Anal. Chem. 1981, 53, 558-560
558 CO(CF,CW),
50’00HZ
250OHz
Figure 2. The 94.1-MHz ‘‘F NMR
0 Hr
spectrum of
0.19 M cobalt(I1)trl-
fluoroacetate In ethanol at 225 K.
fluoroacetate in ethanol (8). This complex dissociates due to the formation of the the mono complex Co(CF,COO)+ and uncoordinated CF3COO- anions. Accordingly three separate signals can be observed at low temperatures in the 19FNMR spectrum, i.e., for the uncoordinated trifluoroacetate anion and the trifluoroacetate anion bound in the mono complex and in the bis complex. In Figure 3 the application of density matrix theory is demonstrated for the sulfur-bridged 1,8perinaphthalene- (3H,7H)-naphthol[1,&de]- 1,Zdithiepin (9). The unsymmetrical twist-boat conformer shows a t low temperatures racemization via boat conformation within the NMR time scale. Therefore at room temperature one averaged signal is obtained whereas a t very low temperatures an AB pattern is shown.
LITERATURE CITED Ullman, A. H.; Pollard, B. D.; Boutilief, G. D.; Bateh, R. P.; Hanley, P.; Winefordnet, J. D. Anal. Chem. 1070, 51, 2382-2387. Martin, C. R.; Frelser, H. Anal. Chem. 1070, 51, 803-807.
150Hz
lOOHz
50tk
0 Hz
Figure 3. The 100-MHz ‘H NMR spectrum of the
methylene protons of 0.1 M 1,8-perlnaphthalene-(3H,7H)-naphthol[1,8-de]-1,2dithlepln at 185 K In acetone-de. Application examples for two cases are shown in Figures 2 and 3. In Figure 2 an example with uncoupled spins is given for three different chemical shifts, the complex cobalt(I1) tri-
Plnnlck, H. R., Jr.; Smith, R. L. Thermochim. Acte 1080, 35. 375-379. McConnel, H. M. J . Chem. Phys. 1958, 28, 430-431. Buckley, P. D.; Joky, K. W.; Plnder, D. N. “Progress in Nuclear M a g netic Resonance”; Emsley. J. W., Feeney, J., Sutcllffe. L. M., Eds.; Pergamon Press: Oxford, 1975: Vol. 10, Part 1, pp 1-26. Sack, R. A. Mol. Phys. 1058, 1 , 163-167. Stelnberg, D. J. ”Computational Matrix Algebra”; McGraw-Hill: New York, 1974; p 92. Dickert, Franz L. 2.Phys. Chem. ( Wlesbaden) 1070, 110. 249-266. Guttenberger, Hans 0.; Bestmann, Hans J.; Dickert, Franz L.; Jerrgensen, Fleming S.; Snyder, James P. J . Am. Chem. Soc.,In
press.
RECEIVEDfor review June 17,1980. Accepted November 12, 1980. This work was supported by “Deutsche Forschungsgemeinschaft” and “Fonds der Chemischen Industrie”.
Long-Range Coupling Constants for Structural Analysis of Complex Polycyclic Aromatic Hydrocarbons by High-Field Proton Magnetic Resonance Spectrometry Frederick E. Evans,” Peter P. Fu, and Thomas Cairns’ Department of Health, Education and Welfare, Food and Drug Administration, National Center for ToxicologicalResearch, Jefferson, Arkansas 72079
Shen K. Yang Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 200 14
Polycyclic aromatic hydrocarbons (PAHs) are an important class of compounds in many fields including chemical carcinogenesis (1-4). ‘H nuclear magnetic resonance (NMR) spectral data can provide unique structural information about PAHs. However, in many instances, the ‘H chemical shifts are difficult to analyze because many resonances are confined to only about 2 ppm in the aromatic region of the spectrum. The relatively large ortho and meta coupling constants do not contain direct information on the relative position of protons on different rings of a PAH. The smaller long-range coupling constants can provide this type of information, but their use in complex PAHs has not previously been practical. We report here the results of high-field ‘H NMR measurements together Present address: Food and Drug Administration, FDA District Laboratory, 1521 W. Pic0 Blvd., Los Angeles, CA 90052.
with a “Lorentzian to Gaussian” resolution enhancement and homonuclear decoupling to the analysis of 8-methylbenz[a]anthracene (&MeBA) and two of its metabolites. The general usefulness of long-range coupling constants for structural studies of complex PAHs and their metabolites is discussed.
