Silicone Membrane Equilibrator: Measuring Chemical Activity of

Jan 15, 2009 - University of Aarhus. , ‡ ... Current address: Department of Agricultural Sciences, Faculty of Life Sciences, University of Copenhage...
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Anal. Chem. 2009, 81, 1536–1542

Silicone Membrane Equilibrator: Measuring Chemical Activity of Nonpolar Chemicals with Poly(dimethylsiloxane) Microtubes Immersed Directly in Tissue and Lipids Philipp Mayer,*,† Lars Tora¨ng,†,§ Nadia Glæsner,†,⊥ and Jan Åke Jo¨nsson‡ Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, University of Aarhus, Frederiksborgvej 399, DK-4000 Roskilde, Denmark, and Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden The chemical activity of organic chemicals directs their diffusion and partitioning and is consequently crucial for their transport, distribution, and toxic effects. A silicone membrane equilibrator is introduced for measuring the chemical activity of nonpolar organic chemicals in lipidrich samples: (I) A 6 m poly(dimethylsiloxane) (PDMS) microtube (300 µm i.d., 640 µm o.d.) was placed in a sample, and a sample-PDMS equilibrium was reached within 10 min for 12 polycyclic aromatic hydrocarbons (PAHs) acting as model compounds. (II) A plug of 100 µL of methanol was pushed through the tube to equilibrate it with the PDMS and thus the sample. (III) This yielded an undiluted methanol extract that was injected into a high-performance liquid chromatograph (HPLC) with multiband fluorescence detection. Quantification limits expressed as unitless chemical activities ranged from 6 × 10-9 to 5 × 10-8, and relative standard deviations were from 6% to 19%. Chemical activities of PAHs in mussels from two polluted sites were measured between 10-7 and 10-5, and activity coefficients for PAHs in vegetable and fish oils hardly differed between oils. This method can be used for internal exposure measurements, for monitoring product safety/conformity, and process control. The method can also be applied to measure total analyte concentrations in lipid-rich samples and oils. Chemical activity and the closely related property of fugacity were suggested as measures of chemical behavior and affinity by Gilbert Newton Lewis in his now century-old formulation of chemical thermodynamics.1 Since then, they have been used to understand, predict, and describe many physicochemical phenomena. It has recently been proposed that the chemical activity of an organic pollutant is important for its bioaccumulation and * To whom correspondence should be addressed. E-mail: [email protected]. Phone: (+45) 4630 1881. Fax: (+45) 4630 1114. † University of Aarhus. ‡ Lund University. § Current address: Sun Chemical A/S, Københavnsvej 112, 4600 Køge, Denmark. ⊥ Current address: Department of Agricultural Sciences, Faculty of Life Sciences, University of Copenhagen, DK-1958 Copenhagen, Denmark. (1) Lewis, G. Proc. Am. Acad. Arts Sci. 1901, 37, 49–69.

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toxicity.2 For instance, diffusion across membranes and phase boundaries occurs spontaneously down gradients of chemical activity, and not concentrations. This fact is important, among other cases, for the distribution of lipophilic chemicals between sediment and sediment organisms3 and between adipose tissue and serum.4 Despite these promising perspectives, we found very few published analytical methods for measuring the chemical activity5-7 or fugacity8,9 of lipophilic organic chemicals in various types of samples. Chemical activity can be measured by equilibrium sampling techniques.5,6,10 Enrichment in a thin polymer is caused by molecular diffusion, which is driven by the chemical activity (i.e., chemical potential) of the analyte in the sample and results in a measurable concentration in the polymer.5,6,11,12 Chemical activity and fugacity are measured with such techniques if certain conditions are fulfilled. Equilibrium (i.e., equal chemical activity) between the sample and polymer must first be reached. Second, the analyte uptake into the polymer must not significantly deplete the sample, as this would reduce the chemical activity in the sample.13 Third, the measurement of the concentration in the polymer must not be affected by adsorption of the analyte or the matrix, and the sorptive properties of the polymer must not (2) Reichenberg, F.; Mayer, P. Environ. Toxicol. Chem. 2006, 25, 1239–1245. (3) Di Toro, D. M.; Zarba, C. S.; Hansen, D. J.; Berry, W. J.; Swartz, R. C.; Cowan, C. E.; Pavlou, S. P.; Allen, H. E.; Thomas, N. A.; Paquin, P. R. Environ. Toxicol. Chem. 1991, 10, 1541–1583. (4) Brown, J. F.; Lawton, R. W. Bull. Environ. Contam. Toxicol. 1984, 33, 277– 280. (5) Reichenberg, F.; Smedes, F.; Jo¨nsson, J. A.; Mayer, P. Chem. Cent. J. 2008, 2, 8. (6) Legind, C. H.; Karlson, U.; Burken, J.; Reichenberg, F.; Mayer, P. Anal. Chem. 2007, 79, 2869–2876. (7) Ossiander, L.; Reichenberg, F.; McLachlan, M. S.; Mayer, P. Chemosphere 2008, 71, 1502–1510. (8) Wilcockson, J. B.; Gobas, F. A. P. Environ. Sci. Technol. 2001, 35, 1425– 1431. (9) Golding, C. J.; Gobas, F.; Birch, G. F. Environ. Toxicol. Chem. 2007, 26, 829–836. (10) Mayer, P.; Tolls, J.; Hermens, J. L. M.; Mackay, D. Environ. Sci. Technol. 2003, 37, 184A–191A. (11) Go´recki, T.; Namiesnik, J. Trends Anal. Chem. 2002, 21, 276–291. (12) Kalua, C. M.; Boss, P. K. J. Chromatogr., A 2008, 1192, 25–35. (13) Vaes, W. H. J.; Urrestarazu Ramos, E.; Verhaar, H. J. M.; Seinen, W.; Hermens, J. L. M. Anal. Chem. 1996, 68, 4463–4467. 10.1021/ac802261z CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

