Novel Hydrophobicity Ruler Approach for Determining the Octanol

Xiang Q. Kong, Damian Shea, Wondwossen A. Gebreyes, and Xin-Rui Xia*. Department of Population .... Zhi Chen and Stephen G. Weber. Analytical Chemistr...
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Anal. Chem. 2005, 77, 1275-1281

Novel Hydrophobicity Ruler Approach for Determining the Octanol/Water Partition Coefficients of Very Hydrophobic Compounds via Their Polymer/Solvent Solution Distribution Coefficients Xiang Q. Kong,† Damian Shea,‡ Wondwossen A. Gebreyes,† and Xin-Rui Xia*,†

Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, and Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695

A novel hydrophobicity ruler approach for determining the octanol/water partition coefficients of very hydrophobic compounds is proposed, which is an indirect method that measures the polymer/solvent solution distribution coefficients (log Kp/s) of reference and unknown compounds. The log Kp/s values of the unknown compounds can be calibrated to their log Ko/w values via the correlation of the log Kp/s values of the reference compounds with their log Ko/w values. An organic solvent was used to increase the solubility of the very hydrophobic compounds in the aqueous solution, so that their concentrations and absorption amounts were high enough to be measured precisely. The solvent also reduced the hydrophobicity scale of the very hydrophobic compounds and controlled the amounts absorbed into the polymer phase, so that compounds spanning a very wide range of log Ko/w values could be measured in a single measurement and the coexisting compounds would not interfere each other. Poly(dimethylsiloxane) (PDMS), aqueous methanol solutions, and a series of 21 PCB (polychlorinated biphenyl) compounds were used to demonstrate the principle of the hydrophobicity ruler approach. The PCB compounds with known experimental log Ko/w values served as reference compounds, whereas the PCB compounds without known log Ko/w values were determined. The log Ko/w values determined for PCB126, PCB187, PCB197, PCB180, PCB170, and PCB195 were 6.94, 7.84, 8.33, 8.17, 7.92, and 8.49, respectively. The correlation of the log Kp/s values of the reference PCB compounds with their log Ko/w values was linear (log Ko/w ) 2.56 log Kp/s + 1.08, R2 ) 0.95). The hydrophobicity ruler approach is also a valuable tool for validating the experimental and theoretical log Ko/w values and identifying outliers in log Ko/w databases.

INTRODUCTION Hydrophobicity is one of the most important physiochemical parameters governing the transport, distribution, and fate of 10.1021/ac048847r CCC: $30.25 Published on Web 01/26/2005

© 2005 American Chemical Society

chemicals in the environment and in biological systems. The octanol/water partition coefficient (log Ko/w) is a quantitative measure of the hydrophobicity of organic chemicals. It is widely used in the development of environmental fate models,1,2 in the estimation of bioaccumulation in animals and plants,3,4 and in the prediction of toxicity and drug absorption.5 Great efforts have been made to develop new methodologies for the determination of the log Ko/w values of chemicals. However, the determination of reliable log Ko/w values is still challenging, particularly for very hydrophobic compounds.1,6 The traditional experimental method for determining log Ko/w is the shake-flask method, which is adapted as the standard OECD (Organisation for Economic Co-operation and Development) method.7 It has been used to determine log Ko/w values for various compounds in the range from -2 to 4. For compounds having higher hydrophobicity, the shake-flask method cannot be used because of the formation of octanol emulsions in water. To overcome the emulsion formation problem, a column generator technique8 and a slow-stirring method9 were developed. The slowstirring method can be used for the determination of very hydrophobic compounds (e.g., log Ko/w as high as 9). It prevents * Corresponding author. Tel.: (919)513-6884. Fax: (919)513-6358. E-mail: [email protected]. Address: Dr. Xin-Rui Xia, North Carolina State University, CCTRP, CVM, Campus Box 8401, 4700 Hillsborough St., Raleigh, NC 276068401. † Department of Population Health and Pathobiology. ‡ Department of Environmental and Molecular Toxicology. (1) Cohen, Y.; Tsai, W.; Chetty, S. L.; Mayer, G. J. Environ. Sci. Technol. 1990, 24, 1549. (2) Mackay, D. Multimedia Environmental Models: The Fugacity Approach; Lewis Publishers: Chelsea, MI, 1991. (3) Briggs, G. G.; Bromilov, R. H.; Evans, A. A. Pestic. Sci. 1982, 13, 495. (4) Veith, G. D.; Defoe, D. L.; Bergstedt, B. V. J. Fish. Res. Board Can. 1979, 36, 1040. (5) Calamari, D.; Vighi, M. Res. Environ. Toxicol. 1990, 4, 1. (6) Finizio, A.; Vighi, M.; Sandroni, D. Chemosphere 1997, 34, 131. (7) Guidelines for Testing Chemicals, Section 1sPhysical-Chemical Properties, 105 n-Octanol/Water Partition Coefficient; Organisation for Economic Cooperation and Development (OECD): Paris, 1981. (8) Woodburn, K. B.; Doucette, W. J.; Andren, A. W. Environ. Sci. Technol. 1984, 18, 457. (9) Brooke, D. N.; Dobbs, A. J.; Williams, N. Ecotoxicol. Environ. Saf. 1986, 11, 251.

