Environ. Sci. Technol. 1984, 18, 457-459
Generator Column Determination of Octanol/Water Partition Coefficients for Selected Polychlorinated Biphenyl Congeners Kent B. Woodburn,+ Wllllam J. Doucette, and Anders W. Andren” Water Chemistry Program, University of Wisconsin, Madison, Wisconsin 53706
A generator column technique has been used to directly
determine log KO,values for 15 PCB congeners and biphenyl. The method circumvents many of the experimental difficulties encountered with the traditional shake-flask system. log KO, values generated by this procedure are compared with data obtained from the shake-flask method, two chromatographic estimation techniques, and the computational method of Hansch and Leo. log KO, values reported for the higher chlorinated congeners, including octa- and decachlorobiphenyl, are some of the highest ever measured directly. Introduction
The 1-octanol/water partition coefficient (KO,) is a useful physicochemical property for characterizing the lipophilicity or hydrophobicity of organic compounds (1). The log KO,value has become an important parameter in environmental fate studies for predicting physical and biological phenomena such as soil sorption (2, 3 ) and bioconcentration in fish (4-6). In addition, the KO,has been correlated to aquatic toxicity ( 7 ) ,biomagnification in beef cattle and swine (B), and aqueous solubility (9,lO). Values for KO,are obtained by direct measurement or by using one of several estimation techniques. The most widely used estimation approach is the Hansch and Leo r-factor method (see, for example, ref 1and 11). A similar approach, called the hydrophobic fragment constant method, has been presented by Nys and Rekker (12). Application of these techniques, however, may only be extended to compounds for which all structural parameters have been determined. (For example, Fujita et al. (13) showed that different fragment values must be applied for ortho, meta, and para substituents in aromatic systems.) Another estimation technique employs reverse-phase high-performance liquid chromatography (RP-HPLC) (14-1 7). In this method, compounds of known KO,values are correlated to their retention times, R,, and a regression equation is obtained. Retention times of compounds with unknown KO,values are determined and KO,values are calculated from the regression equation. Although this technique is rapid and does not require quantitation, it is an indirect method that is dependent on the accurate determination of KO, values by direct methods (18). In addition, the mechanism of partitioning to the CIS stationary phase is not analogous to octanol/water partitioning (19-21). A similar method employing correlations between log KO, and retention times obtained from reversed-phase thin-layer chromatography (RP-TLC) has also appeared in the literature (18, 22, 23). This technique, although rapid and inexpensive to perform, suffers from the same inherent weaknesses as the RP-HPLC method. When exact thermodynamic relationships between KO, and other parameters are explored (for example, KO,and aqueous solubility ( 3 1 ) ) ,it is desirable to use values of highest quality. If such accurate KO,values are required, t Present address: Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL 32610.
0013-936X/84/0918-0457$01.50/0
direct measurement is preferred. Direct measurement of KO,has usually been accomplished by using the conventional shake-flask method (1). This batch technique can be laborious due to the formation of colloidal dispersions, volatilization to the atmosphere, and adsorption of the solute onto surfaces of transfer vessels (2). A rapid, precise coupled-column system has been recently developed for measuring KO,values of hydrophobic substances (24).This technique, termed “the generator column-HPLC method, effectively circumvents many of the experimental difficulties of the shake-flask system. The flow rate of water through the column is slow enough to avoid colloidal dispersions, while the large, interfacial area between phases allows rapid equilibration. The system walls become equilibrated with aqueous solution, and errors from adsorption are avoided. Also, potential losses to the atmosphere are minimized in the closed system. DeVoe’s generator column-HPLC method is experimentally limited to log KO,values less than 6 (21). We have modified the method to extend its applicability to compounds having higher log KO,. The class of compounds chosen for investigation was the polychlorinated biphenyls (PCBs). PCBs are synthetic chlorinated hydrocarbons, possessing physicochemical properties that have given them considerable mobility and stability in natural systems (25, 26). There are 209 possible PCB congeners, and substantial variation in chemical behavior, persistence, and toxicity has been observed among them (27). Although PCBs are widely recognized as serious environmental pollutants, octanol/water partitioning data exist for only a few congeners (9, 18). Both the DeVoe and the modified methods have been used here to determine the log KO,values for a number of PCB congeners, including octa- and decachlorobiphenyl. The log KO,values reported here are some of the highest ever measured directly. Materials and Methods
