Anal. Chem. 2002, 74, 2379-2385
Analysis of Polycyclic Aromatic Hydrocarbons in Soil: Minimizing Sample Pretreatment Using Automated Soxhlet with Ethyl Acetate as Extraction Solvent Oliver H. J. Szolar,* Helmut Rost, Rudolf Braun, and Andreas P. Loibner
Department of Environmental Biotechnology, Institute for Agrobiotechnology, Konrad Lorenz Strasse 20, 3430 Tulln, Austria
A simplified sample pretreatment method for industrially PAH-contaminated soils applying automated Soxhlet (Soxtherm) with ethyl acetate as extraction solvent is presented. Laborious pretreatment steps such as drying of samples, cleanup of crude extracts, and solvent exchange were allowed to be bypassed without notable performance impact. Moisture of the soil samples did not significantly influence recoveries of PAHs at a wide range of water content for the newly developed method. However, the opposite was true for the standard procedure using the more apolar 1:1 (v/v) n-hexane/acetone solvent mixture including postextraction treatments recommended by the U.S. EPA. Moreover, ethyl acetate crude extracts did not appreciably effect the chromatographic performance (HPLC-3DFLD), which was confirmed by a comparison of the purity of PAH spectra from both pretreatment methods. Up to 20% (v/v) in acetonitrile, ethyl acetate proved to be fully compatible with the mobile phase of the HPLC whereas the same concentration of n-hexane/acetone in acetonitrile resulted in significant retention time shifts. The newly developed pretreatment method was applied to three historically contaminated soils from different sources with extraction efficiencies not being significantly different compared to the standard procedure. Finally, the certified reference soil CRM 524 was subjected to the simplified procedure resulting in quantitative recoveries (>92%) for all PAHs analyzed. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic pollutants that are released into the environment as a consequence of incomplete combustion originating from both, natural and anthropogenic processes. The latter involve energy production, waste incineration, and transport, as well as diverse industrial processes such as aluminum or coke production.1,2 Due to their recalcitrance and suspected carcinogenicity,3,4 the U.S. * Corresponding author: (phone) +43 2272 66280 562; (fax) +43 2272 66280 503; (e-mail)
[email protected]. (1) Overall Evaluation of Carcinogenicity: An Updating of IARC Monographs Volumes 1-42; IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; International Agency for Research on Cancer: Lyon, France, 1987; Supplement 7. (2) Wild, S. R.; Jones, K. C. Environ. Pollut. 1995, 88, 91-108. (3) Shaw, G. R.; Connell, D. W. Rev. Environ. Contam. Toxicol. 1994, 135, 1-62. 10.1021/ac015739l CCC: $22.00 Published on Web 04/19/2002
© 2002 American Chemical Society
Environmental Protection Agency (EPA) has identified 16 of these PAHs as priority pollutants. Especially with soils, hydrophobic compounds such as PAHs exhibit strong surface sorption and time-dependent partitioning into organic phases as well as microvoids with the latter being regarded as the predominant process. 5 This phenomenon, referred to as “aging”, is also assumed to be the major obstacle for fully recovering organic pollutants from complex solid matrixes. Thus, proper pretreatment of contaminated soil samples including leaching of strongly bound PAHs from soil is crucial and considered to be the most laborious step of the analytical process.6 As a consequence, a variety of extraction methods have been developed over the past decades with the objectives to improve the extraction performance as well as to reduce overall analysis time and costs. Latest developments include supercritical fluid extraction (SFE),7-10 pressurized solvent extraction (PSE)11-13 and microwave-assisted extraction (MAE).14-16 Whereas the latter technology employs batch extraction, SFE and PSE are continuous leaching techniques providing enhanced mass-transfer rates due to an increase of the concentration gradient between the phases.17 The same principle has been employed by Soxhlet, the standard technique most widely used for more than a century, with the sample repeatedly being brought in contact with fresh, condensated solvent. The apparatus is rather inexpensive compared to (4) Wilson, S. C.; Jones, K. C. Environ. Pollut. 1993, 81, 229-249. (5) Luthy, R. G.; Aiken, G. R.; Brusseau, M. L.; Cunningham, S. D.; Gschwend, P. M.; Pignatello, J. J.; Reinhard, M.; Traina, S. J.; Weber, W. J., Jr.; Westall, J. C. Environ. Sci. Technol. 