Polycyclic Aromatic Hydrocarbons in the Environment: Isolation and Characterization by Chromatography, Visible, Ultraviolet, and Mass Spectrometry Walter Giger' and M a x Blumer2 Woods Hole Oceanographic Institution, Woods Hole, Mass. 02543
Polycyclic aromatic hydrocarbons from many sources accumulate in soils, recent and ancient sediments, and in many other environmental samples. They occur at low concentrations and in the presence of other extractable materials. We describe an isolation technique utilizing a sequence of gel filtration, adsorption chromatography, and charge transfer complexation. It leads rapidly to aromatic ring-type concentrates that are sufficiently pure for UV, visible, and mass spectrometry. In combination, these techniques resolve adequately very complex mixtures of aromatic hydrocarbons, for instance from fossil fuels, and provide a detailed insight into the hydrocarbon composition, on a milligram to microgram scale. Analyses of some recent nearshore marine sediments demonstrate an unanticipated complexity of the polycyclic aromatic hydrocarbon fraction. The principal hydrocarbon series extends over a wide molecular weight range; minor series are the naphthenologs and thienologs of the major series. The analytical and biochemical implications are discussed.
Research during the last decade has demonstrated the ubiquity of hydrocarbons in nature. They occur in great structural variety as contributions from many sources, each source differing in the relative proportions of its component hydrocarbons. Their stability spans a wide range, but most hydrocarbons are sufficiently inert to enter into complex pathways of dispersal, involving transport by air, water, particulates, and by the food web. Much of the research on the origin and fate of hydrocarbons in nature has concentrated on saturated and olefinic hydrocarbons, principally because of the relative ease of analysis by gas chromatography, alone or in conjunction with mass spectrometry. Efforts to study aromatic hydrocarbons in nature are now intensified. The increasing use of fossil fuels and the growing importance of coal and oil shale, whose pyrolysis produces a rich spectrum of aromatic hydrocarbons, pose many environmental and public health questions. However, the formation of aromatic hydrocarbons by geochemical ( I ) , industrial, and life processes and their dispersal through the environment are still poorly understood. In terms of compositional complexity, fossil polycyclic aromatic hydrocarbon (PAH) mixtures range from simple to extremely complex. Thus, nearly pure, crystallized hydrocarbons occur in some mercury ores (2, 3 ) , while petroleum is known for the extreme complexity of its homologous and isomeric compounds. The products of incomplete combustion are of intermediate complexity, though the formation temperature and carbon-to-hydrogen ratio of the l On leave from Swiss Federal Institute for Water Resources and LVater Pollution Control, CH-8600 Duebendorf. To whom requests fur reprints should be addressed.
(1) M. Blumer, Pure Appl. Chem.,34, 591 (1973). (2) T. Geissman. K. Y . Sim, and J. Murdoch. fxperientia, 23, 793 (1967). (3) J. Murdoch and T. A Geissman. Amer. Mineral., 52, 61 1 (1967).
starting material have a marked influence on the composition of the products. Environmental samples may contain PAH assemblages from many of these sources. This implies the need for analytical techniques that can resolve adequately the numerous PAH series in fossil fuels. Specific and relatively simple techniques, such as adsorption chromatography with subsequent spectrophotometry or gas chromatography, determine reliably some aromatic hydrocarbons in simple environmental mixtures ( 4 , 5 ) . However, no existing method separates and resolves adequately the entire PAH fraction on a milligram to microgram scale and on samples that contain a very large excess of nonhydrocarbons. Even in petroleum analysis, where large samples are available, a complete analytical resolution of the PAH fraction exceeds the capability of any existing combination of analytical techniques. However, adequate characterization is possible, even if it is limited to a description of the molecular weight distribution of the many overlapping homologous series (6, 7). We are involved in a continuing study of the origin and fate of hydrocarbons in nature. This paper reports our methods for the isolation, fractionation, and analysis of PAHs from environmental samples. In addition, we present some analyses of near-shore marine sediments which demonstrate a previously undocumented compositional complexity of the PAH fraction.
