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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
Thin-Layer Gas Chromatographic Method for the Determination of Polycyclic Aromatic and Aliphatic Hydrocarbons in Airborne Particulate Matter J. M. Daisey" and M. A. Leyko New York University Medical Center, Institute of Environmental Medicine, 550 First Avenue, New York, New York 10016
during t h e T L C separation. A large number of individual P A H a n d AHC compounds can be resolved a n d quantified by t h e GLC analysis which t h e n follows. T h e potential exists for recovering a n d analyzing a more polar fraction, which is separated during t h e T L C step. Finally, t h e method minimizes sample handling a n d t h e potential for loss of material a n d contamination.
An analytical method involving a single TLC separation of the cyclohexane-soluble fraction of airborne particulate matter into three polycyclic aromatic hydrocarbon fractions and one aliphatic hydrocarbon fraction suitable for GLC analysis has been developed and applied. The method is simple, rapid, and suitable for routine analysis of these compounds in airborne particulate matter. The difficult-to-separate benzopyrene and benzofluoranthene isomers are completely resolved in the TLC separation.
EXPERIMENTAL Reagents. Standard compounds were obtained from Aldrich Chemical Company and Eastman Kodak Company and were purified by recrystallization as required. Burdick and Jackson "distilled in glass" solvents were used. Sampling. Samples of suspended particulate matter were collected on the roof (15th floor, approximately 200-ft elevation) of the NYU Medical Center in mid-hfanhattan. Twenty-four hour samples were collected from 12:00 a.m. of one day to 12:00 a.m. of the next on 20.3 cm X 25.4 cm pre-washed (1-propanol) glass fiber filters, Gelman, Type A, using high-volume samplers. A flow rate of approximately 1.55 m3/min was used. Upon removal from the filter head, the filters were carefully folded into eighths, loaded-side facing in, and placed in a clean glass jar. The jar was slowly flushed with argon, capped with a foil-lined lid and stored in a freezer at -20 "C. Samples were transported from the Medical Center to the A. J. Lanza Laboratory in a Styrofoam box packed with frozen cold packs (-12 "C). The samples were weighed and then extracted with cyclohexane. KO more than 4 days elapsed between sample collection and extraction. Total particulate matter weights on the filters were approximately 100-200 mg. The average volume sampled was approximately 2200 m3. Extraction. The samples (whole filter) were extracted in a Soxhlet extractor with cyclohexane. Eight-hour extractions using 150 mL of solvent were carried out. Samples were run in groups of three plus a blank. Extracts were then filtered and reduced to 10 mL in volume using a rotary vacuum evaporator with a water bath maintained at 35-40 "C. The round bottom flask used in this operation was rinsed with solvent to thoroughly remove all of the sample extract. This brought the evaporated sample to a 20-mL volume. Sample extract solutions were then stored in a freezer until analyzed. As the PAH compounds are sensitive to ultraviolet light, all extractions, evaporations, etc., were carried out with the laboratory lights off, the light being that admitted through north windows. T h i n - L a y e r Fractionation of Cyclohexane E x t r a c t . The cyclohexane extract was chromatographed on 2 0 7 ~acetylated cellulose-coated TLC plates with 1-propanol-acetone-water ( Z : l : l , v/v/v) in sandwich chambers. Aliquots of the extract were spotted on the plate 2.0 cm from the bottom and 1.0 cm apart using a 5.O-pL disposable capillary pipet Fvith a LViretrol dispenser (0.1 to 0.3 mg per site). A mixture of the standards benzo[~]pyrene,perylene. and benzo[ghi]perylene was spotted on each plate for the purpose of marking the limits of each hand on the plate. Standards for the determination of percent recovery were run at levels comparable to those anticipated and subsequently found for ambient samples, i.e., 1-3 p g . After solvent development to a distance of 15 cm (approximately 2 h), the chromatogram was removed from the chamber, dried, and viewed under a long-wavelength ultraviolet lamp. Standards and extracts were marked and the R, values and appearances of the spots on the plates were recorded. The adsorbent in the marked areas was then removed from the plate