Table 1. Activity Coefficients for 11 PAHs in Methanol and Four Fish and Vegetable Oilsa activity coefficient, γ [L/mol]

methanolb

fish oil

olive oil

rapeseed oil

sunflower oil

naphthalene acenapthene fluorene phenanthrene anthracene fluoranthene pyrene benzo[a]anthracene chrysene benzo[k]fluoranthene benzo[a]pyrene

0.6 2.0 1.1 1.7 2.2 2.7 1.8 4.6 7.8 8.9 8.4

0.21 ± 0.01 0.38 ± 0.01 0.23 ± 0.01 0.28 ± 0.02 0.22 ± 0.01 0.29 ± 0.02 0.17 ± 0.01 0.30 ± 0.03 0.48 ± 0.04 0.35 ± 0.03 0.32 ± 0.03

0.21 ± 0.01 0.39 ± 0.02 0.23 ± 0.01 0.26 ± 0.02 0.21 ± 0.02 0.27 ± 0.03 0.16 ± 0.01 0.28 ± 0.03 0.45 ± 0.06 0.34 ± 0.04 0.28 ± 0.03

0.21 ± 0.02 0.38 ± 0.03 0.23 ± 0.02 0.27 ± 0.03 0.21 ± 0.02 0.28 ± 0.03 0.17 ± 0.02 0.29 ± 0.03 0.48 ± 0.06 0.31 ± 0.04 0.31 ± 0.05

0.20 ± 0.03 0.37 ± 0.07 0.23 ± 0.04 0.27 ± 0.05 0.23 ± 0.04 0.30 ± 0.06 0.18 ± 0.03 0.33 ± 0.07 0.54 ± 0.11 0.37 ± 0.10 0.35 ± 0.07

a Standard deviations are shown as ± (n ) 15). Results for dibenzo[a,h]anthracene are not shown because of missing data for γMeOH. b Data from Reichenberg et al. (ref 5).

be affected by the matrix.5,7,14 This matrix-effect criterion is particularly important in complex sample matrixes with constituents that may adhere to the polymer surface or even enter the polymer. The present study provides a new analytical sampling approach to prevent or reduce matrix effects, because the actual measurement is not done on the polymer itself but rather on a solvent that was equilibrated on the back side of the polymer. Membrane extraction is used for the selective enrichment of target analytes, the elimination/suppression of interferences in final extracts, and the reduction of solvent consumption in the analytical laboratory.15 Membrane extraction methods have been reported for many different kinds of analytes in various aqueous sample matrixes.16 Equilibrium sampling through membranes (ESTM) has recently been introduced as an analytical approach that combines equilibrium sampling with the principles of membrane extraction,17,18 but it has so far been limited to aqueous sample matrixes and mainly provides an equilibrium sampling technique suited to polar substances and easily compatible with high-performance liquid chromatography (HPLC). The present study introduces a new ESTM technique whose principal aim is to facilitate the equilibrium sampling of lipophilic substances in lipid-rich complex matrixes such as animal tissue and different types of oils. These matrixes are particularly difficult to sample due to their high content of constituents that can bind or dissolve lipophilic substances and might then adhere to or even enter the polymer. A silicone membrane equilibrator method was developed, optimized, and applied to measure (1) the activity coefficients of polycyclic aromatic hydrocarbons (PAHs) in different types of vegetable oils and fish oil, (2) the partition ratios between the lipids and methanol/1-octanol, and (3) the chemical activity of PAHs in mussel tissue. WORKING PRINCIPLE This new analytical approach is based on a two-step equilibrium strategy. Step 1: a silicone microtube is placed in a sample to establish a thermodynamic equilibrium between the sample and the polymer through its outside surface. The phase ratios are set (14) Mayer, P.; Vaes, W. H. J.; Hermens, J. L. M. Anal. Chem. 2000, 72, 459– 464. (15) Jo ¨nsson, J. Å.; Mathiasson, L. J. Chromatogr., A 2001, 2000, 205–225. (16) Jo ¨nsson, J. Å.; Mathiasson, L. J. Sep. Sci. 2001, 24, 495–507. (17) Liu, J. F.; Jönsson, J. Å.; Mayer, P. Anal. Chem. 2005, 77, 4800–4809. (18) Romero, R.; Liu, J. F.; Mayer, P.; Jo ¨nsson, J. Å. Anal. Chem. 2005, 77, 7605–7611.

