Anal. Chem. 1997, 69, 831-836
Determination of Chlorinated Hydrocarbons in Marine Sediments at the Part-per-Trillion Level with Supercritical Fluid Extraction Dirk Sterzenbach,†,‡ Bernd W. Wenclawiak,*,‡ and Volker Weigelt†
Labor Su¨ lldorf, Bundesamt fu¨ r Seeschiffahrt und Hydrographie, Wu¨ stland 2, 22589 Hamburg, Germany, and Analytische Chemie I, Universita¨ t-GH-Siegen, Adolf-Reichwein-Strasse, 57068 Siegen
An off-line supercritical fluid extraction method has been developed for the determination of polychlorinated biphenyls and organochlorine pesticides at the part-pertrillion level in marine sediments. Four different extraction conditions were evaluated: pure carbon dioxide at 60 °C/220 atm and 100 °C/350 atm; addition of methanol prior to the static extraction at 80 °C/350 atm; addition of methanol prior to the static extraction step and continuously during the dynamic extraction step at 80 °C/ 350 atm. Only the latter condition gave extraction recoveries that were comparable and for some compounds higher than Soxhlet recoveries. The significance of differences between the two extraction methodes was determined in an F- and t-test. The method was validated by analysis of a reference material from an international intercomparison exercise. To determine the distribution, sources, pathways, and fate of hazardous compounds in the environment, it is necessary to detect even the smallest amounts of such compounds in the different compartments of an ecological system.1 For instance, concentrations of chlorinated hydrocarbons in North Sea sediments are usually in the range of a few nanograms or picograms per gram of sediment.2-4 Therefore, an analytical method must be developed in which the concentrations of contaminants inherent in the analytical procedure are much lower than the expected concentrations of the target analytes, which may be very difficult in the case of trace analysis. Supercritical fluid extraction (SFE) has become a technique equivalent to other sample preparation methods such as Soxhlet, sonication, or microwave-assisted extraction.5-9 The use of SFE was limited by the purity of even †
Bundesamt fu ¨ r Seeschiffahrt und Hydrographie. Universita¨t-GH-Siegen. (1) de Boer, J. In Analysis and biomonitoring of complex mixtures of persistent halogenated micro-contaminants; FEBO: Enschede, 1995; pp 11-15. (2) Lohse, J. Water Sci. Technol. 1991, 24, 107-113. (3) Klamer, H. J. C.; Fomsgaard, L. Mar. Pollut. Bull. 1993, 26, 201-206. (4) Bundesamt fu ¨ r Seeschiffahrt und Hydrographie, Annual Report; Hamburg, 1995. (5) Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1994, 66, 4005-4012. (6) Snyder, J. S.; Gropb, R. L.; McNally, M. E.; Oostdyk, T. S. Anal. Chem. 1992, 64, 1940-1946. (7) Lopez-Avila, V.; Young, R.; Benedicto, J.; Ho, P.; Kim, R.; Beckert, W. F. Anal. Chem. 1995, 67, 2096-2102. (8) van der Velde, E. G.; de Haan, W.; Liem, A. K. D. J. Chromatogr. 1992, 626, 135-143. (9) Bowadt, S.; Johannsen, B.; Wunderli, S.; Zenneg, M. F.; de Alencastro, L. F.; Grandjean, D. Anal. Chem. 1995, 76, 2424-2430.