1997, 31, 3341-3347. (6) Luque de Castro, M. D.; Garcia-Ayuso, L. E. Anal. Chim. Acta 1998, 369, 1-10. (7) Test Methods for Evaluating Solid Waste, Method 3561, USEPA SW-846, 1996. (8) Lopez-Avila, V.; Young, R.; Tehrani, J.; Damian, J.; Hawthorne, S.; Dankers, J.; van der Heiden, C. J. Chromatogr. 1994, 672, 167-175. (9) Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1987, 59, 1705-1708. (10) Benner, B. A., Jr. Anal. Chem. 1998, 70, 4594-4601. (11) Test Methods for Evaluating Solid Waste, Method 3545, USEPA SW-846, 1996. (12) Lundstedt, S.; van Bavel, B.; Haglund, P.; Tysklind, M.; O ¨ berg, L. G. J. Chromatogr., A 2000, 883, 151-162. (13) Richter, B. E.; Jones, B. A.; Ezzell, J. L.; Porter, N. L.; Avdalovic, N.; Pohl, C. Anal. Chem. 1996, 68, 1033-1039. (14) Granzler, K.; Salgo, A.; Valko, K. J. Chromatogr. 1986, 371, 299-306. (15) Shu, Y. Y.; Lai, T. L. J. Chromatogr., A 2001, 927, 131-141. (16) Sparr Eskilsson, C.; Bjo ¨rklund, E. J. Chromatogr., A 2000, 902, 227-250. (17) Fick, A. Ann. Phys. 1855, 170, 59-65
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SFE or PSE; however, extraction times of more than 16 h have to be anticipated.18 Among the major Soxhlet improvements, automated Soxhlet extraction,19-21 commercially available as Soxtec or Soxtherm, has proven to be equivalent to Soxhlet in terms of extraction efficiencies; however, extraction time could be reduced by 1 order of magnitude by employing a reflux boiling step preceding the actual Soxhlet extraction. Moreover, up to six parallel samples may be treated simultaneously depending on the model used. This makes automated Soxhlet extraction competitive to PSE, which employs increased temperature as well as pressure to promote analyte transfer into the liquid phase.13 However, most pretreatment strategies for solid environmental samples include drying of samples prior to the extraction as well as cleanup of crude extracts and appropriate solvent exchange preceding the instrumental analysis, which significantly increases the overall analysis time.16,22-24 The predominant extraction solvents in the majority of Soxhletbased applications for PAHs, also recommended by the U.S. EPA,18,19 are hexane, cyclohexane, dichloromethane, and 1:1 (v/v) mixtures of mainly acetone with one of the others. Pure ethyl acetate with a higher polarity index has rarely been used for the extraction of PAHs from solid matrixes although better recoveries were found for a sediment spiked with three PAHs compared to some of the above solvents.25 In some studies, ethyl acetate was used for the extraction of PAHs and their metabolites from soil suspensions (liquid/liquid); however, no detailed performance evaluation is known to the authors.26, 27 In this study, a pretreatment method for industrially PAHcontaminated soil samples is presented by applying automated Soxhlet extraction (Soxtherm) with pure ethyl acetate as the leaching solvent of choice. With the objective to minimize the analytical effort, the effect of sample drying on the extraction efficiency was evaluated in detail for both the newly developed pretreatment method and the standard method applying 1:1 (v/v) n-hexane/acetone. Moreover, the influence of the cleanup of crude extracts as well as interference of the extraction solvent with the chromatographic performance was investigated. Eventually, the simplified pretreatment procedure using ethyl acetate as extraction solvent was applied to three industrially PAH-contaminated soils and to the certified reference soil CRM 524. EXPERIMENTAL SECTION Materials. Standard mixtures of the 16 U.S. EPA priority PAHs with 10 mg/L of each compound dissolved in acetonitrile (for (18) Test Methods for Evaluating Solid Waste, Method 3540C, USEPA SW-846, 1996. (19) Test Methods for Evaluating Solid Waste, Method 3541, USEPA SW-846, 1994. (20) Lopez-Avila, V.; Bauer, K.; Milanes, J.; Beckert, W. F. J. AOAC Int. 1993, 76, 864-880. (21) Giner, L. M.; Rodrigo, J. V.; Navarro, A. C. F.; Burillo, V. L. C. Energy Fuels 1996, 10, 1005-1011. (22) Heemken, O. P.; Theobald, N.; Wenclawiak, B. W. Anal. Chem. 1997, 69, 2171-2180. (23) Berset, J. D.; Ejem, M.; Holzer, R.; Lischer, P. Anal. Chim. Acta 1999, 383, 263-275. (24) Test Methods for Evaluating Solid Waste, Method 3600C, USEPA SW-846, 1996. (25) Ravelet, C.; Grosset, C.; Montuelle, B.; Benoit-Guyod, J. L.; Alary, J. Chemosphere 2001, 44, 1541-1546. (26) Zink, G.; Lorber, K. E. Chemosphere 1995, 31, 4077-4084. (27) Ye, D.; Siddiqi, M. A.; Maccubbin, A. E.; Kumar, S.; Sikka, H. C. Environ. Sci. Technol. 1996, 30, 136-142.