to ensure that the analyte concentration in the sample does not change during this process (Vsample/Vsilicone . Ksilicone, sample). It is known from thermodynamic theory that, at equilibrium, asilicone ) asample

(1)

where asample and asilicone are the chemical activities of the analyte in the donor sample and silicone membrane, respectively. We chose the pure (subcooled) liquid form of the chemical as the reference state at which the chemical activity was set to be 1.2,19 Step 2: a small solvent acceptor plug is pushed through the inside of the tube to establish a thermodynamic equilibrium between the silicone wall and the solvent. The solvent has to be selected with great care, the main criteria being limited absorption in the silicone tubing and compatibility with the instrumental analysis. The phase ratio between silicone and solvent are set to ensure that the analyte concentration in the silicone at the outlet does not change during this process. At equilibrium, the chemical activity of the target analyte in the acceptor equals that of the polymer. This two-step equilibration yields the transfer of the chemical activity of an analyte from the donor sample into a solvent acceptor: aacceptor ) asilicone ) asample

(2)

The chemical activity is proportional to the concentration via an activity coefficient (γacceptor): aacceptor ) γacceptorCacceptor

(3)

The concentration in the solvent acceptor (Cacceptor) is determined by appropriate instrumental analysis (e.g., GC or HPLC), and the activity coefficients (γacceptor) in methanol were taken from another study5 and are listed in Table 1. This method of equilibrating a solvent with a sample through a polymer membrane can be applied to analytical systems to measure (I) the chemical activity (asample ) Cacceptorγacceptor), (II) the chemical activity coefficient (γsample ) γacceptor(Cacceptor/ Csample), (III) partition ratios (Ksample, acceptor ) Csample/Cacceptor), or (IV) the concentration (Csample ) Cacceptor(γacceptor/γsample). (19) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry; John Wiley & Sons Inc.: New York, 1993.

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Very recently, Ooki and Yokouchi reported on a silicone membrane tube equilibrator for measuring partial pressures of volatile organic compounds in natural water.20 The analytical approach of the present study was developed independently and differs in its design, target analytes, measurement end point, and sample matrix. However, we decided to use a similar term, namely, “silicone membrane equilibrator”, because it is very descriptive and both methods are based on equilibrium sampling through silicone microtube membranes. MATERIALS AND METHODS Materials. Twelve PAHs were used as model compounds: naphthalene (>99%, Fluka, Buchs, Switzerland), acenaphthene (>99%, EGA-Chemie, Albuch, Germany), fluorene (>98%, Aldrich), phenanthrene (>98%, Aldrich), anthracene (>98%, Sigma), fluoranthene (>99%, Aldrich), pyrene (>99%, EGA-Chemie), benzo[a]anthracene (>99%, Aldrich), chrysene (>95%, Aldrich), benzo[k]fluoranthene (>98%, Aldrich), benzo[a]pyrene (>98%, Aldrich), dibenzo[a,h]anthracene (>97%, Aldrich). Methanol (>99.9%, Merck) and 1-octanol (>99.5%, Fluka) were used as solvents. The following vegetable and fish oils, all of them commercial products, were used as lipid samples: rapeseed oil (Associated Oil Packers, Belgium), sunflower oil (Associated Oil Packers, Belgium), olive oil (Pietro Coricelli spa, Spoleto, Italy), and purified cod-liver oil (Medic Team, Denmark). The thinnest available poly(dimethylsiloxane) (PDMS) microtubing from A-M Systems, Inc. (Carlsborg, WA) was used to optimize the kinetics of the system. A high surface-to-volume ratio minimizes the equilibration time for partitioning into the PDMS, and the thin wall minimizes the diffusion distance within the polymer.10,21 PDMS was chosen as the membrane material because it provides uniquely high permeability for lipophilic organics,22-25 has been successfully applied in numerous membrane extraction and (ab)sorptive enrichment techniques,5,26-31 and has proved to be suitable for diffusive sampling even in tissues.7,32,33 The microtubing was made of medical-grade and additive-free PDMS (i.d., 300 µm; wall thickness, 170 µm; density, 1.4 g/cm3; interior volume, 71 µL/m; PDMS volume, 251 µL/ m; mass of PDMS, 0.36 g/m). The tubing was precleaned twice in at least 100 mL methanol per gram of PDMS for 24 h just before use. (20) Ooki, A.; Yokouchi, Y. Environ. Sci. Technol. 2008, 42, 5706–5711. (21) ter Laak, T. L.; Busser, F. J. M.; Hermens, J. L. M. Anal. Chem. 2008, 80, 3859–3866. (22) Rusina, T. P.; Smedes, F.; Klanova, J.; Booij, K.; Holoubek, I. Chemosphere 2007, 68, 1344–1351. (23) Kwon, J. H.; Escher, B. I. Environ. Sci. Technol. 2008, 42, 1787–1793. (24) Mayer, P.; Fernqvist, M. M.; Christensen, P. S.; Karlson, U.; Trapp, S. Environ. Sci. Technol. 2007, 41, 6148–6155. (25) Flynn, G. L.; Yalkowsky, S. H. J. Pharm. Sci. 1972, 61, 838–852. (26) Brookes, P. R.; Livingston, A. G. J. Membr. Sci. 1995, 104, 119–137. (27) Cocchini, U.; Nicolella, C.; Livingston, A. G. J. Membr. Sci. 2002, 199, 85–99. (28) Baltussen, E.; Cramers, C. A.; Sandra, P. J. F. Anal. Bioanal. Chem. 2002, 373, 3–22. (29) Weidenhamer, J. J. Chem. Ecol. 2005, 31, 221–236. (30) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145–2148. (31) Doig, S. D.; Boam, A. T.; Livingston, A. G.; Stuckey, D. C. J. Membr. Sci. 1999, 154, 127–140. (32) Bicchi, C.; Cordero, C.; Liberto, E.; Rubiolo, P.; Sgorbini, B.; Sandra, P. J. Chromatogr., A 2007, 1148, 137–144. (33) Riazanskaia, S.; Blackburn, G.; Harker, M.; Taylor, D.; Thomas, C. L. P. Analyst 2008, 133, 1020–1027.