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the formation of an emulsion in the water phase, which is the limiting factor of the shake-flask method. However, the slowstirring method is very labor-intensive and time-consuming; it requires the chemical of interest to reach partitioning equilibrium in a large volume of water and the quantitative measurement of the trace quantity of the chemical in the water phase. Of all experimental methods, the HPLC method has been the most rapid and least expensive method10 and has become the standard method of the OECD and the U.S. Environmental Protection Agency (U.S. EPA).11,12 The HPLC method is an indirect method in which the chemical of interest partitions between a nonpolar stationary phase (C18) and a polar mobile phase. The retention capacity factors of a series of reference compounds are calibrated with their known log Ko/w values. The log Ko/w value of a chemical of interest can be obtained from its capacity factor with the reference correlation. Although the HPLC method theoretically can be used for a wide hydrophobicity range, the reliable log Ko/w range of the HPLC method is 0-6, limiting its use for very hydrophobic compounds.11 Examining the principle of the HPLC method, we realized that the wide hydrophobicity range of the HPLC method is provided neither by the indirect approach nor by the C18 stationary phase, but by the increasing hydrophobicity of the mobile phase. For example, if the mobile phase is limited to water, the HPLC method cannot provide any wider hydrophobicity range than the shakeflask method. The hydrophobic compounds are eluted from the nonpolar C18 column only by increasing the hydrophobicity of the mobile phase. A solid-phase microextraction (SPME) method using poly(dimethylsiloxane) (PDMS) fiber has been used to determine log Ko/w values, but a very large discrepancy was observed for hydrophobic compounds.13 It was concluded that SPME cannot be used to determine log Ko/w values for very hydrophobic compounds.14 Our preliminary experiments with PDMS membranes demonstrated that the absorption amounts of very hydrophobic compounds, such as PCBs (polychlorinated biphenyls), could be differentiated in a methanol-water (50% v/v) solution even though it was difficult to determine PCB distribution coefficients in water solutions. We hypothesized that the capability of a PDMS membrane to differentiate very hydrophobic compounds in an organic solvent-water solution could be utilized to measure log Ko/w values of very hydrophobic compounds. In this paper, a novel hydrophobicity ruler approach is developed for the determination of octanol/water partition coefficients, particularly for very hydrophobic compounds. It is based on the correlation of the distribution coefficients of chemicals between a polymer phase and a solvent phase (log Kp/s) with their octanol/water partition coefficients (log Ko/w). An organic solvent is used to increase the solubility of the compounds to improve quantitative analysis. Taking PDMS and methanol as examples for the polymer and solvent, the basic procedures are as follows: (10) Garst, J. E.; Wilson, W. C. J. Pharm. Sci. 1984, 73, 1616. (11) Guidelines for the Testing of Chemicals, OECD 117; Organisation for Economic Co-operation and Development (OECD): Paris, 2000. (12) Product Properties Test Guidelines. OPPTS 830.7570; U.S. Environmental Protection Agency, U.S. Government printing Office: Washington, DC, 1996. (13) Arthur, C. L.; Pratt, D.; Motlaph, S.; Pawliszyn, J. J. High Resolut. Chromatogr. 1992, 15, 741. (14) Poerschmann, J.; Gorecki, T.; Kopinke, F. D. Environ. Sci. Technol. 2000, 34, 3824.

