Anal. Chem. 2004, 76, 5503-5509
Dialysis of Persistent Organic Pollutants and Polycyclic Aromatic Hydrocarbons from Semipermeable Membranes. A Procedure Using an Accelerated Solvent Extraction Device K.-D. Wenzel,† B. Vrana,‡ A. Hubert,*,† and G. Schu 1u 1 rmann†
Department of Chemical Ecotoxicology, UFZ Centre for Environmental Research, Permoserstrasse 15, D-04318 Leipzig, Germany, and School of Biological Sciences, University of Portsmouth, King Henry Building, King Henry I Street, PO12DY Portsmouth, Hampshire, U.K.
Accelerated solvent extraction (ASE) is one of the most recent solid-phase extraction methods and has caught on all over the world in numerous laboratories. Until now it was not known that this device is also very suitable for performing dialysis. In this study, development of a rapid dialysis procedure (RDP) was described that is based on the dialysis of persistent organic xenobiotics from trioleincontaining semipermeable membrane devices (SPMDs) using ASE. All the operating parameters were optimized within the framework of usage. The RDP procedure was compared with the conventional dialytic recovery of target analytes under atmospheric pressure using spiked analytes and real field samples of SPMDs exposed to urban air. The main advantages of the RDP in comparison to the conventional dialysis are the speed, with up to 70 times faster taking only 40 min, and the considerable reduction in solvent consumption (by two-thirds) when SPMDs with standard configuration are used. Moreover, the RDP is also suitable as an analytical cleanup procedure for the same analytes from various types of lipid samples and other difficult matrixes using semipermeable membranes. In environmental monitoring, two areas of application exist with passive sampler technology and modern cleanup procedures using semipermeable membranes, which refer to the necessity of the realization of dialysis methods. Passive sampling in environmental research is getting increasing attention as an attractive alternative to expensive and more laborious long-term spot-sampling procedures.1-3 In passive sampler technology, triolein-containing semipermeable membrane devices (SPMDs) are preferably used as an innovative technique for detection and assessment of trace concentrations of semivolatile organic pollutants. Passive sampling is based on the free flow of analyte molecules from the sampled medium to a collecting medium as a result of differences in * Corresponding author: (e-mail)
[email protected]; (phone) ++49 341 235 2122; (fax) ++49 341 235 2401. † UFZ Centre for Environmental Research. ‡ University of Portsmouth. (1) Ballesta, P. P.; De Saeger, E.; Kotzias, D. Fresenius’ J. Anal. Chem. 1999, 8, 499-505. (2) Dulson, W. Fresenius’ J. Anal. Chem. 1999, 8, 531-535. (3) Chuang, J. C. Fresenius’ J. Anal. Chem. 1999, 8, 547-556. 10.1021/ac0494073 CCC: $27.50 Published on Web 08/17/2004
© 2004 American Chemical Society
chemical potentials.4 The sampling session is terminated by the user. The amount of analytes collected by the sampler depends on both its concentration in the sampled medium and the exposure time. Time-weighted average analyte concentrations in water and air can be determined easily, if the relationship between the sampling rate and analyte concentration is known.5-11 The determination of pollutants adsorbed on triolein within a semipermeable membrane requires the use of a dialysis method. From an analytical viewpoint, however, the long response time in the sample preparation of ∼48 h for the performance of the conventional dialysis procedure12 (U.S. patent 5,098,573)sthe technique’s used until nowsand the high solvent consumption needed to obtain the pollutant extracts are unfavorable.12-15 In an environmental laboratory, it is of importance to have a rapid method for this purpose to create the precondition for necessary routine operations. The second area of application of dialysis methods is the performance of cleanup procedures using semipermeable membranes. The method is intended for various types of lipid samples and other matrixes difficult to manage in traditional cleanup procedures. It can be used in the analysis of numerous persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs) of different concentrations, molecular shapes, and sizes, using polyethylene membranes with a pore size of ∼50 µm.16 (4) Gorecki, T.; Namiesnik, J. Trends Anal. Chem. 2002, 4, 276-291. (5) Brown, R. H. J. Environ. Monit. 2000, 2, 1-9. (6) Huckins, J. N.; Tubergen, M. W.; Manuweera, G. K. Chemosphere 1990, 20, 533-552. (7) Huckins, J. N.; Manuweera, G. K.; Petty, J. D.; Mackay, D.; Lebo, J. A. Environ. Sci. Technol. 1993, 27, 2489-2496. (8) Ockenden, W. A.; Prest, H. F.; Thomas, G. O.; Sweetman, A. J.; Jones, K. C. Environ. Sci. Technol. 1998, 32, 1538-1543. (9) Ockenden, W. A.; Sweetman, A. J.; Prest, H. F.; Steinnes, E.; Jones, K. C. Environ. Sci. Technol. 1998, 32, 2795-2803. (10) Rantalainen, A.-L.; Hyo¨tyla¨inen, T.; Saramo, M.; Niskanen, I. Toxicol. Environ. Chem. 1999, 68, 335-348. (11) Prest, H. F.; Huckins, J. N.; Petty, J. D.; Herve, S.; Paasivirta, J.; Heinonen, P. Mar. Pollut. Bull. 1995, 31, 306-312. (12) Huckins, J. N.; Tubergen, M. W.; Lebo, J. A.; Gale, R. W.; Schwartz, T. R. J. Assoc. Off. Anal. Chem. 1990, 73, 290-293. (13) Strandberg, B.; Bergquist, P.; Rappe, C. Anal. Chem. 1998, 70, 526-533. (14) Bergqvist, P. A.; Strandberg, B.; Rappe, C. Chemosphere 1999, 38, 933943. (15) Hess, P.; Wells, D. E. Analyst 2001, 126, 829-834. (16) Ahmed, F. E. Trends Anal. Chem. 2003, 22, 170-185.