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Figure 1. Conceptual drawing of a silicone membrane equilibrator and the processes involved to achieve thermodynamic equilibrium between a donor sample and a small acceptor plug pushed through the inside of the PDMS tube: (1) external equilibrium between sample and PDMS; (2) internal equilibrium between PDMS and an acceptor plug; (3) equilibrium across the PDMS membrane. The thicker part of the tube indicates the solvent plug.

Analytical Procedure. Analyses of PAHs in methanol and 1-octanol extracts were carried out by HPLC-fluorescence detection (Agilent 1100 system with G1321A FLD Ex. 260 nm; Em. 350, 420, 440, and 500 nm). The separation column: “CP-Ecospher 4 PAH” (Varian Inc., Palo Alto, CA) was operated at 0.5 mL/min (28 °C, 20 µL injection); mobile phase, HPLC-grade methanol (Merck Darmstadt, Germany) and water (SUPER-Q treated, Millipore, MA), were used as the mobile phase: 50% MeOH at t ) 0-2 min, linear gradient 50-75% MeOH at t ) 2-7 min, linear gradient 75-100% MeOH at t ) 7-35 min, and 100% MeOH at t ) 35-48 min. Gas-tight glass vials with PTFE-lined screw caps were obtained from Supelco. The PAH concentrations in the extracts were quantified by a six-point external standard curve. The HPLC samples were stored at 4 °C and analyzed within 2 weeks after sampling. Signal integration was performed with HP Chemstation software (A.06.03, Agilent Technologies) and corrected by hand as required. Method. A conceptual drawing of the experimental system is shown in Figure 1. The silicone microtube was placed in a 30 mL glass vial with a PTFE-lined screw cap (Microlab Aarhus, Denmark). The donor solution (e.g., fish oil) was added to the vial, and the tube was connected to two needles (BD Micro-Fine syringes, o.d. 0.33 mm, 12.7 mm length, 1 mL, BD Consumer Healthcare) that were inserted through the cap. A microliter volume of methanol could then be pushed through one of the needles with air from a 20 mL syringe, and a methanol extract was harvested from the other needle. The processes involved in achieving equilibrium between the sample () donor) and the small solvent volume () acceptor) are also included in Figure 1. The kinetics of this system toward thermodynamic equilibrium is rate-limited by either (1) uptake from the sample into the PDMS membrane, (2) release from the PDMS membrane to the acceptor plug during its rather short residence time, or (3) diffusion across the PDMS membrane. The silicone membrane equilibrator method was developed and optimized in a number of experiments all performed at room temperature.

