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(7) Tsuda, T.; Novotny, M. Anal. Chem. 19711, 50, 271-275. (8) Hirata, Y.; Novotny, M.; Tsuda, T.; Ishil. iD. Anal. Chem. 1979, 51,
1807-1809.
(9) Hirata, Y.; Novotny, M. J. Chromatogr. 1079, 186, 521-528. (10) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 169, 51-72. (11) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 185, 27-41. (12) Hirata, Y.; Lin, P. T.; Novotny, M.; Wightman, R. M. J. Chromatogr. 1980, 181, 287-294. (13) Brody, S. S.; Chaney, J. E. J. Gas Chrorniatogr. 1966, 4 , 42-48. (14) Juiin, B. G.; Vandenborn, H. W.; Kirkiand, J. J. J. Chromatogr. 1975, 112,443-453. (15) Chester, T. L. Anal. Chem. 1980, 52,638-642. (16) Chester. T. L. Anal. Chem. 1980. 52, 1821-1624. (17) Dagnall, R. M.; Thompson, K. C.; West, T S. Analyst(London) 1967, 92,506-512. (18) Dagnaii, R. M.; Thompson, K. C.; West, T S. Analyst (London) 1988, 93. 72-78 _-, - ._ (19) Aldous, K. M.; Dagnali, R. M.; West, T. S. Analyst(London)1970, 95, 417-424. (20) Jacob, K.; Vogt, W.; Knedel, M. Justus Lleblgs Ann. Chem. 1979, 878-a85. (21) Jacob, K.; Falkner, IC.; Vogt, W. J. Chromatogr. 1978, 167, 67-75. (22) Jacob, K.; Maier, E.; Schwertfeger, G.; Vogt, W.; Knedei, M. Biomed Mass Spectrum. 1978, 5,302-311. (23) Dean, J. A. “Flame Photometry”; McGraw-Hill: New York, 1960;p 134.
95 1
(24) Avni, R.; Alkemade, C. Th. J. Mikrochlm. Acta 1960, 460-471. (25) Wenzel, 6. J. Chromatogr. 1979, 17, 687. (26) St. John, P. A.; McCarthy, W. J.; Winefordner, J. D. Anal. Chem. 1987, 39, 1495-1497. (27) Bevington, P. R. “Data Reduction and Error Analysis for the Physical Sciences”; McGraw-Hill: New York, 1969;p 45. (28) Diebold, G. J.; Zare, R. N. Sclence 1977, 196, 1439-1441. (29) Jones, D. R.; Tung, H. C.; Manahan, S. E. Anal. Chem. 1978, 48, 7-10. (30) Scott, R. P. W. “Liquid Chromatography Detectors”; Elsevier: New York, 1977;p 28. (31) Compton, 6. J.; Purdy, W. C. J. Chromatogr. 1979, 169,39-50. (32) Sydor, R. J.; Hieftje, G. M. Anal. Chem. 1978, 48, 535-541. (33) Pao, Y.-H.; Zitter, R. N.; Griffiths, J. E. J. Opt. Soc. Am. 1966, 56, 1133-1135. (34) O’Haver, T. C. Anal. Chem. 1978, 50, 676-679.
RECEIVED for review December 16,1980. Accepted March 23, 1981. This research was supported by the National Institutes of Health, Grant No. PHS R 0 1 GM 24349. Preliminary results were reported at the 31st Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, 1980.
Identification of Organic Compounds on Diesel Engine Soot Ming-Li Yu and Ronald A. Hites” School of Public and Environmental Affairs and Department of Chemistfy, Indiana University, 400 East Seventh Street, Bloomington, Indiana 47405
Several studies have shown than extracts of soot collected from Ilght-duty dlesel cmgines cause mulatlons In bacteria and mammalian cells both with and wlthouit metabolic activation. To help ldenttfy the speclflc compounds responsible for these biological effects, we studied the detailed Fhemlcal composltlon of one such extract by gas chromatographic mass spectrometry. The iwo most mutagenic fractlons contain alkylated phenanthrenes, fluorenes, fluorenones, and other polycyclic aromatic hydrocarbons, aldehydes, and qulnones. One nitro polycyclic aromatic compound was also identified. The biological Implications of these findings are dlscussed.