Reagents. All mono-, di-, and trichloro PCB congeners, with the exception of 4-chlorobiphenyl,were obtained from Analabs, Inc. (North Haven, CT; 99.9% purity). The 4chlorobiphenyl and decachlorobiphenyl were purchased from Aldrich Chemical Co. (Milwaukee, WI). Penta-, hexa-, and octachloro PCBs were purchased from Ultra Scientific Inc. (Hope, RI). The PCBs were used without further purification. The 1-octanol (Fisher;certified grade) was washed with 0.1 M H2S04,0.1 M NaOH, and HPLCgrade water. It was then dried over CaCl, and distilled at atmospheric pressure. The mobile phases required for liquid chromatography were prepared from HPLC-grade methanol and water (Baker Chemical Co., Phillipsburg, NJ). The HPLC-grade water was also used to generate all aqueous solutions. Pesticide-grade hexane (Fisher Scientific Co., Chicago, IL) was used for gas chromatography. Equipment. The HPLC-generator column system has been described in detail by DeVoe et al. (24) and Woodburn (21). The dimensions of the column were 12 cm X 0.4 cm. This system was used for congeners having log KO,
0 1984 American Chemical Society
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457
Table I. log K , Values for PCB Congeners and Biphenyl
Hansch expt14log
exptl
Knw
log KO,
(generator column)
(shake flask)
biphenyl 2-CB 3-CB 4-CB 4,4'-DCB
3.89 f 0.01 (12) 4.38 f 0.02 (9) 4.58 f 0.03 (6) 4.49 f 0.01 (10) 5.33 f 0.01 ( 5 )
3,4-DCB 2,2'-DCB
5.29 f 0.01 (6) 4.90 f 0.01 (3)
2,4'-DCB 2,2',5-TCB 2,4,5-TCB 2,4',5-TCB 2,2',4,5,5'-PCB 2,2',3,3',6,6'-HCB 2,2',4,4',5,5'-HCB 2,2',3,3',5,5',6,6'-OCB
5.14 f 0.01 (4) 5.60 f 0.01 (3) 5.81 f 0.00 (2) 5.79 f 0.00 (2) 6.50 f 0.13 (6) 6.81 f 0.03 (6) 6.90 f 0.14 (8) 7.14 i 0.17 (9) 8.20 f 0.27 (18)
4.09f 4.598 4.718 4.619 5.36: 5.58h ND~ 5.00: 4.80' 5.10' ND ND ND 6.11h ND 6.72h ND ND
compound
and Leob 1% KO, 4.09 4.80 4.80 4.80 5.51
RP-
RP-
HPLCc log Knw 3.70 3.75 4.35 4.34 4.92
TLC' 1% KO,
4.10 4.56 4.72 4.69 5.28
5.51 5.51
5.10 3.55
ND 5.02
5.51 6.22 6.22 6.22 7.64 8.35 8.35 9.77 11.19
4.48 4.34 5.86 5.30 ND ND ND ND ND
ND 5.64 5.77 5.77 6.85 ND 7.44 8.42 9.60
decachloro-BP "Mean log KO, f standard deviation (number of determinations). bFrom ref 30. Biphenyl = 4.09, rCI= 0.71. "Method of Veith et al. (16) (by Woodburn (21)). dND = not determined. eReversed-phasethin-layer chromatography (18). fLeo et al. (I). gBruggeman et al. (18). shake-flask techniaue. hChiou et al. (9).'Chiou et al. (31). values lower than 6. A modified version was devised for the higher chlorinated congeners and will be briefly described. The length of the generator column in the modified procedure was doubled to enable a larger solute loading. The 24-cm column segment was hand packed with silanized Chromosorb W ("3 g, 60180 mesh) retained by a coarse, ground glass frit at the bottom and a plug of silanized glass wool at the top. The column was thermostated at 25 f 0.1 "C by pumping water from a constant temperature bath through a jacket enclosing the column. A CIESep-Pak (Waters Associates, Milford, MA) was used to extract solute from the aqueous solution generated by the column. A reciprocating HPLC pump (Waters Associates, Model M-45) delivered the octanol-saturated water through the system. A Hewlett-Packard gas chromatograph (5840A),equipped with an electron capture detector (63Ni)was used for quantitation. Procedure. The HPLC-generator column method has been described by DeVoe et al. (24) and was used to determine log KO,values less than 6. The procedure for the modified method is described as follows. Approximately 10 mg of the PCB congener of interest was dissolved in 100 mL of 1-octanol. Fifteen milliliters of this solution was vigorously stirred with 120 mL of HPLC water for 14-18 h. The concentration of PCB in the octanol phase was determined by gas chromatography. The octanol phase was then applied to the generator column and pulled through the dry support with gentle suction until the solid support was saturated as evidenced by the appearance of the 1-octanol at the column base. One experimental difficulty described by DeVoe et al. (24)is the eventual depletion of octanol from the generator column due to its slight solubility in water. In this work, it was found that for PCBs having log KO,> 6 the octanol was depleted before a sufficient amount of water for quantitation could be collected. To slow the depletion of octanol from the column, the water was presaturated with octanol prior to being pumped through the generator column. This greatly extended the life of the generator column and enabled the determination of KO,for all PCB 458
