Anal. Chem. 1987, 59, 1695-1700
matography system as long as the qualifying conditions (see introductory section) are satisfied. In other words, in any system of column chromatography and for any volume of the sample charge, the sensitivity of analyte-mass determination should range between the “extreme” values given by eq 9 and 16 (or by curves 3 and 4 in Figure 2). Although the reducing parameters u, and v,,,,,~ cannot be determined exactly, fair approximations to these may be obtained from a chromatogram of the smallest possible sample charge. Once the volume uc is known, the increase in the sensitivity of analyte-mass determination on concentrating the sample may readily be calculated. T o do so, eq 9 or eq 13 and 16 are applied to the volumes of charges of the concentrated and original samples, respectively. Naturally, the volumes are concerned containing the same amounts of analyte. For the model to be applicable directly, the state of aggregation of both concentrated and original samples should be the same as that of the mobile phase. The increase required is then given by the ratio of the two results. The choice between the two column-inlet profiles depends upon the performance characteristics of the particular sample-introduction device employed. In principle, relationships analogous to eq 9 and 16 may be derived for any other column-inlet concentration profile. It may be shown is that for large values of a, the sensitivity ratio pmi,ap/pmi,c nearly equal to (2a)’i2/a regardless of the column-inlet concentration profile. The use of eq 9,13, and 16 may be managed even with a programmable pocket calculator. Finally, a brief evaluation should be given of the relative significance of qualifying conditions (i-vi) (see introductory section). Obviously, the most severe limitations to the practical applicability of the model are those imposed by conditions iv and vi. The adherence of the behavior of a real instrument to the model described improves on decreasing the dead volume of the chromatographic system and on de-
1695
creasing the time constants of the detection and registration system. For very slightly retained compounds and/or for very quick analyses, condition v may impose significant restriction to the applicability of the model.
LITERATURE CITED Kaimanovskii, V. I.; Zhukhovitskii, A. A. J . Chromatogr. 1965, 18, 243-252. Karger, B. L.; Martin, M.; Guiochon, G Anal. Chem. 1974, 4 6 , 1640-1647. Guiochon, G.; Colin, H. “Analytical Techniques in Environmental Chemistry”; Proceedings of fhe 2nd International Congress, Barcelo na, Spain, November 1981; Aibaiges, J., Ed.; Pergamon: OxfordNew York-Sydney, 1981; pp 169-176. Sternberg, J. C. Advances in Chromatography;Giddings, J. C., Keiier, R. A., Eds.; Marcel Dekker: New York, 1966; Voi. 2, pp 205-270. McWiiiiam, I . G.; Boiton, H. C. Anal. Chem. 1969, 4 1 , 1755-1762. Giadney, H. M.; Dowden, B. F.; Swaien, J. D. Anal. Chem. 1969, 4 7 , 883-888. Anderson, A. H.; Gibb, T. C.; Littiewood, A. B. J . Chromatogr. Sci. 1970, 8, 840-646. Grushka, E. Anal. Chem. 1972, 4 4 , 1733-1738. Pauis, R. E.; Rogers, L. B. Anal. Chem. 1977, 4 9 , 625-628. Barber, W. E.; Carr, P. W. Anal. Chem. 1961, 53, 1939-1942. Foiey, J. P.; Dorsey, J. G. Anal. Chem. 1963, 55, 730-737. Foley, J. P.;Dorsey, J. G. J . Chromatogr. Sci. 1984, 22, 40-46. Anderson, D. J.; Waiters, R. R. J . Chromatogr. Sci. 1984, 22, 353-359. Deiiey, R. Chromafographia 1984, 18, 374-382. Hanggi, D.; Carr, P. W. Anal. Chem. 1985, 57, 2395-2397. Deiiey, R. Anal. Cbem. 1986, 5 8 , 2344-2346. Handbook of Mathematical Functions; Abramowitz, M., Stegun, I. A., Eds.; National Bureau of Standards: Washington, DC, 1964; Applied Mathematics Series No. 55, p 932. van Deemter, J. J.; Zuiderweg, F. J.; Klinkenberg, A. Cbem. Eng. Sci. 1956, 5, 271-289. Porter, P. E.; Deai, C. H.; Stross, F. H. J . Am. Chem. SOC.1956, 78, 2999-3006. Gradshtein, I. S.; Ryzhik, I. M. fablitsy Integralov, Summ, Ryadov i Proizvedenii (fables of Integrals, Summations, Series, and Products), 4th ed.; Fizmatgiz: Moscow, 1982; p 321, formula 3.322.
