Extraction of Organics from Airborne Particulates Effects of Various Solvents and Conditions on the Recovery of Benzo (a)pyrene, Benz( c) acridine, and 7H-Benz( d e )anthracen-7-one Thomas W. Stanley, James E. Meeker, and Myrna J. Morgan Bureau of Disease Prevention and Environmental Control, National Center for Air Pollution Control, Public Health Service, U. S. Department of Health, Education, and Welfare, Cincinnati, Ohio 45226
Recently developed spectrophotometric and spectrophotofluorometric methods of determining several known carcinogens were applied in studies of extraction efficiencies of solvent systems used to obtain the organic fractions of airborne particulates. Some of the solvents investigated were pentane, benzene, cyclohexane, acetone, diethyl ether, methylene chloride, and benzene-diethylamine (4 to 1, v./v.). Weights of organic extracts from known weights of carefully composited air particulates ranged from 20 to 1300 mg. Variations of time, temperature, and method of extraction affect total weights of organic extracts and concentrations. In the analysis of equal weights of air particulates enriched with pure benzo(a)pyrene, benz(c)acridine, and 7H-benz(dr)anthracen-7-one, the percentages of these compounds extracted ranged from 50 to 100, 15 to 100, and 40 to 80, respectively. On the basis of these data extraction efficiency studies are required for the proper selection of solvents used to define the organic extracts of airborne particulates.
0
ver the past few years awareness of atmospheric pollution has increased with increasing urbanization and industrialization of our society. Consequently, much attention has been focused on the chemical assay of biologically active residues from organic extracts of suspended atmospheric pollutants. Many solvents and methods have been used to isolate an organic fraction of particulates into various solubility classes for liquid-liquid extraction and column and thin-layer separation and determination of specific compounds. To obtain fractions for the determination of polynuclear aromatics in air particulates, 2-, 4-, and 5-hour Soxhlet extractions with acetone have proved effective (Cleary, 1962; Cooper, 1954; Waller, 1952). Acetone has also been used to isolate polycyclics from vehicular exhaust particulates by refluxing and subsequent extraction of the residue with petroleum ether (Lyons and Johnston, 1957). Because of the complexity of acetone extracts, some investigators have found benzene extracts more manageable (Kern, 1947; Kuratsune, 1956; Moore and Katz, 1960; Tabor, Hauser, et a/., 1957). An even less complicated extract, rich in polycyclic hydrocarbons, has also been obtained with cyclohexane (Commins, 1958; Harington, 1962; Lam, 1956; Lindsey, 1961; Monkman, Moore, et. al., 1962; Moore, Katz, et a/., 1966). Dimethyl sulfoxide has been used to extract poly-
nuclear hydrocarbons from waxes (Haenni, Howard, et ul., 1962). Mixed solvents such as benzene-methanol (Hoffmann and Wynder, 1962), methanol-ether (Zechmeister and Koe, 1952), and benzene-diethylamine (Sawicki. Stanley, et a/., 1967) have been used for extraction of specific classes of organics. Present efforts to establish rapid screening procedures for benzo(a)pyrene, benz(c)acridine(s), and 7H-benz(de)anthracen-7-one in air particulates have revealed a general lack of information on the extraction efficiencies of various solvents for these compounds. Benzene-diethylamine has been suggested for the quantitative extraction of benz(c)acridine(s) (Sawicki, Stanley, et ai., 1967), and methylene chloride has been reported 86 efficient in extracting benzanthrone (Sawicki, Johnson, et a/., 1967). Recently, rapid and sensitive thin-layer chromatographic methods have been reported for the separation and determination of benzo(a)pyrene (Sawicki, Stanley, et al., 1964), benz(c)acridine(s) (Sawicki, Stanley, et al., 1967), and 7H-benz(de)anthracen-7-one (Sawicki, Johnson, et al., 1967). With some necessary modifications, we applied these methods to determine extraction efficiencies obtained with various combinations of solvents and conditions in recovery of specific compounds from urban air particulates. Experimental
Apparatus. A Cary Model 15 spectrophotometer and 1-ml,, I-cm. path length cells were used for all ultraviolet absorption measurements. For fluorescence determinations a n AmincoBowman spectrophotofluorometer was used with the following settings: slit arrangement, No. 2; sensitivity, 50; and R C A phototube, Type 1P21. Fluorescence of the thin-layer chromatogram was observed with a Chromato-Vue (Ultra-Violet Products, Inc., San Gabriel, Calif.) with a 3600-A. light source. Thin-layer chromatographic apparatus and materials were obtained from Brinkman Instruments, Inc., Westbury, N. Y. Reagents. All solvents used for extractions, elution, and analyses were purchased from nearby commercial sources and purified when necessary by redistillation to a constant boiling point. Purity of solvents was determined by investigation of background absorbance or fluorescence. Benzo(n)pyrene (Aldrich Chemicals) was recrystallized several times from Spectrograde n-hexane and washed with small quantities of redistilled pentane. Volume 1, Number 11, November 1967 927