the best commercially available carbon dioxide.10-12 Recently we optimized a commercial SFE device for the determination of chlorinated hydrocarbons (CHCs) at the ppt level.13 With this optimized SFE system, a method for the determination of polychlorinated biphenyls (PCBs; Ballschmitter Nos. 28, 52, 101, 105, 118, 138, 153, 156, 180, 185) and pesticides [hexachlorobenzene (HCB), p,p′-DDT and its metabolites p,p′-DDE and p,p′-DDD, and R- and γ-hexachlorohexane (HCH)] was developed. The method was validated by comparison with conventional Soxhlet extraction and by analysis of a reference material from an interlaboratory performance study. The choice of the chlorinated hydrocarbons referred to that of the performance study. EXPERIMENTAL SECTION Standards, Chemicals, and Solvents. All solvents (n-hexane, acetone, methanol, ethyl acetate) were of ultraresi-analyzed quality from Baker (Gross-Gerau, Germany); only the standards were prepared in nanograde isooctane (Promochem, Wesel, Germany). Pure standards (Promochem) of the CHCs were used for preparing standard solutions. The silica gel (particle size 0.063-0.200 mm) and the concentrated sulfuric acid (95-97%, analysis grade) were obtained from Merck (Darmstadt, Germany). Gas Chromatography. Extracts were analyzed by gas chromatography on an HT8 column (50 m × 0.22 mm i.d.; film thickness 0.25 µm; Scientific Glass Engineering, Weiterstadt, Germany) installed in a HP 5890 gas chromatograph (HewlettPackard, Hamburg, Germany) equipped with electron capture detector, split/splitless injection port, and an autosampler. The extracts of the reference material were additionally analyzed on a second gas chromatograph (HP 5890 Series II with on-column injector), where a column of different polarity (DB5, 50 m × 0.32 mm i.d.; film thickness 0.25 µm; J & W, Folsom CA) was installed in order to achieve coelution-free performance of each analyte.14-17 Use of two columns of different polarity provides a higher
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S0003-2700(96)00758-5 CCC: $14.00
© 1997 American Chemical Society
(10) Nielen, M. W. F.; Sta¨b, J. A.; Lingeman, H.; Brinkman, U. A. Th. Chromatographia 1991, 32, 543-545. (11) Wallace, J. C.; Krieger, M. S.; Hites, R. A. Anal. Chem. 1992, 64, 26552656. (12) Luque de Castro, M. D.; Valca´rcel, M.; Tena, M. T. In Analytical Supercritical Fluid Extraction; Springer-Verlag: Berlin, Heidelberg, New York, 1994; pp 206-236. (13) Sterzenbach, D.; Wenclawiak, B. W.; Weigelt, V. Anal. Chem. 1997, 69, 965-967. (14) de Voogt, P.; Haglund, P.; Reutergardh, L. B.; de Wit, C.; Waern, F. Anal. Chem. 1994, 66, 305A-311A. (15) Larsen, B. R.; Riego, J. Int. J. Environ. Anal. Chem. 1990, 40, 59-64. (16) Bowadt, S.; Skejo-Andresen, H.; Montanarella, H.; Larsen, B. Int. J. Environ. Anal. Chem. 1994, 56, 87-107. (17) Smedes, F.; de Boer, J. Quim. Anal. 1994, 13, S100-S108.
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997 831
analytical correctness. Therefore, the prescription for the participants of the intercomparison study included the analyses of extracts with two chromatographic columns. For analysis of the SFE extracts, a linear calibration (four-level, 1, 5, 10, and 20 pg/ µL), internal standard method was used. The GC method for the Soxhlet extracts was similar, but the concentration range was different (four-level, 5, 10, 50, and 100 pg/µL). Cleanup Procedure. For the cleanup, a Champagne column (16 cm length, 6 mm i.d., Supelco, Deisenhofen, Germany) with two layers was used. First a plug of clean glass wool was placed into the tip of the column and then the column was prerinsed with a few milliliters of acetone and n-hexane. The first layer consisted of 1 g of silica gel that was precleaned in a Soxhlet apparatus with ethyl acetate and deactivated with 1.5% (by wt) bidistilled water. The second layer was a 1.5 g portion of a mixture of silica gel and concentrated sulfuric acid (50:50, w/w).18 (Caution! The silica powder, which may become airborne, is very corrosive. It should always be handled under a fume hood.) A small amount of anhydrous sodium sulfate was placed onto the top layer in order to exclude moisture from ambient air and solvents. The column was preeluted with 5 mL of the elution solvent, a mixture of n-hexane and dichloromethane (3:1, v/v). The volume of the SFE and Soxhlet extracts was reduced to ∼1 mL with a rotary evaporator and then transferred onto the cleanup column with a Pasteur pipet. For proper purification, it must be ensured that the concentrated extracts consist of pure n-hexane because even small residues of polar solvents perturb the cleanup. The analytes were washed into the column (3 × 1 mL) and totally eluted with a 10 mL portion of the solvent mixture. The same volumes of solvent were also used for rinsing the flask in which the extract was evaporated. This combination of the two layers gave a better purification of the extracts than a cleanup with liquid sulfuric acid or with a silica gel column. Use of the silica gel/ sulfuric acid mixture alone was not sufficient because the extract was still colored, probably by oxidation products from organic matter and sulfuric acid. The improved