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in a previous section. Chromatograms for the sample obtained a t two recorder sensitivities are shown in Figure 6. T h e large peak obtained shortly after sample injection resulted predominately from reduction of Fe(II1) in t h e sample. T h e concentration of Cr(V1) was determined by t h e technique of standard additions to be 1.04 ppm.
ACKNOWLEDGMENT T h e authors are grateful to Ronda Snider and H. Mike Stahr of the Veterinary Diagnostic Laboratory for providing t h e water sample.
LITERATURE CITED Fed. Regisfr., 40, No. 248 (1975). D. C. Johnson and J. H. Larochelle, Talanta, 20, 959 (1973). L. R. Taylor and D. C. Johnson, Anal. Cbem., 46, 262 (1974). R. J. Davenport and D. C. Johnson, Anal. Cbem., 48, 1971 (1974). (5) R. W. Andrews and D. C. Johnson, Anal. Chem., 48, 1056 (1975). (6) V. E. Kazarinov and N. A. Baiashova, Dokl. Akad. Nauk SSSR, 134, 864 ( 1 960). (7) A. T. Hubbard, R. A. Osteryoung and F. C. Anson, Anal. Cbem.. 38, 692 (1966). (8) D. C. Johnson, J , , Nectrochem. Soc., 119, 331 (1972). (9) R. F. Lane and A. T. Hubbard, J . Pbys. Cbem., 79, 808 (1975). (10) F. C. Anson, J . Electrochem. Soc., 110, 436 (1963). ( 1 1) J. P. Cushing and A. T. Hubbard, J. €/ecrroanal. Chem., 23, 183 (1969). (12) A. L. Y. Lan and A. T. Hubbard. J . Nectroanal. Cbem., 24, 237 (1970); . . ibid., 3 3 , 77 (1971). (13) C. N. Lai and A. T. Hubbard, Inorg. Cbem., 11, 2081 (1972). (14) R. F. Lane and A. T. Hubbard. J . Phys. Chern.. 77, 1401, 1411 (1973). (151 R. J. DavenDort and D. C. Johnson. Anal. Cbem.. 45. 1755 11973). ' (16j R. F. Lane and A. T. Hubbard, Anal. Chem., 48, 1287 (1976); (17) D C. Johnson and E. W. Resnick, Anal. Cbem., 49, 1918 (1977). (18) 1. M. Kolthoff, Chern. Weekbiad., 16, 450 (1919). (19) M Kabasakologiu and S. Uneri. Commun. Fac. Sci. Univ. Ankara, Ser. B , 14, 59 (1967); Cbem. Abstr., 70, 173524d (1967). (20) H. A. Laitinen and W. E. Harris, "Chemical Analysis", 2nd ed.,McGraw-Hill Book Co., New York, N.Y., 1975, pp 286-290. (21) C. Liteanu and I. Haiduc, Rev. Roum. Cbim., 14, 1039 (1969). (1) (2) (3) (4)
A
B TIME
+
Figure 6. Chromatogram of water sample. (A) sample injection; (B) eluent switched from 0.18 M HCI to 1.8 M HCI; inset shows only Cr(V1) peak recorded at higher sensitivity
prescribed chromatographic procedure. Precision. Replicate analysis of samples containing Cr(V1) throughout the range 90 ppb-3.5 ppm produced results with average deviations less than 2%. The areas of I-t recordings for this concentration range were measured on t h e recorder chart with a planimeter. Accurate electronic integration was possible for concentrations above 3.5 ppm and the average deviation for replicate analyses was approximately 0.3 %. Water Sample. A water sample containing Cr(V1) was supplied by t h e Veterinary Diagnostic Laboratory of Iowa S t a t e University. T h e sample had been acidified with HC1 prior to receiving and was consequently diluted as described
RECEIVED for review August 29, 1977. Accepted November 14, 1977. Work supported by the National Science Foundation through grant GP-40646X.