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Table 1. log Ko/w Values for PCBs Determined by the Hydrophobicity Ruler Method hydrophobicity ruler compd PCB8 PCB18 PCB28 PCB52 PCB44 PCS66 PCB101 PCB77 PCB118 PCB153 PCB105 PCB138 PCB126 PCB187 PCB128 PCB197 PCB180 PCB170 PCB195 PCB206 PCB209

log

Kp/sa

1.65 1.69 1.96 1.99 1.86 2.11 2.26 1.98 2.35 2.55 2.23 2.42 2.29 2.64 2.30 2.83 2.77 2.67 2.89 3.23 3.52

log

Ko/w*b

5.30 5.42 6.10 6.17 5.85 6.47 6.87 6.15 7.10 7.60 6.79 7.28 6.94 7.84 6.98 8.33 8.17 7.92 8.49 9.36 10.08

log

Ko/w**c

5.38 5.50 6.22 6.28 5.96 6.61 7.03 6.27 7.26 7.79 6.94 7.45 7.09 8.03 7.14 8.55 8.38 8.12 8.72 9.63 10.38

exptl log

Ko/wd

5.09 5.55 5.62 6.09 5.81 6.31 6.8 6.63 7.12 7.75 6.79 7.45 7.31 9.14 8.27

KowWin log Ko/we 5.05 5.69 5.69 6.34 6.34 6.34 6.98 6.34 6.98 7.62 6.98 7.62 8.27 7.62 8.27 8.27 8.91 9.56 10.2

a log K p/s values are distribution coefficients determined in 60% methanol. b log Ko/w* was obtained by the correlation of log Kp/s with experimental log Ko/w values. c log Ko/w** was obtained by the correlation of log Kp/s with KowWin log Ko/w values. d Experimental log Ko/w values were compiled from a handbook; the blanks indicated that experimental log Ko/w values were not available for these PCB compounds.16 The italic experimental log Ko/w value, 8.27, was considered as an outlier in the present hydrophobicity ruler approach. e The italic and underlined values indicate two sets of PCB groups having the same log Ko/w values in KowWin but different log Ko/w by the hydrophobicity ruler approach.

(i) A series of reference compounds with known log Ko/w values and the compounds of interest (unknowns) are prepared in a methanol-water solution. (ii) A PDMS membrane is immersed into this solution and allowed to reach absorption equilibrium with the reference and unknown compounds. (iii) The log Kp/s values for all compounds are determined from their equilibrium concentrations in the solution and the PDMS membrane. (iv) The log Kp/s values of the reference compounds are correlated with their known log Ko/w values, and the log Ko/w values of the unknown compounds are obtained from this correlation using measured log Kp/s values. The hydrophobicity ruler approach is demonstrated with a series of PCB compounds, where the compounds with known log Ko/w values served as reference compounds and the PCB compounds without known log Ko/w values are determined. The critical experimental factors, the advantages and limitations of the method, and its application in validation of experimental or theoretical data are discussed. EXPERIMENTAL SECTION Chemicals and Materials. A standard mixture of PCBs containing 21 compounds in acetone (Table 1) and phenanthrened10 (internal standard) were purchased from AccuStandard, Inc. (New Haven, CT). PDMS (silastic) sheets (1.5 mm in thickness) were purchased from Dow Corning Corp. (Hemlock, MI). All organic solvents were HPLC grade from Sigma-Aldrich (St. Louis, MO). Caution: PCB compounds are carcinogens or suspected