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Although this method is simple and efficient and can dialyze more than 20 g of lipid material as a single sample, it requires a large volume of solvent at very long dialysis times of 40 h and more to be used.13 With the implementation of pressurized liquid extraction (PLE), a remarkable success has been achieved over the past few years regarding extraction techniques. One type of PLE is accelerated solvent extraction (ASE), which has been used internationally for about eight years.17-21 It has been certified by the U.S. Environmental Protection Agency (EPA), e.g., in Method 3545, for use on soils, which covers polychlorinated biphenyls (PCBs), PAHs, organochlorines, and organophosphorus pesticides.22,23 ASE is extraordinarily efficient when the operating variables including solvent, temperature between room temperature and 200 °C, pressure between 3.45 and 20 MPa, number of extraction steps, and extraction time have been optimized for a specific matrix and target analytes. Then ASE provides improved extraction quality for POPs including organochlorine pesticides such as lindane and further hexachlorocyclohexane isomers, p,p′DDT and its degradation products p,p′-DDE and p,p′-DDD as well as chlorobenzenes, PCBs, and PAHs from plants24,25 and soils25-27 as a result of increased solubility, reduced viscosity, and surface tension of the solvent at elevated temperature and pressure, thus better desorption and enhanced diffusion. As far as extraction procedures are concerned, aspects here include shortening extraction times, reducing the amounts of solvents used, and if possible to work solvent-free, automating procedures, ensuring good reproducibility, matrix-specific optimization, and simple handling. The possibility of varying extraction parameters opens up an opportunity to investigate whether a device originally constructed for accelerated extraction procedures can also be used for a rapid dialysis procedure (RDP). The criteria of a modern extraction procedure25,28,29 apply to the rapid dialysis of POPs and PAHs from SPMD membranes. They include the following: (a) fast dialysis, no longer than 30-60 min in total; (b) a reduced solvent consumption, no more than 90 mL in total for a 10-cm SPMD membrane; (c) the ability to fractionate the sample, e.g., at different temperature steps; (d) the automation of dialysis; and (e) a maximum dialysis efficiency in comparison with the conventional dialysis procedure. These criteria were treated as a basis for the development of a new dialysis methodsthe RDP. (17) Windal, I.; Miller, D. J.; De Pauw, E.; Hawthorne S. B. Anal. Chem. 2000, 72, 3916-3921. (18) Tomy G. T.; Stern, G. A. Anal. Chem. 1999, 71, 4860-4865. (19) Vandenburg, H. J.; Clifford A. A.; Bartle, K. D.; Zhu, S. A.; Caroll, J.; Newton, I. D.; Garden, L. M. Anal. Chem. 1998, 70, 1943-1948. (20) Richter, B. E.; Ezzell, J. L.; Knowles, D. E.; Ho ¨fler, F. Chemosphere 1997, 34, 975-987. (21) Preud’homme, H.; Potin-Gautier, M. Anal. Chem. 2003, 75, 6109-6118. (22) Fisher, J. A.; Scarlett, M. J.; Stott, A. D. Environ. Sci. Technol. 1997, 31, 1120-1127. (23) Po ¨rschmann, J.; Plugge, J.; Toth, R. J. Chromatogr. 2001, A909, 95-109. (24) Wenzel, K.-D.; Hubert, A.; Manz, M.; Weissflog, L.; Engewald, W.; Schu ¨u ¨ rmann, G. Anal. Chem. 1998, 70, 4827-4835. (25) Hubert, A.; Wenzel, K.-D.; Engewald, W.; Schu ¨u ¨ rmann, G. Rev. Anal. Chem. 2001, 20, 101-144. (26) Hubert, A.; Wenzel, K.-D.; Manz, M.; Weissflog, L.; Engewald, W.; Schu ¨u ¨ rmann G. Anal. Chem. 2000, 72, 1294-1300. (27) Popp, P.; Keil, P.; Mo¨der, M.; Paschke, A.; Thuss, U. J. Chromatogr. 1997, A774, 203-211. (28) Ho ¨fler, F.; Ezzell, J.; Richter, B. Labor Praxis 1995, 3, 62-67. (29) Ho ¨fler, F.; Ezzell, J.; Richter, B. Labor Praxis 1995, 3, 58-62.
