Anal. Chem. 1982, 54, 709-711
709
Spectrophotometric and Polarimetric Detectors in Liquid Chromatography for the Determination of Enantiomer Ratios in Complex Mixtures W. Boehme,"
Gi.
Wagner, and U. Oehme
Bodenseewerk Perkin-Elmer & Co. GmbH, Ueberlingen, West Germany
U. Priesnitz Bayer AG, Wuppetfal, West Germany
The comblned utlllzatlon of UV and polarlmetrlc LC detectors In serles for the quantltatlon of enantlomers of trans-permethrlnlc acld pentafluorobenzyl ester (PBE) Is demonstrated. The chromatographlc separatlon of the enantlomers Is not required as the response of the UV detector Is proportional to the total amount of enantiomer present, while the response of the polarlmetrlc detector depends on the ratlo of enantlomers present. Therefore, the quantltatlon of the total amount plus the iratlo of R and S trans PBE whlch are coelutlng In a llquld chromatographlc peak Is posslble.
Liquid chromatography has become a powerful separation technique in the last decade ( 1 , Z ) and now has broad utilization in many fields of application ( 3 , 4 ) . Many separations previously not achievable using classical liquid chromatography are now possible due to the utilization of smaller particle supports and their inherently higher efficiency. In most instances, the resolution of cis-trans isomers and diastereoisomers is possible and has been described for various classes of compounds from pharmaceutical chemistry (5),biochemistry (64,and clinical chemistry (3, 9, 10). Enantiomer separations have generally been performed on stationary phases such as starch and cellulose derivatives (I1,12)in low pressure systems. These supports, however, are not suitable for modern high-pressure LC, due to their mechanical instability. In view of this, some investigators have described various approaches to the separation of enantiomers with modern LC, such as the use of chiral stationary phases (13, 14) or the use of chiral additives in the mobile phase (15,16) for the analysis of amino acids. Although modern LC has become an efficient separation technique, there are some areas, such as in the analysis of enantiomers, where improvements in quantitative capabilities and detection limits can be made. In this paper, we describe the combined use of UV and polarimetric detectors for the analysis of chiral compounds in a mixture with other nonchiral compounds, where the chromatographic separation of enantiomers is not required. Since the R and S isomers do not show any difference in their absorption characteristics, only the total amount of R and S enantiomer can be determined with the UV detector. The polarimeter, however, records the ratio of enantiomer as it measures the integral rotation value of the eluting band. The combined use of both detectors in series thus permits the calculation of the enantiomer ratio and therefore the amount of each enantiomer present in a coeluting peak. Permethrinic acid pentafluorobenzyl ester (PBE) is a compound which has two asymmetrical carbon atoms in a cyclopropane ring (Figure 1). The two pairs of enantiomers have different biological activities as an insecticide. Therefore, 0003-2700/82/0354-0709$01.25/0
for synthesis optimization and production control, it is necessary to determine the enantiomer ratios in reaction mixtures and in the final product. The cis and trans isomer pairs are separated by liquid chromatography, while the R and S enantiomer ratio is determined from the UV and polarimeter responses. The UV ieignal determines the total amount of trans isomer, while the polarimeter signal determines the ratio of enantiomers present.
EXPERIMENTAL SECTION Solutions (50 m g / d ) of standards of (R)-and (S)-trans-and (R)-and (S)-cis-PBE and a racemic mixture of the trans compound were prepared in 5% diethyl ether/95% n-hexane. Aliquots of 1-20 pL were injected for chromatographic analysis. The liquid chromatography system utilized consisted of a Series 1pump, an LC-75variable wavelength UV detector, and a Model 241 LC polarimeter all from the Perkin-Elmer Corp. (Norwalk, CT). The polarimeter was equipped with a micro flow cell having a 10 cm optical pathlength and a 0.65 mm internal diameter, thus having a geometrical volume of 33 pL (17). A wavelength of 302 nm was used with a recording range resulting in * 0 . 5 O full scale. The UV detector was operated at either 225 or 280 nm. The detectors were arranged in series, the UV detector followed by the polarimeter, using a 0.38 mm i.d. stainless steel capillary tube between two detectors. The column used was 250 X 4 mm id. packed with 5 pm silica 100-5particles (Bodenseewerk PerkinElmer, Ueberlingen, West Germany). The mobile phase was 5% diethyl ether in n-hexane (Merck AG, Darmstadt, West Germany, Uvasol quality). The analytical flow rate was 2 mL/min which generated a back-pressure of 6.3 MPa. The mobile phase was degassed for 3 min in a Sonorex RK 102 ultrasonic bath (Bandelii, Berlin, West Germany) before use. The samples were injected with a Rheodyne 7105 injector (Rheodyne Corp., Santa Rosa, CA) and a 10 or 25 pL Hamilton syringe (Bonaduz, Switzerland). Chromatograms were recorded on a Perkin-Elmer Model 561 dual-pen recorder. Quantitative determinations and linearity plots were made by measuring peak heights. An optical rotatory dispersion spectrum was obtained by using a Jasco 5-500 spectropolarimeter (Jasco Spectroscopic Co. Ltd., Tokyo, Japan) with sample concentrations of 2 mg/mI, in isooctane.