Determination of Polycyclic Aromatic Hydrocarbons in the Environment by Glass Capillary Gas Chromatography Walter Giger" and Christian Schaffner Swiss Federal Institute for Water Resources and Water Pollution Control (EA WAG), CH-8600 Dubendorf, Switzerland
Polycyclic aromatic hydrocarbons (PAH) are isolated by a sequence of solvent extraction, gel filtration, and adsorption chromatography. The separation of the PAH concentrates into quantifiable individual constituents Is then achieved by glass capillary gas chromatography using 20-m long caplllaries coated with SE-52. Precision and accuracy of the determinatlon of five Individual PAH are presented. Representative analyses are reported for samples of recent lake and river sediments, river particulates, street dust, and airborne particulates. Benzo[ alpyrene levels In surface sediments range from 0.2 to 0.6 pg/g dry material and in rlver particulates and street dust from 2 to 4 pg/g. A sample of airborne particulates contained 40 pg benzo[alpyrene per g.
Polycyclic aromatic hydrocarbons (PAH) have long been recognized as hazardous environmental chemicals. Several members of this class of compounds show considerable carcinogenic activity (1, 2 ) . 0003-2700/78/0350-0243$01 .OO/O
Analytical methods for P A H are basically three-step procedures: (1) Extraction and isolation of the PAH from the sample matrix, (2) Separation of t h e P A H mixture into subgroups or individual constituents, (3) Characterization of these subgroups or compounds (identification and quantitative determination). Two facts call for a quantitative determination of individual P A H in environmental samples. First, t h e biological activity of a particular chemical is very much dependent on its structure. It is well established, for example, t h a t of the two isomeric benzopyrenes, only t h e asymmetric benzo[a]pyrene shows a carcinogenic activity (1,2). Second, if extensive characterization of P A H mixtures is based on analyses of individual constituents rather t h a n unresolved mixtures, we can improve our ability to describe t h e sources a n d t h e fates of these chemicals in t h e environment. We are involved in studies of the origin and fate of a number of different organic compounds in the aquatic environment. Within this area of interest, we have been investigating P A H in recent lake sediments ( 3 ) . In this paper we present our methods which are used to achieve quantitative determi0 1978 American
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nations of individual PAH in various environmental samples. P A H are isolated by a two-step column chromatography procedure a n d are subsequently analyzed by glass capillary gas Chromatography. A limited number of representative analyses will demonstrate the feasibility of our procedures.
SAMPLE
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EXPERIMENTAL Materials. All solvents were of "Nanograde" quality (Mallinckrodt Inc., St. Louis, Mo.) and were used without further purification. Glass-fiber extraction thimbles (Keller, Basel, Switzerland) were cleaned in potassium dichromate-sulfuric acid solution, washed with tap and distilled water, and dried at elevated temperature (approx. 120 "C). Granulated copper was activated by stirring in 2 N hydrochloric acid, washing with water and methanol, and vacuum drying. Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala) was conditioned in benzene-methanol (1:l)and washed with the same solvent after wet packing into the column. Silica gel (Kieselgel 40, 70-230 mesh, Merck) was activated for 17 h at 260 "C. PAH and alkane reference compounds (Fluka, Buchs, Switzerland, and K&K Laboratories, Plainview, N.Y.) and the internal GC-standard 1-chlorotetradecane (Fluka, Buchs) were used as received from the suppliers. Samples a n d Sample Preparation. The lake sediment samples were taken with a gravity coring device; river sediments were collected with a grab sampler. All sediment samples were frozen immediately after recovery. The cores were then sectioned into layers of 3-5 cm. Samples of 30 to 70 g wet sediment were frozen in a cooled isopropanol bath under continuous rotation. Subsequently the samples were evaporated and freeze-dried. Lyophilization times varied between 4 and 14 h a t a pressure of approximately 0.05 Torr. Freeze drying of the samples was chosen because it allows a much cleaner sample storage and handling. No significant losses of the compounds of interest were observed. Air drying at slightly