Environ. Sci. Technol. 2004, 38, 1108-1114
Reverse-Phase HPLC Method for Measuring Polarity Distributions of Natural Organic Matter K S E N I J A N A M J E S N I K - D E J A N O V I C * ,† A N D STEPHEN E. CABANISS‡ Department of Geology, Kent State University, Kent, Ohio 44242, and Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131
A reverse-phase high-pressure liquid chromatography (RP-HPLC) method was developed to measure the polarity distribution of natural organic matter (NOM) samples. The polarity distribution is obtained by calibrating an octadecyl bonded silica phase column and polar eluent with compounds of known octanol-water partition coefficient (Kow) and using this calibration curve to transform NOM retention times into an equivalent Kow. Polarity distributions treat the NOM samples as a complex mixture rather than summarizing the polarity in a single number. The method is sensitive, with UV detection allowing quantitation of samples with 3.5 or are too strongly adsorbed to the column stationary phase to be eluted; this fraction is estimated based on comparison of chromatogram area acquired with and without a column. Data in Table 3 suggest that original water samples have the lowest recovery of all the samples analyzed (48% and 64%). The unrecovered fraction of isolates contains between 5% (Laurentian FA) and 25% (Missouri River FA). Abbt-Braun and Frimmel (47) characterizing a variety of Norwegian NOM samples using gradient RP-HPLC with 0-100% acetonitrile reported 4563% (read off graph) to be hydrophilic, with arbitrary division into hydrophilic and hydrophobic at 4 min retention time. Their hydrophobic fraction corresponds well with our fifth, mostly unrecovered fraction, containing NOM compounds with log Kow >3.5 (Table 3). Comparison with Other Data. We used linear regression to explore possible relationships between our RP-HPLC data
and 13C NMR aromaticity and aliphaticity, molecular weight, and O/C ratio. O/C ratio is commonly taken as an estimate of NOM functionality, and it correlated reasonably well with percemt recovery of NOM; the regression equation was
(O/C) ) 0.0159 (% recovered) - 0.6553
R 2 ) 0.86
This positive correlation supports the assumption that more functionalized NOM should be more hydrophilic. Linear regression of weight-average molecular weights (Mw) with percent recovery gave a weaker relationship:
Mw ) 39.93 (% recovery) - 1168.3
R 2 ) 0.59
This positive correlation suggests that NOM with greater Mw exhibits better recovery. It might be because large size prevents whole-molecule partitioning into the stationary phase or because larger molecules are able to fold into a conformation with hydrophilic groups on the exterior and hydrophobic groups in the interior, which would enhance solubility in the polar mobile phase but decrease partitioning into the nonpolar stationary phase. The relationship between RP-HPLC data and 13C NMR data is more problematic. Although subsets of the samples appear to have positive correlations (R 2 g 0.84) between 13C NMR aromaticity and NOM recovery, suggesting that more aromatic NOM is also more hydrophilic, the correlations are nonexistent (R 2 ) 0.0207) if all of the samples are considered. Similarly, negative correlations of 13C NMR percent aliphatic and NOM recovery for the subsets of samples (R 2 g 0.95) suggest that higher aliphaticity corresponds to poorer recovery and higher NOM hydrophobicity. This correlation is also poor (R 2 ) 0.0005) in the overall data set, giving the overall impression that aliphatic C contributes at least as much to NOM hydrophobicity as aromatic C. Correlations of recovery with the sum of aliphatic and aromatic C are not helpful because six of the samples have (aliphatic + aromatic) C between 57.0 and 58.4% and only two of the other four samples have directly comparable NMR data (Cook and Langford used a different experiment that may well be superior but is not directly comparable to the other measurements; 17). These findings can perhaps be explained by Cook and Langford’s (17) FA model, which is based on solid-state ramp CP-MAS 13C NMR. They proposed that Laurentian fulvic acid consists of three types of units: (i) large relatively immobile units that are mainly aliphatic in nature, (ii) relatively unfunctionalized more mobile units that are mostly aromatic, and (iii) more mobile functionalized units that are mainly carbohydrate in nature. If the aliphatic fraction of NOM is less functionalized and more “compatible” with the C-18 aliphatic stationary phase, it would exhibit longer retention times, lower total recoveries (in 20-min runs), and therefore higher hydrophobicity. Another possible explanation stems from specific interaction of NOM with the stationary phase. Tanaka et al. (48) observed that aromatic stationary phase showed greater retention for aromatic solutes while saturated hydrocarbons stationary phase led to greater retention for saturated (aliphatic) hydrocarbons. They concluded that effects such as steric recognition and π-π interactions between stationary phase and solute are important in addition to solvophobic interaction. Therefore, taking all of the samples into account, we believe that results should be considered as a consequence of polarity/hydrophobicity of NOM caused by both aliphatic and aromatic compounds present and that selective correlations may in fact be misleading. Moreover, this approach agrees with definition of hydrophobic carbons in soil humic substances, which is based on 13C NMR areas of both alkyl and aromatic carbons (49, 50). Gauthier et al. (44) proposed, based on pollutant-DOM interaction that while the degree VOL. 38, NO. 4, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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of aromatic character may be important for planar aromatic PAHs, the degree of aliphatic character of the DOM may be more important for nonplanar, saturated hydrocarbons. The RP-HPLC method developed here reveals NOM polarity distributions in terms of specific log Kow values rather than single-valued quantities such as percent hydrophobic or percent hydrophilic. Consequently, is should provide more detailed insight into the NOM reactions based on polarity.