The corporate average fuel economy (CAFE) standard and the price of gasoline have induced the American automobile industry to manufacture more automobiles with improved fuel economy. Since diesel engines generally give 25% better fuel economy than gasoline engines ( I ) , many automobile manufacturers are increasing the “dieselized” fraction of their product. By 1990, it is expected that 1 5 2 0 % of the automobile fleet in the United States will be powered by diesel engines (1). This is a mixed blessing. Although the nation may be able to conserve petroleum by this change, it might be sacrificing the health of both its people and its eeviroment. These potential problems are dlue to the high emission of particulates and associated organic compounds from diesel engines. On the average, light-duty diesels produce 0.5 g of particulates/mile ( I ) . Although these particulates are mostly carbon, 10-40% by weight can be extracted witlh organic solvents (2). Unfortunately, this extractable material is highly mutagenic both with and without metabolic activation (3, 4). The chemical composition of the material extractable from diesel particulates is clearly important information. Although
several such chemical studies are now in progress (5-8), very little compositional information on diesel exhaust has been published. In this paper, we will discuss the composition of two subfractions of diesel particulate extractables which have been shown to be especially mutagenic (4).
EXPERIMENTAL SECTION A particulate sample was obtained from a 350 CID, 1978, Oldsmobile diesel engine running on commercial diesel fuel. Hot start, federal test procedure cycles were run, and the exhaust was diluted by about a factor of 10 in a dilution tunnel. The total flow rate in this dilution tunnel was 100-200 m3/h. About 0.1-0.3 m3/h were sampled, and the particulates were collected on a 0.2-pm glass fiber fiiter (Pallflex T60-A20). The filter was extracted with methylene chloride for 1-6h, and the solvent was evaporated on a steam bath with nitrogen purging. The final residue was about 2 g. Small amounts (-50 mg) of the crude extract were separated into seven fractions on a silicic acid column (8 cm X 0.8 cm i.d.). The sequence of the eluants was as follows: hexane (3 mL), 1:l hexane/toluene (3 mL), toluene (3 mL), methylene chloride (3 mL), 2:l methylene chloride/methanol(3 mL), 1:2 methylene chloride/methanol (3 mL), and methanol (3 mL). Chemical analyses have focused on the hexane/toluene and toluene fractions because these have been shown to be the most mutagenic (4). Gas chromatography was performed on a Hewlett-Packard 5730 gas chromatograph equipped with a flame ionization detector. A 15 m X 0.26 mm i.d. glass c a p i l l q c o l coated ~ with SP-2100 methyl silicone stationary phase was used. Gas chromatographic mass spectrometrty was done on a Hewlett-Packard 5982A mass spectrometer interfaced to a 5933A data system. The identification of the GC/MS peaks was based on careful comparison of the observed spectra with published spectra and on fundamental interpretation. GC retention
0003-2700/81/0353-0951$01.25/00 I981 American Chemical Society
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9
f3 ,LO
3
Temp ("C) 70
80
90
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t----t+ I 5 25
Time (mid 0
180 1
190 200 I I
210 I
30
220 1
35
230 I
240 I
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250 I
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270 1
1
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L5
Flgure 1. Capillarytolumngas chromatogram of the hexane/tolusne fraction of a diesel particulate extract. Unnumbered peaks c a n be enumerated
by interpolation between numbered peaks.