RESULTS AND DISCUSSION A typical chromatogram from a reaction mixture containing both optically active and nonactive compounds is shown in Figure 2, using both the UV and polarimetric detectors. The cis and trans isomers are separated chromatographically on the system described, however, determining the amount of (1R)-(-)-trans-PBE in the synthesis mixture, in which the R and S cis and R and S trans isomers of the starting material and the reaction products are present, is a major difficulty. Figure 3 demonstrates that the silica stationary phase used has sufficient selectivity in order to separate the trans and cis products which are both in the S configuration. Ten microliters of standard solutions of (S)-cisand (S)-trans-PBE was injected onto the column, corre0 1982 American Chemical Society
710
ANALYTICAL CHEMISTRY, VOL. 54,
NO.4, APRIL
1982
CH3
Flgure 1. Chemical structure of trans-permethrinic acid pentafluorobenzyl ester (PBE)
10’
Flgure 4. ORD spectrum of (1R)-trans-permethrinic acid pentafluorobenzyl ester In Isooctane.
I
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8min7
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6
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5
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2
3
1
I 0
Figure 2. Comparison of the spectrophotometric and polarimetric detector in the analysis of a synthesis mlxture of an optlcally active Insecticide (for condltlons see text).
Jil i >
Lrnin 3
2
1
0
Flgure 3. Separation of (lS)-trans- and (1s)-cis-PBE on a slllca column wlth polarlmetric and spectrophotometric detectlon.
sponding to absolute sample amounts of 500 pg each. With this amount of sample, the capacity of the column, which is approximately 5 x lo4 g/g of stationary phase, is not exceeded (18,19). This was verified by determining that the column efficiency (about 10000 theoretical plates) was not degraded. Due to band broadening in the interconnecting tubing and the relatively large volume of the flow cell in the polarimeter, however, only about 50-60% of the column efficiency is realized from this detector. As indicated in Figures 2 and 3, the sensitivity of the polarimetric detector in terms of signal-to-noise ratio is much
O
K
0
,
100
I
,
200
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I
300 Sampie amount
1
1
400
,
/
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1
/
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119600
Figure 5. Linearity of detector signals for (1S)-trans -PBE.
lower than that of the UV detector. We estimate that the minimum detectable amount of sample is about 50 kg, at a wavelength of 302 nm. The sensitivity of the polarimetric detector using different analytical wavelengths was in good agreement with the ORD spectrum, which is shown for (lR)-truns-PBE in Figure 4. Sensitivity, which is one of the important parameters in modern liquid chromatography, is improved significantly for this compound, when recording at wavelengths below 300 nm. In order to determine the working range of the system, the linearity of both detectors was measured. A major consideration with the peak height measurement is the capacity of the column, which has been previously discussed. Another is the linearity of both detectors, which was determined experimentally. A plot of peak height units vs. amount of sample for (R)-trans-PBE is shown in Figure 5 for both detection systems. The wavelengths used were 302 nm for the polarimetric detector and 280 nm for the spectrophotometric detector. A linear response is obtained up to 600 kg for both systems. At the absorbance maximum of 225 nm, the linear range is only to about 100 pg with the UV detector; therefore, the higher wavelength is utilized. Various enantiomer ratios were injected with the total amount of trans isomer kept constant at 500 p g . The results are plotted in Figure 6 as the peak height ratio of the polarimetric and UV detectors vs. the amount of 1s trans isomer in the sample. The amount of IS trans isomer in the sample is determined by measuring the peak heights from each detector, ratioing those values, and graphically determining the amount of isomer present. By ratioing the peak heights, the plot of the corrected peak heights is free of sampling errors. By use of wavelengths of 302 nm for the polarimetric detector and 280 nm for the UV detector, the upper linear range was experimentally determined to be about 600 pg. The total range of determination of enantiomer ratio is, therefore, from 60 pg, due to the detection limit of the polarimeter, to about 600 pg caused by linearity limitations and the capacity of the
ANALYTICAL CHEMI!STRY, VOL. 54, NO. 4, APRIL 1982
711
detector, however, is its high degree of selectivity (20). These considerations, together with the large cell volume, make the polarimetric detector well suited for preparative separations of optically active compounds. Typical chromatograms showing the traces from both detectors are presented in Figure 7. In sequence are 2-pL injections of a racemic mixture followed by the (1R) trans and (1s)trans isomers. The chromatographic conditions are given in the Experimental Section. The detector parameters are 302 nm, f 0 . 5 O recording range, and 0.5 s response time for the polarimeter and 225 nm, at 5.12 AUFS, for the UV detector.