elevated temperatures (40 "C, over-night) has been checked and can also be used with no detrimental effects. River particulates were obtained by filtering river water through pre-extracted paper filters (2-gm pore size), followed by air drying. Airborne particulate matter was sampled on glass fiber filters in the urban area of Zurich by the Institute of Hygiene of the Swiss Federal Institute of Technology. Street dust was swept from a street with heavy motor traffic in Dubendorf. Extraction and Isolation of PAH (Figure 1). The dry samples (corresponding to approximately 20 g wet sediment) were Soxhlet-extracted in glass-fiber thimbles with methylene chloride (70 mL) for 6 h. Longer extraction times (an additional 20 h) did not provide significantly higher yields (less than 5 % ) . Extraction was performed with methylene chloride because this solvent is low boiling, easily purified, and provides a sufficient extraction yield for the relatively nonpolar substances of interest. In our opinion, methylene chloride can adequately replace the toxicologicallysuspicious benzene. The extracts were concentrated to 3 mL in a rotary evaporator at room temperature. After adjusting the volume of the extract to a known value (1, 2, or 5 mL), the weight of the extractable matter was measured using a Cahn 4100 electrobalance. Small aliquots (lo--40gL) were transferred with a microliter syringe to the aluminum pan of the balance, and weighed after cautious air-drying on a hot plate. Elemental sulfur was then removed from the extracts by percolating through a column of activated copper (12 g). The methylene chloride eluates were carefully evaporated to dryness, taken up in 2 mL of benzene-methanol ( l : l ) , and subjected to gel permeation/adsorption chromatography. For this purpose Sephadex LH-20 (20 g) in benzene-methanol (1:l) was packed in a glass column (50 X 1.6 cm). The same solvent mixture was used to elute two fractions of 50 mL each. Another 50 mL was then used to flush the column before applying the next sample. The column chromatography on Sephadex LH-20 is influenced simultaneously by exclusion and adsorption processes. In the way it is used in our procedure (Le.,as a simple, coarse prefractionation technique), it is favorably characterized by its speed, the high column capacity, and the possibility of reusing the column for many samples. Both fractions were cautiously evaporated to dryness (rotary evaporator, reduced pressure, approximately 30 "C), taken up in 1 mL of n-pentane, and applied to silica gel columns (1-cm id., 10-mL bed volume). Twenty-five mL of
REMOVAL OF ELEMENTAL SULFUR
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pentane and 25 mL of methylene chloride yielded two eluates of low and medium polarity, respectively. Column chromatography was performed under a slight pressure of nitrogen, giving flow rates of approximately 2 mL/min. The cutpoints of these separation steps were established based on the elution volumes of naphthalene and coronene. This separation procedure provided two sufficiently pure concentrates: aliphatic and olefinic hydrocarbons in the pentane eluate of the first Sephadex fraction, and polycyclic aromatic hydrocarbons in the methylene chloride fraction of the material which had been more retained on the Sephadex column (Le., second Sephadex fraction). The PAH concentrates could also be characterized by low voltage mass spectroscopy after probe distillation ( 4 ) . We have not encountered any cases where it would be recommendable to perform the complex formation step with trinitrofluorenone which was included in the method of Giger and Blumer ( 4 ) . The fraction weights were determined by the same procedure used for weighing the total extracts. Gas Chromatography, For subsequent GC analyses, the samples were concentrated at room temperature in a stream of nitrogen to volumes of 0.1-0.2 mL. The internal standard (1chlorotetradecane) was added as a hexane solution yielding a concentration of 1-10 Fg of internal standard per sample. Gas chromatography was performed on a Carlo Erba apparatus (Model 2101 AC) with a flame ionization detector. Two types of inlet ports with built-in septum flushing were used. One type is manufactured by Carlo Erba, Milan, according to Grob and Grob ( 5 ) . The second type (called Model 76) is a modified version (6)and was purchased from Brechbuhler AG, Urdorf, Switzerland. The glass capillary columns (20 m X 0.3 mm) were kindly supplied by K. and G. Grob. SE-52 was the