carcinogens. While their vapor pressures are not high, dermal contact must be avoided during experimental operations. Methanol vapor is toxic, especially to the eyes. All of the solution operations should be performed in a fume hood. A series of calibration solutions with concentrations from 0.01 to 10.0 µg/mL PCBs (each component) with 1.00 µg/mL phenanthrene-d10 internal standard was prepared from the PCB standard mixture. A stock solution containing 1.00 µg/mL PCBs (each component) in methanol was prepared from the PCB standard mixture. A series of aqueous working solutions having a PCB concentration of 20 ng/mL (each component) with different ratios of methanol, namely, 20, 30, 40, 50, 60, 70, 80, and 100% (v/v), were prepared from the stock solution. An acetone extraction solution containing 1.00 µg/mL phenanthrene-d10 was prepared from the internal standard solution. Preparation of PDMS Disks. PDMS disks were punched out from the PDMS sheet (1.5 mm in thickness) with a dermal biopsy punch (Miltex Instrument Company, Inc., Bethpage, NY). The punch was twisted gently to cut the disk having a uniform diameter equal to that of the punch (4.0 mm). The PDMS disks were placed in a Soxhlet thimble and extracted with 300 mL of acetone for 8 h. The PDMS disks were transferred into a glass vial and dried in a glassware oven at 120 °C for 2 h. The PDMS disks were cooled to ambient temperature and sealed in a glass vial until use. Extraction Solvent Selection. A PDMS disk was weighed in a 2-mL vial with a screw cap. The PDMS disk was transferred into another 2-mL vial containing 1.5 mL of pure solvent. The PDMS disk was sealed in the vial, placed on a rocking shaker (Lab-Line Instrument, Melrose Park, IL), and shaken at 200 rpm for 24 h at room temperature (23 °C). The PDMS disk was then removed from the solvent, dried briefly with a piece of filter paper, and reweighed in the same 2-mL vial with the same screw cap. Solvents with different polarity indexes15 were tested: hexane (0.0), toluene (2.3), methylene chloride (3.4), ethyl acetate (4.3), acetone (5.4), and methanol (6.6). Acetone was selected as the extraction solvent for its gas chromatography (GC) compatibility and low extent of absorption into the PDMS disk as discussed in the Results and Discussion section. Kinetic Absorption. Experiments on the absorption of PCBs by PDMS disks from methanol-water solutions were performed in 20-mL glass vials. Fifteen milliliters of the working solution with a given methanol ratio (e.g., 60% v/v) was measured into the 20mL glass vials. A solid stainless steel needle (2 in. long) was inserted through a predrilled hole in the vial cap and tightly held by the cap. The vial caps were lined with aluminum foil. A precisely weighed PDMS disk was pierced by the needle and held in the 20-mL glass vial. The absorption experiment was started by pushing down the solid needle to expose the PDMS disk to the PCB solution. Each vial containing a PDMS disk in the working solution was capped, rapidly placed on the rocking shaker, and shaken at 200 rpm at room temperature (23 °C). After a given absorption time period, the PDMS disk was removed from the solution and dried with a piece of filter paper to terminate the absorption process. The absorption time periods were 0.5, 1, 2, 4, 8, 16, and 24 h. The PDMS disk was then transferred into a 2-mL extraction vial with a conical bottom prefilled with 200 µL of

acetone extraction solution. The extraction vials containing the PDMS disks and acetone extraction solutions were sealed and placed on the rocking shaker at 200 rpm for 2 h. The PDMS disks were removed from the acetone extraction solutions, and the extracts were analyzed directly by GC/MS. Equilibrium Absorption. The equilibrium absorption was performed using the same procedures as the kinetic absorption experiment except that the absorption time was predetermined to ensure absorption equilibrium for all of the PCB components. Six PDMS disks were used to conduct the equilibrium absorption experiments in solutions with a given ratio of methanol to water. The absorption time was 48 h to ensure absorption equilibrium for all compounds. Desorption Kinetics and Efficiency. Twelve PDMS disks were used for desorption kinetics and efficiency experiments. Each PDMS disk was placed in 15 mL of 60% (v/v) methanol solution containing 20 ng/mL PCBs (each component) and shaken at 200 rpm for 24 h to preload the PDMS disk. The desorption kinetics experiments were performed by extracting the preloaded PDMS disks for 1, 5, 10, 20, 40, 60, and 120 min with 200 µL of acetone shaken at 200 rpm. After the first desorption, the PDMS disks were redesorbed with 200 µL of acetone shaken at 200 rpm for 1 h. Desorption efficiency was estimated by comparison to extractions of the preloaded PDMS disks (n ) 5) for multiple times using 200 µL of acetone shaken at 200 rpm for 1 h. GC/MS Analysis. Quantitative and qualitative analyses were performed on an Agilent 6890 gas chromatograph coupled with an Agilent 5973 mass-selective detector. The standard calibration solutions and the acetone extraction solutions were injected with an Agilent 7683 injector. The injection volume was 2 µL, and the injection port was maintained at 280 °C for sample vaporization. Separation was performed on a 30 m × 0.25 mm (i.d.) × 0.25 µm (df) HP-5MS capillary column (Agilent, Palo Alto, CA). The column oven was programmed as follows: the temperature of 100 °C was initially held for 0.5 min; ramped at 40 °C/min to 200 °C, at 6 °C/min to 250 °C, and at 30 °C/min to 300 °C; and then held at 300 °C for 5 min. An electronic pressure control was used to maintain a carrier gas flow of 1.00 mL/min helium. The selectedion-monitoring (SIM) mode was used for quantitative analysis, in which the 21 compounds were grouped according to their retention times and one or two character ions were monitored for each compound depending on the ion intensity produced by the compound. Phenanthrene-d10 was used as the internal standard for quantitative analysis. Data Analyses. The amounts of PCBs absorbed into the PDMS disks from the methanol-water solution were obtained by quantitative GC/MS analysis. When absorption equilibrium was achieved, the equilibrium absorption amount of a given compound was used to calculate its distribution coefficient between the PDMS disk and the methanol-water solution. The distribution coefficient, Kp/s, was calculated from the equilibrium concentrations in the PDMS disk (Cpe ) n°/Vp) and the solution (Cse ) Co - n°/Vs)