Benzanthrone (Matheson, Coleman & Bell) was recrystallized twice from ethylcyclohexane. Benz(c)acridine (Aldrich Chemicals) was used as furnished by the supplier. The aluminum oxide thin-layer plates (20 by 20 cm. by 250 microns) were prepared according to the supplier's directions. Prepared plates were stored in a desiccator adjusted to 5 0 z relative humidity with aqueous sulfuric acid (Lange's Handbook of Chemistry, 1956). The plates gave reproducible chromatograms when the aqueous sulfuric acid was changed every 5 working days. (Precoated plates were inconsistent in activity and contained fluorescent impurities.) Preparation of Samples. Hi-Vol filters were obtained from several urban stations of the National Air Sampling Network and prepared as follows: For the first composite sample, nine filters were equally divided into 10 strips, and one strip from each filter was tied into a bundle for subsequent extraction. For the second composite sample, 18 half-filters were divided into 10 strips each, and one strip from each was placed in a clean glass-fiber filter and tied into a tight bundle. Loose particulates were collected from unoiled filters in a large centrally located urban office building, screened, and thoroughly mixed. A portion was removed and stored in an amber glass container for background determinations. The 100 grams of material remaining was carefully enriched with 2 mg. of benz(c)acridine, 16 mg. of benzanthrone, and 112 mg. of benzo(a)pyrene. The resulting mixture was transferred to an amber glass container, mixed thoroughly, and allowed to equilibrate in a cool dark area for several days before extrac-
Thin-Layer Chromatography. A 1- to 2-mg. aliquot of the filter extracts, proportioned between 10 spots, was carefully added 1.5 cm. from the bottom of a n aluminum oxide thinlayer plate. A 2-pg. aliquot of a methylene chloride solution of pure benzo(a)pyrene was proportioned between two spots at the origin of the same plate. The plate was transferred to a thinlayer chamber, which had equilibrated for 1 hour with 200 ml. of redistilled pentane. After development to 15 cm., the plate was transferred to a Chromato-Vue cabinet (3600 A.). The observed fluorescent areas of the standard and the corresponding fluorescent area in the sample were quickly scored with a sharp stylus and the chromatogram was removed from the light source. The adsorbent within the marked areas was quantitatively transferred to test tubes and stored in a cold box for subsequent elution. An aliquot of the methylene chloride solution of the extracts of enriched particulates was proportioned between six spots added 1.5 cm. from the bottom of a n aluminum oxide thinlayer plate. T o the origin of the same plate were added 2,1, and 2 pg. of benzo(a)pyrene, benz(c)acridine, and benzanthrone, respectively. The plate was transferred to a chamber equilibrated 2 hours with 200 ml. of pentane-ether (19 to 1, v./v.). After development to 15 cm. in semidarkness, the chromatogram was observed under ultraviolet light and then treated in the manner described for filter extracts. Elution and Analysis. Ten milliliters of anhydrous diethyl ether was added to the test tubes containing the adsorbent and mixed mechanically for 3 minutes. The resultant fine suspen-
tion. Extraction of Sample. Normal Soxhlet extractions were done with medium-sized glass units equipped with a Friedrichs condenser and a round-bottomed flask. Four glass beads and 250 ml. of solvent were placed in the flask and 6 hours allowed for extraction. Extraction a t elevated temperature was made with a Soxhlet unit modified with a side arm for temperature measurement and wrapped with heating tape controlled with a Powerstat. This arrangement was used for a benzene extraction at 75 "C. The procedure from this point was the same as for the normal Soxhlet extraction. Room-temperature extractions were made with a reciprocating shaker set at low speed. The filter containing the particulate was placed in a stoppered 250-ml. Erlenmeyer flask and the extraction solvent added. Three 1-hour, 100-ml. extractions were performed for each sample, and each extract was carefully decanted and filtered into a large beaker. All extractions were done in semidarkness; precautions were taken to ensure uniform treatment of each sample and extract. Solvents were carefully evaporated in the dark a t room temperature in a vacuum oven. The residues were weighed by difference and made to an appropriate volume with methylene chloride. 928 Environmental Science and Technology
I5
SOLVENT FRONT
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I 5
0
10
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a
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Figure 1. Ultraviolet absorption spectra of filter extract eluted from thin-layer plate __ Benzo (a) pyrene Benzo ( a ) pyrene area
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Table I. Determination of Benzo(a)pyrene in Extracts of Equal Weights of Airborne Particulates= Wt. of Benzo(a)pyrene Extract, Found in Extract Extraction Solvent Mg. pg./g. Total pg. Cyclohexane 32 5 80 19 Benzene 44 400 17 Benzeneh 49 390 19 46 300 14 Methylene chloride Methylene chlorideC 35 260 10 Acetone 87 220 19 Acetoned 47 230 11 Hi-\ol filter samples collected at a n urban station. Many filters were equally dilided so that each extraction represents about two filters o r d 48-hr. air sample. All samples were extracted for 6 hrs. in a Soxhlet, except as indicated. Benzene held at 7 5 " C. in contact with the particulates. Extracted at room temperature with redistilled methylene chloride. Extracted a t rooin temperature with acetone.