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Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
EXPERIMENTAL SECTION Preparation of SPMDs. SPMDs with a standard configuration of 10 cm in length, containing 0.1 mL (0.092 g) of triolein in a membrane made of a 75-µm-thick low-density polyethylene with a surface area of 54 cm2, supplied by Environmental Sampling Technology (EST; St. Joseph, MO) were used for all the investigations described. SPMD was spiked with environmentally relevant analytes from the groups hexachlorocyclohexane isomers (HCHs) and chlorobenzenes as well as PCBs and PAHs. For spiking, a small cut was made at one end of the SPMD and 50 µL of a solution of test chemicals in n-hexane (0.02 µg µL-1) was injected into triolein phase using a HPLC syringe (volume 100 µL). The punctured SPMD was heat-sealed again using a Sealboy Neo-labfoil welding apparatus 2-1038 (Audion Elektro, Kleve, Germany). The internal fluid was homogenized by squeezing the SPMD content several times from one end to the other using latex gloves. Longer SPMDs up to the most often used size of 91 cm can be dialyzed in the 66 or 100 mL of extraction cells of the Dionex ASE 300 system since developed. For some months, we have also had at our disposal an ASE 300 device, and we could perform a dialysis of 91-cm SPMDs using a stainless steel mesh adapted to the size of a 100-mL cell. The results were comparable with 10cm SPMDs using the ASE 200. The required pressure of 3.45 MPa in the ASE 300 can be set by modification of the system firmware confirmed by personal communication with Dionex. The concentration range of the pollutants analyzed is between 10-9 and 10-12 g (ng up to pg of pollutant/g of matrix). Standards, Materials, and Solvents. The extracts were quantified using p,p′-DDT, p,p′-DDE, γ-HCH, the internal standard mixture containing a 13C-labeled chlorobenzene cocktail (EM-1725A), PCB 28 (EC-1413), PCB 153 (EC-1406), and the deuterated PAH Surrogate cocktail (ES-2044). These standards were supplied by Promochem (Wesel, Germany). The solvents n-hexane, acetone, methanol, dichloromethane, toluene, and diethyl ether used were of analytical grade (Merck Ltd., Darmstadt, Germany). Equipment. The following was used for RDP: (i) ASE 200 extractor (Dionex Corp., Sunnyvale, CA; (ii) ASE 300 extractor (Dionex Corp., Sunnyvale, CA). For size exclusion chromatography (SEC):30,31 (i) HPLC system with ASI-100 autosampler, P580A LPG low-pressure gradient pump, DAD UVD 340 S (Dionex Corp., Idstein, Germany); (ii) Foxy 200 fraction collector (Isco, Lincoln, NE). Columns for SEC cleanup: (i) Lichrogel PS 20, length 250 mm, i.d. 22.5 mm, particle size 10 µm used for spiked samples (Merck); (ii) Nucleogel GPC 50-10, length 250 mm, i.d. 25 mm, particle size 10 µm, exclusion limit 2 kDa used for real environmental samples (Macherey-Nagel, Du¨ren, Germany). For pollutant analysis:24-26 GC/MS system consisting of an HP6890 capillary column gas chromatograph and an HP5971 mass spectrometer fitted with an HP7673 autosampler (Agilent Technologies, Palo Alto, CA). Conventional Dialytic Method.12 The investigations were performed with 10-cm SPMDs containing 0.1 mL of triolein. After spiking with the target analytes, the SPMDs were dialyzed in the dark in glass cylinders covered with aluminum foil-lined lids. The analytes were then dialyzed from the SPMD for 24 h in 130 mL (30) Vrana, B.; Paschke, A.; Popp, P.; Schu ¨u ¨ rmann, G. Environ. Sci. Pollut. R 2001, 8, 27-34. (31) Hubert, A.; Popp, P.; Wenzel, K.-D.; Engewald, W.; Schu ¨u ¨ rmann, G. Anal. Bioanal. Chem. 2003, 376, 53-60.