purification was indicated by better performance of the chromatogram, especially when extracts with a high organic content were cleaned. No losses of analytes caused by the cleanup procedure were observable when the recoveries of the analytes were determined. The purified extract was collected in a 25 mL Kuderna-Danish flask, and the solvent was evaporated with a rotary evaporator to a volume of ∼500 µL and to the final volume of 200 µL with a gentle stream of nitrogen. Soxhlet Extraction. The extraction thimbles (glass fiber) were baked at 450 °C in an oven for 24 h. The 100 mL Soxhlet apparatus including the extraction thimbles was precleaned by first using it empty with 200 mL of ethyl acetate and second with a mixture of 100 mL of acetone and 100 mL of n-hexane for several hours. Then 50 g of the sediment was extracted with a new volume of the latter mixture for 18-24 h. This solvent mixture is very often used for the determination of chlorinated hydrocarbons in marine sediments.19 Thus, it was the recommended solvent for participants of the interlaboraty study. Internal standard solution (50 µL) (-HCH, TCN, and PCB 185, each at 200 pg/µL) and ∼1 g of activated copper powder were added to the solvent flask prior to the extraction procedure. (18) Wells, D. E.; Echarri, I. Anal. Chim. Acta 1994, 286, 431-449. (19) Schantz, M. M.; Benner, B. A.; Hays, M. J.; Kelly, W. R.; Vocke, R. D.; Demiralp, R.; Greenberg, R. R.; Schiller, S. B.; Lauenstein, G. G.; Wise, S. A. Fresenius J. Anal. Chem. 1995, 352, 166-173.
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Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
SFE Equipment. An MPS 225 extraction system (Suprex GmbH, Duisburg, Germany) with 5.5-grade carbon dioxide (Messer Griesheim, Duisburg, Germany) was used for all experiments. Continuous addition of modifier was accomplished with a micro-HPLC pump (Latek P402, Eppelheim, Germany) and a low dead volume T-connector. The modifier was added to the supercritical fluid (SF) immediately before it flowed into the extraction cell. An HPLC valve (Latek HMV-P) downstream of the pump made it possible to rinse the outlet valve, the tubings, and the restrictor with the modifier solvent. This was very useful when concentrations of analytes differed significantly from one sample to another or when samples with high amounts of coextractable compounds had been extracted. In order to avoid sample carryover, this rinsing step was performed every time the SFE was interrupted (e.g., due to leaks) or when the sample character changed (usually after three extractions, because three parallel runs were performed in most cases). The SF flow rate was restricted by means of a heatable, variable restrictor from Isco (A. Semrau GmbH, Sprockho¨vel, Germany). Temperatures of the restrictor were kept at 60 °C (valve body) and 35-45 °C (needle probe). The analytes were trapped in n-hexane; the trapping device is described elsewhere.20 The scheme of the SFE system is shown in Figure 1. The internal standard solution [50 µL of -HCH, tetrachloronaphthalene (TCN), and PCB 185, each at 20 pg/µL] was added to the trapping solvent after SFE. SFE Development for the Marine Sediments. According to the literature, SFE of chlorinated hydrocarbons from soils and sediments has been performed under very different conditions. Some researchers reported successful SFE at relatively mild conditions without modifier, while others used modifiers or very high temperatures.21-24 Therefore, different conditions were compared in SFE of 10 g (8 mL extraction cell) of a freeze-dried mixture of sediments from the German Bight (estuaries of the river Elbe, Weser, Ems). For desulfurization, the sediment was mixed with 0.5 g of activated copper powder.23 After each extraction, two consecutive SFEs were performed, the second one (SFE II) under the same conditions as the first (SFE I) to check whether the extraction time had been long enough, and a third run was performed (SFE III) under more severe conditions (e.g., addition of modifier) to test whether the conditions had been sufficient for quantitative extraction. In all SFE experiments, the sediment was extracted statically for 15 min and dynamically for 30 min. Flow rate was 1-1.2 mL/min CO2 (SF), which led to a total volume of 30-35 mL. Ten grams of sediment containing 100 pg of a PCB per gram of dry sediment will result in a concentration of 5 pg/µL in a final volume of 200 µL. Thus, concentrations of the analytes between 1 and 20 pg/µL in the final extracts (200 µL) were expected from the SFE experiments. RESULTS AND DISCUSSION SFE of Marine Sediments. The results of SFE (n ) 1) of sediment from the German Bight under varying SFE conditions are shown in Table 1. For clearer representation and because (20) Wenclawiak, B. W.; Heemken, O. P.; Sterzenbach, D.; Schipke, J.; Theobald, N.; Weigelt, V. Anal. Chem. 1995, 67, 4577-4580. (21) David, F.; Verschuere, M.; Sandra, P. Fesenius J. Anal. Chem. 1992, 344, 479-485. (22) Lee, H.-B.; Peart, T. E. J. Chromatogr. 1994, 663, 87-95. (23) Bowadt, S.; Johannsson, B. Anal. Chem. 1994, 66, 667-673. (24) Tong, P.; Imagawa, T. Anal. Chim. Acta 1995, 310, 93-100.
