ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
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Discrimination of Monoster eoisomer s in Asymmetric Sohent s by Fourier Transform Infrared Spectrometry David L. Grieble and Peter R. Griffiths" Department of Chemistry, Ohio University, Athens, Ohio 4570 1
Tomas Hirschfeld Block Engineering, Inc., 79 Blackstone Street, Cambridge, Massachusetts 02 139
I n principle, both members of an enantiomeric pair have identical physical properties except to polarized radiation, and thus cannot be distinguished by conventional spectrometric methods. To resolve both compounds, diastereoisomeric derivatives may be prepared by combining them with an asymmetric reagent ("resolving agent"). The resulting change in molecular symmetry then makes both molecules distinguishable. The same effect may be used for analytical purposes through the measurement of spectral perturbations caused by dissolving the enantiomers in an asymmetric solvent. The "virtual diastereoisomers"produced by the solute-solvent interactions will then produce different solvent-induced band shifts which could be used analytically. The procedure is demonstrated by measurements on all the permutations of both stereoisomers of malic acid dissolved in both stereoisomers of 2-oclanol.
T h e analytical differentiation of monostereoisomers is an important aspect of the analysis of natural products, biochemicals, and pharmaceuticals. Such isomers are indistinguishable, in principle, by any physical measurement except those employing polarized radiation. The most common method of distinguishing between monostereoisomers is to determine t h e optical rotatory activity of a solution in an optically inactive solvent ( 1 ) . This measurement is generally performed using ultraviolet or visible radiation, since the optical components necessary for these measurements (achromatic quarter wave plates, rotators, etc.) are not available for infrared radiation. Thus the infrared equivalent of the highly successful ultraviolet-visible optical rotatory dispersion or circular dichroism techniques has not been achieved even though it should have great value in structural analysis. In several analytical and preparative techniques, otherwise identical monostereoisomers are distinguished by combining them with a second monostereoisomer (the "resolving agent"). The resulting diastereoisomeric derivative now shows gross differences in physical properties depending on which initial compound it was obtained from. It should be possible for analytical purposes to dispense with the synthetic step described above, and to study the properties of t h e different "virtual diastereoisomers" formed by solute-solvent interactions between an asymmetric sample and an asymmetric solvent. Fourier transform infrared (FT-IR) spectrometry has been used to distinguish between the spectra of closely related isomers (2) and to detect weak solute-solvent interactions ( 3 ) through the application of absorbance suhtraction routines. A logical extension of these studies was to investigate whether the absorbance subtraction technique could be used t o show specific interactions between asymmetric solutes and asymmetric solvents. In this work we have 0003-2700/78/0350-0415$01.00/0
used the two optical isomers of malic acid for the solute and those of 2-octanol for the solvent.
EXPERIMENTAL Samples of the enantiomers of malic acid, designated +M and -hl, (Fluka AG, Puriss Grade) and of 2-octanol, designated +O and -0, (Fluka AG, Purum Grade) were obtained from Tridom Chemical. Inc. (Hauppage, N.Y.). The octanol samples had been determined as hetter than 9970 pure by gas chromatography, and had specific rotations, [(I];:, of k l l 1 ". The malic acid samples had quite different physical properties. b'hereas the sample of +M was white and apparently quite pure [mp = 98-102 "C. cf. the literature value ( 4 ) of 100-103 "C] and dissolved readily in octanol, the sample of -M was discolored, had a melting range of 92-103 "C, and did not completely dissolve in octanol. The sample of --M was purified by dissolving it in the minimum amount of a heated 1:l mixture of acetone and chloroform, filtering the solution after most of the sample had dissolved, and cooling the filtrate in ice to recover the malic acid. After three such treatments a Khite product was obtained whose melting range was 100-102 "C and whose dissolution properties in octanol were identical to those of +M. The specific rotations of the malic acid samples measured as 1070 (w/w) solutions in water were -1.7' and +1.6O, respectively. A411spectra were measured using a Model FTS-14 FT-IR spectrometer (Digilab, Inc., Cambridge, Mass.) at a resolution of 4 cm-' using double precision (32 bits per word) signal-averaging, FFT, and arithmetic routines. Solutions with 0.100 mole fraction of malic acid in 2-octanol were prepared and held in a precision sealed cell (ffilks Scientific Corp., South Norwalk, Corin.). This cell was the thinnest we could obtain commercially, hut even though its pathlength was specified as 15 pm, it was measured as 25 pm. At this pathlength, one band in the spectrum of malic acid and three bands due to 2-octanol had peak absorbances greater than 2.5. The absorbance of all the other bands in the spectra of the solutions studied were of the correct magnitude to allow spectral subtraction techniques to be used to differentiate the spectra. All measurements were made without dismantling the cell holder, and we believe the pathlength to be constant to within 1YC.