sion was transferred to a fine-porosity fritted glass funnel positioned in a 50-ml. filtering flask attached to the vacuum line, and contained in a 50" C. water bath. Rinsing of the test tube, funnel, and adsorbent was continued a t a rate approximately equal to evaporation until 100 ml. of ether had been used. As soon as evaporation was complete, the residue was quantitatively transferred to a test tube and reduced carefully to dryness. The ultraviolet absorption between 400 and 240 m p was determined in l ml. of pentane for the eluted benzo(a)pyrene standard and area. The amount of benzo(a)pyrene present in the sample was determined by comparison at wavelengths of 390,382, and 375 mp (Cooper, 1954; Commins, 1958). The fluorescence (excitation 290 m p and emission 470) was determined in pentane-trifluoroacetic acid (49 to 1, v.lv.) and the eluted benz(c)acridine present in the sample was then determined by comparison of meter-multiplier times transmission values. The fluorescence (excitation 480 m p and emission 555 mp) was determined in sulfuic acid for the eluted benzanthrone standard and area. The amount of benzanthrone present in the sample was then determined by comparison. Resiilrs rind Discussion
Benzo(a)pyrene. The thin-layer separation and analysis of extracts of urban filter particulates gave the characteristic chromatogram and absorption spectra shown in Figure 1. The lack of tailing and the well-shaped spot for the 2-pg. standard show the ability of the aluminum oxide layer to support fairly large samples of organic residues-e.g., the 1- to 5-mg. samples used for this investigation. The benzo(ghi)perylene (Gee), which would interfere in this determination, was well separated from the benzo(a)pyrene fraction. Most of the benzo(k)fluoranthene, normally found in the benzo(a)pyrene fraction,
was in an area just below, as determined from fluorescent measurements. Solvents most often suggested for extraction of organics from air particulates were used to extract an equal composite of filter samples with results shown in Table I. The range in weight of extractable material is reflected in the great differences for the calculated micrograms of BaP found per gram of extract. The values for total micrograms of benzo(a)pyrene found in the sample were essentially the same for normal Soxhlet extractions with cyclohexane, acetone, and benzene. The value shown for the normal extraction with methylene chloride is lower than would be expected, although care was taken to ensure equal treatment of samples and to avoid decomposition. A second series of normal Soxhlet extractions was made with equal weights of Hi-vol filter composites to give the results shown in Table 11. The filters composited to obtain these data were from a second urban site; this difference is reflected in the weight of residue. The values for total micrograms found should be used to compare efficiencies of extraction. Normal 6-hour Soxhlet extractions of the nonenriched particulates with benzene, benzene-diethylamine, 4 to 1 (v./v.), acetone, and methylene chloride produced residues which gave negative results when 5-mg. samples were analyzed in this procedure. By use of many solvents, 6-hour Soxhlet extractions of particulates enriched with pure benzo(u)pyrene gave the recovery data shown in Table 111. Duplicate extractions were made with cyclohexane, benzene, benzene-diethylamine, and methylene chloride. The results given for the recovery of benzo(a)pyrene are mean values from six or more determinations for each extract. The weight of extracts obtained with these solvents reflects an increasing solubility for the more polar organic compounds and many inorganics. Acetone, ethanol, and acetic acid contained some material that was water-soluble and could not be redissolved in the methylene chloride. The acetic acid extract was redissolved in methanol; an increase in precipitate and color was observed before analysis could be completed. Benzo(a)pyrene could not be found in this sample nor in a sample from a duplicate extraction. The weight of extract obtained with toluene probably reflects some error in treatment of the sample. No sample was available for a duplicate extraction. A select group of solvents was used for room-temperature extractions of equal weights of enriched particulates (Table IV). The weight of extracts ranged very widely, but only acetone proved 100% efficient. Benz(c)acridine. A 6-hour Soxhlet extraction of particulates enriched with benz(c)acridine gave the characteristic thin-layer chromatogram and excitation and emission spectra shown in Figure 2. Water in the samples, resulting from elution on the water bath, caused low fluorescence intensities. Methylene chloride solutions of the residues gave values 10% lower after standing 3 days in a cold box. Volume 1, Number 11, November 1967