Figure 1. Design of the SFE system: A, adsorption device; B, tubing coil; C, inlet valve; D, low dead volume T-connector; E, extraction cell; F, outlet valve; G, outlet filter; H, oven; I, restrictor; J, trapping device; K, HPLC valve; L, micro-HPLC pump. Pathway of the SF is indicated by the continuous lines. Table 1. Concentration of PCB 153 Extracted from a Marine Sediment Mixture under Different SFE Conditions addition of methanol conc of static dyn sum PCB 153 (pg/g) T P extr extr SFE I-III (°C) (atm) (mL) (mL/min) SFE I SFE II SFE III (pg/g) 60 100 80 80
220 350 350 350
1 1
0.1
123 192 217 269
11
143 99 8
266 291 236 269
this compound is quantified correctly without coelution on the HT8 capillary column, only the concentration of PCB 153 is shown. Concentrations of the other compounds showed a similar behavior. The second SFE was performed under the same conditions as the first, while the third extraction was performed under more severe conditions.25 For the extractions without modifier, the extraction conditions were rendered more severe by adding 1 mL of methanol to the sediment in the extraction cell, prior to the third SFE. The conditions for SFE where modifier had already been used were intensified by increasing the pressure and temperature to 400 atm and 100 °C, respectively, and by adding more methanol to the sediment. The results clearly demonstrate that only continuous addition of modifier allows quantitative extraction of chlorinated hydrocarbons. It must be mentioned that marine sediment is a highly variable matrix. Therefore, it is important to develop a “hard” method that ensures feasibility also for other samples of a different composition. A slightly “weaker” method especially developed for a particular sediment might fail (25) Hawthorne, S. B.; Miller, D. J.; Burford, M. D.; Langenfeld, J. L.; EckertTilotta, S.; Louie, P. K. J. Chromatogr. 1993, 642, 301-317.
Table 2. Results of Soxhlet Extraction of the Marine Sediment and Blank Value of the Method compd
conca (pg/g)
% RSD
blank valueb (pg/g)
R-HCH HCB γ-HCH PCB 28 PCB 52 PCB 101 DDE PCB 118 DDD PCB 153 PCB 105 DDT PCB 138 PCB 156 PCB 180
34.4 133.5 83.0 75.6 116.4 97.9 63.8 93.3 233.9 195.2 39.7 210.1 143.4 28.9 66.3
10.5 21.7 17.7 19.3 15.4 20.1 19.5 16.4 28.0 18.8 12.0 21.7 17.9 11.9 18.7
5.4 3.5 4.5 4.2 4.2 2.5 1.0 1.8 6.0 2.7 1.9 10.8 3.6 1.0 1.7
a
n, ) 6. b n ) 3.