Initial Experiment. The rate limitations for the mass transfer kinetics were investigated with PDMS microtubes 1 and 6 m in length and 28 mL of a standard methanol solution containing a mixture of 12 PAHs at approximately 50 µg/L. The tubes were pre-equilibrated in the solution for 1 day. Two different test systems were compared in order to identify the rate-limiting step and possible optimization options: (A) The microtubes remained in the standard methanol solution while small solvent acceptor plugs were pushed through the tube. (B) The methanol solution was removed before the acceptor plugs were pushed through the tube. Five small acceptor plugs of 200 µL of methanol were pushed through the microtubes 5 min apart. The solvent was pushed with air from a 20 mL syringe, resulting in a residence time of 2-3 min, and the methanol extract was collected in small glass vials (100 µL, Microlab Aarhus). Triplicate samples were also collected from the standard methanol solution (donor) at the end of the experiment. Optimization of Acceptor-Plug Volume. The acceptor-plug volume must be sufficiently large to yield a methanol extract for the instrumental analysis and sufficiently small to avoid depletion of the PDMS in the microtube effluent. A 6 m microtube was immersed in purified cod-liver oil spiked with a mixture of PAHs to approximately 85 µg/L. After 1 day, the microtube was sampled five times with an acceptor plug of 50 µL of methanol while the tube remained in the cod-liver oil. The solvent residence time ranged from 1.5 to 9 min, and the system was allowed to re-equilibrate for 5 min between each sampling. The sampling was subsequently repeated with acceptor-plug volumes of 100 and 200 µL of methanol. Release Kinetics. The release kinetics of the PAHs from the PDMS membrane to the acceptor plug during its residence time in the tube was also investigated. Seven microtubes were closed by knots at both ends and then placed in 500 mL of methanol containing PAHs at approximately 140 µg/L. After 1 h, the microtubes were removed from the spiking solution and samples of this solution were taken for analysis. The tubes were washed briefly with demineralized water and transferred to a round-bottom flask that was rotated slowly for 2 days to establish thermodynamic equilibrium between all the tubes. One tube at a time was removed from the flask, the knots were cut off, and two acceptor plugs of 50 µL of methanol were passed through the tubing. The solvent residence time in the tubing was measured and ranged from the fastest practically possible time of 39 s up to 8 min. Sampling Kinetics. The kinetics of the partitioning from the sample and into the PDMS were investigated in a methanol donor solution with a mixture of PAHs spiked to approximately 50 µg/ L. After approximately 5, 10, 15, 20, and 25 min, plugs of 100 µL of methanol were pushed through the tubing. Triplicate samples were also collected from the donor sample at the end of the experiment. Such partitioning kinetics were also studied in other matrixes: (A) various oils spiked to known concentrations of PAHs; (B) directly in tissue samples of homogenized blue mussels (Mytilus edulis) with a lipid content of 10-20% dry weight. METHOD APPLICATION Activity Coefficients and Partition Ratios. Rapeseed oil, sunflower oil, olive oil, and purified cod-liver oil were used as lipid samples, and methanol and 1-octanol were used as reference

solutions. To measure the activity coefficients of the PAHs in the different lipids, a PDMS microtube was placed in the glass vial, connected to needles through the cap, and 28 mL of lipid samples with a known PAH concentration of 330 µg/L was added. Triplicate vials of each lipid sample were made together with a background sample not spiked with PAHs. After 1 and 21 h, three plugs, each of 100 µL of methanol, were pushed through the tubes. A small volume of 1-octanol was then pushed through the tubes to remove any traces of methanol and to prepare for a change of solvent. The tubes were sampled again after 2 weeks, this time with two acceptor volumes of 150 µL of 1-octanol. Chemical Activities of PAHs in Mussels. Blue mussels (M. edulis) from two locations with known moderate PAH pollution were used to investigate the possibilities of measuring the chemical activities of PAHs directly in the tissue. Mussel samples from an earlier study were available in a -18 °C freezer, where the mussels had been stored for 1-2 years. Mussels A had been collected in an inlet near an asphalt production site (Tarco nord, Nyborg), and mussels B were from a shallow inlet near an old dump site (Seden Strand, Odense). The mussel tissue was homogenized in a blender, and approximately 25 g was added to a glass vial together with a PDMS microtube. The tube was connected to two needles through the cap. After 1 and 7 days, two 100 µL plugs of methanol were pushed through the tube. Between day 1 and day 7 the mussel samples were stored in a refrigerator at 4 °C. RESULTS AND DISCUSSION Precleaning of Microtubes and Constant Donor Concentrations. Methanol plugs pushed through precleaned PDMS microtubes showed only trace amounts of PAHs (data not shown), confirming that the precleaning procedure was sufficient. PAH concentrations in the sample solutions (donor) remained constant, which was analytically confirmed over periods of up to 3 months. Loss processes, including photochemical degradation and evaporation, can thus be neglected. The only identified loss process concerned the transfer of small acceptor plugs through the microtubes. All experiments were designed and operated in such a way that only a very small fraction of the PAHs was removed with the acceptor solution. Choice of Solvent. The uptake of methanol, 1-octanol, and other solvents into the PDMS was determined gravimetrically by immersing PDMS microtubing in the appropriate solvent. At equilibrium, the PDMS absorbed only 1.5% w/w methanol, an amount that should not affect its sorptive properties.34 This limited swelling makes methanol an attractive acceptor solvent. For comparison, the PDMS absorbed 5.5% w/w of 1-octanol, 129% w/w of pentane, and 136% w/w of toluene at equilibrium. Such high sorption means that (most of) the solvent is lost to the silicone, and this would also affect the sorptive properties of the silicone. The purging of 100 µL of methanol through a 6 m PDMS microtube yielded a final extract of approximately 45 µL, meaning that more than 50% of the methanol remained in the tubing (surface film and absorption in the polymer). The purging of 200 µL of methanol yielded an extract of 106 µL. Higher losses were observed when 1-octanol was used as the solvent, making it necessary to increase the acceptor volume to 150 µL to obtain a (34) Gill, K.; Brown, W. A. Anal. Chem. 2002, 74, 1031–1037.