of n-hexane as solvent. The solvent was replaced by a second portion of 130 mL of n-hexane and the dialysis was repeated. The two fractions in a total 260 mL were combined, the internal standards were added, and the solution was reduced by rotary evaporation to ∼2 mL and transferred to a sample vial. This extract, still containing ∼4-5% triolein, was concentrated under nitrogen to near-dryness and filled to 500 µL. SEC Cleanup11 for Conventional and RDP Method. The final extract of 500 µL was injected into the HPLC system. The mobile phase for the SEC was dichloromethane with a flow rate of 5 mL/min. The collected fraction containing the compounds of interest extended from 85 to 195 mL. The eluate from SEC was concentrated to ∼10 mL by rotary evaporation. To the concentrated eluate, 100 µL nonane was added as a keeper and the dichloromethane was evaporated using high-purity nitrogen. The residue was redissolved in n-hexane to 1 mL final volume, transferred to a vial, evaporated under nitrogen, and redissolved in 200 µL of toluene for a GC/MS analysis. Analysis of Organic Analytes from SPMDs. GC/MS analysis was carried out using an Agilent HP5971 mass spectrometer, coupled to a HP6890 capillary column gas chromatograph equipped with a programmed temperature vaporization inlet system. Analysis of target analytes was performed in SIM mode. A HP Ultra 2 capillary column, 25 m × 0.32 mm i.d. × 0.52 µm film thickness, was used. The carrier gas was helium. A 1-µL sample was injected in the splitless mode. GC temperature program conditions were 60 °C isothermal for 1 min, ramped at 10 °C min-1 to 260 °C, and then isothermal for 1 min. The sample was identified and quantified by matching each substance retention time with the retention time of an internal standard mix containing a 13C-labeled organochlorine mix and the deuterated Surrogate PAH cocktail ES-2044. Concentrations of the analytes were calculated based on the external and internal standards. The detection limit using 10cm SPMD samples was between 0.1 pg/0.1 mL of triolein for chlorobenzenes and 1 pg/0.1 mL of triolein for the other organochlorines and PAHs analyzed. The detection limit decreases with increasing volume of triolein, e.g., at the use of 91-cm SPMDs containing ∼1 g of triolein. There were no differences in the detection limits between the conventional method and RDP. In this case, some higher triolein portions are also in extracts after HPLC cleanup and can lead to overlapping of substance peaks in the GC/MS chromatogram. Recovery values according to the RDP method for chlorinated organics ranged from 88% for δ-HCH up to 100% for the other ones and for PAHs from 89% for benzo[a]pyrene up to 100% for the other ones. For the HCH group, using the conventional procedure the recovery rate was only between 68 and 88%. The RSD varied between 5.1 and 19% without significant differences between the both methods. Analysis of variance was done in order to find out whether the average values of the two dialysis methods in Tables 4 and 5 are significantly different. According to the data in Table 4, there is a good agreement of data obtained by two different dialysis methods. The mean recoveries obtained by the two methods were statistically compared using Student’s t-test at the level of significance p ) 0.05. No significant difference of the mean recoveries was observed for most compounds excepting PCB congeners 153 and 180. Also, in Table 5 there is in general a very
good agreement of data obtained by two different dialysis methods. The mean concentrations obtained by the two methods were statistically compared using Student’s t-test at the level of significance p ) 0.05 assuming unequal variance of both sample sets. No significant difference of the mean concentrations was observed for individual polycyclic aromatic hydrocarbons (excepting fluoranthene and benzo[e]pyrene), hexachlorocyclohexane isomers, and chlorobenzenes. The mean concentrations of PCBs differed significantly for all congeners with the exception of PCB 28; however, the concentrations determined were very low near the limit of quantitation. RDP Optimization. To demonstrate the universal applicability of a PLE device and to exploit the wide range of some of its operating parameters, we investigated whether ASE is also suitable for performing a rapid dialysis of persistent organic pollutants from semipermeable membranes. For this purpose, we decided to compare conventional dialysis6,7 with ASE dialysis using SPMDs spiked with a selected range of analytes. The studies were concluded by comparing conventional dialysis and the ASE dialysis method on a real environmental sample. The aims of the investigations were to optimize all the operating parameters of the ASE for a rapid dialysis method, to achieve at least the same recovery rates with distinctly shorter dialysis times, and to reduce solvent consumption. The results for pressure optimization at 22 °C were transmitted to higher temperature ranges and then accepted if the recovery rate was in the expected field, thus making it at least comparable with the conventional procedure. The next steps were planned in the following order. Effect of Extraction Pressure. The pressure range of the ASE device was between 3.45 and 20 MPa. It was very important to start by finding the suitable pressure conditions for dialysis depending on the device parameters. How does the pressure influence the membrane pores? The following studies on parameter optimization could then be performed at this pressure. Dialysis Efficiency Depending on the Ambient Medium in the Cell. The next step was to ascertain whether dialysis using a polyethylene membrane in a cell is possible. Did the ambient medium need to be changed and, if so, how? Effect of Temperature. Depending on the outcome of the first two points, the effect of temperature on the permeation of molecules through the membrane at a fixed optimized pressure was investigated. Any enlargement of the membrane pores potentially leads to the higher unintentional permeation of matrix constituents. Effect of Solvent Composition. We needed to examine whether the recovery rate of the analytes, especially of the HCH group, which had recovery rates of just 68-88%, could be further improved by adding a polar component such as acetone. HCH isomers with a partition coefficient of ∼3.9 were the most polar pollutants investigated in these studies. Effect of Time and Number of Dialysis Cycles. Once the influence of the individual parameters had been investigated (according to points 1-4) and the results proved positive, more technical questions needed to be addressed to conclude the studies. Dialysis Efficiency of the Optimized Rapid Procedure Using Spiked Analytes. The ASE dialysis procedure was Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
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Figure 1. Dialysis recovery depending on the dialysis pressure. Dialysis conditions: solvent, n-hexane; temperature, room temperature (22 °C); three 10-min static cycles.