As p a r t of a program t o investigate t h e organic compound fraction of t h e New York City aerosol, a method was sought for t h e separation and analysis of t h e polycyclic aromatic hydrocarbon (PAH) a n d aliphatic hydrocarbon (AHC) classes of compounds in airborne particulate matter. A method which would minimize sampling handling and t h e potential for loss of material a n d contamination was required. A review of the literature indicated t h a t a method combining the best features of thin-layer chromatography (TLC) a n d of gas-liquid chromatography (GLC) would be advantageous. As a first step, a suitable one-step thin-layer chromatographic (TLC) separation of t h e cyclohexane extractable material from airborne particulate matter was sought. Brocco e t al. ( 1 ) had reported t h e use of silica gel t o separate organic material in cyclohexane extracts into a P A H a n d a n AHC fraction prior t o GLC analysis. However, separation of certain isomers within t h e P A H fraction. benzo[a]- a n d benzo[elpyrene in particular, is difficult by GLC. Pierce and Katz (2) reported a method for t h e analysis of P A H in particulate m a t t e r which involves two T L C separations followed by fluorescence spectrometry. T h e P A H compounds are first separated as a class by T L C o n aluminum oxide. Twelve individual P A H within t h e class a r e then further separated by T L C o n acetylated cellulose, extracted individually. a n d t h e n measured by fluorescence spectrometry. While t h e difficult-to-separate isomers such as benzo[a]- a n d benzo[elpyrene a n d benzo[k]- a n d benzo[b]fluoranthene are sepa r a t e d , two T L C separations a n d considerable sample handling are involved. I n addition, T L C resolution is inherently less t h a n what can be achieved by GLC. As a result of our investigations, we have found t h a t , with some modification, t h e second stage of t h e T L C system used by Pierce a n d Katz (2) can be used in a more general application for t h e separation of t h e P A H a n d AHC compound classes within t h e cyclohexane-soluble fraction of airborne particulate matter. Based on this finding, we have developed a simplified T L C / G L C analytical method for t h e separation a n d determination of P A H a n d AHC compounds present in suspended particulate matter. T h e method employs a single T L C separation of t h e cyclohexane-soluble compounds into one AHC a n d three P A H subfractions which can b e suitably re-extracted for subsequent GLC analysis. T h e difficultto-separate isomeric pairs, benzo[a]- and benzo[e]pyrene and benzo[b]- a n d benzo[k]fluoranthene are completely resolved 0003-2700/79/035 1-0024501 O O / O
C
1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 1 , J A N U A R Y 1979
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Table I. R f and R, Values of Some Polycyclic A r o m a t i c Hydrocarbons Separated by Thin-Layer Chromatography compound no. Band I 1
PAH II
I
,
~
e Perylene
e
e 8enzolk)fluoranthene Dibenz lohlanthracene
Chrysene-• e-Benro(b)fluoranthene e-Benzo(a)pyrene
I
2 3
4 Band I1 I
I
e-Triphenylene
Figure 1. Thin-layer chromatogram showing the separation of the
polycyclic aromatic hydrocarbons, aliphatic hydrocarbons, and other polar aromatic hydrocarbons into different regions of the plate and placed in a chromic acid-washed scintillation vial for extraction. Samples and standards of PAH were extracted with 15-20 mL of 407' (by volume) diethylether in cyclohexane at 40 "C for 30 min. Recoveries of PAH standards were determined on a Perkin-Elmer Model 203 fluorescence spectrophotometer with a mercury lamp and grating monochromators in the excitation and emission modes. The aliphatic hydrocarbons were extracted with 15-20 mL of cyclohexane at 40 "C for 30 min. Recoveries of AHC compounds were determined by GLC analysis. Gas-Liquid Chromatography. A dual column Varian Model 2740 Gas Chromatograph was used for sample separation and analysis, equipped with a 3.66 m X 3.18 mm stainless steel column packed with 6 % Dexsil300 on 8OjlO0 mesh Chromosorb If' (HP) and two flame ionization detectors (FID). Operating conditions were as follows: injector temperature, 325 "C; detector temperature, 325 "C; temperature program. 165 "C for 2 min, 165 "C to 285 "C a t 4O/rnin, hold at 285 "C for 30 min; carrier gas, N2,40 mL/min; HS,30 mL/min; air, 300 mL/min. n-Tetracosane, n-eicosane, or p-terphenyl was used as an internal standard. Final sample volumes were approximately 30-50 p L .