Acknowledgments We thank George Aiken and Mike Perdue for generous gifts of well-characterized NOM isolates. We also give thanks to Patricia A. Maurice and Mike Pullin for their work in collection, extraction, and characterization of NOM samples from McDonalds Branch and for reviewing the manuscript. This research was funded by NSF Grant EAR-0106752.
Literature Cited (1) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry; John Wiley and Sons: New York, 1993; p 681. (2) Landrum, P. F.; Nihart, S. R.; Eadle, B. J.; Gardner, W. S. Environ. Sci. Technol. 1984, 18, 187-192. (3) Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E. Environ. Sci. Technol. 1986, 20, 502-508. (4) Kile, D. E.; Chiou, C. T. Water Solubility Enhancement of Nonionic Organic Contaminants; In Aquatic Humic Substances: Influence on Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; ACS Symposium Series 219; American Chemical Society: Washington, DC, 1989; Chapter 10; pp 131157. (5) McCarthy, J. F.; Roberson, L. E.; Burrus, L. W. Chemosphere 1989, 19, 1911-1920. (6) Davis, J. A.; Gloor, R. Environ. Sci. Technol. 1981, 15, 12231229. (7) Tomaic, J.; Zutic, V. J. Colloid Interface Sci. 1988, 26 (2), 482492. (8) Ochs, M.; Cosovic, B.; Stumm, W. Geochim. Cosmochim. Acta 1994, 58 (2), 639-650. (9) Wang, L. L.; Chin, Y. P.; Traina, S. J. Geochim. Cosmochim. Acta 1997, 61 (24), 5313-5324. (10) Meier, M.; Namjesnik-Dejanovic, K.; Maurice, P. A.; Chin, Y. P.; Aiken, G. R. Chem. Geol. 1999, 157 (3-4), 275-284. (11) Namjesnik-Dejanovic, K.; Maurice, P. A.; Aiken, G. R.; Cabaniss, S. E.; Chin, Y. P.; Pullin, M. J. Soil Sci. 2000, 165, 545-559. (12) Leenheer, J. A.; Huffman, E. W. D., Jr. J. Res. U.S. Geol. Surv. 1976, 4 (6), 737-751. (13) Leenheer, J. A. Environ. Sci. Technol. 1981, 15, 578-587. (14) Croue, J.-P.; Benedetti, M. F.; Violleau, D.; Leenheer, J. A. Environ. Sci. Technol. 2003, 37, 328-336. (15) Perminova, I. V.; Grechishcheva, N. Y.; Petrosyan, V. S Environ. Sci. Technol. 1999, 33, 3781-3787. (16) Peuravuori, J. Anal. Chim. Acta 2001 29, 75-89. (17) Cook, R. L.; Langford, C. H. Environ. Sci. Technol. 1998, 32, 719-725. (18) Traina, S. J.; Novak, J.; Smeck, N. E. J. Environ. Qual. 1990, 19, 151-153. (19) Braun, D. V.; Floyd, A. J.; Sainsbury, M. Organic Spectroscopy; John Willey: New York, 1988; pp 3-23. (20) Hammers, W. E.; Meurs, G. J.; De Ligny, C. D. J. Chromatogr. 1982, 247, 1-13.
