Application of a Rapid Thin-layer Chromatographic Procedure to the Determinaton of Benzo(u)pyrene, Benz(c)acridines, and 7H-Bemz(de)anthracm-7+ne in Airborne Particulates from Many American Cities
carefully weighed, and dissolved in a small volume of dichloromethane. A 2- to 5-mg. aliquot of the sample was added 1.5 cm. from the bottom of the thin-layer plate. A 2-pg. aliquot of BaP and a 1-pg. aliquot of BcAcr and BO were added to the origin of the same plate. (All standards were dissolved in dichloromethane, kept refrigerated, and prepared fresh weekly.) The plates were developed to 15 cm. (45 minutes) in a chromatographic chamber equilibrated with 200 ml. of pentaneether (19 to 1, v./v.). The chromatograms were observed under a 3600-A. light source, and the fluorescent areas of the standards and corresponding areas of the sample were marked quickly with a sharp stylus. The adsorbent containing standard and unknown was removed from the plate with two flat spatulas and placed in test tubes. Exposure of the chromatogram to ultraviolet radiation was kept to a minimum to avoid photodecomposition. Elution and Analysis. Adsorbent containing sample and unknown was eluted thoroughly with 100 ml. of anhydrous diethyl ether. After evaporation of the ether the ultraviolet absorption between 400 and 240 mp 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, by the base-line technique. Samples containing very low concentrations of benzo(a)pyrene, or samples of questionable separation, were determined spectrophotofluorometrically in concentrated sulfuric acid (F470/540). The BcAcr in the sample area and the BcAcr standard were measured spectrophotofluorometrically (F290/470 mp) in trifluoroacetic acid. The amount of BcAcr was then determined by comparing values of meter-multiplier times transmission. The BO standard and sample area were also determined spectrophotofluorometrically (F480/555 mp) in trifluoroacetic acid, and the amount of BO present in the sample was determined by comparison of relative intensities. More detailed instruction for the elution and determination of these compounds has been presented (Stanley, Meeker, et ai., 1967). Results and Discussion
The thin-layer chromatographic separation of the benzenesoluble fraction of particulates collected from Birmingham, Ala., gave a characteristic chromatogram (Figure 1, A ) . Complete separation of benzo(a)pyrene and benzo(ghi)perylene (Gee) was required for the spectrophotometric determination of BaP at a wavelength of 3820 A. When separation of the BaP and Gee areas was questionable, fluorometric measurement (F470/540) in concentrated sulfuric acid was used to determine the extent of mixing and the necessity of repeating the spectrophotometric procedure. Most separations were good, and all data reported are from the spectrophotometric determination in pentane. The more sensitive fluorometric procedure for the determination of BaP in sulfuric acid was not applied because it was impossible to maintain routinely the specified ultraclean laboratory conditions. When the eluted BaP was rechromatographed on a layer of 20% acetylated cellulose, five distinctively different fluorescent spots were observed (Figure 1,B). Fluorometric examination of the eluted areas in pentane revealed benzo(e)pyrene and an unknown in spot 5 ; perylene and some benzo700
Environmental Seience and Technology
(e)pyrene in spot 4; benzo(k)fluoranthene in spot 3; unknown in spot 2 ; and benzo(a)pyrene in spot 1. The fluorescence and the ultraviolet absorption of the eluate of spot 1 corresponded to pure BaP. mution from cellulose acetate with this procedure was only 55% efficient; therefore, the amount of BaP present in the sample was calculated by comparison with the relative fluorescence intensity of pure compound recovered from the same plate. After rechromatographing of the eluted BaP area, the amount of BaP in the Birmingham sample was calculated to be 850 pg. per gram of extract. Data obtained from the separation and analysis of BaP in samples of the air of many American cities are shown in Table I. AU values reported represent an average of four to six separate determinations. Samples that gave very high or very low values were rechecked and analyzed again by both spectrophotometric and fluorometric measurement. Benz(c)acridine was separated from the crude benzenesolubles on alumina (Figure 1,A). The BcAcr area exhibited a blue or blue-green fluorescence, which changed to yellowgreen on exposure to trifluoroacetic acid fumes. This change of fluorescent color was used effectively to improve definition of areas on chromatograms containing very low concentrations of BcAcr. Removing some of the adsorbent in the benzo(ghi)perylene area of the chromatogram did not interfere in the analysis. Traces of water interfered with the fluorometric determination of BcAcr in pentane containing 2 % trifluorciacetic acid, but in concentrated acid as much as 10% water showed no interference. Relative fluorescence intensities in concentrated acid were comparable to those in acidic pentane. When an eluate of a BcAcr area from Birmingham was rechromatographed on cellulose, further separation occurred (Figure 1,C). Most of the BcAcr was concentrated in spot 2. The fluorescence spectrum of an eluate of spot 2 was determined in trifluoroacetic acid and compared with that of the original BcAcr area (Figure 1, S2). The BcAcr in spot 2 when calculated by comparison with a pure standard eluted from the same plate gave 87 pg. of BcAcr per gram of benzene extract. Spots 1 and 3 exhibited a pale blue fluorescence, which changed to light yellow-green on exposure to acid vapors. Eluates from spots 1 and 3 gave an emission wavelength at 465 mp when excited at 290 mp. The data shown in Table I reported as benz(c)acridine probably reflect some contritution from alkylated derivatives. 7H-Benz(de)anthracen-7-one (BO) was found in all Eamplts and at easily measured concentrations. The chromatogram of benzene-solubles from Birmingham showed the charccteristic green fluorescent area of BO, which changed to bright orange with acid vapors (Figure 1, 0 ) . The eluate of this original BO area separated into four distinctly different areas on cellulo:e layers. Most of it was concentrated in spot 2. Spot 1 was thought at first to be tailing, but did not give the characteristic change of fluorescent color with acid vapors. Spots 3 and 4 exhibited pale blue-green and blue fluorescence, respectively ; the change of fluorescent color with acid was not the same as for BO. The spectrum of the eluate of spot 2 was compared with that of the original area (Figure 1, S3). The concentration of BO in the Birmingham sample was determined by comparison to a pure standard eluted from the same plate; the concentration calculated was 370 pg. per gram of benzene extract. The amounts of BO found in the air of many American cities (Table I) show fairly high concentrations of BO even when values for BaP are low.