the goal of quantitative extraction of the next, slightly different sample. Selectivity is lost anyway because coextraction of matrix compounds occurs each time modifiers are used in SFE. Cleanup procedures thus can be hardly avoided, especially in trace analysis. Comparison of SFE and Soxhlet Extraction. The same sediment was extracted with SFE under all conditions indicated above (three parallel runs). The Soxhlet extraction was performed six times. Three blank runs of Soxhlet extraction were also carried out. Table 2 shows the results with their relative standard deviation (RSD) of Soxhlet extraction and the blank values. The deviations are in an acceptable range, considering the low concentrations. Only the RSD of the p,p′-DDD was higher than the other values, which was due to one outlying observation (the concentration of p,p′-DDD determined in the fourth Soxhlet Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
833
Figure 2. Percent recoveries of the different SFE conditions relative to Soxhlet extraction. Error bars indicate the standard deviation. Table 3. F- and t-Values for the Comparison of SFE (n ) 3) and Soxhlet Extraction (n ) 6) compd
F
t
compd
F
t
R-HCH HCB γ-HCH PCB 28 PCB 52 PCB 101 DDE PCB 118
1.35 1.54 1.41 1.47 5.11 14.24 20.74 6.76
6.84 0.08 2.88 2.39 1.22 1.48 3.70 2.97
DDD PCB 153 PCB 105 DDT PCB 138 PCB 156 PCB 180
1.36 1.06 1.86 3.08 5.17 3.33 5.18
3.27 2.30 3.25 2.25 2.44 4.36 3.21
F (P ) 0.95; 3; 6) ) 8.94, f1 ) 3 and f2 ) 6 F (P ) 0.95; 6; 3) ) 4.76, f1 ) 6 and f2 ) 3 F (P ) 0.99; 3; 6) ) 27.91 F (P ) 0.99; 6; 3) ) 9.78 t (P ) 0.95; 7) ) 2.36, n1 ) 6; n2 ) 3 t (P ) 0.99; 7) ) 3.50, f1 ) 5; f2 ) 2; f ) 7
analysis was 367 pg/g). Figure 2 presents the results of the SFE experiments expressed as percent recovery relative to Soxhlet extraction. Error bars indicate the standard deviations. As expected, SFE with continuous addition of modifier gave the best recoveries, often much better than the Soxhlet results. These results must be interpreted carefully. To determine the analytical significance, optimized SFE and the Soxhlet extraction were compared in an F-test and t-test.26 The data and the F- and t-values for the probability of 95 and 99% are listed in Table 3. In (26) Doerffel, K. Statistik in der analytischen Chemie; Deutscher Verlag fu ¨r Grundstoffindustrie: Leipzig, 1990; pp 110-120.
834 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
comparison of the standard deviations (F-test), a significant difference based on the probability of 99% was found only for PCB 101 and p,p′-DDE. Thus, the reproducibility of SFE is at least comparable to, and probably better than, the Soxhlet performance, taking in account the fact that only 10 g of sediment was used for SFE but 50 g was used for the Soxhlet extraction. The higher recoveries had no statistical significance, which was confirmed in the t-test. Only for R-HCH, p,p′-DDE, and PCB 156 were the t-values higher than the value for the probability of 99% (P ) 0.99; F ) 7). Use of a t-test is not allowed for the comparison of mean values if the F-test showed a difference in reproducibility (p,p′DDE). PCB 156 and R-HCH were detected at concentrations of 42.7 and 51.1 pg/g, respectively. The blank value of the SFE method for R-HCH is ∼20 pg/g, and for PCB 156 ∼10 pg/g. The small difference between determined values and blank value concentrations allows no conclusions as to the significance of these results. Many more analyses of similarly low contaminated sediments would be needed to determine the difference between the two methods. For higher accuracy, the extracts of SFE and Soxhlet should be analyzed with the same GC method and, if possible, in the same GC sequence. An improved blank value could be achieved by (inert gas) distillation of the solvents prior to use.13 Another experiment was carried out to find out whether SFE would give higher extraction efficiencies for the marine sediment than the Soxhlet procedure. Sediment that had already been extracted by a Soxhlet was removed from the Soxhlet thimble, spread on cleaned aluminium foil, and dried for some hours in a