RESULTS A N D D I S C U S S I O N T h i n - L a y e r Chromatographic Separation. A separation of t h e P A H a n d AHC classes of compounds from each other a n d from other materials present in t h e cyclohexane filter extract can be achieved on silica gel-coated T L C plates (I). T h e P A H fraction thus obtained, however, is not suitable for direct GLC analysis as t h e benzopyrene a n d benzofluoranthene isomers are not readily resolved by conventional GLC columns. Consideration of t h e polarity of t h e materials present in t h e cyclohexane extract led to t h e conclusion t h a t a reverse-phase T L C system might be more suitable. Our investigations have shown t h a t t h e PAH and AHC classes of compounds can be separated from each other and from other classes of compounds on 20% acetylated cellulose-coated TLC plates with 1-propanol-acetone-water (2:1:1, v/v/v). T h e P A H compounds are simultaneously separated t o yield subfractions which are suitable for subsequent GLC analysis. We have found t h a t t h e nonpolar aliphatic hydrocarbon compounds remain a t t h e origin with this reverse-phase TLC system a n d a r e visible during solvent development as transparent oils on the adsorbent. Analysis of the region above t h e origin indicated t h e presence of only background AHC, Le., 51-2 pg per 30 cm2. Aza-arene compounds and other more polar compounds which may be present move with the solvent t o the top portion of the TLC plate while the PAH compounds on t h e plate and spread through the region of R f 0.15-0.80 can be subdivided into three P A H fractions or bands as shown in Figure 1. R f a n d RB values for t h e standard compounds used in this study are reported in Table I. (Ri = Distance of compound from t h e origin/distance of solvent from t h e origin a n d RB = Distance of compound from t h e origin/
5
6 7 8 Band I11 9 10 11
12 polar compounds 13
14 15 16 17 18
compound
Rf
RB
triphenylene benzo[a] pyrene an thanthrene chrysene
0.16 0.23 0.31 0.36
0.8 1.0 1.4 1.6
benz[a] anthracene dibenz[a, h ] anthracene perylene 7,12-dimethylbenz[a]anthracene
0.57 2.5
benzo[ e 3 pyrene benzo[ghi] perylene fluoranthene pyrene
0.70 0.70 0.73 0.73
benzanthrone benzo[h] quinoline benzo[f] quinoline benzo[c] cinnoline acridine carbazole
0.85 3.7 0.88 3.8 0.89 3.9
0.59 2.6 0.58 2.5 0.66 2.9 3.0 3.0
3.2 3.2
0 . 9 2 4.0 0.96 4 . 2 0.97 4.2
distance of benzo[a]pyrene from t h e origin.) The two isomers of benzopyrene which are found in airborne particulate matter a n d have proved to be very difficult t o separate on a GLC column are readily separated by thin-layer chromatography, since isomers of benzopyrene are distinguished by spreading t o different regions of t h e plate. Similarly, benzo[b]- and benzo[h]fluoranthene, as shown by Pierce a n d Katz ( Z ) , can b e separated, although these standards were not available during this study. I n addition, we have found t h a t benz[a]anthracene, a weak carcinogen, is separated from chrysene and triphenylene which are not carcinogens. Following T L C separation, three "bands" of P A H compounds and one "band" of AHC compounds are removed from t h e plate for subsequent extraction and GLC analysis. Band I, containing benzo[a]pyrene, chrysene, a n d triphenylene, is taken from R, N 0.15 to 0.36 ( - 2 - 5 cm); Band 11, containing benz[a]anthracene, perylene, a n d dibenz[a,h]anthracene, is taken from R, -0.36-0.6 (-5-9 cm); and Band 111, containing benzo[e]pyrene, benzo[ghi]perylene, fluoranthene, and pyrene, is taken from R, -0.6-0.75 (-9-11 cm). T h e aliphatic hydrocarbons are removed with the origin. Standard compounds are run in t h e center of each plate t o determine where each cut should be made. E x t r a c t i o n . Several solvents a n d solvent mixtures were investigated for t h e extraction of F A H from t h e 20% acetylated cellulose. Diethylether, which was used by Pierce and Katz (2) for t h e extraction of P A H was found t o give good recoveries of P A H as measured by spectrofluorometry, b u t proved unsuitable for GLC analysis as a high background, possibly due t o breakdown of acetylated cellulose, was observed. Cyclohexane gave very low recoveries of t h e P A H compounds. A mixture of 40% diethyl ether in cyclohexane (by volume) was eventually found to give good recoveries of P A H as measured by spectrofluorometry, (reported in Table 11) a n d a low background for GLC analysis. The aliphatic hydrocarbons were extracted with cyclohexane a n d recoveries were determined by GLC. Recoveries ranged from a few percent for CI8 t o approximately 30% for CZ6 through CS2, T h e low recoveries may be due to t h e 30-min extraction time and are presently being investigated. A fifth fraction consisting of unidentified slightly polar compounds is potentially recoverable from the T L C plate. It