(15) Godfrey, N. B. CHEMTECH 1972, 359.

where Co is the initial concentration in the solution; n° is the

Kp/s )

Cpe n°Vs ) Cse Vp(VsCo - n°)

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Figure 1. Desorption profiles in acetone extraction solution. The PDMS disks were preloaded in 15 mL of 60% (v/v) methanol solution containing 20 ng/mL PCBs shaken at 200 rpm for 24 h. The desorption was performed with 200 µL of acetone extraction solution shaken at 200 rpm for given periods of time.

equilibrium absorption amount in the PDMS disk; and Vp and Vs are the volumes of the PDMS disk and the solution, respectively. The same volume of the methanol-water solution (Vs ) 15.00 mL) and initial concentration (Co ) 20 ng/mL PCBs) were used throughout the experiments. The weights of the PDMS disks were in the range of 19.1-20.2 mg. The absorption amount of each PDMS disk was calibrated to an equivalent of a 20.0-mg PDMS disk. The volume of the 20.0-mg PDMS disk was 18.8 µL from its geometry (1.5 mm thickness and 4.0 mm diameter). RESULTS AND DISCUSSION Extraction Solvent Selection. Quantitative desorption of the absorbed PCBs from PDMS disks was a critical step in the method development. PDMS is a hydrophobic material and has a strong affinity to very hydrophobic compounds such as PCBs. Therefore, a hydrophobic solvent is needed for quantitative desorption, but too much hydrophobicity could result in a large amount of solvent being absorbed by the PDMS disk and even swelling the PDMS disk. The ideal extraction solvent should have a high extraction strength but low absorption into the PDMS disk. Solvents with different polarity indexes15 were tested: hexane (0.0), toluene (2.3), methylene chloride (3.4), ethyl acetate (4.3), acetone (5.4), and methanol (6.6); the amounts absorbed into a 20.0-mg PDMS disk were 19.3, 25.1, 17.1, 16.1, 4.6, and 0.5 mg, respectively. The PDMS disks were swollen in hexane, toluene, methylene chloride, and ethyl acetate. Acetone was selected as the extraction solvent for its superior GC compatibility and relatively low absorption into PDMS. Desorption Kinetics. After the PDMS disks were preloaded in 60% methanol solutions, they were desorbed in 200 µL of acetone for different time periods. The desorption profiles for six PCBs, selected to represent the range of PCBs, are shown in Figure 1 and illustrate the rapid attainment of equilibrium; PCB8 reached equilibrium within 10 min; PCB44, PCB118, PCB126, and PCB180 within 20 min; and PCB209 within 1 h. To ensure 1278 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005

Figure 2. Desorption efficiency with acetone extraction solutions. The PDMS disks were preloaded in 15 mL of 60% (v/v) methanol solution containing 20 ng/mL PCBs shaken at 200 rpm for 24 h. The desorption was performed multiple times, each with 200 µL of acetone extraction solution shaken at 200 rpm for 1 h. The desorption efficiencies were about 96% in the first desorption; the PCBs remaining in the PDMS disks were fully recovered in the second desorption. PCB compounds were not detectable in the third desorption.