Environ. Sci. Technol., Vol. 18, No. 6, 1984
congeners, including decachlorobiphenyl which required collecting 10 L of aqueous phase. The aqueous phase, generated by pumping octanolsaturated water through the coated generator column, was pumped through a CI8 column (Sep-Pak). The column efficiently extracted PCBs from the aqueous solution (greater than 90%). The liquid leaving the column was collected in a tared weighing flask. When an amount of PCB sufficient for analysis was collected on the Sep-Pak, excess water was removed by purging with a gentle stream of N2 and the solute eluted with 10 mL of hexane. The amount of PCB was then quantitated with gas chromatography. The KO, was calculated by dividing the PCB congener concentration in the octanol phase by that in aqueous phase. DeVoe et al. (24) demonstrated that the aqueous solute concentration exiting the generator column was independent of flow rate (0.5-2.0 mL/min), of the volume of water passed through the column, and of the concentration of solute in octanol. Experiments in this laboratory verified this (21), and all determinations were carried out in the flow-independent range. Results and Discussion
The log Knwvalues measured for 15 PCB congeners and biphenyl using the generator column technique are given in Table I. In addition, available log Ko,'s determined by the shake-flask method and by several commonly used estimation techniques are presented in Table I for comparison purposes. The confidence limits on measured log KO, in this study are within 1.5% of the average values, with the exception of octa- (2.4%) and decachlorobiphenyl (3.3%). The extremely low aqueous concentrations of these congeners made analytical quantitation less precise. Although the number of log KO,values determined by the shake-flask method are limited, data in Table I show that values agree closely with those determined by the generator column method. Close agreement between shake-flask and generator column methods has been reported previously by DeVoe et al. (24) and Wasik et al. (28). To our knowledge, a log KO,value of 8.20 (that of
decachlorobiphenyl) is the highest measured value ever reported. Analytical problems with the shake-flask technique would make a log KO, determination of this magnitude exceedingly difficult. log KO,values estimated by using the Leo and Hansch ?r-factormethod are compared to our measured values in Table I. The method, while simple and widely used, is best applied when all necessary structural factors have been experimentidly determined. This is not the case for PCBs. The ?rcl factor most commonly used for PCBs (0.71) is derived from chlorobenzene and benzene and is assigned regardless of the position on the biphenyl ring. Tulp and Hutzinger (29) and Woodburn (21)attempted to improve this approach using different ?rc1factors for ortho-, meta-, and para-substituted chlorines. However, until the substituent effects are completely understood, these predicted structural application techniques may yield values which differ by several orders of magnitudes from the directly measured values. The deviations seem especially large for compounds having high log KO,values. Results of two indirect chromatographic estimation techniques using RP-HPLC (16)and RP-TLC (18)are also presented in Table I for several PCB congeners. Although rapid and inexpensive, the Km values estimated from these chromatographic techniques deviate considerably from the generator column measurements, especially for the higher chlorinated PCBs. Conclusions Direct measurement is preferred when accurate KO, values are required. The generator column method used here provides several advantages over shake-flask techniques, especially for compounds with log KO,> 6. The generator column method for determining Kow’shas been applied to PCB congeners having log KO,in the range 4-8 and is shown to be the method of choice for direct measurement of KO,values for highly lipophilic compounds. Acknowledgments We gratefully acknowledge Tom Stolzenburg for his critical review of the manuscript. Jean Schneider and Helen Grogan provided expert typing assistance. Diane Norback, Department of Pathology, University of Wisconsin, kisdly provided the two hexachlorobiphenyl congeners. Registry No. 2-CB, 2051-60-7; 3-CB, 2051-61-8; 4-CB, 205162-9; 4,4’-DCB, 2050-68-2; 3,4-DCB, 2974-92-7; 2,2’-DCB, 13029-08-8; 2,4’-DCB, 34883-43-7; 2,2’,5-TCB, 37680-65-2; 2,4,5TCB, 15862-07-4; 2,4‘,5-TCB, 16606-02-3; 2,2‘,4,5,5‘-PCB, 37680-73-2; 2,2’,3,3’,6,6’-HCB, 38411-22-2; 2,2’,4,4’,5,5’-HCB, 35065-27-1; 2,2,3,3’,5,5’,6,6‘-OCB, 2136-99-4; decachloro-BP, 2051-24-3; biphenyl, 92-52-4; octanol, 111-87-5;water, 7732-18-5.