indices (9) were also used, especially for the hexaneltoluene fraction. The following compounds were synthesized to confirm identifications: 6-methyl-2-naphthaldehyde, 2methylphenanthrenecarboxaldehydes, and 2-methylfluorenone. 6-Methyl-2-naphthaldehydewas synthesized from 2,6-dimethylnaphthalene by reacting it with N-bromosuccinimide and benzoyl peroxide; 2-(bromoethyl)-6methylnaphthalene resulted (10). The bromomethyl group was subsequently converted to an aldehyde by heating it with hexamethylenetetramine in glacial acetic acidlwater solution (11). 2-Methylphenanthrehecarboxyladehydes were synthesized by the reaction of 2-methylphenanthrene with Nmethylformanilide and phosphorus oxychloride (12). Five isomers were observed by GC/MS analysis. 2-Methylfluoreneone was synthesized from 2-rnethylphenanthrene through its epoxidation with sodium hypochlorite (13), followed by photolysis (14). The mass spectrum of this compound was identical with published data (15). All of these synthesized compounds were used as GC/MS standards.
RESULTS AND DISCUSSION The compounds in the hexane/toluene fraction are polycyclic aromatic hydrocarbons (PAH). Oxygenated polycyclic aromatic compounds such as aldehydes and ketones are found in the toluene fraction. We will discuss these two fractions in the following sections. Polycyclic Aromatic Hydrocarbons. The gas chromatogram of the hexaneltoluene fraction i s shown in Figure 1, and the compound identities are given in Table I. The compounds in this fraction are PAH containing three to five aromatic rings. Phenanthrene and alkylated phenanthrenes are the major components. Fluoranthene, pyrene, and their methyl homologues were also detected. A group of compounds with mass Spectra showing a strong ion at mle = 165 were identified as alkylated fluorenes. For example, the mass spectrum of dimethylfluorene (peak 8) is shown in Figure 2. The ion a t mle = 179 results from the loss of a methyl group forming a tropylium ion. The fragment at m / e = 165 corresponds to the loss of CW2, also forming a tropylium ion.
Table I. Compounds Found in the Hexane/Toluene Fraction of the CH,CI, Extract of Diesel Exhaust Particulates peak noes compound 192 3 4 5-9 10 11
12 13 14, 15, 17, 18 16 19-25, 27, 31 26 28, 32, 34, 35 29 30, 33 36, 37, 39, 40 38, 41, 43, 45 42, 44, 46, 47 48-52 53,54 55 56 57 58 59,60 61 62 a
methylfluarenes dibenzothiophene b phenanthrene C,-fluorenes 3-methylphenanthrene 2methylphenanthrene 9-and 4-methylphenanthrene l-methylphenanthrene C, -fluorenes phenylnaphthalede C, -phenanthrenes fluoranthene C,-fluorenes pyrene methylphenylnaphthalenes
C,-phenanthrenes methylfluoranthenes and methylpyrenes C,-pheny lnaphthalenes C,-phenanthrenes C,-phenylnaphthalenes benzo[ghi] fluoranthene benzo [ a 3 anthracene chrysene or triphenylene nitropyrene or nitrofluoranthene benzofluoranthenes benzopyrenes perylene
Numbers refer to Figure 1.
Tentative identification.
20 0
80
100
120
1LO
160
, / I I1 ' 180
1
200
Flgure 2. Mass spectrum of dimethylfluorene.