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1, JANUARY 1979
Table 11. Recoveries of PAH from 20% Acetylated Cellulose with 40% Diethyl Ether in Cyclohexane wavelengths used for measurement A excitation, A emiscompound recovery, % nm sion, nm benz[a] anthracene benzo [ a ] pyrene benzo[e] pyrene benzo[ghi] perylene chrysene dibenz[a,h 3 anthracene fluoranthene perylene pyrene
71 + 90 t_ 72101 = 88 * 82 t 86 2 90 i 89 t
280 380 326 278 280 31 1 382 27 3 328
8
5 5 22 9 5 9 4 10
390 406 391 420 385 400 46 5 467 389
Table 111. Results of PAH Analysis of Some Airborne Particulate Matter
compound
concentration ," ng/m3 8117- 8124R R T a 1 8 / 7 6 25/76
fluoranthene 1.32 0.4 0.6 pyrene 1.42 0.2 0.5 benz [ a ]anthracene 1.81 1.6 2.4 chrysene/triphenylene 2.00 1.6 3.0 benzo[e] pyrene 2.58 3.0 4.4 benzo[a] pyrene 2.60 1.6 2.9 benzo[ghi] perylene 3.63 1.3 1.1 a R R T = retention time relative to C 2 0 , Concentrations corrected for recoveries. Table IV. Results of AHC Analysis of Some Airborne Particulates concentration, ng/m3 alkane c 22
C24 C2 C26 5
c,
-
RRTa,b 0.90 1.09 1.17 1.24 1.31
811718/76 5
812425/76 32
4
18
10 18 100
47 52 84 76 121 97 159
1.40 47 1.50 224 C," 1.61 61 C,, 1.76 81 c 3 2 1.94 54 100 a R R T = retention time relative to p-terphenyl. Temperature program described in Experimental modified to 8 "Cimin. c 2h
c 29
24
?i
Le
^L
6, .,'?I
Figure 2. Typical sample chromatogram: PAH Band 111 shown
is likely t h a t both oxidized hydrocarbons and aza-arenes are present in the upper portion of the chromatogram near the solvent front. Examination of this region of the chromatogram indicates the presence of fluorescent compounds. Attempts to extract these more polar compounds from the acetylated cellulose with 40% diethylether in cyclohexane, cyclohexane, and ethanol have been unsuccessful. Gas-Liquid Chromatographic Analysis. Following solvent extraction from the acetylated cellulose, the three PAH subfractions and the AHC fraction are reduced in volume under argon and analyzed by GLC using n-eicosane, n-tetracosane, or p-terphenyl as a n internal standard. A typical chromatogram of a sample is shown in Figure 2 for the PAH Band 111 fraction. Chromatograms for the other fractions, while not identical, are very similar. Results of the analyses of samples taken during a study of t h e New York City aerosol in the summer of 1976 ( 3 ) are reported in Tables I11 and IV along with retention times relative to the internal standard.
CONCLUSION A simple and rapid analytical method for the separation and analysis of PAH and AHC compounds present in airborne particulate matter has been developed. This method was
developed t o reduce the amount of sample handling; consequently, the potential for loss of material and possible contamination are minimized. Isolation of many individual PAH compounds is achieved by resolving the sample extracts on T L C followed by further resolution and quantitation on GLC. This analytical procedure can discriminate between benzo[a]- and benzo[e]pb~ene.The method is both convenient and versatile since PAH, AHC, and polar organic compounds can be analyzed after a single T L C separation.
ACKNOWLEDGMENT T h e authors thank Theo. J. Kneip for helpful discussions and comments and Robert J. Mallon for the collection of the air samples.
LITERATURE CITED (1) D. Brocco, V. Cantuti, and G. P. Cartoni. J . Chromatogr., 49, 661 (1970). (2) R. C. Pierce and M. Katz. Anal. Chem.. 47, 1743 (1975). (3) B. Leaderer, D. M. Bernstein, J. M. Daisey, M. T. Kleinman, T. J. Kneip, E. 0. Knutson, M. Lippmann, P. J. Lioy, K. A. Rahn, D. Sinclair,R. L. Tanner, and G . T . Wolff, J . Air Pollut. Control Assoc., 28, 321 (1978).
RECEREDfor review June 19,1978. Accepted October 4,1978. This work was supported by Grant No. RP439-1 of the Electric Power Research Institute and is part of a Center Program supported by the National Institute of Environmental Health Sciences, Grant No. ES00260, and the National Cancer Institute, Grant No. CA 13343.