*
RESULTS AND DISCUSSION The spectra of all four solutions (-M in +0,+M in +0,-hf in -0, and + M in -0) showed appreciable differences in overall intensity as shown in Figure 1. All bands in the spectrum of -M in +O were substantially weaker than the corresponding bands in the spectra of any of the other solutions, and the spectrum of +M in -0 was somewhat more intense than that of the other two solutions. No obvious spectral shifts are seen from these spectra before spectral subtraction routines are applied, but the relative intensities of several bands can be seen to have changed. The most obvious region where this effect can be observed is between 1100 and 1000 cm-' (see Figure 2), where the intensity of the band a t 1040 cm-' relative to the intensity of the band at 1070 cm-l can be seen to change markedly. Before the result of applying spectral subtraction routines is discussed, it should be recognized that for these routines C 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
1800
1600
1400
1200
FREQUENCY ( C M - ' )
I ooa
800
Figure 1. Absorbance spectra of the malic acid/2-octanol solutions.
0.25
W
z 4
(L
0 v) m 4
0 0 0 II'
Figure 2. Scale-expanded absorbance spectra of malic acid/2-octanol solutions between 1100 and 1000 cm-'. (A) (+M in +O); (6) (-M in -0); (C) (SM in -0); (D) (-M in +0)
to be effectively used, the peak absorbance of all bands in the spectrum should be small, preferably less than 0.7 (5-8). However both malic acid and octanol are such strong infrared absorbers that we were unable to keep the peak absorbance of the strongest bands in the solution spectra (at 1715, 1455, 1375 and 1110 cm-') below 2.5 with the IR cells available t o us a t the moment. If absorbance spectra containing such intense bands as these require multiplication by a large scaling factor before subtraction, it is known that artifacts will be introduced into the difference spectrum due to the effect of "resolution errors" (5). Thus all difference spectra are plotted only at frequencies where the absorbance is less than a certain value, which was arbitrarily selected as 2.5. In addition no difference plots involving strong bands in the spectrum of -M in +O are shown, since the scaling factor would have to be so great that enormous resolution errors are incurred and meaningful conclusions cannot be drawn from the data. The three difference spectra showing the result of subtracting each combination of two of the other three spectra are shown in Figure 3. Two features, centered at 1715 cm-' and 1035 cm-', are prominent in these spectra. The band at 1715 cm-' may be unequivocally assigned to the carbonyl
2000
FREQUENCY ( C M - ' )
700
Figure 3. Unscaled difference spectra of malic acid/2-octanol solutions. (A) (+M in +O) - (SM in -0); (6)(+M in S O ) - (-M in -0); (C) (-M in -0) - (+M in -0). Note evidence of a shift in the carbonyl band of (+M in +0) and (+M in -0) (spectrum A), but the similarity between the spectra of (-M in -0) and (+M in -0) (spectrum C). The variation in intensity of the 1035 cm-' band is very apparent from these spectra. Frequency regions where the absorbance of either spectrum exceeds 2.5 are left blank
stretching vibration of malic acid, while the 1035 cm-' band is present at medium intensity in the spectrum of octanol and weakly in the spectrum of malic acid. Since octanol is the major component, it is most likely that perturbations to this band indicate an interaction with the octanol molecule. However, we are not certain of the vibrational mode to which this band can be assigned; initially we believed it to be due to the C-0 stretching vibration, but the strong band a t 1110 cm-' is better assigned to this mode for a secondary alcohol (9). Small shifts and intensity changes associated with other bands are evident from the difference spectra, but the effects
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
0.10
Flgure 4. Scaled difference spectra, (+M in -0) - x(-M in +O),between 900 and 700 cm-'. (A) x = 1.15: (B) x = 1.20; (C) x = 1.25: (D) x = 1.30
