araki,‡ and Nobuo Tanaka*,‡
Nacalai Tesque, 17 Ishibashi, Kaide-cho, Muko-shi, Kyoto 617, Japan, and Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan
Direct separation was achieved between two isomers which are enantiomeric to each other by virtue of the presence of hydrogen/deuterium isotopes. A racemic mixture of the R- and S-isomers of phenyl(phenyl-d5)methanol was separated by high-performance liquid chromatography using cellulose tribenzoate-coated silica as a stationary phase and a 2-propanol/hexane (5/95) mixture as a mobile phase, and the absolute configuration of each separated isomer was identified. The cellulose derivative showed preferential retention of (R)-(-)-phenyl(phenyld5)methanol compared to the (S)-(+)-isomer, with a separation factor of 1.0080 based on the preferential binding of a C6D5 group over a C6H5 group to the primary binding site. Separation of compounds which owe their chirality to isotopic substitution is one of the extremes in separation science. Since the beginning of large-scale production of deuterium oxide, separation on the basis of the chirality created by the presence of deuterated and protiated groups of the same functionality on one carbon atom has been drawing attention.1-5 Earlier results on the separation of diastereomers on the basis of isotopic chirality by fractional recrystallization1-3 were disputed by later studies.4-6 Thus, differentiation on the basis of isotopic chirality had been achieved only by spectroscopic methods6-8 and thought to be not amenable to usual separation methods such as fractional recrystallization and chromatography.8 We recently reported an indirect separation on the basis of isotopic chirality by showing the separation of diastereomers created by the presence of C6H5 and C6D5 groups on one of the two chiral carbons of methyl 3-phenyl3-phenyl-d5-glycidate with an achiral reversed-phase liquid chromatography (RPLC) system.9 The presence of an ordinary chiral group next to the isotopic chirality resulted in the difference in †
Nacalai Tesque. Kyoto Institute of Technology. (1) Clemo, G. R.; McQuillen, A. J. Chem. Soc. 1936, 808-810. (2) Clemo, G. R.; Raper, R. Proc. Chem. Soc. 1961, 333. (3) Pocker, Y. Proc. Chem. Soc. 1961, 140-141. (4) Adams, R.; Tarbell, D. S. J. Am. Chem. Soc. 1938, 60, 1260-1262. (5) Coppock, J. B. M.; Kenyon, J.; Partridge, S. M. J. Chem. Soc. 1938, 10691074. (6) Makino, T.; Orfanopoulos, M.; You, T.-P.; Wu, B.; Mosher, C. W.; Mosher, H. S. J. Org. Chem. 1985, 50, 5357-5360. (7) Parker, D. Chem. Rev. 1991, 91, 1441-1457. (8) Meddour, A.; Canet, I.; Loewenstein, A.; Pechine, J. M.; Courtieu, J. J. Am. Chem. Soc. 1994, 116, 9652-9656. (9) Kimata, K.; Kobayashi, M.; Hosoya, K.; Araki, T.; Tanaka, N. J. Am. Chem. Soc. 1996, 118, 759-762. ‡
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Figure 1. Structures of phenyl(phenyl-d5)methanol, R-1 and S-1.
the hydrophobic properties between the diastereomers, which were differentiated through the interaction with the aqueous mobile phase. The purpose of this communication is to report direct separation between the R- and S-isomers of phenyl(phenyld5)methanol (benzhydrol-2,3,4,5,6-d5, 1, Figure 1) on the basis of the favorable binding of the C6D5 group of the R-isomer (R-1) to the cellulose tribenzoate stationary phase. EXPERIMENTAL SECTION A racemic mixture of phenyl(phenyl-d5)methanol (1) was prepared by reduction of benzophenone-2,3,4,5,6-d5 with LiAlH4. Di(phenyl-d5)methanol was prepared similarly. For the purpose of identification, R-1 and S-1 were prepared individually from optically active precursors. 4-Bromophenyl phenyl-d5 ketone was reduced with NaBH4 to produce 4-bromophenyl(phenyl-d5)methanol. The enantiomers of 4-bromophenyl(phenyl-d5)methanol were separated by using a cellulose tribenzoate-coated silica column (20 mm i.d., 25 cm in length) in hexane/2-propanol (80/ 20) mobile phase at room temperature, and the configuration of each separated enantiomer was identified.10 Treatment of (R)(+)- and (S)-(-)-4-bromophenyl(phenyl-d5)methanol with LiAlH4 in tetrahydrofuran under reflux afforded (R)-(-)-phenyl(phenyld5)methanol (R-1) and the (S)-(+)-isomer (S-1), respectively. The structures of the products were confirmed by NMR (200 MHz, CDCl3) and MS. The optical rotations, [R]20D ) -1.05 (c 12, CHCl3) for R-1 and [R]20D ) +0.95 (c 12, CHCl3) for S-1, agreed well with the reported values.6 HPLC separation was carried out by using a pump with two 10 µL displacement heads (Shimadzu LC-9A), a variable-wavelength UV detector (Shimadzu SPD-6A) having an 8-µL cell, and a recycle column system consisting of four columns of 6 mm i.d., 15 cm in length. Cellulose tribenzoate was coated onto silica particles (Nucleosil, pore size 100 nm, particle size 5 µm), as described previously,11 and used as a stationary phase. The solutes were monitored at 220 nm in each cycle. (10) Wu, B.; Mosher, H. S. J. Org. Chem. 1986, 51, 1904-1906. (11) Kimata, K.; Tsuboi, R.; Hosoya, K.; Tanaka, N. Anal. Methods Instrum. 1993, 1, 23-29. S0003-2700(97)00338-7 CCC: $14.00
© 1997 American Chemical Society
Figure 2. Separation of isotopic chirality obtained with phenyl(phenyl-d5)methanol(R-1 and S-1) by using four columns (6 mm i.d., 15 cm) of cellulose tribenzoate-coated silica in 2-propanol/hexane (5/95) mixture at 40 °C, 65 cycles.