Although methylfluorenes have been detected in cigarette smoke (16), the presence o f alkylfluorsnes in combustion
emissions is generally rare. In this case, we suggest that alkylfluorenes are formed from the abundant alkylphenanthrenes; see pathway I in Figure 3. 9,lO-Epoxyalkylphenanthrenes, which presumably result from the at-
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Table 11. Compounds Found in the Toluene Fraction of the CH,Cl, Extract of Diesel Exhaust Particulates compound peak no.a 2 3-6, 8, 9
2-naphthqldehyde 1-naphthaldehyde methylnaphthaldehydes
7
6-methyl-2-naphthaldehyde
10-1 2
14-19 19,22 20, 24, 26 21, 23, 27-29 25 30, 31 32, 33, 35-42 34 43 44-48 49
biphenylcarboxaldehydes fluorenone b C,-naphthaldehydes methylbiphenylcarboxaldehydes methylfluorenones C,-naphthaldehydes 2-methylfluorenone C, -biphenylcarboxaldehydes C,fluorenones 9,lO-anthracenedione 4H-cyclopenta[def]phenanthren-4-oneC methyl-9,lO-anthracenedione C,-naphthaldehyde and methyl-9,lOanthracenedione
50,52
phenanthrenecarboxaldehydes 2-phenanthrenecarboxaldehyde
13
51
53-59 60, 69 61-68 70 71-76
C,-fluorenones and C,-fluorenones C,-fluorenones methylphenanthrenecarboxaldehydes
81,83 84
C,-fluorenones C,-naphthaldehydes benzanthrone/ll-benzo[a]fluorenonec benzo[ b]naptho[ 2,1-d]thiophen/ other isomersC 'IH-benz[de]anthrone-7-one pyrenecarboxaldehyde or
86-97
unknown
77
78-80, 82, 85
fluoranthenecarboxaldehyde
Numbers refer to Figure 5. Standards used to confirm GC/MS analyses. C Tentative identification. a
100
J
+
M-GO-H
165 I
M+ 194
! I
Figure 8. Mass spectrum of 2-methylfluorenone(peak 25.)
By considering several previously investigated reaction mechanisms for the combustion of hydrocarbons (22,23), a reasonable mechanism for the formation of polycyclic aromatic aldehydes in diesel combustion is shown in Figure 3, pathway 11. Initially oxygen could abstract a vinylic hydrogen from an alkylated PAH thus giving a vinylic free radical. Subsequently, the capture of a second oxygen could give a peroxide free radical. Finally, the peroxide would decompose into an aldehyde. Substantial amounts of ketones were also detected in the toluene fraction. Fluorenone (peak 13) and its alkyl homologues (C,-C,) are the most abundant aromatic ketones. On the basis of a comparison with the authentic compound, synthesized in our laboratory, peak 25 was positively identified as 2-methylfluorenone. Its mass spectrum is shown in Figure 6. The ion at mle = 165 is due to the loss of a hydrogen and CO group. Fluorenone and alkylated fluorenones have been found in crude oil (24) and cigarette smoke condensate (15). Erickson et al. (25)have also demonstrated the existence of alkylfluorenones (C,-C4) in diesel particulate extracts by using infrared analysis. As shown in Figure 3, these compounds can be formed directly from the oxidation of fluorenes. The diketones, 9,lO-anthracenedione and methyl-9,lOanthracenedione, were also detected. The presence of a strong
molecular ion for 9,lO-anthracenedione allows u9 to distinguish this species from g,lO-phenanthrenedione, which normally shows a weak molecular ion. Furthermore, our silicic acid fractionation eluted these two compounds in different fractions; the more polar 9,lO-phenanthrenedione was found in the methylene chloride fraction. The most intense ion in the mass spectra of the methyl-9,lO-anthracenedionesis also at mle = 165, due to the loss of a hydrogen and two CO groups; the molecular ions are weak (about 20% relative intensity). These characteristic ions were used to differentiate methyl9,lO-anthracenediones from trimethylfluorenones. The mutagenic activities of several aldehydes have been tested in a Salmonella typhimurium system (20). Both 1and 2-naphthaldehyde were toxic but nonmutagenic; phenathrene-9-carboxaldehydeand pyrene-1-carboxaldehyde were slightly mutagenic after enzymatic activation. No mutagenicity was observed for 6-methyl-2-naphthaldehyde. Fluorenone has been reported to be a stimulator of microsomal epoxide hydrase (26). Whether fluorenone exhibits this biological activity in the s. typhimurium system and how this biological activity may affect the mutagenicity of the diesel extracts are open questions.