Table I. Analysis of Benzene-Soluble Fractions of Suspended Particulates Collected January to June
(1
c
_
City
State
Birmingham Anchorage Phoenix Little Rock Los Angeles Oakland San Francisco Denver Hartford Wilmington Washington Atlanta Honolulu Chicago Indianapolis Davenport Kansas City Ashland New Orleans Portland Baltimore Detroit Minneapolis Jackson St. Louis Helena Omaha L a s Vegas Concord Jersey City AI buquerque New York Charlotte Cincinnati Youngstown Oklahoma City Portland Philadelphia Pittsburgh Providence Greenville Sioux Falls Nashville Houston Ogden Burlington Norfolk Seattle Charleston Milwaukee Cheyenne
Ala. Alaska Ariz. Ark. calif. Calif. calif.
colo. COM. Del. D. C. Ga . H. I. Ill. Ind. Iowa Kan. KY. La. Me. Md. Mich. Minn. Miss. Mo. Mont . Neb. Nev. N. H. N. J. N. M. N. Y. N. C. Ohio Ohio Okla. Ore. Pa. Pa. R. I.
s. c.
S. D. Tenn. Tex. Utah
vt. Va . Wash. W. Va. Wis. wyo.
BenzO(0)PYrene Extract Air, pg./lOOO cu. m. a./g.
700 490 180 240 150 290 150 210 370 400 530 280 190 550 910 850 190 1700 200 600
360 650 400 140 710 280 800 150 190 570 310 450 1200 700 1800 290 490 410 900 320 410 390 600 210 370 390 570 390 500 800 120
5.6 5.1 2.2 2.0 2.2 2.4 0.85 2.2 2.3 2.9 3.2 2.1 0.6 3.9 11 5.5 1 .o 18 1.4 1.7 2.6 4.0 2.0 0.6 6 1.1 4.3 1.1 0.6 5.0 1.9 3.2 13 5.1 13 1.9 2.6 3.4 5.6 2.6 3.8 0.8 4.4 0.9 1 .o 1.1 4.0 3.0 2.7 4.1 0.2
Benz(C)aaidine
Extract, a/g.
Air, lrg./lOOO cu. m.
100 84
0.8 0.9
420
b
b
42 16 43 20 20 41 130 50 48 40 100 120 54 43 60
0.3 0.2 0.4 0.1 0.2 0.3 0.9 0.3 0.4 0.1 0.7 1.5 0.4 0.2 0.6 0.4 0.3 1.4 0.5 0.4 0.1 0.7 0.3 0.1 0.4 0.1 1 .o 0.2 0.3 0.2 0.5 0.8 0.3 0.2 0.7 0.6 0.2 0.3 0.1 0.6 0.2 0.2 0.2 0.8
310 770 290 620
64 110 200 86 83 27 83 67 14 50
46 140 30 37 75 70 110 50 56 80 90 30 29 50 80 35 100 70 120 C
65 55 78
cu. m.
420
400
320 490 600
730 500 340 770 690 702 390 740 450 1200 650 970 450 400
680 900
860 470 450 loo0 667 350 360 740 1800 440
800 530 960
430 1200 480 1600 590 480 420 1100
C
C
0.4 0.3 0.1
550 lo00
640
3.4 4.4 3.7 6 3 4.2 5.1 2.3 3.4 3.1 4.4 4.4 37 1 .o 5.4 8.5 4.5 1.7 7.6 3.1 3.4 4.7 6.0 2.3 1.8 5.8 3.4 4.7 3.6 1.3 8.6 4.0 2.5 3.7 5.4 13 2.8 4.3 4.3 6.0 3.5 11 0.9 12 2.5 1.2 1.2 7.7 C
3.0 5.1 1 .o
January--June NASN composite benzene extracts. Less than detection limit of method used. Sample lost after benzo(o)pyrene was determined.
_
_
~
Volume 2, Number 9, September 1968 701
X
X
X
FRACTION
A . Crude beozene extract
B. Eluate of BaP C. Uuate of BcAcr D. Uuate of BO
Conclusions The data reported have been corrected for losses during extraction of the partidam with beuzme, but not losses during collection and storage. AU samples used for this study represented an average composite benzene extract of particulates collected during the first two quartem of 1966. Because hydrocarbon concentrations decrease during the spring quarter, values would be higher for individual samples collected during the winter quarter. The results of this investigation cannot be used to cofilpare the quality of air over specific urban areas,primarily because the air quality ofa city cannot be represented by one sample from a single sampling site. These data have been obtained to demonstrate the applicability of a simple thin-layerchromatographic procedure to the routine determination of three different compounds representing three Merent classes of poilutants associated with airborne particulates. The measurements are of interest in terms of relative contributions of the three pollutants and the o v e r 4 ranges of concentrations of these pollutants in the United States.
--... Eluate dcrurbe6raerim
Liter& Cited Badger, G. M.,Brit. 1.Cmras 2, Meso (1948). Falk, H. L., Kotin, P., Mehler, E, Amk Emham. H d h 8, 721-30 (1%). M d m L. S., Ar& Bid-Wmck 60,10&12 (1940).