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(21) Hansch, C.; Gao, H.; Hoekman, D. In Comparative QSAR; Devillers, J., Ed.; Taylor and Francis: Washington, DC, 1998; pp 285-371. (22) Lyman, W. J.; Reehl, W. F.; Rosenblatt, D. H. Handbook of Chemical Property Estimation Methods; American Chemical Society: Washington, DC, 1990. (23) Leo A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71 (6), 525-613. (24) Hansch, C.; Hoekman, D.; Leo, A.; Zhang, L. T.; Li, P. Toxicol. Lett. 1995, 79, 45-53. (25) Braumann, T. J. Chromatogr. 1986, 373, 191-225. (26) Martin, A. J. P.; Synge, R. L. M. Biochem. J. 1941, 35, 1359. (27) Consden, R.; Gordon, A. H.; Martin, A. J. P. Biochem. J. 1944, 38, 224. (28) Tomlinson, E. J. Chromatogr. 1975, 113, 1-13. (29) Braumann, T. J.; Weber, G.; Grimme, L. H. J. Chromatogr. 1983, 261, 329-343. (30) Finizio, A.; Vighi, M.; Sandroni, D. Chemosphere 1997, 34, 131161. (31) Skoog, D. A.; Holler, F. J.; Nieman, T. A. Principles of Instrumental Analysis; Saunders College Publishers: Philadelphia, 1998; p 849. (32) Klein, W.; Kordel, W.; Weis, M.; Poremski, M. J. Chemosphere 1988, 261, 361-386. (33) Roses, M.; Canals, I.; Alleman, H, Siigur, K.; Bosch, E. Anal. Chem. 1996, 68, 4094-4100. (34) Martell, A. E.; Smith, R. M.; Motekaitis, R. J. Critically Selected Stability Constants of Metal Complexes Database, Version 5.0; NIST CSC: Washington, DC, 1998. (35) Maurice, P. A.; Pullin, M. J.; Cabaniss, S. E.; Zhou, Q.; NamjesnikDejanovic, K.; Aiken, G. R. Water Res. 2002, 36, 2357-2371. (36) Chin, Y. P.; Aiken, G. R.; O’Loughlin, E. Environ. Sci. Technol. 1994, 28, 1853-1858. (37) Huizen, G. A New Approach to Fractionation of Natural Organic Matter. Ph.D. Dissertation, 1994, p 186. (38) Karanfil, T.; Kilduff, J. E.; Schlautman, M. A.; Weber, W. J. Environ. Sci. Technol. 1996, 30, 2187-2194. (39) Saleh, F. Y.; Ong, W. A.; Chang, D. Y. Anal. Chem. 1989, 61, 2792-2800. (40) Zhou, Q.; Cabaniss, S. E.; Maurice, P. A. Water Res. 2000, 34 (14), 3505-3514. (41) O’Loughlin, E.; Chin, Y.-P. Water Res. 2001, 35 (1), 333-338. (42) Egeberg, P. Kr.; Alberts, J. J. Water Res. 2002, 36, 4997-5004. (43) Raber, B.; Kogel-Knabner, I.; Stein, C.; Klem, D. Chemosphere 1998, 36, 79-97. (44) Gauthier, T. D.; Seitz, W. R.; Grant, C. L. Environ. Sci. Technol. 1987, 21, 243-248. (45) Chin, Y. P.; Weber, W. J., Jr. Environ. Sci. Technol. 1989, 23, 978-984. (46) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Technol. 1981, 14 (4), 463-466. (47) Abbt-Braun, G.; Frimmel, F. H. Environ. Int. 1999, 25 (2-3), 161-180. (48) Tanaka, N.; Tokuda, Y.; Iwaguchi, K.; Araki, M. J. J. Chromatogr. 1982, 239, 761-772. (49) Piccolo, A.; Conte, P. In Structure and Surface Reactions of Soil Particles; Huang, P. M., Senesi, N., Buffle, J., Eds.; John Wiley & Sons: New York, 1998; pp 184-250. (50) Piccolo, A.; Conte, P.; Cozzolino, A. Soil Sci. 2001, 166 (3), 174185.
Received for review April 30, 2003. Revised manuscript received October 9, 2003. Accepted November 19, 2003. ES0344157