equilibrium desorption for all other experiments, a desorption time of 1 or 2 h was selected. Desorption Efficiency. The desorption efficiency was studied by multiple extraction of a PDMS disk. The PDMS disks were preloaded in 60% (v/v) methanol solutions containing 20 ng/mL PCBs (each component). The first desorption with 200 µL of acetone yielded 95-97% of the total amount of PCBs in the disk (Figure 2). The desorption efficiency decreased slightly with increasing hydrophobicity, from PCB8 (97%) to PCB209 (95%). The remaining 3-5% of PCBs in the PDMS disks was fully recovered in the second desorption. PCB compounds were not detectable in the third desorption. Therefore, the desorption amounts from the first and second desorptions were combined to yield the absorption amounts in the rest of the experiments. Absorption Profiles. In the present hydrophobicity ruler approach, the equilibrium concentrations in the solution and PDMS disk are required for calculating the distribution coefficient. The time to reach equilibrium for different compounds can be obtained from their absorption profiles. The absorption profiles for six PCBs are shown in Figure 3. PCB8 and PCB44 reached absorption equilibrium within 2 h in 60% (v/v) methanol solution. PCB118 and PCB 126 reached equilibrium within 4 h, and PCB180 and PCB209 reached absorption equilibrium in 8 and 16 h, respectively. Thus, the concentrations in the solution and PDMS disk determined after 16 h could be used for calculating their distribution coefficients. Methanol Effects. The main difficulty in the determination of octanol/water partition coefficients of very hydrophobic compounds using traditional methods is their extremely low solubility in water. Our approach uses a solvent added to water to increase the solubility of the very hydrophobic compounds to overcome this problem. However, octanol cannot be used as an organic sorptive phase because the added solvent will partition into the octanol phase. We hypothesized that a polymer such as PDMS could be substituted as the organic phase and allow determination

Figure 3. Absorption profiles of PCB compounds. Each PDMS disk was held on a stainless needle in 60% (v/v) methanol-water solution containing 20 ng/mL PCBs shaken at 200 rpm for given periods of time. Each PDMS disk was briefly dried with a piece of filter paper and transferred into an extraction vial filled with 200 µL of acetone extraction solution shaken at 200 rpm for 2 h.

of the distribution coefficients of very hydrophobic compounds between the polymer phase and the solvent solution. The organic solvent not only increased the solubility of the very hydrophobic compounds, but also reduced the distribution coefficients of the very hydrophobic compounds between the PDMS disk and the solution and, consequently, reduced the absorption amounts into the PDMS disk. These three effects of the solvent are critical factors in the proposed hydrophobicity ruler approach: (1) it increases the solubility of the very hydrophobic compounds, so that the concentrations and absorbed amounts can be measured precisely; (2) it reduces the hydrophobicity scale of the very hydrophobic compounds, so that compounds with a very wide log Ko/w range can be measured in a single measurement; and (3) it controls the absorbed amounts of the very hydrophobic compounds into the PDMS disk and reduces the potential for coexisting compounds to interfere with each other in the absorption process. These three solvent effects must be balanced for optimal results. If the solvent proportion is too high, the distribution coefficient could be very small, and consequently, the absorption amount into the PDMS disk could be too low to be accurately determined. Methanol is a common organic solvent with high polarity and was selected because the methanol/water ratio can be adjusted in a wide range to balance these three solvent effects. The solvent effects of methanol on PCB solubility were investigated to find the optimal methanol proportion. If the methanol proportion was higher than 50% (v/v), all of the PCB compounds at the concentration of 20 ng/mL were dissolved completely. If the methanol proportion was under 40% (v/v), some of the higher PCBs were not completely dissolved. Their absorption rates were lower because of undissolved proportions. Thus, the methanol concentration should be higher than 40% (v/v) to study the absorption equilibrium of the PCB compounds at the concentration of 20 ng/mL. The absorption profiles (Figure 3) for all of the compounds (reference and unknown) were obtained in 50% and 60% methanol