EXPERIMENTAL Sediment Samples. Samples were taken in Buzzards Ray, Mass., with a 1/25 m2 Van Veen Grab, which was cleaned between uses with water and redistilled methanol, acetone, and pentane. T h e grab was suspended from polypropylene rope; neither steel cable nor a mechanical winch was used; this prevented sample contamination with lubricants. T h e sediments were sealed in clean stainless steel containers and frozen until analysis. Results from two stations in Buzzards Bay are described here, Station A, 1.3 miles from shore a t West Falmouth, Mass., in 15 m of water, [location 90 of Blumer and Sass ( 8 ) ] ,and Station B, 1.8 miles off the entrance to New Bedford Harbor and 0.5 mile from the nearest shore, outside the dredged channel in 7.5 ni of water. Both samples were collected in July 19'73. Buzzards Bay is a typical Northeastern U.S.coastal estuary, stretching between the mainland to the northwest and Cape Cod and the Elizabeth Islands to the southeast. Domestic and industrial wastes from the population centers of New Bedford-Fairhaven, Mass., to the north and the commercial and pleasure craft moving through the Cape Cod Canal are the principal sources of pollution. T h e southern shore of the bay has less or no influx from domestic or industrial wastes, especially along the islands, but it was the site of a 600-700 ton spill of diesel fuel in 1969 (9).Except for the polluted areas near New Bedford and the area affected by Grimmer and H. Hildebrandt, J. Chromatogr.,20, 89, (1965). (5) G. Grimmer and H. Boehnke, Fresenius' 2. Anal. Chem., 261, 310 (4) G.
(1972). (6) H. J. Coleman, D. E. Hirsch. and J. E. Dooley, Anal. Chem., 41, 800 (1969). (7) H. J. Coleman, J. E. Dooley, D. E. Hirsch, and C. J. Thompson, Anal. Chem., 45, 1724 (1973). (8)M. Blumer and J. Sass, Woods Hole Oceanogr. lnst. Tech. Rept., 72-19 (1972). (9) M. Blumer and J. Sass, Science, 176, 1120 (1972).
A N A L Y T I C A L CHEMISTRY, VOL. 46. N O . 12. OCTOBER 1974
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SAMPLE
L_rl J/
Extraction. . .
Isolation..
. . . . . . . . . . ., . . ...
. . .. . . , . . . . . . . . . . . . .
Methanol-benzene, Soxhlet Partitioning into mpentane Sa removal on Cu column Gel permeation/adsorption chromatographyon Sephadex LH-20 Chromatography on alumina/ silica gel Charge transfer complexation, with 2,4,7.trinitro-9-fluorenone
I
,
HYDROCARBONS
Chromatographic Fractionation.
Chromatography on alumina, Fractions I-VI1
Polychlorinated Biphenyls Phenanthrene
I II
thene Chrysene, Benzanthracene Benzopyrenes, Perylene Benzoperylene, Anthanthrene Coronene
IV V VI VI1
Fractions 1-7.
Analysis.. . . . . . . . . . . .UV and visible spectrophotometry, mass spectrometry, probe distillation Figure 1. Analysis of polycyclic aromatic hydrocarbons in environmental samples
the 1969 spill, the bay presents a relatively normal bottom ecology ( I O ) . The biology a t Station A was not noticeably affected by the 1969 spill, nor was the fuel oil from that spill detectable by gas chromatography of sediment extracts from that location. T h e GCs indicate the presence of homologous straight and branched ( e g . , pristane) paraffins and of several olefins that exceed all other individual hydrocarbons in concentration. Above CIS, odd carbon numbered n-alkanes exceed the even homologs. The chromatograms from both stations have a slowly rising base line, rather than the large unresolved envelope common to the sediments contaminated by the diesel fuel ( 8 ) . While this suggests the absence of major low boiling oil contamination, Station B yielded larger hydrocarbon extracts (84 mg/100 g dry sediment compared to 10 mg/100 g dry sediment a t Station A) and a much . a n odd carbon more complex chromatogram above C ~ ONeither predominance nor an excess of n-alkanes over iso- and cycloalkanes is observed there. This suggests possible hydrocarbon pollution, more likely by lubricants or partly degraded oils, than by a single or recent spill (11). .Materials. Analytical grade solvents were used throughout; they were redistilled in an all-glass still. Extraction thimbles, cotton, and anhydrous sodium sulfate ("Baker Analyzed") were extracted with benzene-methanol (1:lJand dried. the latter a t 120