-
RECEIVED for review October 10, 1986. Accepted March 3, 1987.
Identification of Mutagenic Methylbenz[ a ]anthracene and Methylchrysene Isomers in Natural Samples by Liquid Chromatography and Shpol’skii Spectrometry Philippe Garrigues,*’Marie-Pierre Marniesse,’ Stephen A. Wise: Jacqueline Bellocq,’ and Marc Ewald’ Groupe d’OcCanographie Physico-chimique, LA 348 C N R S , Universitd de Bordeaux I , 33405 Talence Cedex, France, and Organic Analytical Research Division, National Bureau of Standards, Gaithersburg, Maryland 20899
Chromatographic extracts of natural samples (rock and air partlculate matter) have been examlned by high-resolution Shpoi’skll spectrometry (HRS) at 15 K In n-alkane polycrystalline frozen solutlons for the ldentificatlon of the 12 methylbenr[a ]anthracenes (MBA) and the six methylchrysenes (MC). This Is the flrst report on the unamblguous identlflcation of each MBA Isomer in real samples which wlll provide a better understanding of carcinogenic potency and further quantlflcation of these compounds in tetraaromatic fractlons.
Polycyclic aromatic hydrocarbons (PAH) and their alkylated derivatives are well recognized as ubiquitous contamiUniversitB de Bordeaux. 2 N a t i o n a l Bureau of Standards.
nants of the environment. The major analytical problem in the determination of PAH in complex natural mixtures is the separation and the identification of individual components in the presence of the numerous other isomeric parent and alkyl-substituted PAH. Since the biological activity of aromatic compounds is isomer specific, the identification of each compound in an alkylated aromatic series is a vital part of understanding the carcinogenic activity of PAH mixtures. Methylbenz[a]anthracenes (MBA) and methylchrysenes (MC) are among the most biologically active alkylated aromatic series found in man’s environment (Figure 1) (1-6). There are 12 possible isomers in the MBA series which vary significantly with respect to carcinogenicity(Figure 1). 7-MEiA has been recognized as the most tumorigenic compound, followed by 6-, 8-, and 12-MBA, which are of equal carcinogenicity, while 9- and 11-MBA are the next most carcinogenic compounds. The low tumorigenicity of the 1-,2-, 3-, and 4-MBA has been generally cited in support of the bay region
0003-2700/87/0359-1695$01.50/0 0 1987 American Chemical Society
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13, JULY 1, 1987
I/ --3 MBA I
1
. Figure 1. Relative carcinogenic activity of methylbenz[a ]anthracene (MBA) and methylchrysene (MC) isomers on a scale of zero to AAA.
I
d
Wavelength (nr?)