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HPLC analysis) and 1:1 (v/v) benzene/dichloromethane (for GC analysis), respectively, were obtained from Supelco (Bellefonte, PA). Ethyl acetate (Baker analyzed), acetonitrile (ultragradient HPLC grade), n-hexane (for organic residue analysis), and acetone (HPLC grade) were used as received from J. T. Baker (Deventer, The Netherlands). Water was obtained from a MilliporeQ plus PF system (Molsheim, France) with a specific resistivity of 18.3 MΩ‚ cm. Al2O3 (90, neutral, activity 1, 70-230 mesh) obtained from Merck (Darmstadt, Germany) and anhydrous sodium sulfate purchased from J. T. Backer were heated overnight at 130 and 640 °C, respectively, prior to use. Extraction thimbles (Ederol) were used as received from Sto¨lzle-Oberglas AG (Vienna, Austria). Industrially contaminated soil samples were obtained from a former railroad sleeper preservation plant (AS1: 20.3% (w/w) water, 8.3% (w/w) organic dry mass), an integrated steelmaking site (BS1: 10.3% (w/w) water, 32.8% (w/w) organic dry mass), and a former manufactured gas plant (ES1: 11% (w/w) water, 12% (w/w) organic dry mass). After arrival, soil samples were passed through a 2-mm sieve and eventually stored at 4 °C in the dark prior to further manipulation. Certified reference soil CRM 524 was purchased from Sigma-Aldrich (St. Louis, MO). Sample Pretreatment. All extractions were performed on an automated Soxhlet extractor (Gerhardt Soxtherm extractor model 2000 automatic, Bonn, Germany). For standard sample pretreatment, triplicate 5-g (dry matter) subsamples were mixed and ground with 10 g of anhydrous sodium sulfate (unless indicated otherwise), transferred into the extraction thimbles, and extracted using 100 mL of 1:1 (v/v) n-hexane/acetone. The Soxtherm apparatus was programmed to start with a 60-min boiling phase of the sample being immersed in the solvent, followed by a 90min Soxhlet extraction. A solvent reduction routine allowed the final volume to be reduced to about 20-25 mL. The obtained crude extract was transferred into a 50-mL volumetric flask and filled to the mark using the extraction solvent. Subsequently, a 1-mL aliquot of the crude extract was passed through a Pasteur pipet filled with Al2O3 for sample cleanup. The cleanup column was then rinsed with 10 mL of 1:1 (v/v) n-hexane/acetone to allow a quantitative recovery of the target compounds in the eluate. Eventually, the cleaned sample was solvent exchanged into acetonitrile using a Heidolph VV 2011 Rotavap equipped with a Heidolph WB 2001 water bath (Schwabach, Germany) and a Vacuubrand diaphragm vacuum pump (Wertheim, Germany) at 40 °C and 350 mbar. The final volume was 5 mL, and samples were stored at 4 °C in the dark prior to HPLC analysis. For sample pretreatment using ethyl acetate as extraction solvent, aliquots of triplicate 5-g (dry matter) subsamples were transferred into the extraction thimbles without mixing/grinding using anhydrous sodium sulfate (unless indicated otherwise) and extracted as described above. No cleanup/solvent exchange was performed on crude extracts, which were directly diluted in acetonitrile for HPLC analysis and ethyl acetate for GC/MS analysis. Analysis. For HPLC analysis, a HP 1050 series highperformance liquid chromatograph (Hewlett-Packard) equipped with a HP 1100 series three-dimensional fluorescence detector (Hewlett-Packard) was used. Sample aliquots and calibration standards of 20 µL were injected on to an ODS Hypersil guard column (20 × 4 mm, particle size 5 µm, Hewlett-Packard) followed