stationary phase chosen for quantitative determinations, whereas many qualitative analyses have been performed with OV-101 columns. Columns prepared by the barium carbonate procedure (7) were best suited for quantitative work. The lifetime of the capillary columns was four to six months of daily use, corresponding to about 240 GC runs. Aliquot samples of 1-2 p L were injected without stream splitting onto the column at ambient temperature. The injector temperature was held a t 270 "C. After 30 s, the split valve was opened, allowing the septum and injection port to be purged a t a flow rate of 50 mL/min. Subsequent to the elution of the solvent, the oven temperature was raised rapidly to 60 "C and then at 2.5 OC/min up to 250 "C. Hydrogen was used for carrier
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gas with the flow adjusted so that coronene eluted at about 250 "C from columns with O.l-pm thick films. Back pressures of approximately 0.5-1.0 atm were needed depending on the particular column dimensions. Our GC procedure basically follows the description given by Grob and Grob (5). Electronic integration of the gas chromatographic peak areas was performed by a digital integrator (Minigrator, SpectraPhysics). Procedure B l a n k a n d Recoveries. An empty thimble was extracted in the Soxhlet apparatus with 70 mL of methylene chloride for 6 h. The extract was then carried through the entire procedure as described above. The resulting gas chromatograms of several such blank analyses did not display any peaks interfering with the compounds of interest. The recovery studies were performed using a standard mixture containing approximately 5 bg of each constituent. This solution was analyzed omitting the extraction step. Gas Chromatography/Mass Spectrometry. For mass spectrometric identification and mass specific detection, a Finnigan GC/MS system (Model 1015 D)combined with an on-line computer (Model 6000) was used. The glass capillary column was directly coupled to the mass spectrometer by means of a platinum capillary. Helium was used for carrier gas. The MS operating conditions were: electron energy, 70 eV; emission current, 350 PA; preamplifier sensitivity, low8A/V.
Table I. Polycyclic Aromatic Hydrocarbons Identified in Environmental Samples. Numbers Refer to Figures 2-4 No. Compound 1 Biphenyl Acenaphthene 2 3 Fluorene Phenanthrene 4 Anthracene 5 Methylphenanthrenes 6 7 4,5-Methylenephenanthrene 8 Fluorant hene 9 Pyrene 10 Benzo[a] fluorene Benzo[b]fluorene 11 12 Benz[a]anthracene 13 Chrysene/triphenylene 14 Benzofluoran thenes Benzo[e]pyrene 15 16 Benzo[o] pyrene 17 Perylene 18 Dibenzanthracenes 19 Indeno[ 1,2,3-cd]pyrene 20 Benzo[ghi] perylene 21 Anthanthrene 22 Coronene
RESULTS A N D DISCUSSION Q u a l i t a t i v e A n a l y s i s . Figure 2 shows a typical gas chromatogram of a P A H mixture isolated from recently deposited lake sediment. T h e major peaks were found to be unsubstituted P A H ranging from two-ring (biphenyl) to seven-ring compounds (coronene). Constituents positively identified by GC/MS and co-injection of authentic reference compounds are listed in Table I. Two and more isomers with the same number of condensed aromatic rings occur in this PAH mixture. Phenanthrene and anthracene can readily be separated by the glass capillary column used in Figure 2. Some other isomeric unsubstituted PAH, however, are not as well resolved, such as triphenylene and chrysene (peak 13) or t h e several benzofluoranthenes (peak 14). No significantly better separation of these isomeric P A H was achieved by using longer columns (50 m). These
isomeric P A H can be resolved on more polar columns with higher selectivities (coated e.g. with polyphenyl ether, carbowax, or silar phases). However, we did not routinely use such columns because of their reduced practical temperature range and because of their lower intrinsic separation efficiency (due to coating on a more intensely roughened support surface). Thus, our choice of 20-m long SE-52 columns is a compromise between resolution efficiency and practicality (analysis time and column temperature). T h e gas chromatogram is also characterized by a large number of poorly resolved minor peaks which are due to substituted PAH. A detailed qualitative analysis by mass chromatographic methods ( 3 ) has shown that these P A H mixtures contain, in addition, great numbers of homologous and isomeric alkylated PAH. Methylphenanthrenes (peaks
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