929
Table 11. Determination of Benzo(a)pyrene in Extracts of Equal Weights of Airborne Particulates" Weight of Benzo(a)PYreIle Extract,h Found in Extract Extraction Solvent Mg. pg./g. Total pg. Cyclohexane Benzene Toluene Ethyl acetate Chloroform Benzene-diethylamine Acetic acid EthanolC
4.3 7.3 9.7 11 12 49 105 111 . 0
760 460 530 310 340 217 30 23
3.3 3.4 5.1 3.4 3.9 4.9 3.2 2.6
a Hi-vol filter samples collected at a n urban site during winter. Each extract represents 0.9 of one filter extracted i n a Soxhlet for 6 hours. c Residue was very dark with a n asphalt-like appearance.
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Table 111. Recovery of Benzo(a)pyrene (BaP), Benz(c)acridine (BcAcR), and Benzanthrone (BO) from the Extraction of Equal Weights of Enriched Particulates. Wt. of Extract,b Recoveryc Solvent Mg. BaP BcAcR BO
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,
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Figure 2. Fluorescence excitation and emission spectra of extract of enriched particulates eluted from thin-layer plate -Benz (c) acridine --- Benz (c) acridine area
Investigation of many solvents produced only three that were 100% efficient for the extraction of benz(c)acridine. A mean value of 63% for duplicate extractions with benzene indicates that a correction factor greater than 1.5 is needed to correct results of present analyses for benz(c)acridine. Chloroform, acetone, and benzene-diethylamine (4 to 1, v./v.) were 100% efficient, and the results for the latter were very reproducible for duplicate extractions. Room-temperature extractions with various solvents were very low in recovery of benz(c)acridine (Table IV). The methanol extraction recovered 37 % from the enriched particulates, best recovery of the six solvents investigated. 7H-Benz(de)anthracen-7-one (Benzanthrone). Extraction of particulates with the various solvents gave the typical thinlayer chromatogram and fluorescence excitation and emission spectra shown in Figure 3. The precision of the method for the determination of benzanthrone was the best of any used in this study, with a calculated relative standard deviation of k 4 z ; however, none of the solvents or conditions investigated proved 100 % efficient for the extraction of benzanthrone from particulates. Among solvents that proved best for the recovery of benzanthrone were Soxhlet extractions with acetone and methylene chloride, which gave 71 and 80% recovery, respectively (Table 111).
930 Environmental Science and Technology
Pentane Cyclohexane Toluene Benzene Ether Methylene chloride Chloroform Ethyl acetate Acetone Benzenediethylamine Ethanol Waterd Acetic acide
79 120 150 160 160
54 76 77 95 99
10 36 27 63 44
40 46 56 42 48
170 180 180 310
95 90 100 100
40 100 34 100
80 58 32 71
530 560 590 1300
100 95 0.01 . . .e
100 60 Trace 40
46 54 0.21 42
' I Homogeneous mixture of air particulates enriched n ith benzo(a)pyrene, benz(c)acridine, and benzanthronr and extracted in Soxhlet for 6 hours. Residue after evaporation of solvent. Average of six determinations. Methylene chloride, cyclohexane, benzene, and benzetle-diethylamine represent mean values from two extractions and six determinations for each extract. From analysis of 3.9 mg. of residue from a n ether extract of the water. Weight given includes ether-solubles. e Drastic change in color indicated decomposition of residue.
Table IV. Recovery of Benzo(a)pyrene (BaP), Benz(c)acridine (BcAcR), and Benzanthrone (BO) from Equal Weights of Enriched Particulates Extracted at Room Temperaturea Wt. of Extract, % Recoveryb Solvent Mg. BaP BcAcR BO Cyclohexane Benzene Chloroform Isooctane Acetone Methanol
22 27 29 25 38 140
68 80 75 72 100 95
15 22 17 10 17 37
38 46 40 35 63 64
a One gram of particulate: enriched with 1.12, 0.02, and 0.16 mg. of benzo(a)pyrene, benz(c)acridlne, and benzanthrone, respectively. Each 1-gram portion extracted six times with 50 ml. of solvent and 30 minutes' shaking time per extraction. Mean values from six determinations.