by a C-18 Vydac separation column (250 × 4.6 mm, particle size 5 µm; Vydac, Hesperia, CA) using a HP 1050 autosampler (Hewlett-Packard). The flow rate was set to 1.5 mL/min, and the column temperature was 26 °C. A gradient profile was employed using a water/acetonitrile mobile phase starting with 60% acetonitrile for 2.5 min followed by a 9.5-min linear gradient to 90% acetonitrile and a final 8-min linear gradient to 100% acetonitrile for 2.5 min. A linear gradient (2.5 min) back to starting conditions followed by a 5-min prerun allowed the separation column to equilibrate prior to each subsequent run. For each run, four wavelengths were recorded quasi-simultaneously with excitation being set to 260 nm and emissions to 350, 420, 440, and 500 nm, taking into account the various fluorescence properties of individual PAHs. In addition, spectra of peaks being identified as PAHs in real samples (by the HP-Chemstation based on retention time) were acquired using the three-dimensional fluorescence detector and compared to spectra of a library that was generated using a PAH standard mixture. For quantification, a multilevel calibration ranging from 10 to 800 µg/L was performed at the beginning of each chromatographic sequence with a maximum of 60-70 samples/sequence. For quality control, a 100 µg/L standard was analyzed every 15 samples. Guard columns were replaced after ∼250 injections, to prevent potential clogging of the separation column. For GC/MS analysis, a Hewlett-Packard 6890 Series gas chromatograph interfaced to a HP 5972A mass-selective detector (70 eV) was used with a HP 6890 Series autosampler. The GCMSD system was equipped with a 30-m HP5MS column (0.25mm i.d., 1-µm film thickness), and helium with a linear velocity of 35 cm/s (constant flow) was used as carrier gas. Splitless injection with a sample volume of 1 µL was applied. The injector temperature was set at 320 °C, and the transfer line was maintained at 300 °C. The GC oven was programmed as follows: 50 °C for 2 min, followed by a 17 °C/min ramp to 270 °C (13-min hold). The program was completed with a ramp of 10 °C/min to a final temperature of 320 °C (3-min hold). The mass spectrometer was operated using the single ion monitoring (SIM) mode with time-based ion switching according to the molecular ion (M+) of the respective PAH. An external multilevel calibration was carried out for quantification ranging from 0.5 to 10 mg/L. With both, HPLC and GC/MS, the following PAHs were analyzed for all soils in this study: Fluorene (flu), phenanthrene (phe), anthracene (ant), fluoranthene (flt), pyrene (pyr), benz[a]anthracene (baa), chrysene (chr), benzo[b]fluoranthene (bbf), benzo[k]fluoranthene (bkf), benzo[a]pyrene (bap), benzo[ghi]perylene (bp), indeno[1,2,3-cd]pyrene (ip). Statistical Evaluation. For statistical evaluation of results, SPSS 10.0 for windows (SPSS Inc., Chicago, IL) was used. For testing differences of means, Student’s t test and ANOVA were applied. Levene’s test was used to test homogeneity of variances. Safety. Several components of soils and chemical standards being used in this study have been identified as potential carcinogens; thus, special precautions have to be taken when working with these materials. Unexceptionally, all manipulations with known or suspect hazardous materials are carried out in ventilated hoods in a specially designated and spatially separated laboratory compartment. Protective clothing (the use of laboratory coats and safety goggles is obligatory for all laboratories of our
Figure 1. Effect of sodium sulfate addition on the extraction efficiency of PAHs in soil AS1 using ethyl acetate as extraction solvent. Error bars are standard deviation of the mean, n ) 3.