developed in the laboratory using spiked analytes. The parameters were optimized in line with the possibilities of the ASE device and incorporated into a new dialysis procedure. Fivefold determinations were performed to determine the recovery rates and the RSD to enable comparison with the conventional method. Comparison between Conventional Dialysis and ASE Dialysis Using a Real Environmental Sample. The investigations were completed by comparing the conventional method and the newly developed dialysis method using real air samples. These samples were taken near a busy crossroads in the city of Leipzig to demonstrate the practicability of the method. RESULTS AND DISCUSSION Effect of Extraction Pressure. The extraction pressure of 10-15 MPa is important to keep the solvent or mixture of solvents used at temperatures higher than the boiling point in the liquid state required for extraction.28,29 It is reported that reducing the pressure to 10 MPa or raising it to 20 MPa does not affect the extraction efficiency.32 Therefore, most of the previous extraction processes using the ASE had been performed between 10 and 15 MPa. The influence of the pressure on dialysis efficiency was investigated at room temperature (22 °C) in the range 3.45-15 MPa using n-hexane as solvent and three 10-min static cycles. The results are listed in Figure 1. The distinct increase in dialysis efficiency with decreasing pressure is clearly apparent. The highest efficiency was attained at a pressure of 3.45 MPa. Since the device only works under elevated pressure, we decided to use the lowest adjustable pressure for dialysis. The initial pressure of the ASE 200 is 3.45 MPa, and so the following investigations were performed at this pressure. It is possible that a lower pressure could be even more favorable. However, another device would have to be developed that is used only for the realization from dialysis procedures. It is to be considered that at atmospheric pressure the dialysis time is 48 h. Dialysis Efficiency Depending on the Ambient Medium in the Cell. Initial dialysis attempts in a 33-mL ASE extraction (32) Saim, N.; Dean, J. R.; Abdullah, M. P.; Zakaria, Z. Anal. Chem. 1998, 70, 420-424.
5506 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
Figure 2. Stainless steel mesh including the SPMD; ASE cell including the mesh; closed ASE cell including the mesh with the SPMD (from left to right).
cell were conducted with the pollutant-spiked triolein in SPMDs without the addition of any special mechanical support matrix. In comparison to the conventional dialysis procedure, under these conditions, the recovery values were clearly lower. When the 33mL cell was opened, it was obvious that the SPMD was pressed up against the cell wall, preventing the complete solvent analyte infiltration of all sides into the SPMD. This needs to be avoided so that the solvent can access the surface of the membrane and the analytes can freely diffuse through the membrane to the solvent. In a second variant, the SPMD was embedded in sea sand in the cell, the temperature was varied between room temperature and 40 °C, and the pressure was maintained at the initial pressure of 3.45 MPa. The solvent used was n-hexane, which is given in the U.S. patent specification for the dialysis of pollutants such as organochlorines and PAHs from triolein-containing SPMDs.12 Although the dialysis time was 3 × 10 min, the recovery rate were only in the range of ∼50% or less in comparison to the conventional method. The sand prevents a sufficient solvent convection near the surface of the membrane, which is necessary for a rapid dialytic analyte partitioning between the solvent and the membrane. As a further possibility, we used a stainless steel mesh with the shape and size of the 33-mL cell (Figure 2). This ensures the optimal, reproducible fixing of the SPMD in the cell and that the SPMD does not come into contact with the inner wall of the cell. Even at room temperature, recovery rates were distinctly higher when a mesh was used in comparison to sand as ambient medium (Figure 3). Effect of Temperature. The recovery rate of the RDP generally increases with increasing temperature (Table 1). When n-hexane is used as the dialysis solvent, an increase in coextraction of triolein with increasing temperature from the SPMD is observed (coextraction of up to 23% of the total amount in the membrane at 60 °C), but it cannot be completely separated from the analytes by subsequent SEC cleanup. The presence of triolein in the dialysate interferes with the quantification of the target analytes using GC/MS and prevents a correct determination of the dialysis efficiency. Therefore, a maximum temperature of 50 °C is regarded as optimal for the dialysis. Effect of Solvent Composition. Favored solvents in dialysis are nonpolar organic solvents such as n-hexane and toluene or
Table 2. Recovery Values (in Percent) Depending on the Extraction Solventa solvent/solvent mixture n-hexane/acetone compd R-HCH β-HCH γ-HCH δ-HCH
Figure 3. Dialysis recovery depending on the surrounding matrix in the cell. Dialysis conditions: solvent, n-hexane; three 10-min static cycles. Table 1. Recovery Values (in Percent) Depending on Temperaturea
n-hexane
90:10
70:30
50:50
74 88 871 87
89 90 119 90
88 81 114 81
58 87 75 85
hexaCB
88
107
110
62
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180
82 135 101 107 106 120
113 105 99 98 98 99
113 101 95 94 94 91
84 79 85 91 90 91
PHEN ANT FLUOR PYR BaP
98 93 96 93 76
130 110 114 114 99
130 110 115 110 84
78 68 91 90 85
a Temperature is 50 °C. Pressure is 3.45 MPa. Three 10-min static cycles.
temp (°C) RTb (22)
40
50
60
R-HCH β-HCH γ-HCH δ-HCH
29 9 28 11
58 42 63 41
74 88 81 87
101 172 161 61
hexaCB
52
74
88
79
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180
42 35 34 35 36 34
63 68 66 67 67 65
82 135 101 107 106 120
74 82 82 83 83 85
PHEN ANT FLUOR PYR BaP
56 48 53 53 50
86 73 82 82 111
100 93 96 93 76
429 119 200 222 171
compd
a Solvent is n-hexane. Pressure is 3.45 MPa. Three 10-min static cycles. b RT, room temperature.