theory of PAH carcinogenesis (7-11). In the methylchrysene series, 5-MC is one of the most carcinogenic compounds while 2-, 3-, 4-, and 6-MC are moderately active ( I , 12). However, identification of methyl tetraaromatic compounds is particularly difficult because of their similar behavior in chromatographic techniques (liquid or capillary gas chromatography) (13, 14). Capillary gas chromatography on conventional nonpolar phases (e.g., SE-54) and liquid chromatography are unsuccessful in the separation of all MBA and MC isomers (12,13). Recent reports on liquid crystalline stationary phases in capillary gas chromatography demonstrated improved separation of these isomers (15);however, several isomers were still unresolved. Whereas the identification of MC isomers in natural extracts has been realized (12, 14, 16) only one tentative identification of MBA isomers was reported previously by 'H NMR (3, 14). One way to overcome the limitation of classical analytical techniques in the differentiation of isomeric compounds is to use high-resolution spectrometry (HRS) in n-alkane matrices (Shpol'skii effect) which has been shown to be adequate for solving such problems (16-18). This technique takes advantage of the sharpening of the fluorescence emission spectra when aromatics are incorporated into an appropriate n-alkane matrix a t low temperature (19). During the past decade, Shpol'skii spectrometry has attracted the attention of analytical chemists considering the numerous publications recently reviewed (20). In this paper, we report the low-temperature emission properties of MBA and their identification in rock and air particulate samples which also contained MC. Such studies will lead to the quantification of individual MBA and MC isomers, which is of particular interest in environmental chemistry and organic geochemistry (21). EXPERIMENTAL SECTION Chemicals and Samples. The MBA were purchased from the chemical repository of the National Cancer Institute (Bethesda, MD). The purity of each MBA was reported as greater than 96%. The six MC were purchased from the Community Bureau of Reference (BCR, Commission of the European Community, Brussels, Belgium). The purity of each MC was certified to be greater than 99.6%. All the solvents (spectroscopic grade from Fluka and Merck) used for HPLC fractionation and Shpol'skii analysis were purified by distillation and then dried and kept over molecular sieves. Residual fluorescence emission of solvents was verified by roomtemperature spectrofluorometry. The sedimentary rock sample came from a well in an Indonesian petroleum field (21)and was provided by J. L. Oudin (TOTALCFP, Pessac, France). The tetraaromatic fractions from a Philadelphia, PA, air particulate sample and from a Washington, DC, air particulate sample (Standard Reference Material, SRM 1649, Urban Dust/Organics) were obtained according to the analytical procedure reported previously (22, 23).
Figure 2. Fluorescence emission spectra of some individual reference MBA in n-octane at 15 K (c = lo-' M). Excitation was at 294 nm. Note the important diffuse base for the 3-MBA. Isolation of Monomethyl Tetraaromatic Isomers. The fractionation procedure of aromatics, proposed by Wise et al. (24) has been modified and used as applied previously to the isolation of alkylated PAH (16, 18, 21). Briefly, the first normal-phase high-performance liquid chromatography (HPLC) step on an aminosilane column separates the aromatics into ring classes. A two-step reversed-phase LC fractionation procedure then utilizes the differences in selectivity between monomeric and polymeric CIS columns to isolate fractions suitable for HRS analysis. The separation on a monomeric reversed-phasec18 