department) is specially labeled and only used in this area to avoid contamination. Nitrile gloves are used for the handling of toxic materials and disposed of separately with other contaminated disposables by incineration of hazardous materials. Contaminated glassware is rinsed thoroughly with organic solvents prior to general cleaning. All solvents are collected and disposed of appropriately. RESULTS AND DISCUSSION Influence of Soil Moisture on Extraction Efficiency. To ensure optimum extraction efficiencies for nonpolar, non(semi)volatile organic compounds such as PAHs, soils and sediments are recommended to be finely ground with a drying agent such as sodium sulfate prior to Soxhlet extraction.18,19,28 This manipulation, however, is relatively time-consuming especially when series of soil samples have to be treated. Furthermore, if not performed appropriately, this procedure may result in a significant loss predominantly of the low molecular weight PAH fraction due to volatilization as a consequence of the heat introduced during grinding. Thus, samples of the industrially PAH-contaminated soil AS1 were extracted with and without sodium sulfate addition using ethyl acetate as extraction solvent. Figure 1 shows a comparison of the results obtained by the analysis of the crude extracts using HPLC-3DFLD. No significant difference (n ) 3, P > 0.05) for the two treatments were observed for any of the PAHs analyzed. The relative standard deviation (RSD) for the extraction without sodium sulfate addition was in the range of 6.8-12% and, employing the drying/grinding process, 2.2-5.5% for all PAHs except fluorene, phenanthrene, and anthracene, respectively. These low molecular weight compounds exhibited up to 3 times the RSD of the larger PAHs. This phenomenon has been observed regularly with soils contaminated with PAHs and may be a result of their increased volatility, variably affecting parallel subsamples in the course of the manipulation. As mentioned earlier, the sodium sulfate addition resulted in an overall decrease in variance which may be due to the extra homogenization by means of grinding. However, Levene’s test of homogeneity of variances showed no significant difference at P ) 0.05 for any of the PAHs analyzed. (28) Soil quality-Pretreatment of samples for the determination of organic contaminants, E DIN ISO 14507, 1996.
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Table 1. Extraction of PAHs from Soil AS1 at Different Water Contents Using Ethyl Acetate as Extraction Solvent compound fluorene
phenanthrene
anthracene
Figure 2. Effect of sodium sulfate addition on the extraction efficiency of PAHs in soil ES1 using 1:1 (v/v) n-hexane/acetone as extraction solvent. Error bars are standard deviation of the mean, n ) 3.
The influence of the sodium sulfate addition was also tested for the standard sample pretreatment including aluminum oxide cleanup of crude extract and solvent exchange into acetonitrile. Using 1:1 (v/v) n-hexane/acetone as extraction solvent, the addition of sodium sulfate resulted in a significant increase of extraction efficiency (n ) 3, P < 0,05) for all PAHs of the industrially contaminated soil ES1 (Figure 2). Unlike with ethyl acetate, drying of soil samples prior to extraction using a less polar solvent such as the n-hexane/acetone mixture is assumed to be unavoidable for exhaustive extraction. The recovery for samples without sodium sulfate addition was in the range of 74-83% compared to extraction yields of the dried soil samples. Similar findings were reported earlier for the same solvent mixture in combination with ASE22 as well as with Soxtec20 and may predominately be due to insufficient contact of the relatively apolar solvent with the analytes trapped in water-sealed soil pores.12 However, the more hydrophilic ethyl acetate (1 mL dissolves in 10 mL of water at 25 °C)29 is capable of overcoming the “water barrier” and still providing sufficient solvation power for PAHs. Moreover, the boiling temperature of ethyl acetate (77 °C) is considerably higher compared to the n-hexane/acetone mixture (∼50 °C) increasing its dissolving capacity for both analytes and water in the first step of the Soxtherm process.13 Since soil samples received at field moisture may show considerably high water content, the current extraction method for PAHs using ethyl acetate was applied to soil AS1, adjusted to various moisture levels ranging from 20 to 35%. Using one-way ANOVA, no significant difference of extraction efficiency due to the varying water content of the soil samples was observed for any of the PAHs at P ) 0.05 with n ) 3 (Table 1). As for the previous experiment, three-ring PAHs resulted in about 2-3 times the RSD of the high molecular weight PAHs (four rings and higher). In general, the lowest RSD were found for the soil with the highest moisture content (35%); however, differences were not significant for any of the PAHs analyzed based on Levene’s test of homogeneity at P ) 0.05. Effect of Crude Extract Cleanup. To avoid interference from the soil matrix with the analytical method, the U.S. EPA suggests (29) The Merck Index, 11th ed.; Merck & Co., Inc.: Rahway, NJ, 1989.