mixtures of well-miscible nonpolar and polar solvents as well as cyclohexane and ethyl acetate, cyclohexane and acetone, n-hexane and acetone, or dichlormethane and acetone. For the dialysis of POPs and PAHs, a solvent mixture of n-hexane and acetone in the ratio of 90:10 and also 70:30 or 50:50 (Table 2) proved useful in specific cases. Dialysis efficiency was found to slightly decrease again with an increasing proportion of acetone in the mixture, e.g., 30 or 50%. Yet, simultaneously the lipid content of triolein in dialysate was reduced from 4.5 (10% acetone) to 2.5 (30% acetone) and then 0.8% (50% acetone) of the total amount in SPMD. The best solvent mixture is n-hexane/acetone 90:10, but in the case of chromatogram interferences, a 70:30 or 50:50 mixture of n-hexane/acetone can be used. Effect of Time and Number of Dialysis Cycles. The efficiency of dialysis was tested performing 4 × 5 and 4 × 10 min dialysis cycles. The contingent at the total recovery rate was determined separately for each cycle (Table 3). It can be seen
Table 3. Time-Dependent Dialysis in Four Dialysis Stepsa dialysis time 4 × 5 min steps compd
4 × 10 min steps
1
2
3
4
1
2
3
4
R-HCH β-HCH γ-HCH δ-HCH
38.5 23.5 38.0 29.0
29.0 27.5 30.0 29.5
21.0 25.5 19.5 25.5
11.5 23.5 12.5 16.0
53.5 29.0 50.0 34.5
29.0 29.0 30.0 32.5
14.0 27.0 15.0 21.5
3.5 15.0 5.0 11.5
hexaCB
72.5
22.0
4.5
1.0
86.0
13.0
1.0
0
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180
60.5 45.5 44.0 43.0 44.5 40.5
27.5 31.0 30.0 28.5 29.0 29.5
9.5 16.0 18.0 19.0 18.0 19.0
2.5 7.5 8.0 9.5 8.5 11.0
75.0 63.0 59.0 54.0 56.0 53.0
20.0 24.5 26.5 28.0 25.5 27.5
5.0 9.0 11.5 13.0 13.0 14.5
0 3.5 3.0 5.0 5.5 5.0
PHEN ANT FLUOR PYR BaP
69.5 77.5 69.0 71.5 80.5
20.0 18.5 22.5 21.5 19.5
6.5 3.5 6.5 5.5 0
4.0 0.5 2.0 4.5 0
78.5 88.0 81.0 82.5 94.5
14.0 11.0 15.5 14.5 5.5
4.5 1.0 2.5 2.5 0
3.0 0 1.0 0.5 0
a Average recovery values are based on double determinations. Recovery values per dialysis step in percent refer to total recovery of 100%. Temperature is 50 °C. Pressure is 3.45 MPa. Solvent is n-hexane/ acetone 90:10.
that the recovery values using the 4 × 10 min procedure are excellent for most analytes. In the fourth cycle, the percentage of the analyte concentrations at total recovery is distinctly lower in comparison to 4 × 5 min. Only for δ- and β-HCH is the recovery of the fourth cycle somewhat higher (11.5 and 15.0%). A fifth cycle could under certain circumstances lead to a further improvement in recovery regarding the HCH group. However, the total recovery of 88-100% (Table 4) is certainly sufficient and far better than the recovery efficiency of the conventional procedure. Dialysis Efficiency of the Optimized Procedure Using Spiked Analytes. The investigations resulted in the following Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
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The results of 5-fold determination using the optimized RDP conditions are listed in Table 4. The average recovery values for the HCH group were between 88% for δ-HCH and 100% for γ-HCH, >100% for hexachlorobenzene, ∼100% for the PCBs, and for the PAH group between 89% for benzo[a]pyrene and 123% for phenanthrene. Using the RDP method, the RSD values depending on the analytes (∼5 up to 19%) are acceptable and also comparable to the conventional procedure. The recovery for the HCH group is higher in comparison to the conventional procedure (between 68% for β-HCH and 86% for δ-HCH) using a mixture of n-hexane/ acetone, 90:10, as dialysis solvent instead of only n-hexane. Comparison between Conventional Dialysis and RDP Using a Real Environmental Sample. To examine practicability, a comparison was carried out between the traditional method and RDP using real air samples. Six 10-cm SPMDs manufactured by EST and filled with 0.1 mL of triolein were exposed in exposure boxes for 14 days near a busy crossroads in the city of Leipzig, Germany. Triple determinations were performed for each dialysis method. The results of the analysis including 11 organochlorines and 20 PAHs are listed inTables 5 