Analysis of Mixtures of Isomeric Polynuclear Hydrocarbons by Nuclear Magnetic Resonance Spectrometry Methylated Derivatives of Anthracene, Benz [a] anthracene, Benzo [c] phenanthrene, and Pyrene Larry K. Keefer,’ Lawrence Wallcave, James Loo, and Ruth S. Peterson The Eppley Institute f o r Research in Cancer, University of Nebraska Medical Center, Omaha, Neb. 68105 NMR spectrometry i s used for the determination of specific methylaromatic hydrocarbons in mixtures of structurally similar compounds. Identifications are made on the basis of relative chemical shifts, and confirmed using T~ values methyl chemical shift at infinite dilution in a speci ied solvent system) and peak multiplicity information. Mixtures containing as little as 30-40 pg of each component are quantified by spectral integration. Chemical shift data for authentic specimens of all monomethyl derivatives of anthracene, benz[a]anthracene, benzo[c]phenanthrene, and pyrene are presented. While such reference data are often essential for positive identifications, satisfactory assignments can sometimes be made on the basis of values predicted from structureshift correlations. t h e method is applicable to the determination of methylaromatic hydrocarbons in complex environmental mixtures, and the characterization of some petrolatum and asphalt fractions is described.
Chart I
I
SINCE THE ISOLATION of benzo[a]pyrene (I) from coal tar in 1932 and its identification as a powerful agent for the production of malignant tumors in laboratory animals (I), a great deal of effort has been devoted to searching for (I) and related compounds in the human environment (Chart I). The importance of this attempt to establish the “total load of carcinogens” in a given locale as a prerequisite to “planning an effective reduction of the exposure of man to carcinogens” has recently been underscored by the International Union Against Cancer (2), and the chromatographic-spectrophotometric procedures (3) employed in these studies have accordingly been refined to a level of sophistication such that (I) and several other known polynuclear carcinogens can be routinely determined at levels of a few parts per billion or less. Nevertheless, the procedures described in the literature as applicable to the study of the distribution of environmental carcinogens all seem to be deficient in at least one critical respect. The chromatographic resolution of most environmental hydrocarbon samples ultimately results in the isolation of many different “alkylaromatic” fractions, each of which may be homogeneous with respect to “ring content” and molecular weight, but which contains a mixture of isomeric compounds. The close similarity of the constituents of these fractions with respect to chemical and physical properties normally precludes analyzing them by the usual chromatoAuthor to whom inquiries may be addressed, c/o The National Cancer Institute, Bethesda, Md. 20014 ~
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(1) J. W. Cook, C. L. Hewett, and I. Hieger, Nature, 130, 926 (1932). (2) P. Shubik, D. B. Clayson, and B. Terracini, Eds., “The Quantification of Environmental Carcinogens,” UICC Technical Report Series, Vol. 4, International Union Against Cancer, Geneva, Switzerland, 1970, p 1. (3) E. Sawicki, Chemist-Analyst, 53, 24, 56, and 88 (1964).
Benzo [a]pyrene (I)
@ ‘ 6
5
Pyrene (111)
Benzo [c] phenanthrene (V)
Benz [ a ] anthracene (E)
m Anthracene
(m)
Fluoranthene (VI)
fi
Triphenylene (VU)
Chrysene (VIII)
graphic-spectral methods. Of the numerous reports describing analyses of environmental substances for carcinogenic hydrocarbons, only a few include reliable identifications of specific alkylated polynuclear compounds, and virtually all of these identifications have involved tedious, nonquantitative isolation procedures. The great majority of authors have either drawn conclusions not supported by their data, or have chosen noncommittally to report the total quantities of each compound type (e.g., “alkylpyrene” or “methylpyrene”) present in the sample. The inadequacy of this latter type of analytical information for the purpose of establishing or rationalizing carcinogenicity data becomes readily apparent when one considers the remarkable dependence of the biological properties of these compounds upon the precise nature of their alkyl substitution pattern. Methylation of benz[a]anthracene (11) at both the 7 and 12 positions converts a compound which is at most only mildly carcinogenic to one of the most potent tumor-producing agents known. Of the twelve possible monomethyl derivatives, the 6-, 7-, 8-, and 12-isomers are carcinogenic, while the remaining eight have been reported to be inactive (4). (4) C . B. Huggins, J. Pataki, and R. G . Harvey, Proc. Naf.Acad. Sci., 58, 2253 (1967).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 11, SEPTEMBER 1971