"C. Silica gel (Grade 923, Davison Chemical Company, Baltimore, Md. 22126) and alumina (Catalyst, AI 0102-P, Harshaw Chemical Company, Cleveland, Ohio 44106) were activated for twenty-four hours a t 210 O C ; subsequently, they were deactivated with various amounts of water to reduce their catalytic activity. Sephadex L H 20 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J. 08854) was conditioned in benzene-methanol (1:l) and washed with the same solvent, after packing into the column. PAH references, platinum oxide (K & K Laboratories, Plainview, N.J. 11803) and 2,4,7-trinitro-9-fluorenone ( T N F , Eastman Organic Chemicals, Rochester, N.Y. 14650) were used as received, except for phenanthrene, pyrene, fluoranthene, and chrysene, which were purified by column chromatography. (10) H. Sanders, Woods Hole Oceanographic Institution, personal communication, 1974. (11) J. W. Farrington and J. G. Quinn, Estuarine Coastal Mar. Sci., 1, 71
(1973). 1664
Extraction and Isolation of the PAHs (Figure 1).Wet sediments (100-150 g) were Soxhlet-extracted with methanol (275 ml) for twenty-four hours. Then benzene (75 ml) was added and the extraction was continued (24 hours). The extracted samples were dried to constant weight a t room temperature and weighed. T h e hydrocarbons were partitioned from the wet benzene-methanol extract into n-pentane (125 ml, 3 X 75 ml). After washing with tap water (2 X 250 ml) and drying (anhydrous sodium sulfate), the pentane extracts were concentrated to 1 ml in a rotary evaporator a t room temperature. Elemental sulfur was then removed by percolation through a column of copper ( 1 2 ) ,using benzene-pentane (1:l) as eluent. T h e eluate was evaporated to dryness and subjected to gel permeation chromatography. Sephadex LH-20 (20 g) in benzene-methanol (1:l) was packed in a column (1.6-cm i.d., 38-cm bed height). Samples were applied in a minimum volume of that solvent, which was also used as eluent a t a flow rate of 6 ml/min under slight pressure. The first eluate, 50 ml of colored material, was discarded. The next 50 ml were evaporated to dryness in the rotary evaporator a t room temperature. T h e column was reused after washing with 100 ml of solvent; occasional repacking maintained the flow rate. Reference standards, especially phenanthrene and coronene, were used to establish the cut points and the volume of solvent needed for the regeneration of the column. The aromatic concentrate from Sephadex in 1 ml of pentane was transferred to a column (1.2-cm i.d., alumina over silica gel, both 4 ml, deactivated with 3% water). T h e first eluate (20 ml of n-pentane) was discarded. T h e flask which had contained the PAH concentrate from Sephadex was washed with 2 ml of methylene chloride. This, and an additional 13 ml of the same solvent, was added to the alumina-silica gel column, eluting the PAHs. Trinitrofluorenone (20 mg) was added to the methylene chloride (CH1C12) eluate. This solution is evaporated to complete dryness in a 100-ml round-bottom flask. The uncomplexed material was removed by washing the walls of the flask with n-pentane ( 5 x 2 ml). These solutions were withdrawn from the flask with a pipet through a cotton pad and discarded. Excess T N F and the complexes were dissolved in methylene chloride and percolated through a silica gel bed (12-ml, 1.2-cm i.d. column. activated, packed in pentane). The PAHs eluted in 75 ml of methylene chloride, which was evaporated to near dryness in a rotary evaporator a t room temperature. A small aliquot of the CH2C12 eluate (1-596) was transferred with a Hamilton 100-gl syringe to the aluminum pan of a Cahn electrobalance, and weighed after air-drying. The main fraction of PAHs was dried under a stream of nitrogen at room temperature. Fractionation of the PAH Concentrate and UV Analysis. T h e PAH fraction in methylene chloride-pentane (0.5 ml. 1:4J was applied to an alumina column (0.6-cm i d . , 5-ml. lolo water). A column load of 200 gg should not be exceeded if optimum resolution is desired. Elution with pentane containing an increasing percentage of methylene chloride (4, 15, 20, 30, 1000/0,v/v) separated the PAHs into eight ring-type concentrates. Reference standards (12 compounds, 1-3 gg each) have been used to establish the cutpoints (Figure 2 ) . The resulting fractions were adjusted to 25 ml for the C Y measurement (230-450 nm) by partial evaporation or by dilution with the same solvent. Spectra were measured with a Cary Model 14 recording spectrophotometer, using a logarithmic slide wire and 10cm quartz cells. Absorptivities were measured a t those wavelengths (Table I) which are most specific for each hydrocarbon type in the presence of PAHs with comparable chromatographic retention. Peak absorbances were measured a t the wavelengths given in Table I from a base line obtained by straight line interpolation. Specific absorbancies were obtained by spiking sample eluates with known amounts of pure standards; spiking also aided in the qualitative interpretation of the spectra. The UV spectrum of fraction one was usually poorly defined. The next fractions contained the following major (hl) and minor ( m ) components: Fraction 11: phenanthrene (hl);Fraction 111: pyrene and fluoranthene ( M ) , anthracene ( m ) ; Fraction IV: chrysene and benz[a]anthracene (M), triphenylene and benzofluorenes were suggested. Fraction V: benzo[a]pyrene. benzo[e]pyrene and pervlene ( M ) , Fraction VI: Benzo[ghi]perylene (M). anthanthrene ( m ) . picene and benzo[k]fluoranthene were suggested; Fraction YII: corPnene (MI. (12) M