ACKNOWLEDGMENT The authors thank the Exxon Research and Engineering
Co. (Linde, NJ) for the diesel particulate extract. LITERATURE CITED Dimick, D. L. "Prospects for Diesel Passenger Cars and the Need for an Improved Fuel"; API Automotlve & Industry Forum, Jan 23, 1980. Hites, R. A. "Compositlon of Organics in Dlesel Exhaust"; Environmental Impacts Panel of the Diesel Impacts Study Committee, Natlonal Research Council: Washington, DC, Sept 1980. Wei, E. T.; Wang, Y. Y.; Rappaport, S.M. J . Air Pollot. Control Assoc. 1980, 30, 267.
Liber, H. L.; Andon, B. M.; Hltes, R. A; Thilly, W. G. "Diesel Soot: Mutation Measurements in Bacterial and Human Cells"; Proceedings of the Conference on Health Effects of Dlesel Engine Emissions: Cincin-
nati, OH, 1979. Schuetzle, D.; Lee, F. S. C.; Prater, T. J.; Tejada, S. B. "The Identification of Polynuclear Aromatic Hydrocarbon Derlvatlves in Mutagenic Fractions of Diesel Particulate Extracts"; presented at the 10th Annual Symposium on the Analytical Chemistry of Pollutants, Dormund, Germanv. Mav 1980. Rappapori, S.M.; Wang, Y. Y.; Wei, E. T.; Sawyer, R.;Watkins, 8. E.; Rapoport, H. Environ. Sci. Techno/. 1980, 14, 1505. Rodriguez, C. F.; Fischer, J. B.; Johnson, D. E. "Characterization of Organic Constituents in Diesel Exhaust Particulates"; Proceedlngs of the Conference on Health Effects of Diesel Engine Emlssions: Cincln-
nati, OH, 1979. Erickson, M. D.; Pellizzari, E. D. 'Characterization of Diesel Exhaust Particulate Extracts"; Research Trlangie Insitute. 1978. Lee, M. L.; Vassilaros, D. L.; White, C. M.; Novotny, M. Anal. Chem. 1979, 51, 768. Wenner, W. J. Org. Chem. 1952, 17, 523. Angyal, S. J.; Tetaz, J. R.; Wilson, J. G. "Organlc Synthesls"; Wiley: New York, 1963; Collect. Voi. IV, p 690. Fieser, L. F.; Hartwell, J. L.; Jones, J. E. "Organlc Synthesis";Wiley: New York, 1955; Collect. Vol. 111, p 98. Krlshnan, S.;Kuhn, D. G.; Hamilton, G. A. J . Am. Chem. SOC. 1977, 99, 8121.
Shudo, K.; Okamoto, T. Chem. Pharm. Bull. 1973, 21, 2809. Bell, J. H.; Ireland, S.;Spears, A. W. Anal. Chem. WSg, 41, 310. Levlns, R. J. Chromatographla 1978, 1 1 , 736. Yu, M. L., unpublished work, 1980. Pitts, J.; VanCauwenberge, K. A,; Groslean, D. Science 1978, 202, 515.
Searle, C. E. "Chemical Carbinogens"; American Chemical Society: Washington, DC, 1976. Barfknecht, T. R.; Andon, B. M.; Thiily, W. G.; Hltes, R. A., presented at the Fifth Symposium on PAH, Battelle Columbus Labortories, Oct 1980.
LaVole, E. J.; Tulley, L.; Bederko, V.; Hoffman, D. Mutat. Res., in press. Braddley, J. N. "Flame and Combustion Phenomena"; Butler and Tanner Ltd.: Frome and London, 1969. "Literature of the Combusion of Petroleum"; American Chemical Soclety: Washington, DC, 1958. Latham, D. R.; Ferrin, E. R.; Ball, J. S,Anal. Chem. 1962, 3 4 , 311. Erickson, M. D.; Newton, D. L.; Pellizzarl, E. D.; Tomer, K. B. J . Chromatogr. 1979, 17, 449.
Ganu, V. S.;Alworth, W. L., Blochemistry 1978, 17, 2876.
RECEIVED for review December 11,1980. Accepted March 2, 1981. This work was supported by the U S . Department of Energy (Grant No. AC02-80EV-10449).