and 6. It can be seen that the RDP method is quite comparable to conventional dialysis in terms of the measured values, the recovery rate, and the RSD. Consequently, the optimized RDP method with its multiple advantages such as short dialysis time and low solvent consumption provides a very good alternative to the conventional method. Reasons for a RDP Using an Accelerated Solvent Device. The patented conventional method was performed at atmospheric pressure and room temperature. Temperature rises or pressure changes may also increase the permeability of the polyethylene membrane for large molecules, influencing the permeation of analytes as well as coextraction of lipids or other undesired matrix constituents. However, in the conventional method, a long time
Table 4. Recovery Values of the Conventional Dialysis Procedure and of the Optimized Rapid Dialysis Procedure (RDP) recovery (%) dialysisa
RDPb
av value
RSD
1
2
3
4
5
av value
RSD
R-HCH β-HCH γ-HCH δ-HCH
82 68 75 86
10.8 8.7 11.4 11.6
85 83 85 77
90 95 94 91
96 96 97 80
89 90 119 90
103 95 103 102
93 92 100 88
10.8 12.0 19.0 13.6
hexaCB
103
7.8
109
120
106
107
89
106
16.0
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180
118 113 119 123 145 120
2.9 1.8 4.5 5.4 1.7 5.9
112 105 104 99 98 93
122 118 112 110 104 102
110 107 95 97 100 98
113 105 99 98 98 99
87 93 111 102 95 97
109 106 104 101 99 98
11.9 12.3 8.7 8.9 5.1 5.1
PHEN ANT FLUOR PYR BaP
137 95 129 120 79
8.5 16.8 20.2 11.4 13.3
123 100 118 116 82
134 92 127 124 84
121 87 116 112 85
130 110 114 114 99
105 95 100 90 93
123 97 115 111 89
14.6 13.4 13.0 18.9 11.2
compd
a Conventional dialysis procedure. Average recovery values are based on three determinations. Dialysis time is 2 × 24 h. Solvent is n-hexane. b Average recovery values are based on five determinations. Temperature is 50 °C. Pressure is 3.45 MPa. Solvent is n-hexane/ acetone 90:10. Four 10-min static cycles.
optimized dialysis conditions: (i) insertion of an SPMD within a stainless steel mesh into a 33-mL cell of the ASE 200 (cf. Figure 2); (ii) adjusted pressure, 3.45 MPa; (iii) adjusted temperature, 50 °C; (iv) flush volume, 100%; (v) dialysis solvent mixture, n-hexane/acetone 90:10; (vi) dialysis time of 4 × 10 min.
Table 5. Comparison of PAH Concentrations between Conventional Dialysis Procedure and Rapid Dialysis Procedure Considered as an Example of Real Samplesa conventional dialysisb
RDPc
compd
1
2
3
av value
RSD (%)
1
2
3
av value
RSD (%)
acenaphthene fluorene phenanthrene anthracene 3-methylphenanthrene 2-methylphenanthrene 4-/9-methylphenanthrene 1-methylphenanthrene fluoranthene pyrene benz[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[e]pyrene benzo[a]pyrene perylene indeno[1,2,3]pyrene dibenz[a,h]anthracene benzo[ghi]perylene
4.58 20.5 115 5.78 14.3 14.6 9.95 7.63 34.8 14.3 1.22 3.36 0.72 0.57 1.14 0.42 0.10 1.36 0.20 1.53
5.28 25.0 122 5.26 13.5 14.2 9.26 7.16 28.6 8.64 0.89 2.85 0.77 0.57 1.02 0.42 0.12 1.48 0.20 1.48
3.94 17.7 120 5.30 15.4 16.3 10.5 8.27 34.2 9.51 1.10 3.52 0.90 0.68 1.11 0.41 0.12 1.54 0.22 1.58
4.60 21.1 119 5.45 14.4 15.0 9.89 7.68 32.5 10.8 1.07 3.24 0.80 0.61 1.09 0.42 0.11 1.46 0.21 1.53
14.7 17.3 3.1 5.3 6.7 7.3 6.1 7.3 10.4 28.0 15.4 10.7 11.8 10.4 5.8 0.7 7.0 6.2 5.1 3.3
4.35 17.9 134 5.75 16.0 16.5 11.4 8.55 38.7 13.9 0.99 3.17 0.63 0.54 0.78 0.46 0.14 1.00 0.16 1.05
4.68 17.5 118 6.03 15.5 16.2 10.4 8.26 39.0 14.6 1.13 3.11 0.59 0.61 0.80 0.37 0.08 1.04 0.16 1.05
3.09 14.5 122 4.43 15.0 15.3 9.82 7.94 37.8 12.9 1.10 3.49 0.55 0.47 0.81 0.37 0.08 0.84 0.13 1.00
4.04 16.6 124 5.40 15.5 16.0 10.5 8.25 38.5 13.8 1.07 3.26 0.59 0.54 0.79 0.40 0.10 0.96 0.15 1.03
20.7 11.0 6.6 15.8 3.2 4.0 7.4 3.8 1.5 6.2 6.8 6.3 7.0 13.7 1.6 13.2 35.7 11.0 10.0 2.5
∑ PAHs
252
249
252
251
276
259
251
262
0.7
4.8
a Standardized SPMDs of the company EST, 10 cm in length, filled with 0.1 mL of triolein. Values in ng/0.1 g of triolein. Exposure time is 14 d. b Dialysis time is 2 × 24 h. Solvent is n-hexane. c Temperature is 50 °C. Pressure is 3.45 MPa. Solvent is n-hexane/acetone 90:10. Dialysis time is 4 × 10 min.