LITERATURE CITED 0.4
+ 0
I
I
I
IO 20 30 40 50 IS-trans in racemate
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I
I
60
70
80
I
I
90 %lo0
Flgure 6. Peak height ratlos vs. composltlon of enantiomers for trans-PBE (total amount 500 pg).
11
Figure 7. Typical chromatograms of trans enantiomers and a racemlc mixture.
analytical column. This sensitivity limitation of the polarimetric detector restricts ita use somewhat, as relatively large amounts of sample must be injected in order to obtain adequate signal-to-noise ratios. The primary advantage of this
(1) Snyder, L. R.; Klrkland, J. J. "Introduction to Modern Liquid Chromatography"; Wliey-Interscience: New York, 1979. (2) Engelhardt, H. "High Performance Liquid Chromatography, Chemical Laboratory Practice"; Springer: Berlin, Heidelberg, New York, 1979. (3) Hawk, G.,Ed. "Chromatographic Sclence Series"; Marcel Dekker: New York, 1979;Vol. 10. (4) Heftmann, E. J . Chromafogr. Llbr. 1976, 8. (5) Ali, S. L.; Strittmatter, T. I n f . J. Pharm. 1978, 1 (3),185. (6) Barnett, S. A.; Frick, L. W.; Baine, H. M. Anal. Chem. 1960, 52,610. (7) Hara, S.; et ai. Anal. Chem. 1980, 25,429. (8) Drlnkwine, A. D.; Bristol, D. W.; Fleeker, J. R. J . Chromafogr. 1879, 174, 264. (9) Shallch, B.; Hallmark, M. R.; Issaq, H. J.; Rlsser, N. H.; Kawalek, J. C. J . Llq. Chromafogr. 1979, 2 (7),943. (IO) Wooldrldge, T. A.; Llghtner, D. A., J . Ll9. Chromafogr. 1978, 1 ( 5 ) , 653. (11) HBkl, H.; Mannschreck, A. Angew. Chem. 1977, 89,419. (12) Rogozkin, S. V.; Davandov, V. A. Russ. Chem. Rev. 1968, 30 (7), 565. (13) Davankov, V. A.; Zolotarev, Y. A. J . Chromafogr. 1978, 755,303. (14) Lefebre, B.; Audebet?, R.; Quivlron, C. J . Ll9. Chromafogr. 1978, 7 , 761. (15) Hare, P. E.; Gii.-Av., E. Science 3979, 204, 1226. (16) Llndner, W.; Le Page, J. N.; Davles, G.; Seltz, D. E.; Karger, B. L. J. Chromafogr. 1978, 1'85,323. (17) Bbhme, W. Chromafogr. News/. 1980, 8, 38. (18) Done, J. N. J . Chromafogr. 1978, 725,43. (19) Endele, R.; HalBsz, I.; Unger, K. J . Chromafogr. 1974, 99,377. (20) Di Cesare, J. C.; Ettre, L. S., presented at the 5th International Sym,. posium on Column I-lquid Chromatography, Avignon, France, May
1981.
RECEIVEDfor review August 18, 1981. Accepted December. 21, 1981.