solutions. The equilibrium absorption amounts of all PCBs were differentiated and measured accurately. If the methanol amount was too high (e.g., 80%), the amounts absorbed into the PDMS disks were too similar to be differentiated for different compounds. For example, the amounts of PCB8 and PCB18 absorbed into a 20-mg PDMS disk were 2.2 and 3.5 ng, respectively. The amounts absorbed into the polymer phase can be controlled via proper selection of the solvent proportion in the solution. This allows many compounds coexisting in solution to be studied without interfering with each other. For example, the maximum absorption amount of PCB209 into a 20-mg PDMS disk was 241 ng in 15 mL of 60% methanol solution containing 20 ng/mL PCBs (each component), equivalent to 12 ppm in the PDMS disk by weight. Distribution Coefficients, log Kp/s. In consideration of the solubility, distribution coefficient, and absorption time, the aqueous solution with 60% (v/v) methanol was selected for determination of the distribution coefficients of the 21 PCB compounds. The absorption time was 48 h to ensure equilibrium absorption for all of the PCB compounds. The first desorption and second desorption were combined to yield the equilibrium absorption amount (n°) for each compound. The relative standard deviations of Kp/s values were less than 12% in the six replicated measurements. The logarithms of the distribution coefficients of the PCBs between the solution and the PDMS disk were in the range of 1-4. This log Kp/s range is similar to the optimal log Ko/w range for the shake-flask method. This is not a coincidence; it is the result of the strategy behind the present hydrophobicity ruler approach. It is in this range that the distribution coefficients (log Kp/s) can be determined experimentally with high accuracy. Correlation with Experimental log Ko/w Values. The present hydrophobicity ruler approach is not a direct method for the determination of the octanol/water partition coefficients. Instead, it is an indirect method similar to the HPLC method for log Ko/w estimation.10 When the log Ko/w values of a series of reference compounds are determined experimentally, the distribution coefficients (log Kp/s) of the reference compounds and the unknown compounds can be determined simultaneously in methanol-water solution. The log Ko/w values of the unknowns are then estimated using the measured log Kp/s values of the unknowns and the correlations between the log Kp/s values of the reference compounds and their log Ko/w values. Figure 4 shows the correlation between the log Kp/s values of the reference PCBs and their experimental log Ko/w values from a reference handbook,16 yielding the regression equation: log Ko/w ) 2.56 log Kp/s + 1.08, R2 ) 0.95. The log Ko/w values estimated by using this hydrophobicity ruler approach are listed in Table 1 (log Ko/w*); values are provided for each of the PCB compounds regardless of whether their log Ko/w values were known or not. The log Ko/w values for the reference PCB compounds were regression results from all of the experimentally determined log Ko/w values compiled from the literature.16 If the experimental determinations were not biased, these regression values should bear better biological meaning than the parent values because their individual deviations were corrected. log Ko/w values were also obtained for the PCB compounds without known experimental log Ko/w values in the literature. The log Ko/w values determined (16) Howard, P. H.; Meylan, W. M. Handhook of Physical Properties of Organic Chemicals; CRC Press: Boca Raton, FL, 1997.

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Figure 4. Correlation of log Kp/s with experimental log Ko/w.

Figure 6. Differentiation of relative hydrophobicity for closely related PCB isomers.

Figure 5. Correlation of log Kp/s with theoretical log Ko/w (KowWin).

for the unknown compounds PCB126, PCB187, PCB197, PCB180, PCB170, and PCB195 were 6.94, 7.84, 8.33, 8.17, 7.92, and 8.49, respectively. Correlation with Theoretical log Ko/w Values. For very hydrophobic compounds, it is hard to determine log Ko/w experimentally, but their log Ko/w values can be estimated using different algorithms. Theoretically calculated log Ko/w values often differ substantially from experimentally determined ones, particularly at high log Ko/w values. There is an excellent correlation (Figure 5) between the measured log Kp/s values for PCBs reported here and the theoretical log Ko/w values calculated with KowWin software (Syracuse Research Corp., North Syracuse, NY), where log Ko/w ) 2.68 log Kp/s + 0.964, R2 ) 0.96. Many individual PCB congeners have the same log Ko/w values when calculated by KowWin (Table 1). For example, the log Ko/w values for PCB44, PCB52, PCB77, and PCB66 were calculated to be 6.34. The log Ko/w values for PCB128, PCB138, and PCB153 were calculated to be 7.62. These data could result from the fragment additive principle of the KowWin program, i.e., the isomers have similar fragments. The hydrophobicity ruler method presented here differentiates among these closely related isomers. This is demonstrated in Figure 6, where the absorption profiles and equilibrium absorption amounts are differentiated for these two groups of PCB isomers. A set of log Ko/w values (log Ko/w**) was also obtained from the correlation curve of the log Kp/s values with the KowWin values 1280 Analytical Chemistry, Vol. 77, No. 5, March 1, 2005