allows the collection of the methyl tetraaromatic fraction. This fraction is then s e p arated on a polymeric reversed-phase c18 column which has high selectivity for the separation of PAH isomers (Supelcosil, type LC-PAH) (13,18,25,26). A mixture of 70% acetonitrile in water was used as the mobile phase to yield fractions of one to four methyl tetraaromatic isomers that were amenable to identification by HRS. High Resolution Shpol'skii Spectrometry (HRS). Lowtemperature luminescence experiments were performed with a homemade spectrofluorometer described previously (16). Fused silica tubes containing the solutions were attached to the cold head of a closed cycle cryogenerator (CTI, Cryodyne 21 SC) operating at 15 K. Excitation was provided by a xenon lamp (450 W). Emission spectra were recorded and stored on a hard disk of a microcomputer (IBM/XT). Processing of the spectra gives emission peak wavelengths with a precision of 0.1 nm. R E S U L T S A N D DISCUSSION Low-Temperature Spectrofluorometry of MBA Reference Compounds. Some previous studies have already reported the Shpol'skii emission spectra of some MBA isomers (27-30). The site multiplet structure for the 0-0 transition of the 12 MBA in n-octane a t 15 K covers a spectral region of about 10 nm (Table I and Figure 2). Each compound exhibits sharp fluorescence emission spectra with a multiplet structure containing up to four quasi-line emission peaks arising from several different orientations of the MBA guest molecules in the frozen n-octane host (29,31). The maximum long-wavelength displacement relative to the emission peak of the parent compound (benz[a]anthracene, BA) is observed when the methyl group is introduced a t position 7 or 12, whereas 1-, 2-, 3-, 4-, 5-, 6-, lo-, and 11-methyl derivatives exhibited a shift of about 2-3.5 nm. The 9-MBA exhibits a slight short-wavelength displacement while the 8-MBA gives rise to a quasi-line structure in the same spectral area as the BA. All these observations are in good agreement with previous studies (27, 28). When the low-temperature fluorescence emission spectra on the complete series of the 12 MBA are obtained, some interesting observations can be made. Some isomers (1-,2-,
ANALYTICAL CHEMISTRY, VOL. 59,
Table I. Characteristic Shpol'skii Fluorescence Emission Peaks of MBA and MC Isomers in n -Octane at T = 15 K"
compound
fluorescence emission peak, nm
AA, nm
benz[a]anthracene(BA) 384.0 (+++), 384.3 (+++) 1-MBA 2-MBA 3-MBA 4-MBA 5-MBA 6-MBA 7-MBA 8-MBA 9-MBA 10-MBA 11-MBA 12-MBA
chrysene (C) 1-MC 2-MC 3-MC 4-MC 5-MC 6-MC
386.7 (+++), 387.0 386.7 (+++I, 387.0 (+++), 387.9 (+) 385.3 (+++), 385.8 (+++) 386.2 (+++), 388.5 (+++), 387.3 (+++) 387.6 (+++), 387.9 (+), 388.1 (++I, 388.5 (+) 385.0 (++), 386.3 (++), 386.5 (+++I, 386.8 (+) 389.2 (+++), 390.9 (+) 384.1 (+++), 384.3 (++), 385.0 (++), 385.4 (++) 383.1 (+), 384.0 (+++), 384.5 (+) 386.1 (+), 387.0 (+++), 387.1 (++), 387.4 (+) 384.3 (+), 385.6 (+++), 385.9 (++), 386.7 (++) 390.6 (+++I, 390.8 (+++), 391.4 (++), 392.1 (+) 360.5 (+++), 361.1 (+++), 362.2 (++) 361.3 (++), 361.9 (++), 362.2 (++), 362.5 (+++) 361.2 (+++), 361.5 (+++) 362.0 (+++), 362.2 (++), 362.6 (++), 363.1 (+) 365.4 (+++), 366.0 (+), 366.9 (++) 367.2 (+++), 367.6 (++), 367.9 (+) 362.6 (+), 363.3 (+++), 364.6 (++)
2.7; 2.4 2.7; 2.4 1.3; 1.0 2.2; 2.9 3.6; 3.3 2.5; 2.2 5.2; 4.9 0.1; 0.2 -0.9; -1.2 2.1; 1.8
1.6; 1.3 6.6; 6.3
0.8; 0.2 0.7; 0.1 1.5; 0.9 4.9; 4.0 6.7; 5.8 1.9; 1.3
"Excitation at 294 nm for MBA and at 274 nm for MC. AA represents the peak shifts (in nm) of the major fluorescence peak of each methylated derivatives relative to the two major fluorescence peaks of the respective parent compound. Intensity scale is from zero to +++.