EXPERIMENTAL SECTION The 8-methyl-BA was synthesized according t o published procedure (5). The preparation of 8-hydroxymethyl-BA and 8-hydroxymethyl-BA-trans-5,6-dihydrodiol will be described elsewhere. Samples were dissolved in 99.97% acetone-& Concentrations ranged from 0.01 to 0.04 M. The NMR spectra were obtained on Bruker WM500 or WH270 spectrometers in the ‘H configuration at ambient probe (5 mm) temperature. The chemical shifts were reported in parts per million downfield from tetramethylsilane (Me,Si) by assigning the acetone-d, resonance as 2.056 ppm. The Lorentzian to Gaussian multiplication and
This article not subject to U S . Copyright. Publislhed 1981 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
I
a
, ,, 9.35
, .. 9.25
ii
11
,
, I
I64
,
8.6 8.1
559
I
8.0
7.9
7.8
7.7
7.6
7.5
7.4
PPM
Flgwe 1. (a) 500-MHz 'H N M I spectrum of the aromatic region of 8-methylbenz[a]anthracene (0.04 M) in acetonede with ring numbering system. Data conditions: data size, 64K; sweep width, 3900 Hz; number of scans, 128; relaxation delay, 0. (b) A Lorentzian to Gaussian multiplication of the free induction decay with line broadening of -1.4 Hz and Gaussian broadening of 0.2 of the acquisition time.
Table I. Proton Chemical Shifts in ppm of 8-Methylbenz[a]anthracene and Metabolites B-hydroxymethyl8-methyl- 8-hydroxyBA-trans-5,6assignment BA methyl-BA dihydrodiol 8.960 8.958 7.705 7.706 7.641 7.641 7.919 7.921 7.726 7.724 7.915 6 7.946 7 8.617 8.713 5.293 Ba 2.826 9 7.422 7.667 10 7.468 7.545 11 8.078 8.140 12 9.365 9.385 Protons of the substituents. 61
2 3 4 5
8.024 7.434 7.405 7.766 4.690 4.768 8.431 5.51 7 7.603 7.464 7.904 8.352
the computer simulation were carried out on a Bruker Aspect 2000 computer using Bruker software. An eight-spin program was used for the simulations.
RESULTS AND DISCUSSION The 'H NMR spectrum of the aromatic region of 8methyl-BA is shown in Figure la. All 11 aromatic protons were confined to just 2.0 ppm (Table I), yet a t 500 MHz it was possible to distinguish each resonance along with the ortho coupling constant pattern simply by inspection (Figure la). Some fine splitting was also present. The assignments were determined by homonuclear decoupling. The free induction decay was not multiplied by a weighting function. A "Lorentzian to Gaussian" resolution enhancement (6, 7) dramatically increased the number of resolvable lines (Figure l b ) which arose primarily from small long range coupling constants. The resolution enhancement was carried out by multiplication of the free induction decay by a two-term exponential of the form eatdt*,where t was time and a and b were the resolution-enhancement parameters which were optimized empirically. This function generally enabled better resolution of fine splittings and with less loss of signal to noise compared t o the traditional one-parameter exponential resolution enhancement function (eat). Homonuclear decoupling of the
Table 11. Proton-Proton Coupling Constants of 8-Methylbenz[a]anthracene -1.1 0.65 0.26 8.9 0.15 -0.8 0.3 5Ji-4 'Js-7 6.8 0.65 0.8 5J~-s 4J6-, 1.15 0.55 0.6 '51-12 sJ,-12 8.5 0.3 7.1 '57-9 "2-3 0.65 1.4 0.8 sJ7-11 4J2-4 7.8 0.5b 'J7-,, "3-4 a Coupling constants (J) in Hz were accurate t o kO.15 Hz unless indicated otherwise. The minimum detectable J value was 0.3 Hz. The sign of J was not determined except that negative values were used for ortho and para benzylic coupling (8). Fine splitting was insufficient to determine these coupling constants directly. Values were estimated on the basis of simulation and comparisons of coupled and decoupled resolution-enhanced spectra. 3J1-2
4J1-3
8.2
1.15
4J4-,
'JJ,-,