ANALYTICAL CHEMISTRY, V O L . 46, N O . 12. OCTOBER 1974
Blumer, Anal. Chem., 29, 1039 (1957)
50
ELUTION VCL
c1.1
FRACTIONS
ELUENT
%cn2ci2
0. r - C g H t z 1
Figure 2. Chromatography of aromatic hydrocarbons on alumina, 1 O h water: 5 ml in 6-mm i.d. column
~~
~
Table I. Polycyclic Aromatic Hydrocarbons in S e d i m e n t s at S t a t i o n s A a n d Bo
Compound
Phenanthrene Anthracene Pyrene Fluoranthene B e n z \a ] a n t h r a c e n e Chrysene Perylene B e n z o /aIpyrene B e n z o [e Ipyrene Anthanthrene B e n z o [ g hIperylene Coronene T o t a l , f r o m UV Total, actual weight of P A H concentrate T o t a l actual weight of all f r a c t i o n s in final c h r o m a t o g r a m
Station A
Station B
33
n.d.c
8
100 110 41 40 26 75 59 7 66 5 570
170 960 790 330 240 94 370 310 34 280 20 3,600
6,700
63,000
Std dev %b
24 37 13 14 27 15 14 19 8 34
19 34
Wavelength used, nm
29 3 252 333 286 287 267 435 383 331 428 382 338
1,630
" Weight calculated froin VV-spectra,
pg kg dry sediment. Calculations hased on spectra and molecular weight of unsubstituted hydrocarbons. Three analyses, Station -4. Fraction contains much material, h u t spectra unsuited for quantitative analysis because of excessive peak broadening.
T h e spectra of fraction VI1 and more markedly of VI11 were poorly defined and may contain contributions from overlapping spectra of' unidentified hydrocarbons. Rechromatography of the Phenanthrene-Pyrene Fraction. T h e combined phenanthrene-pyrene fractions from several batches of sediment (Station A) were rechromatographed on alumina (with 1% water in 14-mm i.d. column) and eluted consecutively (Fractions 1-81. with 40 ml of pentane, four 25-ml portions of pentane with 2. 2, 3. and 3% CHZC12, 5 ml of pentane with 3?h CH2C12, followed by 25 ml of pentane with 4% CHZC12 and, finally, 50 ml of CHICI~. T h e UV spectra indicated no absorbing material in Fraction 1 and weak absorption in Fraction 2; Fractions 3 and 4 contained phenanthrenes, Fraction 5 pyrene and phenanthrene with minor amounts of anthracene and fluoranthene. Fractions 6 and 7 con-
tained pyrenes with increasing amounts of fluoranthenes. The presence of benzofluorenes was suggested in 7 and 8 (weak spectrum). Hydrogenation. Platinum oxide (1-2 mg) in 100 p1 isooctane was reduced to platinum under a hydrogen atmosphere with stirring at room temperature. T h e sample in isooctane was added and hydrogenated for one hour. Olefins are quantitatively reduced a t room temperature; a t boiling temperatures, the aromatic rings are partially hydrogenated. Mass Spectrometry. Spectra were obtained on a CEC 21-104 mass spectrometer (Du Pont Instruments, Monrovia, Calif. 910161, using the low mass heated probe for sample introduction. Samples not exceeding 1 pg total solids in 1-3 pl solvent were injected into a closed-end capillary (15 X 2-mm 0.d.; 1-mm i d . ) ; the solvent was evaporated a t room temperature in a stream of notrogen. T o minimize premature evaporation during sample introduction. samples were exposed for the shortest possible time to the fore-vacuum in the vacuum lock (30 seconds) and to the high vacuum (40 seconds) before initiating the first scan. The instrument recorded one magnetic scan of 20 seconds duration, every 40 seconds. from 12-3.50 amu at approximately 500 amu resolution. Radiative heating from the source (200 "C) raised the sample temperature at a rate near 7 OC/minute; after reaching 50 "C, the probe temperature was raised a t the same rate by means of the probe heater. Operating conditions are: 1600-V acceleration voltage, 2720 V on the electron multiplier (17 stages), 35 PA source current at 12 eV ionizing voltage. The detection limit for 4-methyl pyrene at 12 eV g [(signal noise)/noise = 2)]. is 4 x Procedure Blank. A blank of methanol (275 ml) and benzene (75 ml1 was refluxed in the Soxhlet apparatus and then carried through the entire procedure, excluding chromatogram B. The fractions from chromatogram A had no measurahle LV/Vis absorbance. The fractions were then combined for greater sensitivity in mass spectral analysis, and subjected to probe distilllation. Ko PAH series was recognizable in the mass spectra.