2382 Analytical Chemistry, Vol. 74, No. 10, May 15, 2002
fluoranthene
pyrene
benz[a]anthracene
chrysene
benzo[b]fluoranthene
benzo[k]fluoranthene
benzo[a]pyrene
benzo[ghi]perylene
indeno[1,2,3-cd]pyrene
dry massa meanb SEMb RSDa P valuec 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65 80 74 69 65
17.1 25.0 20.1 27.2 59.3 79.5 62.3 84.6 72.9 102 88.1 102 179 191 173 193 117 117 105 120 24.0 23.4 21.4 23.3 23.0 25.4 23.3 24.8 9.82 9.97 9.13 9.78 5.62 5.63 5.35 5.56 9.89 11.4 10.6 9.81 4.22 4.93 4.58 4.15 5.09 5.44 5.29 5.02
3.0 3.4 1.6 2.8 8.7 11 8.1 6.4 15 13 8.2 12 8.2 5.3 9.6 4.6 5.9 3.4 7.4 1.9 1.2 0.75 1.5 0.33 1.2 0.40 1.8 0.48 0.42 0.30 0.60 0.08 0.22 0.14 0.34 0.18 0.50 0.27 0.73 0.14 0.29 0.11 0.31 0.08 0.35 0.13 0.16 0.16
30 24 14 18 25 24 22 13 36 22 16 21 7.9 4.8 9.6 4.1 8.7 5.0 12 2.8 8.9 5.5 12 2.4 8.7 2.7 14 3.4 7.5 5.2 11 1.4 6.8 4.4 11 5.7 8.8 4.1 12 2.5 12 4.0 12 3.5 12 4.2 5.1 5.5
0.12
0.19
0.36
0.23
0.26
0.40
0.42
0.50
0.81
0.15
0.12
0.55
a Dry mass and relative standard deviation (RSD) in percent. Concentrations of mean (n ) 3) and standard error of the mean (SEM) in mg/kg dry mass. c Observed P value as a result of one-way ANOVA. Significance level set to P < 0.05. b
cleanup of crude extracts,24 selectively isolating target compounds of interest. Figure 3 shows two chromatographic traces (excitation 260 nm; emission 350 and 420 nm) from HPLC-3DFLD analysis of an ethyl acetate crude extract (2a/b) as well as a cleaned up 1:1 (v/v) n-hexane/acetone extract (1a/b), both from soil AS1. Ethyl acetate crude extract samples did not notably impact the chromatographic performance in terms of retention times shifts, effects on peak areas and shapes, the occurrence of ghost peaks or baseline elevations for any of the fluorescence traces even after multiple injections. In addition, spectra of PAH peaks from both pretreatment methods were compared to those of a library generated using a
Figure 3. Fluorescence traces (excitation 260 nm) from ethyl acetate crude extracts (2a, emission 350 nm; 2b, emission 420 nm) and from cleaned up and solvent-exchanged 1:1 (v/v) n-hexane/ acetone extracts (1a, emission 350 nm; 1b, emission 420 nm). Table 2. Comparison of Spectra Matches (Purity) for Different Pretreatment Methods compound fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
solventa
meanb
SEMb
P valuec
etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace
99.9 99.9 98.7 98.8 94.2 90.8 99.4 99.4 98.4 98.1 99.6 99.5 99.9 99.8 89.0 95.6 99.4 99.3 99.8 99.8 91.3 93.9 45.2 41.5
0.03 0.03 0.13 0.06 1.10 2.08 0.06 0.03 0.31 0.18 0.10 0.06 0.00 0.03 1.02 1.97 0.09 0.03 0.07 0.03 1.22 1.44 7.70 5.74
0.23 0.67 0.22 0.64 0.49 0.44 0.06 0.04 0.74 1.00 0.24 0.72
a Etac, ethyl acetate; hexace, 1:1 (v/v) n-hexane/acetone. b Spectra match and standard error of the mean (SEM) in percent. c Observed P value (two-tailed) as a result of Student’s t test. Numbers with statistical difference (n ) 3, P < 0.05) are in italics.