resolution-enhanced spectra was very useful for detecting and assigning the long-range coupling constants. The fine splittings from protons 1,2, 3,4,6,9, 10, and 11 of 8-methyl-BA were sufficiently well-defined to match their frequencies with corresponding second-order computer-generated transition frequencies. Virtually all peaks were fit t o within 0.1 Hz, which showed that the frequencies in the enhanced spectra were reliable and that the second-order measurement was accurate. The magnitude of the long-range coupling constants depended on the specific pathway between protons. For example, all protons located peri to each other (Table 11) had four-bond coupling constants (4J)ranging from 0.65 to 0.8 Hz. Coupling constants of 0.6-0.8 Hz were observed between all epi protons, which had in common a 5J zigzag pathway. The bay coupling (5J1_12) was 0.55 Hz.These data were consistent with the partial analysis of some simpler PAHs (8). The couplings 5J5-7and 5J7-9, which had similar bond pathways, were 0.3 Hz, in agreement with data obtained for partially deuterated naphthalenes (9). Possible 6J or ' J zigzag coupling was too small to be detected. The 8-methyl protons were coupled to protons a t the ortho, meta, and para positions as expected. In addition selective decoupling of the methyl protons were carried out in order to determine whether the
560
Anal. Chem. 1981, 53, 560-562
methyl protons were coupled to protons on other rings. This possibility was double checked by observing the effect of selective decoupling of the aromatic protons on the resolution-enhanced spectrum of the methyl group. No other benzylic couplings were detected. The results for %methyl-BA (Table 11) were the first measurement of a complete set of long-range coupling constants of an asymmetrically substituted PAH containing four or more fused rings. I t was made possible by combining the new software technology with high-field instrumentation. The analysis of %methyl-BA can be a basis for the structure elucidation of other substituted PAH derivatives. We have applied the results (Tables I and 11) to the analysis of the 500-MHz spectrum of 8-hydroxymethyl-BA and found that all coupling constants were the same within experimental error. The chemical shift differences of the aromatic protons compared to 8-methyl-BA were small (Table I). The results were also applied to the analysis of the 270-MHz spectrum of &hydroxymethyl-BA-trans-5,6-dihydrodiol.Tight coupling prevented an accurate measurement of the coupling constants. However, long-range coupling of approximately 1 Hz was found for 6J2-5, 4J4-5 and 4J6_7. These were analogous to the benzylic coupling measured in 8-methyl-BA and 8-hydroxymethyl-BA and consequently could be conveniently used to assign H5 and H6 (Table I). The dihydro protons had not previously been assigned for any dihydrodiols of PAHs. The large chemical shift differences compared to 8-hydroxymethyl-BA, especially for H 1 and H12, were due mainly to loss of ring current shielding from the puckering of the ring system and the loss of aromaticity of one of the rings. These compounds are of interest because they are metabolites of the carcinogen, 8-Me-BA in microsomal system (10). The same approach should be useful for analysis of diolepoxides, which
are thought to be the ultimate carcinogenic form of carcinogenic PAHs. The dependence of long-range coupling constants on bond pathway and substitution is useful for structural studies of unknown PAHs in general because it can be related to the relative position of protons on different rings and to benzylic substituents. This information is unavailable from the more easily measured ortho and meta coupling constants. The long-range coupling constant data complement other NMR parameters in structural studies of PAHs. Their use is now practical with the new technology.