+
RESULTS Isolation Technique. Most e n v i r o n m e n t a l s a m p l e s c o n t a i n h y d r o c a r b o n s o n l y as t r a c e s and i n t h e p r e s e n c e of many other e x t r a c t a b l e m a t e r i a l s . These h y d r o c a r b o n s h a v e t o be separated and r e c o v e r e d in s u f f i c i e n t p u r i t y for subsequent UV and mass s p e c t r o m e t r i c analysis. The c a r e ful application of a s i n g l e s e p a r a t i n g t e c h n i q u e ( e . g . , colu m n chromatography) m a y sometimes yield a sufficiently p u r e c o n c e n t r a t e . H o w e v e r , o f t e n m i x t u r e s r e s u l t which r e sist f u r t h e r r e s o l u t i o n b y t h e s a m e m e t h o d . I n g e n e r a l . p u -
A N A L Y T I C A L C H E M I S T R Y , V O L . 46. NO. 12, OCTOBER 1974
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Table 11. Recovery o f Reference Hydrocarbons a t Microgram Levels Recovery, Compound
Present, p g
Found, p g
70
Phenanthrene Pyrene Perylene Benzo [alpyrene Benzo [elpyrene Coronene
3.1
1.2 1.8 2.3 2.3 2.5
39 75 77 88 78
1.3
54
2.4 3 .O
2.6 3.2 2.4
rity is more rapidly approached through a combination of techniques that respond to different aspects of the physical and chemical properties of the samples. Our isolation technique utilizes a sequence of gel filtration, adsorption chromatography, and charge-transfer complexation. The resulting analytical scheme (Figure I ) , though involving numerous steps, is basically simple and rapid. In all operations, except the final fractionation, only two consecutive fractions are collected. One of these contains the PAH concentrate; the other is rejected. The initial isolation steps are conventional. Wet sediments are Soxhlet-extracted with methanol, and benzene is added to the refluxing solvent after the initial extraction of the water. This eliminates previous drying of the sample, possible loss of volatile hydrocarbons, and changes the refluxing solvent from methanol to the benzene-methanol azeotrope, which is a good hydrocarbon solvent. From the extract, elemental sulfur, which would chromatograph together with the PAH fraction, is removed by percolation over precipitated copper (12). The subsequent chromatographic separation, on Sephadex LH-20, utilizes the particular properties of that rather polar gel permeation substrate (13). True gel permeation chromatography of the nonpolar lipids in the sample is superimposed over the adsorption, and therefore greater retention, of the more polar materials, including the PAH fraction (14-16). Most lipids and pigments elute near the column void volume. The greatly retarded PAH fraction elutes later, in order of increasing molecular weight and polarity rather than in the reverse order that would be expected for gel permeation chromatography. Other authors have used gel permeation substrates to fractionate PAHs, either in an adsorptive separation on Sephadex LH-20 with isopropanol as solvent (14-16) or in a gel permeation chromatogram on polystyrene gels in benzene (6, 7 ) .However, such a one-step purification is inadequate for complex environmental samples, and the final ring-type separation is more easily and more rapidly accomplished on alumina. Therefore, and because of the high sample capacity of Sephadex LH-20, we apply it in the initial separation of the PAH fraction, a t flow rates and with a solvent that favor rapid elution in a single fraction, rather than resolution into ring-type concentrates. T h e second chromatography on silica gel-alumina separates the PAH fraction from saturated hydrocarbons and most olefins, which are eluted more rapidly, and from more polar materials, which remain on the column. This separation provides adequate purity of the PAH concentrate for the following TNF-adduction; if the chromatography is omitted, lipids remaining in the PAH fraction solubilize the TNF-adduct or prevent its precipitation entirely. Many soils are rich in long chain methyl ketones (17, 18). These (13) (14) (15) (16) (17) (18)
M. Wilk, J. Rochlitz, anx. Bende, J. Chromatogr., 24, 414 (1966). C. A. Streuli, J. Chromatogr.,56, 219 (1971). H. J. Kisch and D. Reese, J. Chromafogr., 80, 266 (1973). H. H. Oelert. Fresenius'Z. Anal. Chem., 244, 91 (1969). M. Streibl and K. Stransky, Fette, Seifen, Ansfrichsm., 74, 566 (1972) R . I. Morrison and W. Bick, Chem. lnd. (London),596 (1966).