NO. 13, JULY 1, 1987
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3-, lo-,and 12-MBA) exhibit quasi-linear emission peaks that emerge from a broad diffuse base (Figure 2) which could be attributed to a strong interaction between aromatic molecules and the n-octane chains. Such interaction could be due to the position of the methyl group which leads to a nonplanar character for some MBA molecules (32), e.g., the sterically hindered MBA with substituents in position 1 and 12. The presence of this diffuse base is also observed for MBA molecules for which the substituted carbon is bonded to carbon atoms not engaged in ring structure (2-, 3-, and 10-MBA). On the contrary, very nearly planar structures such as 4- and 11-MBA (32) do not exhibit such a diffuse base. As shown in Table I, the multiplets of the MBA are subject to spectral interferences in the emission region of the 0-0 transition. Particularly 1-and 2-MBA exhibit major emission a t the same wavelengths. However, as presented in Figure 3, the most carcinogenic compounds (6-, 7-, 8-, and 12-MBA) are easily identified in the synthetic mixture of the 12 isomers. The other MBA isomers could be identified tentitatively by two different analytical approaches: (a) by using selective excitation with a laser (29)or (b) by HRS analysis of fractions collected with a highly selective reversed-phase column (18). This second approach has been developed for the identification of MBA and MC as described in this paper. Analysis of MBA and MC in Natural Extracts. Specific emission and excitation wavelengths of tetraaromatic parent compounds are listed in Table 11. Despite numerous tetracyclic alkylaromatic derivatives, the selectivity of spectrofluorometry allows the observation of each series (chrysene, benz[a]anthracene, triphenylene, naphthacene, benzo[c]phenanthrene) without interferences between each other (Table I and 11) (33). Methyltriphenylenes have also been observed as minor components of this fraction: indeed triphenylene exhibits fluorescence emission in n-octane at about 350 nm (30) and fluorescence emission peaks corresponding to possible methyltriphenylenes have been observed at about 355 nm; but without reference standards a true identification was not possible. Naphthacene (linear) and benzo[c]phenanthrene (sterically hindered) derivatives have not been detected and are most likely minor contributors according to
Table 11. Characteristic Room-Temperature Fluorescence Properties of Tetraaromatic Parent Compound (31, 33)"
tetraaromatic compounds benz[a]anthracene
max emission wavelengths, nm
max absorption wavelengths, nm
385, 405, 430 222, 268.5, 278, 299
chrysene
361, 380, 402
218, 259,269
triphenylene
355, 362,373
284, 273, 258, 249
structure
@&
possible methyl derivatives
l2
@ p 6 2 @ @
naphthacene
482.5, 513, 551
294, 275, 265
3
benzo[clphenanthrene
broad band centered at 400
218.5, 222, 272, 283
6
The most intense band is italicized.
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
1698
T
5 MEA
I
5 MBA
7 MEA
12 MBA
3
4 u R e t e n t i o n t i m e (mn)
+J > .6Y c
+J W
v,
c ._ W 0
e
W 6Y 0
Figure 5. Reversed-phase LC analysis of C, tetraaromatic fraction from Washington urban dust (SRM 1649). Absorption detection was centered at 288 nm. Compounds have been identified on the basis of HRS spectra of reference compounds. Note the lack of 1-, 7-, and 12-MBA.
5 MBA
L W
3 0 -
LL
sedimentary r o c k C,-tetraarornatics
1
7 MBA
oi
390
Wavelength (nrn)
Figure 3. Fluorescence emission spectra of MBA isomers (excitation at 294 nm): (a) in an equimolar synthetic mixture of the 12 MBA (each at c = lo-' M); (b) in a C, tetraaromatic rock extract. Note the lack of 12-MBA and the presence of the very carcinogenic 7-MBA.
+
fraction 2
I 0
0
i
385
sedimentary r o c k
5 MEA
o 1 1 MBA X
1 MBA
0
7 MBA
Wavelength (nrn)
Figure 6. Fluorescence spectra of some MBA in subfraction 2 of C, tetraaromatic fraction from the sedimentary rock. Excitation was at 294 nm.
R e t e n t i o n t i m e (mn)
Figure 4. Reversed-phase LC analysis of C, tetraaromatic fraction from a sedimentary rock. Absorption detection was centered at 288 nm. Compounds are identified on the basis of HRS spectra of reference compounds. Note the lack of 12-MBA. the predominance of angular over linear annelated and sterically hindered structures (34). Therefore, the distribution of monomethyl tetraaromatics is dominated by the methylbenz[a]anthracenes and methylchrysenes.