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Figure 2. Sampling a 1 and 6 m long PDMS microtube 5 min apart with a 200 µL plug of methanol in (A) a tube remaining in the standard methanol solution and (B) a tube pre-equilibrated in, and then removed from, this standard solution.

final extract of approximately 30 µL. A number of other solvents were also tested, including ethanol amine, dimethyl sulfoxide, isooctane, 1,4-dioxan, tetrahydrofuran, ethyl acetate, toluene, pentane, and cyclohexane. Unfortunately, none of these solvents could be used either because they were completely lost to the polymer or because their viscosity was too high. This is a significant limitation of the method, because typical gas chromatography (GC) solvents cannot be used directly. Length of Microtubing. The length of the tubes required to obtain thermodynamic equilibrium between the silicone tubing wall and the acceptor solution was studied. Methanol was used as the donor (spiked) as well as the acceptor so that the concentration ratio should be 1 at equilibrium. The acceptor concentration ratios in the 6 m tubes were generally close to 1 (concentration in acceptor 76-104% of donor except for dibenzo[a,h]anthracene (65%)), whereas they were lower in the 1 m tubes (Figure 2). The acceptor to donor concentration ratios slightly decreased with increasing hydrophobicity of the analytes, which we at present cannot explain. Upon repeated elution, the acceptor concentrations remained constant in the 6 m tubes that remained in the donor solution. In contrast, the acceptor concentrations decreased steadily with repeated elution of tubes that were taken out of the donor solution (Figure 2). This shows that partitioning of PAHs into the polymer, diffusion across the PDMS, and partitioning into the acceptor solvent were all fast. Acceptor-Plug Volume. A too large acceptor-plug volume would lead to depletion of the silicone tube, and the optimal volume was determined in PAH-spiked fish oil. PAH concentrations in the acceptor methanol solution were very similar for acceptor volumes of 50 and 100 µL; they increased on average by 9% (Figure 3). However, when the acceptor volume was increased to 200 µL, the concentration only reached approximately 80% of that for the smaller plug volumes, which indicates depletion. A plug volume of 100 µL of methanol was selected for all later experiments, because this was convenient and ensured a final sample plug large enough to be injected into the HPLC. When 1-octanol was used as the solvent, the acceptor volume was increased to 150 µL to obtain a final extract of approximately 30 µL. A control vial with octanol as the donor and acceptor resulted in acceptor concentrations between 72-104% of the donor value. This shows that 1-octanol can also be used as an acceptor solvent. However, its use is much more difficult from a practical point of view due to its higher viscosity, which results in increased 1540

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Figure 3. Concentration in acceptor solution after sampling PAHspiked fish oil (≈85 µg/L) with acceptor-plug volumes of 50, 100, and 200 µL of methanol.

Figure 4. Elution kinetic from pre-exposed PDMS microtubes to an acceptor of 50 µL of methanol fitted to a simple one-phase exponential function. No measurements in the kinetic regime were obtained, and the fitted curves are thus only suggestive. Data for longer residence times were constant between 80% and 120% of the donor concentration, and they were omitted from the graph.

backpressure and sometimes to high losses of 1-octanol to the silicone tube. Residence Time. Equilibrium partitioning between the PDMS tubing and the methanol acceptor was reached for all PAHs even at the shortest residence time of 39 s (Figure 4). All concentrations in the acceptor solution were in the range of 80-120% of the methanol donor. The results show that the residence time is not crucial for the analytical results, so there is no need to control it. Sampling Kinetics. The kinetics of the partitioning from donor sample to PDMS was investigated in experiments with varying contact times between the sample and PDMS tubing. A

Table 2. Chemical Activities of Selected PAHs in Blue Mussels (M. edulis) and Total Concentration Measured with Traditional Biota-Solvent Extraction and GC/MS Analyses (Ref 40) chemical activity (× 106) (unitless) compound