of these changes are generally of much smaller magnitude than the effect of the band shift on the carbonyl band of malic acid and the intensity change in the 1035 cm-' band of 2-octanol. Therefore it appears that infrared difference spectrometry can indeed detect spectral changes due to interactions near the chiral center, and thus differentiate between monostereoisomers for analytical purposes. It is also apparent that much more work is needed for a theoretical interpretation of these perturbations in terms of sample structure. An interesting question is raised by the intensity difference between t h e spectrum of -M in +O and that of + M in -0. This anomaly, which occurs for the bands due t o both components of t h e solutions, could not be traced to air bubbles, cell thickness variations, sample preparation, or other such trivial causes. It is also of interest that whereas some bands in the spectra of the other three solutions were as much as twice as intense as the corresponding bands in the spectrum of -M in +0, other bands are only slightly stronger. For example, in the region between 900 and 700 cm-', there are two bands of medium intensity (at 840 cm-' and 722 cm-I). Each of these bands in t h e spectrum of -M in +O is apparently slightly shifted from the corresponding bands in the spectra of the other three solutions, while also exhibiting a relatively small intensity change. Figure 4 shows the result of multiplying the spectrum of -M in +O by various scaling factors and subtracting the result from the spectrum of + M in -0. The intensity of the 840 cm-l band is equalized by applying a scaling factor of 1.20, as evidenced by the most symmetrical feature in the series of difference spectra, while the intensity of the 722 cm-' band is equalized by applying a scaling factor of 1.25. Other bands in the spectrum require scaling factors
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of 2.0 or more to minimize the difference band, although it should be stressed that the effect of resolution errors is very noticeable when such large scaling factors are applied. The explanation of these phenomena seems to lie in t h e nature of the residual polarization in Michelson interferometers. I t has been shown that the beamsplitter acts differently on both polarizations, so that a weak residual polarization develops in the output beam (10). However, since a t all scan locations except the center fringe there is a phase delay between the interfering beams, this residual polarization will be not linear but elliptical (11). The spectral. degeneracy between monostereoisomers will, of course, break down in elliptically polarized light. The effect has not yet been calculated quantitatively because of the multicyclic nature of the delay and the mathematical consequences of the Fourier transformation, which lead to fairly complex equations. The complementary o d p u t emerging from the interferometer will show a residual ellipticity of the opposite sense, raising the possibility of infrared circular dichroism measurements using a dual-beam (optical subtraction) FT-IR system of the type described recently (12), and an attempt to build a spectrometer for this purpose is now under way. In summary it can be stated that the solute-solvent interactions between a monostereoisomeric solute and a chiral solvent lead to the formation of a virtual diastereoisomer capable of breaking the degeneracy between the spectra of the enantiomers. These interactions, picked up by absorbance subtraction FT-IR spectrometry allow analytical differentiation of monostereoisomers. The behavior of different bands can be used to study molecular structure and behavior, although much work will have to be done to clarify these phenomena.
ACKNOWLEDGMENT We gratefully acknowledge the advice for the purification of malic acid given to us by William Huntsman of Ohio University.
LITERATURE CITED (1) C. Djerassi, "Optical Rotatory Dispersion", McGraw-Hill, New York, N.Y., 1960. (2) R. M. Gendreau and P. R. Griffiths, Anal. Cbem., 48, 1910 (1976). (3) T. Hirschfeld and K. Kizer, Appl. Spectrosc., 29, 345 (1975). (4) "CRC Handbook of Chemisby and physics", 46th ed.,The C h e m i i l Rubber Co., Cleveland, Ohio, 1967. (5) R. J. Anderson and P. R. Griffiths, Anal. Cbem., submitted for publication (1977). (6) T. Hirschfeld, Appl. Specfrosc., 29, 523 (1975). (7) T. Hirschfeld, Appl. Spectrosc., 30, 550 (1976). (8) T. Hirschfeld, Anal. Cbem., submitted for publication (1977). (9) K. Nakanishi, "Infrared Absorption Spectroscopy-Practicai", Holden-Day, San Francisco, Calif., 1962. (10) T. Hirschfeld, Appl. Spectrosc., 29, 192 (1975). (11) M. J. Block, J . Opt. SOC. Am., (advertisement), April 1960. (12) D. Kuehl and P. R. Griffiths, Anal. Chem., 50, this issue
RECEIVED for review August 22, 1977. Accepted December 2 , 1977.