5508 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
Table 6. Comparison of Organochlorine Concentrations between Conventional Dialysis Procedure and Rapid Dialysis Procedure Considered as an Example of Real Samplesa conventional dialysisb compd
av value
RSD (%)
12.1 nd nd nd
11.3
7.3
12.1
11.3
1
2
3
R-HCH β-HCH γ-HCH δ-HCH
11.4 nd nd nd
10.5 nd nd nd
∑ HCHs
11.4
10.5
1245tetraCB pentaCB hexaCB
0.07 0.13 0.87
0.09 0.20 1.15
RDPc
0.06 0.12 0.91
av value
RSD (%)
9.72 nd nd nd
10.3
12.6
9.72
10.3
1
2
3
9.47 nd nd nd
11.8 nd nd nd 11.8
7.3
9.47
0.07 0.15 0.98
19.3 26.5 15.6
0.04 0.09 0.91
0.07 0.15 0.95
0.06 0.13 0.98
0.06 0.12 0.95
12.6 27.1 20.5 3.4
∑ CB
1.07
1.44
1.09
1.20
17.0
1.04
1.17
1.17
1.13
6.0
PCB 28 PCB 52 PCB 101 PCB 138 PCB 153 PCB 180
0.25 0.42 1.40 0.51 0.64 0.06
0.25 0.39 1.18 0.46 0.57 0.06
0.24 0.40 1.33 0.50 0.60 0.06
0.25 0.40 1.30 0.49 0.60 0.06
3.3 3.7 8.6 5.3 5.3 6.0
0.38 0.65 1.86 0.82 1.06 0.09
0.27 0.54 1.63 0.71 1.01 0.07
0.34 0.75 1.97 1.03 1.26 0.10
0.33 0.65 1.82 0.85 1.11 0.09
17.6 15.8 9.5 18.6 11.8 19.0
∑ PCBs
3.28
2.91
3.13
3.10
5.6
4.86
4.23
5.45
4.85
12.4
a Standardized SPMDs of the company EST, 10 cm in length, filled with 0.1 mL of triolein. Values in ng/0.1 g of triolein. Exposure time is 14 d. nd, not detectable. b Dialysis time is 2 × 24 h. Solvent is n-hexane. c Temperature is 50 °C. Pressure is 3.45 MPa. Solvent is n-hexane/acetone 90:10. Dialysis time is 4 × 10 min.
period of ∼24 h is needed to reach the analyte partitioning equilibrium between the lipid phase in the interior of the membrane and the dialytic solvent. To increase the dialytic recovery, the solvent is refreshed and is dialyzed for 24 h to reach equilibrium again. This is a very simple procedure and is feasible in every laboratory. On the other hand, an increase in the dialysis temperature leads to faster permeation of the molecules through the semipermeable membrane. The recovery rates at 50 °C exceeded the values at 22 °C (room temperature) by factors of 2-3 (cf. Table 1). At 60 °C, increased membrane permeability caused an increased permeation of matrix constituents, too. That means that the dialysis time of the conventional procedure could be shortened at higher temperatures of 50 °C to about half. However, a closed system must be used in order to set up pressure and to avoid vaporization of the dialysis solvent. The problem can by solved by using a cell of an ASE device. This is a closed system, and the dialysis solvent can be automatically added and released. At a low pressure of 3.45 MPa, a series of short static cycles of 4 × 5, 10, or 15 min can be performed back to back in which the state of equilibrium can be reached far more rapidly than in the conventional procedure. The multiple repetitions of these cycles presumably ought to be the essential factor for acceleration of the dialysis. CONCLUSIONS The applicability of the ASE device for the dialysis of organic environmental pollutants from semipermeable membranes has
been demonstrated in this study. The operating variables of a Dionex ASE 200 device, pressure, temperature, solvent, number of dialysis cycles, and dialysis time were optimized for this purpose. To avoid the polyethylene membrane being pressed up against the cell wall, a stainless steel mesh adapted to a 33-mL cell of an ASE 200 for 10-30-cm length of semipermeable membranes or to a 100-mL cell of an ASE 300 for 50-91-cm length of membranes is required. The RDP developed greatly shortened the dialysis time from 48 h to 40 min and clearly reduced solvent consumption by two-thirds depending on the size of the membrane used. The RDP method was accepted for German (10219554) and international (PCT/EP03/04320) patents in 2003. ACKNOWLEDGMENT We also thank both Mrs. B. Mothes, Mrs. M. Heinrich (Dipl.Ing.), and U. Schro¨ter (Dipl.-Ing.) for their commitment and excellent technical assistance in carrying out the extensive investigations. This paper is dedicated to Prof. Dr. habil. Werner Engewald, University of Leipzig, on the occasion of his 65th birthday in 2002, in recognition of his significant contributions to analytical chemistry.
Received for review April 20, 2004. Accepted July 8, 2004. AC0494073
Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
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