Literature Cited (1) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71,525. (2) Karickhoff, S. W.; Brown, D. S. “Determination of Octanol/ Water Partition Coefficients, Water Solubilities, and Sediment/ Water Partition Coefficients for Hydrophobic
(3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23)
Organic Pollutants”. U.S. Environmental Protection Agency, Washington, DC, 1979, U S . EPA Report 600/479-032. Schwarzenbach, R. P.; Westall, J. Enuiron. Sei. Technol. 1981, 15, 1360. Neely, W. B.; Branson, D. R.; Blau, G. E. Enuiron. Sci. Technol. 1974, 8, 1113. Sugiura, K.; Ito, N.; Matsumoto, N.; Mihara, Y.; Murata, K.; Tsukakoshi, Y.; Goto, M. Chemosphere 1978, 9, 731. Konemann, H.; van Leeuwen, K. Chemosphere 1980,9,3. Konemann, H. Ecotoxicol. Enuiron. Saf. 1980, 4, 415. Kenaga, E. E. Environ. Sei. Technol. 1980, 14, 553. Chiou, C. T.; Freed, V. H.; Schmedding, D. W.; Kohnert, R. L. Enuiron. Sci. Technol. 1977, 11, 475. Mackay, D.; Bobra, A.; Shiu, W. Y.; Yalkowsky, S. H. Chemosphere 1980,9, 701. Hansch, C.; Fujita, T. J. Am. Chem. SOC.1964,86, 1616. Nys, G. G.; Rekker, R. F. Chim. Ther. 1973,5, 521. Fujita, T.; Iwasa, J.; Hansch, C. J. Am. Chem. Soc. 1964, 86, 5175. Carlson, R. M.; Carlson, R. E.; Kopperman, H. L. J. Chromatogr. 1975, 107, 219. McCall, J. M. J. Med. Chem. 1975, 18, 549. Veith, G. D.; Austin, N. M.; Morris, R. T. Water Res. 1979, 13, 43. McDuffie, B. chemosphere 1981, 10, 73. Bruggeman, W. A.; Van der Stenen, J.; Hutzinger, 0. J. Chromatogr. 1982,238, 335. Horvath, C.; Melander, W. J. Chromatogr. Sci. 1977, 15, 393. Berendsen, G. E.; de Galan, L. J. Chromatogr. 1980,196, 21. Woodburn, K. M.S. Thesis, University of Wisconsin, Madison, 1982, pp 1-171. Ellgehausen, H.; D’Hondt, C.; Fuerer, R. Pestic. Sci. 1981, 12, 219. Papp, 0.;Valko, K.; Szasz, Gy.; Hermecz, I.; Vamos, J.; Hanko, K.; Ignath-Halasz, Zs. J. Chromatogr. 1982,252, 67. DeVoe, H.; Miller, M. M.; Wasik, S. P. J . Res. Natl. Bur. Stand. (U.S.) 1981, 86, 361. Hammond, P. B.; Nisbert, I. C. T.; Sarofim, A. F.; Drury, W. H.; Nelson, N.; Rall, D. P. Environ. Res. 1972,5, 249. National Academy of Sciences “Polychlorinated Biphenyls”; National Academy of Sciences: Washington, DC, 1979; pp 1-182. Hutzinger, 0.;Safe, S.; Zitko, V. “Chemistry of PCBs”; CRC Press: Cleveland, OH, 1974; pp 1-252. Wasik, S. P.; Tewari, L. B.; Miller, M. M.; Martire, D. E. Natl. Bur. Stand., [Tech. Rep.] NBSIR ( U S . ) 1981, NBSIR-82-2406, 1-56. Tulp, M. Th. M.; Hutzinger, 0. Chemosphere 1978,10,849. Hansch, C.; Leo, A. J. “Substituent Constants for Correlation Analysis in Chemistry and Biology”; Wiley: New York, 1979; pp 1-339. Chiou, C. T.; Porter, P. E.; Schmedding, D. W. Enuiron. Sci. Technol. 1983, 17, 227.
Received for review July 27,1983. Accepted December 22, 1983. This work was funded by the University of WisconsinSea Grant College Program under grants from the Office of Sea Grant, National Oceanographic and Atmospheric Administration, U S . Department of Commerce, and from the State of Wisconsin. Federal Grant NA800AA-D-00086, Project RIMW-21.
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