Figure 5. Uptake kinetics from a spiked methanol solution into a PDMS microtube were studied by varying the contact time (donor, tube) and then purging an acceptor plug of 100 µL of methanol. The uptake into the PDMS was studied by plotting the measured concentration in the acceptor plug against the contact time and then fitting the data to a simple exponential function.

contact time of less than 10 min was sufficient to obtain constant PAH concentrations in the acceptor methanol for all tested matrixes. The time profiles can be seen in Figure 5. These results demonstrate that the overall mass transfer, including partitioning from donor to PDMS tubing, diffusion through the PDMS, and partitioning into the solvent plug, was sufficient to transfer the chemical activity from donor to acceptor within less than 10 min. However, these results were obtained in lipid-rich matrixes with a high solubilizing capacity and a high permeability for the PAHs so that these kinetics should only be extrapolated to other matrixes with caution. However, similar kinetics can be expected in all kinds of lipids and fat, in solvents, and in tissues with a high fat content, whereas tissues with low fat content will have significantly slower uptake kinetics.35 Analytical Performance Data. The quantification limits of the PAHs were 0.2-3 µg/L (S/N ) 10) in the acceptor solution that was directly injected into the HPLC. They can be multiplied by the analyte-specific activity coefficients in methanol in order to obtain quantification limits in terms of chemical activities, which ranged from 6 × 10-9 for anthracene to 5 × 10-8 for chrysene. The relative standard deviations at a 50 µg/L level were 619% (n ) 11) for the various PAHs, including the deviation between the sampling devices and the HPLC analysis. The quantification limits can be compared with the chemical activity of 1 at the reference state, which is the pure compound in its (subcooled) liquid state.

fluorene phenanthrene anthracene fluoranthene pyrene benzo[a]anthracene chrysene benzo[k]fluoranthene benzo[a]pyrene dibenzo[a,h]anthracene ∑PAH

concentration (µg/kgww)

mussel A mussel B mussel A mussel B 0.32 5.80 1.02 12.42 5.16 6.86 11.57 2.44 1.03 n.d. 46.6

0.11 0.80 0.31 1.66 0.74 0.73 0.56 0.23 0.20 n.a. 5.3

20.2 176.6 123.5 308.5 93.5 129.3 230.1a 118.0b 43.4 9.9 1253

38.9 121.0 36.0 71.7 37.8 13.1 24.2a 14.7b 9.9 1.6 369

a Sum of chrysene and triphenylene. b Sum of benzo[k]fluoranthene and benzo[b]fluoranthene.

APPLICATION OF METHOD Chemical Activity. The chemical activities of 10 selected PAHs were measured directly in homogenized samples of blue mussels (M. edulis) (Table 2). Measurements from the two moderately polluted sites were significantly higher than the detection limits except for dibenzo[a,h]anthracene. Naphthalene and acenaphthene could not be quantified due to interference in the chromatograms and uncertainties in the peak assignment. The total concentrations of the PAHs are also reported for comparison. The chemical activities of the PAHs (10-7 to 10-5) as well as their sum were well below the level for baseline toxicity (10-2 to 10-1),2 which suggests their rather limited contribution

to the baseline toxic potential. Such measured chemical activities in the mussels can also be compared to those in the water column, the sediment, or predator fish in order to study and understand uptake routes, diffusive mass transfer direction, and bioaccumulation.5,7,36 Equilibrium partitioning and simple bioconcentration would be indicated by equal chemical activities, whereas differences would indicate the direction of diffusive mass transfer. Activity Coefficient. This method can be used to determine analyte-specific activity coefficients in the donor medium by using activity coefficients in the acceptor medium and the donor-toacceptor partition ratio as input parameters. The activity coefficients of the PAHs in the various lipids were determined in this manner and are listed in Table 1. PAH specific activity coefficients were very similar for all four oils with differences of only a few percent. This means rather constant partition ratios, lipid solubilities, and fugacity capacities for the tested different oils. The activity coefficients were also rather constant between the PAHs, since they only ranged from 0.2 to 0.5 L/mol. This are very low activity coefficients that are equivalent to subcooled liquid solubilities of 2-5 mol/L. The measured activity coefficients can be used to translate between chemical activities and lipid concentrations (Clipid ) Cacceptor(γacceptor/γlipid)). There are at least two important application areas: (1) Concentrations of lipophilic organic chemicals in the lipids of animal and human tissue are of great importance in environmental monitoring, bioaccumulation research, and (environmental) toxicology. (2) They can also be used for the product and process control of various types of oils such as vegetable oil and fish oil. The novelty of this approach is to measure the concentrations in oils without exhaustive extraction and without an extensive cleanup procedure for the extract. The acceptor solution of such applications can be injected into an HPLC for conventional analysis or be monitored on-line by fluorescence detection, diode array detectors, or even toxicity tests. This allows any unintended

(35) Trapp, S.; Cammarano, A.; Capri, E.; Reichenberg, F.; Mayer, P. Environ. Sci. Technol. 2007, 41, 3103–3108.