ACKNOWLEDGMENT We thank William E. Hull of Bruker Instruments, Inc., for providing the 500-MHz NMR spectra. LITERATURE CITED (1) Dipple, A. ACS M n o g r . 1976, No. 173, 245. (2) Gelboin, H. V., Ts’o, P. 0. P., Eds. “Polycyclic Hydrocarbons and Cancer: Environment, Chemistry, and Metabolism”; Academic Press: 1978; Vol. I, p 173. (3) Clar, E. “Polycyclic Hydrocarbons”; Academic Press: New York, 1964; Vol. I, p 133. (4) ?mas, R. S.; Lao, R. C.; Wang, D. T.; Robinson, D.; Sakuma, T. Carcinogenesis, Vol. 3: Polynuclear Aromatic Hydrocarbons”; Jones, P. W., Freudenthal, R. I., Eds.; Raven Press: New York, 1978; p 9. (5) Newman, M. S.; Gaertner, R. J. Am. Chem. SOC. 1950, 72, 284. (6) Ernst, R. R. Adv. Magn. Reson. 1966,2, 1. (7) Ferrige, A. G.;Linden, J. C. J. Magn. Reson. 1978, 31, 337. (8) Bartle, K. D.; Jones, D. W.; Matthews, R. S. Rev. Pure Appl. Cbem. 1969, 19,191. (9) Jarvis, M. W.; Moritiz, A. G. Aust. J . Chem. 1971, 24,89. (10) Chou, W. et al., unpublished results, Uniformed Services Universtty of the Health Sciences, Bethesda, MD.
RECEIVED for review June 13,1980. Resubmitted October 16, 1980. Accepted December 2,1980. This work was presented at the American Chemical Society Meeting in Las Vegas, NV, August 1980.
Microcomputer Interface for Calculation of Autocorrelation Functions of Electron Spin Resonance Spectra Gunter Grampp” and Carl A. Schiller Institute of Physical and Theoretical Chemistry, University of Erlangen-Nurnberg, Egerlandstrasse 3, 0-8520 Erlangen, West Germany
For a great number of organic radicals, the many-line ESR spectra are very complicated due to overlapping and multiple splittings. Often it is not possible to extract a plausible set of coupling constants aidirectly out of the spectra. As is well-known, the autocorrelation function A(m
A(AH) =
S+=Z(H) Z ( H - AH) dH -m
(1)
predicates the periodical line distances of the spectrum, where I ( H ) is the intensity of an ESR spectrum as a function of the magnetic field H. A(AH) has a relative maximum, if AH is equal to one of the line distances. Hence maxima of A(m appear as a result of the coupling constants ai and of their integer multiples and combinations of ai as well. But the numbers of combinations are not as large as the numbers of simple coupling constants themselves; therefore, the A ( W maxima are more significant. The autocorrelation procedure has been well-known for a long time ( I ) , but it has seldom been applied to ESR spectroscopy (2-4). One of the reasons has been the complicated procedure of storing the spectra on a magnetic tape or on a punched tape.
Furthermore, the calculations had to be done with a big computer. In the following it is shown that all the fussy procedures are avoidable if an inexpensive microcomputer is used in combination with a special AD/DA converter.
EXPERIMENTAL SECTION Figure 1shows the arrangement between an ESR spectrometer (JEOLJES-PE3X) and a cbm-3032 microcomputer with 32 kbyte memory. The main component of the arrangement is a 12-bit AD/DA-converter “andi” (now manufactured by (including software) Zahner-Elektrik, Kronach, West Germany), developed in our laboratory. “Andi” is a 16-channel converter with two handshake lines and is fully compatible with the cbm computer. In the position “AD” the cbm computer draws data from one of the 16 analog input channels. In the position “DA” the converter works as an analog output device. To take advantage of the dynamic range of the converter (-2047 to +2047 mV) the input channel in use is connected to a differential amplifier (Tektronix 3-A9). In this way the noise is reduced to a small amount. The trigger pulses for the converter are generated by the pulse control circuit of the ESR recorder. The control circuit generates 8192 square waves to drive the stepping motor of the ESR recorder. These trigger pulses can be subdivided in the ratio 1:2:48:16 by a frequency divider. The
0003-2700/81/0353-0560$01.00100 1981 American Chemical Soclety