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follow the PAH fraction through the separation until they are rejected, together with other uncomplexed materials, during the formation of a PAH-trinitrofluorenone chargetransfer complex (19,201. The TNF-adduct is purified by solvent washing; after dissolution, the adduct is split by percolation over a silica gel column. Column dimensions, elution volumes, and full activity of the silica gel are critical for optimal separation between T N F and especially coronene. Under our experimental conditions, 10 pg coronene is separated from 20 mg T N F with a 97% yield. The total PAH fraction from the T N F separation is still too complex for UV spectroscopy, but mass spectra recorded during gradual evaporation from a temperature-programmed probe will provide a first indication of sample composition and molecular weight distribution. Much more complete insight into the presence of overlapping homologous series can be obtained after a further chromatographic ring-type separation, by correlation of the UV and mass spectra. Many published procedures are available for such a final fractionation. We prefer column chromatography on partly deactivated alumina a t high adsorbent-to-sample ratio because of the good resolving power (Figure 21, the high flow rate, and the fact that the separation is carried out a t room temperature. These features make column chromatography superior to gel permeation and gas Chromatography. For safety reasons (dust of PAH loaded silica particles!), we reject thin layer chromatography in spite of its excellent resolution. Recoveries for six reference PAHs at microgram levels (Table 11) and the reproducibility of recoveries from environmental samples (Table I) are adequate for most geochemical and environmental considerations. The quantitative interpretation of the data is limited more by the uncertainties in the evaluation of the UV and mass spectra than by the sample recovery. The low recovery of phenanthrene in the artificial standard may be due to partial volatilization. Actual environmental samples contain lipids in addition to PAH which lower the vapor pressure of the PAH fraction, especially in the early steps of the separation. Ultraviolet Analysis. Electronic spectra of aromatic hydrocarbons exhibit characteristic absorption bands with considerable fine structure, especially in nonpolar solvents and in the vapor phase (21). The spectra are often used for qualitative and quantitative analysis. However, UV analysis, especially of mixtures rich in alkyl derivatives, has severe limitations. Alkylation of the aromatic ring systems leads to bathochromic shifts in the spectra (22): the peak displacement increases with the number of substituents. Thus, absorption measurements on mixtures of substituted aromatics with the same basic ring system at a single wavelength assess the contributions from different homologs a t different sensitivity levels. An integration of the absorbance over a wider wave length range would provide a more accurate analysis. However, the spectral overlap between various aromatic systems precludes this, except in simple mixtures. In complex PAH fractions ( e g . from fossil fuels). numerous alkyl and cycloalkyl derivatives are present and the spectral overlap is severe; in the resultant spectra, most of the fine structure is lost. The interpretation of such spectra (19) M. Orchin and E. 0. Woolfolk, J. Amer. Chem. SOC.,68, 1727 (1946). (20) G. H. Schenk, P. W. Vance, J. Pietrandrea, and C. Mojzis, Anal. Chem., 37, 372 (1965). (21) R. A. Friedel and M. Orchin. "Ultraviolet Spectra of Aromatic Compounds,"J. Wiley, New York. N , Y 1951. (22) E. Clar, "Polycyclic Hydrocarbons," Academic Press, New York, N.Y., 1964.