fume hood to remove the acetone/n-hexane mixture. A 10 g portion of this sediment was filled into an extraction cell and extracted again by the SFE method. The chromatogram still showed signals of target analytes. For example, PCB 153 was determined with a concentration of 58.3 pg/g. With the Soxhlet method, the same compound was found at a concentration of a 95.2 pg/g and with SFE at 154.5 pg/g. This experiment was only carried out once, so the result may have been coincidence, but for some other compounds the balance was similar (e.g., p,p′DDE, p,p′-DDE, and PCB 138). Since other analytes showed a large difference when that comparison was made and because more statistically significant data were not available, the results from this experiment could only be a vague indication that SFE may be the more complete extraction method for a low contaminated marine sediment. SFE of a Reference Material from an Intercalibration Exercise. A (new) analytical method can be validated by comparing it to other established methods or by analyzing a commercial reference material. Such materials for marine sediments are available from several institutions, e.g., the Community Bureau of Reference (BCR, Brussels, Belgium), the National Institute of Standards and Technology (NIST, Gaithersburg MD), and the National Research Council (NRC, Ontario, Canada). Unfortunately, these sediments contain chlorinated hydrocarbons at the ppb level or even higher. Besides, these reference materials are slightly artificial because they have been grinded, sieved, sterilized, and homogenized.27-29 Nevertheless, they are very useful in method testing, validation, and intercalibration projects.1,30-32 Our laboratory participates in the European QUASIMEME project.33,34 QUASIMEME stands for Quality Assurance of Information for Marine Environmental Monitoring in Europe. Part of this project includes a regular interlaboratory performance study of a marine sediment. Normally these sediments are contaminated in the ppb range, but in round 2 of the project, the sediments had low contamination levels (sediments QOR006MS and QOR007MS).35 The values obtained in three parallel extractions, analyzed on two different chromatographic columns, are shown in Table 4. As has been mentioned above, the lower value is the better one in terms of analytical correctness because a higher concentration of an analyte on one of two columns indicates coelution of the analyte peak with another compound. The assigned values, the mean values, and the relative standard deviations of six reference laboratories are presented as well. In QUASIMEME, the performance of a laboratory is accepted if the values determined are in a range of (25% of the (27) Schantz, M. M.; Benner, B. A.; Chesler, S. N.; Koster, B. J.; Hehn, K. E.; Stone, S. F.; Kelly, W. R.; Zeisler, R.; Wise, S. A. Fresenius J. Anal. Chem. 1990, 338, 501-514. (28) Rebbert, R. E.; Chesler, S. N.; Guenther, F. R.; Koster, B. J.; Parris, R. M.; Schantz, M. M.; Wise, S. A. Fresenius J. Anal. Chem. 1992, 342, 30-38. (29) Wise, S. A.; Schantz, M. M.; Benner, B. A., Jr.; Hays, M. J.; Schiller, S. S. Anal. Chem. 1995, 67, 1171-1178. (30) de Boer, J.; Duinker, J. C.; Calder, J. A.; der Meer, J. Van J. AOAC Int. 1992, 75, 1054-1062. (31) Alford-Stevens, A. L.; Budde, W. L.; Bellar, T. A. Anal. Chem. 1985, 57, 2452-2457. (32) de Boer, J.; van der Meer, J.; Reutergardh, L.; Calder, J. A. J. AOAC Int. 1994, 77, 1411-1422. (33) Wells, D. E. Mar. Pollut. Bull. 1994, 29, 143-145. (34) Bailey, S.; Wells, A. S.; Wells, D. E. Mar. Pollut. Bull. 1994, 29, 187213. (35) Aminot, A.; de Boer, J.; Cofino, W.; Kirkwood, D.; Pedersen; B.; Wells, D. E.; Bailey, S.; Keay, K. Report on the Laboratory Performance Studies 1994.
Table 4. Results of SFE of Two Reference Sediments from an Intercalibration Exercise Analyzed on Two Different Chromatographic Columnsa concentration (ng/g) column type HTB DB5
compd
result
assigned value
mean value
SDb
R-HCH HCB γ-HCH PCB 28 PCB 52 PCB 101 DDE PCB 118 DDD PCB 153 PCB 105 DDT PCB 138 PCB 156 PCB 180
0.05 0.12 0.04 0.14 0.10 0.18 0.09 0.08 0.15 0.46 0.21 0.38 0.25 0.06 0.29
(A) Sediment QOR006MS 0.06 0.05
