342
Anal. Chem. 1901, 53, 342-343
(52) Templeton, G. D., 111; Chasteen, N. D. Geochim. Cosmochim. Acta i a m 44. . , 741-752 . . . . - -. (53) Saai,’R. A.; Weber, J. H. Can. J . Chem. 1879, 57, 1263-1268. (54) Baes, c. F., Jr.; Mesmer, R. E. “The Hydrolysis of Cations”; Wiiey-Interscience: New York, 1976; pp 267-274. (55) Stumm, W.; Morgan, J. J. ”Aquatic Chemistry”; Wiiey-Interscience: New York, 1970: pp 268-271. (56) Truitt, R. E. Ph.D. Dissertation, University of New Hampshire, Durham,
NH, 1980,
Received for review August 1, 1980. Accepted for publication November 6, 1980. Partially supported by Office of Water Resources Technology Grant B004-NH through the University of New Hampshire Water Resources Research Center.
CORRESPONDENCE ~
~~~
Identification of Acephenanthrylene in Combustion Effluents Sir: Acephenanthrylene (I) has crept into the polycyclic aromatic hydrocarbon (PAH) literature. It has been identified several times with apparent certainty by gas chromatographic mass spectrometry even though neither its GC retention characteristics nor its mass spectral behavior are known. For example, Oro and Han ( I , 2) first “identified” acephenanthrylene as a product of the silica-catalyzed pyrolysis of methane. It has also been “identified” in carbon black extracts (3),cigarette smoke condensate (4),tobacco pyrolyzate (5,6),and the combustion products from kerosene (7,8),wood (7), and coal (7).
Table I. Retention Indexes for Fluoranthrene, Acephenanthrylene, and Pyrene Measured in This and Other Studies retention indexa fluoran- acephenansource thene thrylene pyrene this study: synthetic 344.7 347.9 351.3 mixture (Figure 2) coal combustion 344.0 347.6 350.9 products ( 7 ) wood combustion 344.2 348.2 352.0 products ( 7 ) kerosene so0 t ( 7) 344.0 347.7 carbon black ( 3 ) standard ( 15) a
344.7 344.01
348.6
352.1 351.22
Based on phenanthrene = 300 and chrysene = 400; see
ref 16.
I11
CpJ \
X
In all these instances, the identification of acephenanthrylene was based on its characteristic gas chromatographic elution between fluoranthene (11)and pyrene (1111, combined with its electron-impact mass spectrum which indicated a polycyclic aromatic hydrocarbon structure with a molecular weight of 202 (C16Hlo). From this information together with the drastic conditions (pyrolysis or combustion) under which the compound was formed, structures were proposed with the largest number of six-membered aromatic rings: acephenanthrylene (I) or aceanthrylene (IV). The recognition that angular, phenanthrene-like compounds are thermodynamically more stable (9) than linear, anthracenelike isomers was invoked in favor of acephenanthrylene. Despite such circumstantial evidence, however, acephenanthrylene has yet to be positively identified in combustion effluents. We undertook the preparation of an authentic sample of acephenanthrylene in order to determine its gas chromatographic and mass spectral behavior, to confirm its formation in combustion systems, and to assess its mutagenic activity 0003-2700/81/0353-0342$01.00/0
in a quantitative mutation assay (10, 11). Synthesis. The synthetic scheme (see Figure 1) used to prepare acephenanthrylene (I) was based on Haworth’s synthesis (12)of phenanthrene. The initial step of the sequence is the succinoylation (13)of acenaphthene (V). The resulting 3-(5-acenaphthoyl)propionic acid (VI) was reduced to 445acenaphtheny1)butyricacid (VII) by using tin and hydrochloric acid. This latter acid was cyclized to the tetracyclic ketone VI11 under acid catalysis (12).This ketone (VIII)was reduced (NaBHJ to the corresponding alcohol (IX) which was dehydrated with toluene sulfuric acid and then dehydrogenated by using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in refluxing benzene to yield acephenanthrylene. Final purification of the product was achieved by alumina column chromatography using hexane as the eluant. The sample was found pure by high-resolution gas chromatographic analysis. Electron impact mass spectrum: m / e (relative intensity) 203 (17), 202 (loo), 201 (14), 200 (15), 101 (24), 100 (17). Ultraviolet spectrum (hexane): A, (nm), 227,254, 286,298,317, 328,346,364. This UV spectrum agrees with those reported previously (6, 14). GC Retention. A solution (in hexane) containing phenanthrene, fluoranthene, acephenanthrylene, pyrene, and chrysene was prepared. A partial gas chromatogram (SE-52 WCOT column, 10 m X 0.025 cm i.d., He carrier gas, 100 “C initial temperature, 4 OC/min temperature program, flame ionization detection) of this mixture is shown in Figure 2. The retention indices (Ri)of fluoranthene, acephenanthrylene, and pyrene were calculated on the basis of phenanthrene (Ri = 300) and chrysene (Ri= 400) as standards (15). The retention index of acephenanthrylene was also calculated from the gas chromatograms available in the literature which alleged to 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL.
n
n
kn/HCI
-
n
n in
ether
H3C- C6H,,-S03 H
/
Figure 1.
pared.