PAH standard mixture to evaluate the effect of extract cleanup on the peak purity of target analytes. Except for indeno[1,2,3-cd]pyrene, all PAHs exhibited excellent spectra matches of >89% for both pretreatment methods with no significant difference (n ) 3, P > 0.05) for any of the matches besides the one for benzo[b]fluoranthene (Table 2). Indeno[1,2,3-cd]pyrene, however, as a consequence of its generally poor peak shape (relatively strong fronting) only resulted in a 45 and 42% match, respectively, assuming superimposing/coeluting compounds. This was confirmed using GC/MS, with indeno[1,2,3-cd]pyrene yielding ∼73% of the concentration measured by HPLC-3DFLD. The findings above reveal that, even for complex matrixes such as soil, cleanup of crude extracts may be bypassed without
Figure 4. HPLC-3DFLD chromatograms (excitation 260 nm, emission 420 nm) of a PAH standard mixture in (a) pure acetonitrile, (b) 20% (v/v) ethyl acetate in acetonitrile, and (c) 20% (v/v) n-hexane/ acetone in acetonitrile. Peak labels correspond to (1) anthracene, (2) fluoranthene, (3) pyrene, (4) benz[a]anthracene, and (5) chrysene.
appreciable loss in analytical performance by applying the current ethyl acetate extraction method in combination with HPLC-3DFLD. Effect of Sample Solvent on the Chromatographic Performance. HPLC requires method-compatible sample solvents for reproducible analytical performances. Samples from ethyl acetate were simply diluted up to 20% (v/v) in acetonitrile with a notable influence on neither peak area and shape nor retention time compared to a standard solution of PAHs in pure acetonitrile (Figure 4a,b). Higher concentrations of ethyl acetate in acetonitrile (up to 60%) had no significant impact on peak area or on retention time; however, peaks broadened somewhat with increasing concentrations of ethyl acetate possibly leading to quantification difficulties in real samples. A 1:5 (v/v) sample dilution provided sufficient final concentrations for all PAHs and soils analyzed in this study. The less polar solvent mixture 1:1 (v/v) n-hexane/acetone only diluted 1:5 (v/v) in acetonitrile already resulted in significant retention time shifts for most of the analytes of interest (Figure 4c). Thus, straightforward allocation of PAHs especially with real samples was considerably restricted, making solvent exchange into acetonitrile inevitable for accurate analysis of chromatograms. Conventional versus Simplified Pretreatment Method. The simplified sample pretreatment method using ethyl acetate as extraction solvent was compared to the standard procedure (1:1 (v/v) n-hexane/acetone) including addition of sodium sulfate, aluminum oxide cleanup of crude extract, and solvent exchange into acetonitrile prior to instrumental analysis using HPLC-3DFLD covering three industrially PAH-contaminated soils (AS1, ES1, BS1) from different sources (Table 3). The obviously more laborious standard pretreatment method did not prove superior to the simplified ethyl acetate extraction in terms of PAH extraction efficiency for any of the PAHs and soils, respectively. The only significant difference (n ) 3, P < 0.05) was found for benzo[ghi]perylene in soil AS1 with the standard method resulting in ∼86% of the extraction efficiency of the simplified ethyl acetate extraction. Extraction of Certified Reference Soil CRM 524. Eventually, the simplified sample pretreatment method using ethyl acetate Analytical Chemistry, Vol. 74, No. 10, May 15, 2002
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Table 3. Standard (Hexace) versus Simplified (Etac) Pretreatment Method for the Analysis of PAHs in Soil soil ES1 compound fluorene phenanthrene anthracene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene benzo[ghi]perylene indeno[1,2,3- cd]pyrene
soil AS1
soil BS1
solventa
meanb
SEMb
P valuec
mean
SEM
P value
mean
SEM
P value
etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace etac hexace
90.8 80.0 335 303 316 334 393 402 314 345 199 208 212 225 149 163 81.4 91.0 152 149 84.7 99.6 128 148
11 5.4 36 19 64 33 38 26 21 23 17 14 22 16 13 9.9 4.4 5.1 14 10 7.1 5.8 4.3 9.6
0.42
90.6 79.8 235 235 145 109 200 216 127 135 23.8 26.0 25.1 28.1 9.83 10.7 5.40 6.17 10.7 10.3 5.00 4.29 5.72 6.25
5.0 3.3 15 7.7 10 7.0 6.4 3.1 4.6 1.8 0.6 0.5 0.9 1.0 0.3 0.3 0.1 0.3 0.3 0.5 0.1 0.1 0.2 0.1
0.15
6.19 5.36 50.8 40.5 20.5 18.4 64.2 57.8 56.6 52.9 41.1 33.8 34.8 30.8 35.6 31.1 21.4 19.1 38.5 32.6 30.2 26.5 43.5 36.5
0.2 0.5 1.5 4.3 0.5 2.0 1.4 5.5 2.3 5.5 0.8 3.3 0.4 2.7 1.1 2.8 0.4 1.4 1.1 3.5 1.3 1.6 0.8 3.2
0.17
0.48 0.82 0.85 0.37 0.70 0.67 0.45 0.23 0.84 0.18 0.13
1.00 0.05 0.09 0.18 0.06 0.09 0.11 0.09 0.50 0.02 0.09
0.09 0.37 0.33 0.57 0.10 0.27 0.21 0.18 0.18 0.15 0.10
a Etac, ethyl acetate; hexace, 1:1 (v/v) n-hexane/acetone. b Concentrations of mean and standard error of the mean (SEM) in mg/kg of dry mass. c Observed P value (two-tailed) as a result of Student’s t test. Numbers with statistical difference (n ) 3, P < 0.05) are in italics.