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 12. OCTOBER 1974
is difficult. Thus, a crude oil sample, carried through our separating procedure, yielded fractions with smoothed UV spectra that revealed only the presence of the most abundant ring types. This is attributed not to an inadequate separation, but to excessive spectral overlap. Mass spectra confirm both this wide range of alkyl homology and the success of the chromatographic separation. The quantitative interpretation of the UV spectra requires knowledge of the molecular weights of the PAH. Ir. simple systems, mass spectrometry provides this and, with sufficiently large sample, a direct measurement of the molecular weight or its estimate from the boiling range is possible. In small and complex environmental samples, a reliable determination of the average molecular weight is difficult, even by mass spectrometry, which may not resolve the many overlapping series of homologs into their individual contributions. In particular, the higher ring-number concentrates contain unknown and unidentified PAH components or UV-absorbing non-PAH impurities. Their contribution to the measured absorbance leads to errors that cannot be eliminated by the base-line technique and the incremental addition of standards. The quantitative UV analysis of PAH in complex environmental samples is limited by the above errors; and the heavy reliance on the UV data in much of the previous work in this area may not be warranted. UV analysis is most reliable where mass spectrometry confirms a simple composition or the predominance of very few components; neither is realized in the samples whose analyses are discussed here. In our analyses, the summed weights of PAH derived from the UV measurements account for 6-9% of the actual weight of the total PAH fraction or 29% of the weight of the combined fractions of the final PAH chromatogram (at Station A, Table I). The total fraction weight and the weight after the final chromatographic separation step may include non-PAH impurities that have not been rejected by the separating procedure; on the other hand, our UV measurements are based on the absorptivity of the unsubstituted hydrocarbons. Also, the spectra indicate the presence of additional, unidentified aromatics, whose presence is neglected in the sum of weights derived from the UV data. Therefore, the true PAH weight lies between the actual weight of the chromatographic fractions and the weight that has been reconstructed from the individual UV contributions. In spite of these limitations, omission of the UV measurements from the analytical scheme would severely limit the interpretation of the data. The spectra establish the order of elution, the chromatographic resolution, and the recoveries. They supplement the mass spectral interpretation and often reveal significant trends in analyses of related samples. Thus, the spectrum of the phenanthrenes from Station A exhibits the typical fine structure of that hydrocarbon; however, a t Station B, the fine structure is smoothed out and the normally sharp peak a t 293 nm is too indistinct to be used for a quantitative measurement of the phenanthrene concentration. This suggests the presence of a wider range of alkylated phenanthrenes a t Station B than a t Station A. Similarly, a t Station B (Table I), the decreasing trend in the concentrations from low to high ring-number aromatics suggests a relatively greater contribution of three- and four-ring aromatics. Not surprisingly, this trend, and that noted above for the alkane distribution, suggests a greater contamination from fossil fuels near New Bedford Harbor. Mass S p e c t r a l Analysis. Mass spectrometry provides much information rapidly, even on small and complex samples, especially in conjunction with ancillary equipment for
fractionation during sample introduction-e.g., gas chromatography (GC-MS, mass chromatography) or gradual evaporation from a temperature programmed probe. In this initial investigation, we preferred not to use GCMS, since the thermal stability of highly substituted PAH derivatives under GC conditions is poorly known. Pyrolysis of geochemical products occurs often under surprisingly mild thermal conditions ( 2 3 , 2 4 ) .Programmed temperature probe distillation, though providing a much less efficient separation than GC-MS, fractionates mixtures according to their volatilities, with much shorter and less severe thermal exposure. The separation is performed in the high vacuum of the spectrometer rather than a t higher pressure as in GC. Considerable advantage is retained over bulk sample introduction a t a fixed inlet temperature; a volatility profile is obtained and good spectra of the more volatile PAH can be measured before the higher boiling non-PAH impurities distill from the probe. In order to simplify the spectra and their interpretation, we operate