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Thus an analytical method capable of identifying and quantifying specific alkylaromatic carcinogens in the presence of their biologically inactive isomers on a routine and efficient basis must clearly be made available before the contributions of various environmental factors to the total carcinogenic load can be meaningfully predicted. We have found that nuclear magnetic resonance (NMR) spectrometry, with its nearly unique sensitivity to subtle differences in molecular architecture, goes a very long way toward meeting this requirement. Specifically, information on the chemical shifts, multiplicities, and relative integrals of the peaks found in the methyl region of a sample’s NMR spectrum provides a convenient basis for analyzing mixtures of alkylated polynuclear hydrocarbons. EXPERIMENTAL
Materials. The three methylpyrene samples were purchased from L. Light & Co., Ltd. (Colnbrook, England). 2-Methylanthracene was obtained from Aldrich Chemical Co., (Milwaukee, Wis.), and the 9-methylanthracene was a product of Eastman Organic Chemicals (Rochester, N. Y,). All other methylaromatic compounds were kindly provided by M. S. Newman, of Columbus, Ohio. The amber petrolatum was an N. F. grade obtained from Arthur S. La Pine and Co., Chicago, Ill. It had a total methylpyrene content of 25 ppm, a dimethyl (and/or ethyl)pyrene content of 15 ppm, and a methyltriphenylene content of 6 ppm (Unpublished analyses in this laboratory using chromatographic fractionation and UV and mass spectrometry). The asphalt was an 85/lOO penetration sample of the grade commonly used in road paving and corresponds to “Asphalt F” of ref. 5. It had a monomethylpyrene content of 59 pprn and a dimethyl (and/or ethy1)pyrene content of 20 ppm. Procedure. The entire spectral region of interest was investigated in a single (left-to-right) scan with a Varian Model HA-100 NMR Spectrometer operating in the frequency sweep mode. The sweep width was usually set at 50-100 Hz for maximum horizontal sensitivity. Normal precautions were taken to avoid saturation effects. The center of each observed peak was located visually, and the pen was swept to that point from left to right. The chemical shift was taken as the average of five successive readings of the V-4315 Frequency Counter, which was set to read the difference to the nearest 0,001 ppm between the pen position and the reference lock signal. Each set of concentration us. chemical shift data was fitted to a best straight line using a standard least-squares program, which furnished values for the intercept ( T ~ ) ,slope, standard error around the line, and the variances in the intercept and slope. The error limits given in Tables I-V are the square roots of these variances. Decoupling experiments were conducted with the aid of a Hewlett-Packard Model 200 AB Audio Oscillator. Irradiation frequency was determined with the V-4315 Frequency Counter in External mode, and intensity was estimated from the amplitude of the sine wave on the oscilloscope. Time-averaged spectra were accumulated on a C-1024 Time Averaging Computer. Error Analysis. Chemical shift values were critically dependent upon solvent effects. The T O value of 4-methylpyrene, for example, was 0.013 ppm smaller in chloroform-d than in carbon tetrachloride (cf. Table I). The shift data also depended intimately upon the concentration of tetramethylsilane (TMS), which was carefully maintained at 5 % of the solvent by weight. ( 5 ) L. Wallcave, H. Garcia, R. Feldman, W. Lijinsky, and P.
Shubik, Toxicol. Appl. Pharmacol,, 18, 41 (1971). 1412
In raising the temperature from -45” to $45 “C, the approximately linear increase in 7 for each of the rnethylpyrenes was only 0.03 Hz/degree, on the average. Therefore, variations in chemical shift due to fluctuations in normal probe temperature (which ranged from 3&36 ”) were neglected, The importance of other potential sources of systematic error, e.g., differences among operators in locating centers of peaks, might largely be compensated for by adjusting a given set of readings to a pair of calibration points. The chemical shift of TMS was defined as 10.000 r by the electronic lock component of our spectrometer, and the chloroform in 95.0:5.0 (w:w) DCC13:TMS furnished a second reference point of 2.744 T . Random errors were estimated by repeatedly determining the chemical shifts for methylpyrene mixtures of four different concentrations throughout the course of a week. The standard deviation for each compound was found to be 50.15 Hz. This value was taken as an estimate of the standard deviation of chemical shift measurements throughout this study. The uncertainty in the intercept position could be made as small as necessary. The variance of the T O values can be estimated from the equation
where V, is the variance for the N chemical shift readings, Ci is the concentration coordinate of the ith reading, and is the arithmetic mean of the C;s. The standard deviation of a r 0 value calculated from only two chemical shift readings (for concentrations x and 2x) can thus be approximated as