Table 4. Recoveries of PAH from Reference Soil CRM 524 Applying the Simplified Sample Pretreatment Method Using Ethyl Acetate as Extraction Solvent measured values
certified values
compound
meana
recoveryb
uncertaintya,c
meana
uncertaintya,c
pyrene benz[a]anthracene benzo[a]pyrene benzo[b]fluoranthene benzo[k]fluoranthene indeno[1,2,3-cd]pyrene
179 22.8 7.9 14.1 6.4 6.4
103 101 92 104 103 126
2.1 0.06 0.05 0.11 0.15 0.17
173 22.5 8.6 13.5 6.2 5.1
11 1.8 0.5 1.6 0.6 0.4
a Concentrations of mean and uncertainty in mg/kg of dry mass. b In percent of certified concentrations (n ) 4) as measured using the simplified ethyl acetate pretreatment method. c Half-width of the 95% confidence interval.
as extraction solvent was applied to the reference soil CRM 524 with results being compared to the certified concentration of six high molecular weight PAHs. Four of the six PAHs analyzed in this reference soil exhibited excellent recoveries ranging from 101 to 104% of the certified concentrations, all being within the 95% confidence interval (CI) of the certified concentrations (Table 4). Only benzo[a]pyrene yielded a lower recovery (92%), being slightly outside the CI. The latter is also true for indeno[1,2,3c,d]pyrene, with a recovery of 126%, a phenomenon that may be attributed to superimposing compounds as was observed to the same extend for soil AS1 when comparing results from GC/MS to HPLC-3DFLD (see above). Moreover, very low RSDs were found for PAHs in soil CRM 524 ranging from only 0.18 to 1.56%, which is most probably due to the high degree of homogenization of this reference material. These deviations were appreciably lower compared to those of the industrially contaminated soils (AS1, ES1, BS1), which were subjected to a significantly less intensive sample treatment prior to the extraction. 2384 Analytical Chemistry, Vol. 74, No. 10, May 15, 2002
CONCLUSION In general, the analysis of pollutants such as PAHs in complex solid matrixes such as soils or sediments requires a chain of sample treatments prior to the final analytical method. Since cost effectiveness and high sample throughput are of major concern to many routine laboratories, the present study has focused on the development of a convenient, cost- and time-efficient sample pretreatment method using automated Soxhlet extraction (Soxtherm) with ethyl acetate as extraction solvent. Laborious treatment steps such as the addition of a drying agent, sample cleanup, and solvent exchange for analytical compatibility could successfully be bypassed without significantly affecting the measurements. Moreover, ethyl acetate is by far less toxic compared to hexane, which may show central nervous system toxicity after chronic exposure and exhibits TLV-TWA values of ∼1 order of magnitude less than ethyl acetate.30 On the basis of the results of this study, (30) American Conference of Governmental Industrial Hygienists (ACGIH), TLVs and BEIs; ACGIH, Cincinnati, OH, 1998.
ethyl acetate has proved to be applicable for the extraction of aged PAHs from complex matrixes such as soils considering both economic efficiency and environmental and safety requirements.
wirtschaft’ administered by the Kommunalkredit Austria AG (Project A020006).
ACKNOWLEDGMENT This research project was enabled by funds of the ‘Bundesministerium fu¨r Land- und Forstwirtschaft, Umwelt und Wasser-
Received for review December 17, 2001. Accepted March 26, 2002. AC015739L
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