(Table 1). The log Ko/w values from this correlation should provide a better representation of the relative hydrophobicities of the compounds than the original data. As shown in Figure 6, the PCB isomers having the same theoretical log Ko/w values actually have different hydrophobic strengths. Therefore, the log Ko/w values determined by the present method for these isomers could provide a better prediction of their environmental and biological fates.1-4 Moreover, the log Ko/w values obtained for the unknown compounds should offer the same quality of data as the reference compounds. Advantages of the Hydrophobicity Ruler Approach. The use of a polymeric hydrophobic phase (PDMS) allowed for the addition of a solvent (methanol) to the aqueous phase to increase the solubility of all compounds and reduce the hydrophobicity scale of the very hydrophobic compounds. Consequently, this allowed compounds with log Ko/w values ranging from approximately 5 to 10 to be measured in one analysis with a single PDMS disk and in a single methanol-water solution. In this experimental approach, all of the experimental factors were identical for all of the compounds (references and unknowns). The absorption amounts at equilibrium were determined only by the relative hydrophobicities of the compounds. The PDMS disk serves as a hydrophobicity ruler to measure the relative strengths of all of the compounds, and the methanol solvent reduces the hydrophobicity scale of the very hydrophobic compounds. The reduction of the affinity difference between the two partition phases PDMS vs methanol-water in comparison to octanol vs water is the central principle of the present approach. Only this makes it possible to measure such a wide range of hydrophobicities. This principle is reflected in the high value of the slope of the linear correlation between log Ko/w and log Kp/s (Figures 4 and 5). For example, the log Ko/w range for the 21 PCB compounds was 5 orders of magnitude from 5.30 to 10.08, whereas their log Kp/s range was only 2 orders of magnitude from 1.65 to 3.52 (Table 1).

The hydrophobicity ruler approach is a high-throughput method and is easily automated. The reference and unknown compounds coexist in the same solvent solution, so their log Kp/s values are determined in one measurement. Absorption equilibrium in the solvent solution is achieved much faster than that in the water phase, and experimental artifacts due to emulsions are eliminated. For example, absorption equilibrium was obtained within 24 h in 60% methanol solution for all of the PCB compounds (Figure 3). Of the existing methods, only the slow-stirring method can be used to determine the octanol/water partition coefficients for very hydrophobic compounds (log Ko/w > 6), and that method requires weeks to reach equilibrium and also requires quantitative analysis of extremely low concentrations of the compound in the water phase.17 This hydrophobicity ruler approach is an indirect method. Its accuracy depends on the accuracy of the log Ko/w values of the reference compounds. Moreover, the reference and unknown compounds should have similar chemical functional groups as the log Kp/s-log Ko/w correlation could be different for compounds with significantly different chemical functional groups. This makes the hydrophobicity ruler approach particularly useful for the determination of log Ko/w values for very hydrophobic compounds, such as the PCB compounds reported here. Another important application of the hydrophobicity ruler approach is in identifying outliers resulting from previous studies. The experimental value for PCB209 is far off the correlation line (17) De Bruijn, J. H. M.; Busser, F.; Seinen, W.; Hermens, J. Environ. Toxicol. Chem. 1989, 8, 499. (18) Mackay, D.; Shiu, W. Y.; Ma, K. C. Illustrated Handbook of Physical-Chemical Properties and Environmental Fate for Organic Chemicals; Lewis Publishers: Chelsea, MI, 1992.

as shown in Figure 4. It was treated as an outlier and not included in the present correlation. This is an important application of the present method, because the experimental difficulties and complex error sources involved in the determination of the log Ko/w values have made it difficult to identify outliers with reasonable confidence as large discrepancies in log Ko/w values have been reported in the literature, even in reference handbooks.16,18 CONCLUSION The hydrophobicity ruler approach can be used for determining the log Ko/w values of very hydrophobic compounds. The novel strategy of this approach was to utilize a polymeric hydrophobic phase and to add an organic solvent to the aqueous phase so that the solubilities of all compounds (references and unknowns) in the aqueous solution were increased, the hydrophobicity scale was reduced for all of the compounds, and the equilibrium absorption amounts were controlled for optimal quantitative analysis. The log Kp/s values of all of the compounds can be determined with high precision in one measurement. The accuracy of this indirect method mainly depends on that of the determinations of the reference compounds. The log Ko/w values of the unknowns obtained by the hydrophobicity ruler approach should be of the same quality as those of the reference compounds. The hydrophobicity ruler approach is also a valuable tool for validating experimental and theoretical log Ko/w values and identifying outliers in log Ko/w databases. Received for review August 5, 2004. Accepted November 29, 2004. AC048847R

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