Normal Soxhlet extractions with benzene showed a mean recovery of 42 % from duplicate extractions and 12-spectrophotofluorometric determinations. This value indicates that a correction factor of 2.38 is necessary to correct present analyses for benzanthrone in benzene extracts. Room-temperature extractions with acetone and methanol gave recoveries of 63 and 64 %, respectively (Table IV). Conclusions Before this investigation methodology was not available for the rapid analysis of small quantities of organic extracts of airborne particulates. This study represents a first effort toward better understanding of organic extracts of air particulates and losses due to extraction. Data reported for the determination of benz(c)acridine and benzathrone in benzene extracts require use of large correction factors; therefore, a more efficient method of extraction would be necessary for making routine determinations. Cyclohexane, benzene, and acetone were nearly 100 % efficient in the extraction of benzo(a)pyrene from air particulates ; however, none of these proved to be quantitative for all three compounds investigated. Individual advantages and disadvantages must be carefully considered in making the proper selection of solvents. Also, to have meaning in terms of
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500
500
550
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Figure 3. Fluorescence excitation and emission spectra of extract of enriched particulates eluted from thin-layer plate -7H-Benz(de)anthracen-7-one 7H-Benz(de)anthracen-7-one area
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human exposure, all data resulting from the analysis of air particulates should be reported in terms of volume of air sampled and the efficiency of the method of extraction should be determined. It is assumed that homologous members of polynuclear aromatics, ring carbonyls, and aza heterocyclics will behave similarly and these data should be applicable. Further study is needed to ensure proper selection of extraction solvents and corrections in reporting data from the determination of specific compounds in polluted atmospheres. Acknowledgment The authors acknowledge the conscientious efforts of Ethel Grisby throughout this study. They are indebted to George B. Morgan for his glass-blowing services. Literature Cited Cleary, G. J., J . Chromatog. 9,204-15 (1962). Commins, B. T., Analyst 83,386-9 (1958). Cooper, R. L., Analyst 79,573-9 (1954). . Haenni. E. 0..Howard. J. W.. Joe. F. L.. Jr.. J . Aswr. Ofic. ”< Agr. Chemis’ts 45,67-?0 (1962). ‘ Harington, J. S., Nature 193,43-5 (1962). Hoffmann, D., Wynder, E. L., Cancer 15,93-102 (1962). Kern, W., Helv. Chim. Acta30,1595-9 (1947). Kuratsune, M., J. Natl. Cancer Inst. 16,1485-96 (1956). Lam, J., Acta Pathol. Microbiol. Scand. 38:39,198-206 (1956). “Lange’s Handbook of Chemistry,” 9th ed., p. 1423. McGrawHill, New York, 1956. Lindsey, A. J., Combust. Flame 4,261-4 (1961). Lyons, M. J., Johnston, H., Brit. J. Cancer 11, 60-6 (1957). Monkman, J. L., Moore, G. E., Katz, M., Ani. Ind. Hyg. ASSOC.J . 23,487-95 (1962). Moore, G. E., Katz, M., Intern. J. Air Pollution 2, 221-4 (1960). Moore, G . E. Katz, M., Drowley, W. B., J . Air Pollution Control Assoc. 16,492-7 (1966). Sawicki. E., Johnson., H.., Mornan, , M. J., Mikrorhim. Acta 2,297-306 (1967). Sawicki, E., Stanley, T. W., Elbert, W. C., J . Chromatog. 26,72-8 (1967). Sawicki, E., Stanley, T. W., Elbert, W. C., Pfaff, J. D., Anal. Chem. 36.497-502 (1964). Tabor, E. C., Hauser;T R.,Lodge, J. P., Burttschell, A.M.A. Arch. Ind. Health 17,58-63 (1957). Waller, R. E.. Brit. J . Cancer 6, 8-21 (1952). Zechmeister, L , Koe, B. K., Arch. Biochein. Biop/iys. 35:36, 1-11 (1952). Receiced Jiir review July 21, 1967. Accepted October 4 , 1967. Division of Water, Air, and Waste Chemistry, 153rd Meeting, ACS, Miami, Fla., April 1967. Mention of commercial products does not constitute endorsement by the Public Health Sercice. I
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