RESULTS AND DISCUSSION Separation of isotopic chirality had been attempted in earlier studies with compounds having C6H5 and C6D5 groups on a chiral carbon,1-4,6,8,9 and this approach was thought to be promising, based on the greater CH/CD isotope effects with aryl groups on chromatographic retention than with alkyl groups.12 Phenyl(phenyl-d5)methanol (1) was selected as a substrate because this compound was one of the targets of earlier studies.3,8 It is known that a chiral stationary phase is effective for the separation of certain types of enantiomers. We selected a cellulose-based stationary phase which showed high selectivity for chiral aromatic alcohols.13 Cellulose tribenzoate-coated silica, prepared as previously described,11 can easily separate the racemates of substituted diphenylmethanols having ordinary chirality with a 4-methyl, 4-chloro, or 4-bromo substituent on one of the two phenyl groups. Compound 1 (a mixture of R-1 and S-1) was subjected to recycle chromatography by using cellulose tribenzoate-coated silica as a stationary phase in a 2-propanol/hexane (5/95) mixture at 40 °C. After 65 recycles with about 350 000 theoretical plates, separation into two peaks was obtained, with a separation factor of 1.0080 and resolution of about 1.0, as shown in Figure 2. The resolution was poorer at lower temperatures due to the lower column efficiencies. The elution order of R-1 and S-1 and the effect of each deuterated phenyl group on retention were studied by chromatographing the enantiomers 1 with diphenylmethanol (C6H5CHOHC6H5, 2) and di(phenyl-d5)methanol (C6D5CHOHC6D5, 3). Figure 3a shows the separation of 1, 2, and 3, and parts b and c of Figure 3 show the separation between R-1 and S-1 and between 2 and 3, respectively, at 29 recycles. The compound with isotopic chirality with one deuterated phenyl group (R-1 and S-1) were eluted between 2 and 3. Figure 3d,e indicates that R-1 is retained longer than S-1 on the cellulose benzoate-coated silica. Figure 3 suggests the additivity in the isotope effect in the direct separation of isotopic chirality with the elution order 2, S-1, R-1, followed by 3. The more deuterated species underwent the more favorable binding. The C6D5 group in R-1 accounted for about two-thirds of the total isotope effect, the increase in retention due to the presence of deuterium, observed between 2 and 3, showing much greater effect than the C6D5 group in S-1. The difference in the effect of a C6D5 group between R-1 and S-1 is greater than 30% of the total isotope effect of the two C6D5 groups observed between 2 and 3, as shown by the separation factors, (12) Tanaka, N.; Thornton, E. R. J. Am. Chem. Soc. 1977, 99, 7300-7307; 1976, 98, 1617-1619. (13) Wainer, I. W.; Stiffin, R. M.; Shibata, T. J. Chromatogr. 1987, 411, 139151.
Figure 3. Chromatography of phenyl(phenyl-d5)methanol (1), diphenylmethanol (2), and di(phenyl-d5)methanol (3): (a) 1, 2, and 3; (b) 1; (c) 2 and 3; (d) 2 and R-1; and (e) S-1 and 3. Conditions are similar to those in Figure 2 except for the number of recycle operations, 29 cycles.
k′(R-1)/k′(S-1) ) 1.0080 and k′(3)/k′(2) ) 1.022. When bound to the stationary phase, the two phenyl groups of a diphenylmethanol molecule must be in significantly different environments, one in a site differentiating CH and CD and the other in a site not so differentiating, leading to the easier direct separation of the racemates having isotopic chirality (in terms of a separation factor) than expected considering the difficulty experienced with the separation of the diastereomers in RPLC.9 The difference in the environments of the two phenyl groups of diphenylmethanol upon binding to a helical structure of a polypeptide was mentioned in a NMR study.8 It has been reported that cellulose tribenzoate stationary phase can provide a chiral separation for secondary aromatic alcohols on the basis of two points of attachment, the hydrogen bonding between the hydroxyl group of a solute and the ester carbonyl group of the stationary phase, and the insertion of a phenyl group into a cavity of a stationary phase, while the other groups provide additional steric interactions.13 The present results suggest that the binding of a C6D5 group is more favorable than that of a C6H5 group at the primary binding site, implying the tight fit of one phenyl group, which seemed to provide the greater separation factor for direct separation of isotopic chirality than the diastereomer separation in RPLC,9 where the differentiation was provided by the difference in the hydrophobic properties. The cellulose tribenzoate stationary phase showed the preferential retention for R-1 for the isotopic chirality, the R-isomer of 1-phenylethanol,13 the S-isomer of (4-bromophenyl)phenylmethanol, (4-chlorophenyl)phenylmethanol, and (4-methylphenyl)phenylmethanol. The preferred configurations have an aromatic group, which is the smaller in the case where there are two, at the same position relative to hydrogen and a hydroxyl group on Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
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the chiral carbon, although the discrimination mechanism for the isotopic chirality is not totally clear. Examination of enantiomer separations on the basis of isotopic chirality will contribute to the understanding of chiral discrimination processes, because isotopic species are expected to assume the same complexation geometry upon binding to a chiral selector. The direction (preference for either a protiated or a deuterated moiety) and the magnitude of the isotope effect on binding may provide information on the properties of binding sites as well as the type of interactions involved.
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ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture. The authors thank Prof. Volker Schurig of University of Tubingen for his helpful suggestions. Received for review March 31, 1997. Accepted April 7, 1997.X AC970338K X
Abstract published in Advance ACS Abstracts, May 15, 1997.