(36) Lohmann, R.; Burgess, R. M.; Cantwell, M. G.; Ryba, S. A.; MacFarlane, J. K.; Gschwend, P. M. Environ. Toxicol. Chem. 2004, 23, 2551–2562.

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Figure 6. Partition ratios between (A) methanol and (B) 1-octanol and four different fish and vegetable oils for 12 PAHs.

pulses of contaminants to be detected with almost no delay. In the process control of food or feed production, it is recommended to test the suitability of ethanol as an acceptor solvent in order to prevent methanol traces entering the production stream. Partition Ratios. The silicone membrane equilibrator facilitates the precise measurement of partition ratios between (partly) miscible phases, which is difficult to accomplish with most other methods. In the present study, the partition ratios between various types of oils and either methanol or 1-octanol were measured (Figure 6). The concentrations of the smaller PAHs were approximately 1-2 times greater in these oils than in 1-octanol, increasing to a factor of 10 for the largest PAHs (Figure 6B). Octanol is often used as a surrogate for how a chemical will distribute in biota.37 The results clearly show that the partitioning behaviors of lipids, 1-octanol and methanol are significantly different for the largest PAHs. These lipid-to-methanol partition ratios can be divided by each other in order to obtain lipid-to-lipid partition ratios for PAHs. These were in all cases very close to unity, which is in good agreement with a very recent study on polychlorinated biphenyls and organochlorine pesticides.38 Evaluation of these observations in a bioaccumulation context shows that equilibrium partitioning of neutral lipophilic environmental contaminants into the lipids of organisms from different trophic levels will be very similar.38 CONCLUSIONS AND PERSPECTIVES The silicone membrane equilibrator was successfully used to measure chemical activities and activity coefficients of PAHs in different types of vegetable oils, fish oil, and mussel tissue. It performed physical separation of the sample and solvent and allowed direct measurement of partition ratios between miscible phases. The liquid extract was obtained with little effort and no dilution and was ready for instrumental analyses. The method worked very well with methanol and is expected to be suited to other polar solvents, which makes it very useful for HPLC analysis, for instance. The present format could not be operated with nonpolar solvents, which makes it less suitable for GC analysis. The main perspectives of this approach are summarized in Table 3. They include measurements of internal exposure in animal tissue (e.g., (37) Mackay, D.; Fraser, A. Environ. Pollut. 2000, 110, 375–391. (38) Jahnke, A.; McLachlan, M. S.; Mayer, P. Chemosphere 2008, 73, 1575– 1581. (39) Lee, J. N.; Park, C.; Whitesides, G. M. Anal. Chem. 2003, 75, 6544–6554. (40) Pecseli, M.; Pritzl, G.; Thomsen, M.; Asmund, G.; Christensen, J. T. Polycyclic Aromat. Compd. 2002, 22, 689–702.

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Table 3. Silicone Membrane Equilibrator Can Be Used To Determine Chemical Activities (a), Activity Coefficients (γ), Partition Ratios, and Concentrations measurement end point

needed input data

examples of applications

asample

γMeOH

γsample

γMeOH

K1,2

none

Csample

none

exposure assessment, flux direction comparing matrixes, e.g., different lipids partition ratios between miscible liquids, e.g., lipid/octanol product and process control

toxicology), studying the partitioning properties of matrixes (e.g., bioaccumulation), determining the partitioning ratios between miscible phases (e.g., transport models), and finally the on-line monitoring of contaminants in lipid-rich products (e.g., product safety and process control). Additionally, total analyte concentrations in lipid-rich samples and oils can also be measured with this analytical principle. There are at least three different calibration strategies for this purpose: (1) The concentration in the acceptor extract is measured and then multiplied with the donor-to-acceptor partition ratio. (2) The method is applied to the sample matrix spiked to known concentrations, and these measurements are then used for external calibration. (3) Finally, a sample can also be measured and then remeasured after addition of an analyte spike (i.e., standard addition). The compatibility of methanol to PDMS-based microfluidic devices has recently been demonstrated,39 and such devices offer a very interesting, compact, and practical format for the proposed analytical concept. The PDMS part of the microfluidic device would then act as a passive sampling phase that could be “read” at intervals by passing a small acceptor solution through its microchannels. ACKNOWLEDGMENT We gratefully acknowledge the assistance of Margit Møller Fernqvist for her skilled HPLC analyses and Fredrik Reichenberg for his valuable comments and suggestions. The study was funded by MISTRA, the Swedish Foundation for Strategic Environmental Research, and the EU Commission (NOMIRACLE, GOCE-003956). Received for review December 3, 2008. AC802261Z

October

27,

2008.

Accepted