The total C, tetraaromatic fraction of the sedimentary rock which was isolated by reversed-phase LC on the monomeric C18 column was analyzed by HRS for MBA determination. Despite MBA emission interferences, HRS allows a partial identification of selected MBA isomers (Figure 3). In particular, it is possible to observe the lack of 12-MBA and the presence of 7-MBA in the C1 tetraaromatic rock extract which are two of the most carcinogenic compounds of the series. The C1 tetraaromatic fractions from the sedimentary rock and air particulate samples were submitted to an HPLC step on the highly selective polymeric CIS column (Figures 4 and 5 ) . As indicated by Wise et al. (13) and in Table 111, only part of the methyl tetraaromatic isomers could be cleanly separated from each other. The identification of MBA and MC isomers in HPLC subfractions is demonstrated in Figures 6-8. Indeed, despite the use of an observation centered at an absorption of 288 nm that favors MBA detection, MC
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
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Table 111. LC Retention Index (log I , ref 13) of MBA and MC and Their Identification in Natural Sample Fractions (See Figures 4 and 5) Philadelphia air particulate compound
retention index (log I)
sedimentary rock
Washington urban dust
matter
2-MBA 12-MBA 6-MBA 11-MBA 1-MBA 5-MC 6-MC 7-MBA 4-MC 8-MBA 10-MBA 5-MBA 3-MC 4-MBA 3-MBA 9-MBA 1-MC 2-MC
4.09 4.10 4.10 4.13 4.14 4.14 4.14 4.14 4.18 4.19 4.24 4.88 4.29 4.33 4.39 4.39 4.43 4.52
fraction 1
fraction 1
fraction 1
fraction 1 fraction 2 fraction 2
fraction 1 fraction 2
fraction 1 fraction 2
fraction 2 fraction 2
fraction 2
fraction 2
fraction 3 fraction 4 fraction 4
fraction 3
fraction 3 fraction 4 fraction 4 fraction 4 fraction 5
fraction 4 fraction 4 fraction 4 fraction 5 fraction 6 fraction 6
fraction 4 fraction 5 fraction 6 fraction 6 fraction 6 fraction 7
fraction 6 fraction 6 fraction 6 fraction 7
fraction 6 fraction 7
4 MBA
I
e
r e f e r e n c e compound
?
z. c ZI .rn
h
7
c
&.
c a?
.-
E
+ .-
Q
v)
c
0
c a?
c
t
.-c
a? 0
ln
a? 0
Philadelphia a i r
L a? 0 3
c
0 a?
particulate matter
i i
u)
a? L
fraction
Washington urban dust fraction 5
0
a LL
11;1, 362
365
2
360
W a v e l e n g t h (nm) 3a5
390
fnm)
Wavelength (nm) Figure 7. Fluorescence spectra of 4-MBA in n-octane at T = 15 K (excitation at 294 nm): (a) reference compound (c = lo-' M); (b) Washington urban dust subfraction.
isomers contribute to the HPLC fingerprint (see Figures 4 and 5 where peak 7 is due only to 2-MC). The 11MBA and four MC presented in Table I11 could be identified by comparison of their HRS emission spectra with that of the respective standard spectra. Both a coincidence in the position of the quasi-lines and also a good agreement in the relative intensities of the quasi-lines provide a definitive identification of each compound (Figures 7 and 8). 12-MBA is absent from the three natural extracts. Steric hindrance could be responsible for the lack of 12-MBA as well for the lack of 4-and 5-MC, which has been previously mentioned
Fluorescence spectra of 6-MC in n-octane at T = 1 (excitai I at 274 nm): (a) reference compound (c = lo-' M); subfraction from Philadelphia air particulate matter. Note the lac1 the highly carcinogenic 5-MC which would be eluted with 6-MC, present.