Synthetic scheme by which acephenanthrylene was p r a
53, NO. 2, FEBRUARY 1981 343
Bacterial Mutation Assay. Acephenanthrylene was assayed for its ability to induce mutation to 8-azaguanine resistance in Salmonella typhimurium as described by Skopek et al. (10, 11). Exponentially growing cultures of S. typhimurium were incubated for 2 h at 37 OC with 0.08,0.25,0.5, and 1.0 mM concentrations of acephenanthrylene dissolved in dimethyl sulfoxide. A drug metabolizing system derived from the livers of Aroclor 1254 induced rats (10) was added to a set of duplicate cultures to allow for the possible metabolic activation of the compound. Following the incubation, bacteria were centrifuged (2000 rpm for 15 min), resuspended in phosphate buffered saline, and plated under selective (50 pg/mL 8-azaguanine) and permissive conditions. Colonies were counted after 48 h of growth at 37 "C, and the mutant fraction for each culture was calculated (10)from the number of colonies observed under selective conditions divided by the number of colonies observed under permiasive conditions times the appropriate dilution factors. The bacterial mutation m y shows that acephenanthrylene is not active in the range of concentrations studied both with and without metabolic activation. It is interesting to note that a structurally related PAH, cyclopenteno[cd]pyrene (X),induced significant bacterial mutation with metabolic activation (16,17). It was proposed (16) that the formation of an epoxide at the ethylenic bridge may transform this hydrocarbon into a bacterial mutagen. Clearly, a similar metabolic activation is also possible in the case of acephenanthrylene. The lack of mutagenic activity for this compound indicates that structure-mutagenicity correlations ought to be continually supported by experimental observations. ACKNOWLEDGMENT Acknowledgments. We are grateful to William G. Thilly and Barbara M. Andon for the bioassay experiments. LITERATURE CITED (1) Oro, J.; Han, J. Science, 153, 1966, 1393. (2) Oro, J.; Han, J. J . Oes Chromatogr. 1067, 5 , 480. (3) Lee, M. L.; HIes R. A. Anal. Chem. 1976, 48, 1890. (4) Severson, R. F.; Snook, M. E.; Arrendaie, R. F.; Chortyk, 0, T. Anal. Chem. 1976, 48, 1866. (5) Severson, R. F.; Schlotzhauer, W. S.; Anendale, R. F.; Snook, M. E.;
Hlgman, H. C. Bietr. Tabakforsch. 1977, 9 , 23. (6) Snook, M. E.; Severson, R. F.; Arrendale, R. F.; Hlgman, H. C.; Chortyk, 0, T. Bletr. Tabaktorsch. 1077, 9 , 79. (7) Lee, M. L.; Pracb, G. P.; Howard, J. B.; Hites, R. A. B&med. Mass Spectrom. 1977, 4 , 182. (8) Laflamme, R. E.; Hites, R. A. Geochlm. Cosmochlm. Acta 1976, 42,
12
I
I
I
1
I
16
20
2L
28
32
289. (9) Paullng, L. "The Nature of the Chemical Bond", 3rd ed.;Cornell Unhrersky Press: Ithaca, NY, 1960,p 199. (10) Skopek, T. R.; Llber, H. L., Krolewskl, J. J.; Thllly, W. 0.Proc. Net/. Acad. Scl. U.S.A. 1976, 75, 410. (11) Skopek. T. R.; Uber, H. L.; Kahn, D. A.; Thllly. W. 0.Proc. Natl. Acad. Scl. U.S.A. 1978, 75, 4465. (12) Haworth, R. D. J. Chem. SOC. 1932, 1125. (13)Fieser, L. F. "Organic Syntheses"; Wiley: New York, 1055; Collect. Vol. 111, p. 6.
Time iminutes)
Figure 2. Gas
chromatogram of a mixture of five authentic
PAH
standards.
identify this compound. Table I contains these data. The various retention index values for acephenanthrylene are consistent with one another and (given the approximations that are involved in retention time measurements from the miniaturized chromatograms available in the literature) are well within the expected limits of experimental error. The mass spectrum of authentic acephenanthrylene was identical with those of "alleged acephenanthrylene" (3,7,8).Thus, the identity of acephenanthrylene was confirmed through gas chromatograpahic and mass spectral analysis of an authentic sample.
(14) Laarhoven, W. H.; Cuppen, Th. J. H. M. Red. Trav. Chlm. Pay-Bes 1976, 95, 165. (15) Lee, M. L.; Vasslleros, D. L.; White, C. M.; Novotny, M. Anal. Chem. 1979, 51, 768. (16) Eisenstadt, E.; Gold, A. Pfoc. Natl. Acad. Scl. U.S.A. 1976, 15, 1667. (17) Kaden, D. A.; Hites, R. A.; Thilly, W. 0. CancerRes. 1979, 39, 4152.
Subramanian Krishnan Ronald A. €lites* School of Public and Environmental Affairs and Department of Chemistry Indiana University 400 East Seventh Street Bloomington, Indiana 47405
RECEIVED for review August 4, 1980. Accepted October 27, 1980. This work was supported by the US. Department of Energy (Grant No. EE 7743-02-4267).