consistently a t low electron beam energy. At 12 eV, the ionization of impurities and the fragmentation of the alkyl-substituted PAH are minimized. Though this eliminates nearly .all except the molecular ions from the spectra, much additional compositional information is deduced from the volatility profiles and from previous sample knowledge, especially from chromatographic behavior and UV spectra. The mass spectra, even of the chromatographically well separated ring type concentrates, show an abundance of peaks and a wide molecular weight range (Table 111). Intense ions a t even masses represent principally molecular ions, and weaker peaks a t odd masses represent isotope peaks. Nitrogen compounds, which would also give rise to odd mass peaks, are largely removed in the separating procedure, and no major odd-mass series are evident in the spectra. Most mass spectral peaks contain contributions from overlapping homologous series which represent isomeric hydrocarbons with the same number of aromatic rings ( c g . , chrysene, triphenylene, benzanthracene) or aromatic ring structures combined with sulfur containing or naphthene rings [“thienologs” and “naphthenologs” (711. Neither low voltage mass spectra or UV spectra can resolved this overlap, though the latter permit the identification of the basic isomeric series. An actual measurement of some of t.he components may be possible by gas chromatography. a t least for the lowest boiling memhers but. a t higher molecular weights, the multiple contributions t o most masses are unlikely to be resolved by any existing technique. In probe distillation, the onset of a new homologous series, or of overlapping isomeric series, is often sharp and evident from the sudden appearance of an intense peak at the mass of the unsubstituted parent hydrocarbon of ihe series (Table IV-V). At the temperature of their first kippearance, the contributions from higher homologs are small, but gradually their abundance increases and i he maxima of the distribution curves shift toward higher molecular weights. The first members disappear, unless they have been present, initially, in such concentrations that their pump-out is sluggish. The major hydrocarbon series are readily identified from their mass and UV spectra. They appear chromatographically in the proper elution sequence and, a t least the first members of most series, have been identified in environmental samples before. However, a much wider range of alkyl homologs is now described. (23) M. Blurner and M. Rudrurn. J. lnst Petrol.. 56, 99 (1970) (24) M. Blurner, Pure Appl Chem., 34, 591 (1973)
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Table 111. Homologous Series of Polycyclic Aromatic Hydrocarbons in Sediments at Station A Tentative identification"
Series'
Fraction
First mass
- 14
2
168 182 as Fraction 3 180 as 4
294
Biphenyl, Acenaphthene Tetrahydrophenanthrene, Tetrahydroanthracene
236
Fluorene, Acenaphthylene, Biphenylene, Benzoindene
166
306 262
As 11, also Dinaphthenenaphthalenes (?) Phenanthrene
3 4
- 16
I1 3 4
- 18
V
178 weak 178 178 178 178 weak 190 190 weak 230 202 230 202 230 202 216 242 228 228 184 254 184 240 280 252 252
IV VI VI VI1 VI1 VI11 VI1 VI11
278 278 276 276 302 302 300 326
I1 2 3 4 5 6
- 20
2
- 22
4 2
3
3
4 4 5 5 6
IV 5
- 24
6
IV - 16s - 26
or
3
3 4 IV 4
- 28
IV - 30 - 32 - 34 - 36 - 38
Last mass
290 276 318 318
+
Phenanthrene Phenanthrene Phenanthrene, Anthracene Phenanthrene, Anthracene
330 344
Naphthenephenanthrene Naphthenephenanthrene
328
Dinaphthenephenanthrene Pyrene Dinaphthenephenanthrene Pyrene/Fluoranthene Dinaphthenephenanthrene Pyrene/Fluoranthene Benzofluorene
328 328 342 230 284 312 338
Trinaphthenephenanthrene
Chrysene, Triphenylene, Benzanthracene Chrysene, Triphenylene, Benzanthracene Dibenzothiophene, Naphthothiophene Tetranaphthenephenanthrene
338 322 336 320 348 346 346 344 344 342
Dibenzothiophene, Naphthothiophene Naphthenechrysene Pen tanapht henephenanthrene Dinaphthenologs of Tetracyclic Aromatics Benzo [aIpyrene, Benzo [elpyrene, Perylene, Benzofluoranthene Trinaphthenologs of Tetracyclic Aromatics Picene, Dibenzanthracene, Dibenzophenanthrene Benzo [ghilperylene, Anthanthrene, Indenopyrene Benzo [ghiJperylene, Anthanthrene, Indenopyrene Dibenzofluoranthene Dibenzofluoranthene Coronene Dinaphthenedibenzofluoranthene, Naphthenecoronene
Z- number; see text. Listed are identifications which are most consistent with spectra (UV, VIS, MS), chromatographic mobility, and relative volatility. Many series extend beyond the scan limit (350 amu). No last mass is listed if a single peak predominates (as in the first eluates of each major series) or where severe overlap between series occurs.
Table IV. Mass Distribution of t h e Nominal -12, -26 Series in F r a c t i o n 3 n
Table V. Mass Distribution of t h e Nominal -22 Series in Fraction 3
Probe temperature
Probe temperature
amu