Figure
K 3)
f, if
(21). 1-and 7-MBA have been specifically identified in the rock extract but where not found in the air particulate extracts. These two compounds are presumably less stable than the other isomers. Methyl shift reactions could occur at higher temperatures in aerosol formation than in the sedimentary matter and would favor the formation of more stable methyl isomers. The absence of 7- and 12-MBA in the atmospheric particulate samples is in good agreement with the only previously reported determination of these compounds in air particulate matter ( 3 ) . These studies on the methylbenz[a]anthracene and methylchrysene series demonstrate the capability of HRS coupled with HPLC separations for the differentiation of very closely
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
related compounds such as MBA and MC. Positive identification of the 12 MBA isomers has not been reported previously due to the chromatographic interferences of MBA from each other or from other methyltetraaromatic isomers. This work is only the first step toward a better understanding of MC and MBA behavior in environmental samples. Further developments will lead to the relative quantification of these methyl-PAH in various natural samples to give a real evaluation of biological activity of PAH fractions. ACKNOWLEDGMENT
We thank J. Joussot-Dubien for his continuous interest and support. Thanks are due to J. L. Oudin (CFP-TOTAL) for providing sedimentary rock samples and to J. Lewtas (U.S. EPA) for providing the Philadelphia air particulate sample. The authors also wish to thank R. De Sury and N. Chedozeau for technical assistance with the analytical work. Registry No. 1-MBA, 2498-77-3; 2-MBA, 2498-76-2;3-MBA, 2498-75-1;4-MBA, 316-49-4; SMBA, 2319-96-2; 6-MBA, 316-14-3; 7-MBA, 2541-69-7; 8-MBA, 2381-31-9; 9-MBA, 2381-16-0; 10MBA, 2381-15-9 11-MBA, 6111-78-0; 12-MBA,2422-79-9; 1-MC, 3351-28-8; 2-MC, 3351-32-4; 3-MC, 3351-31-3; 4-MC, 3351-30-2; 5-MC. 3697-24-3: 6-MC. 1705-85-7. LITERATURE CITED Hoffman, D.: Schmeltz, I.; Hecht, S. S.; Wynder, E. L. Polycyclic Hydrocarbons and Cancer; Gelboin. H. V., P.O.P.T.'s 0, Eds.: Academic: New York, 1978: Vol I, pp 85-117. Thomas, R. S.;Lao, R. C.; Wang, D. T.; Robinson, D.; Sakuma, T. Carcinogenesis Vol. 3: Polycyclic Aromatic Hydrocarbons ; Jones, P. W., Freudenthal. R. I., Eds.; Raven Press: New York, 1978;pp 9-19. Bartle, K. D.; Lee, M. L.; Novotny, M. Analyst (London) 1977, 702,
731-738. Carruthers, W.; Stewart, H. N. M.; Watkins, D. A. M. Nature (London) W87. 273, 691-692. Grimmer, G.;Jacob, J.; Naujack, K. W.: Dettbarn, G. Fresenius' 2. Anal. Chem. 1981,309, 13-19. Lee, M. L.; Novotny, M.; Bartle, K. D. Anal. Chem. 1976, 48,
1566- 1572. Morgan, D. D.: Warshawsky, D.: Atkinson, T. Photochem, Photobiol.
1977,25,31-3a. Snook, M. E.; Severson, R. F.: Higman, H. C.; Arrendale, R. F.; Chortyk, 0. T. Polycyclic Aromatic Hydrocarbons; Jones, P. W., Leber, P., Eds.; Ann Arbor Science Publishers: Ann Arbor, MI, 1979; pp
231-260. Smith, I. A.: Seybold, P. G. Int. J. Quantum Chem.. Quantum Biol. Symp. 1978, No. 5 , 311-320. Wlsiocki, P. G.;Florentinl. K. M.; Fu, P. P.; Yang, S. K.; Lu. A. Y. H. Carcinogenesis 1982,3 , 215-217.
(11) Newman, M. S. Carcinogenesis Vol. I , Polycyclic Aromatic Hydro-
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RECEIVED for review November 20, 1986. Accepted March 2, 1987. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
