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Response to Comment on “Noncovalent Interactions between Acenaphthenone and Dissolved Fulvic Acid As Determined by 13C NMR T1 Relaxation Measurements” SIR: We appreciate the comments of Jayasundera et al. regarding our paper (1). Based on our 13C NMR T1 relaxation measurements of acenaphthenone (13C-labeled in the carbonyl position) with Suwannee River fulvic acid in a methanol/ D2O solvent, we proposed that acenaphthenone was involved in three noncovalent interactions that were a function of the acenaphthenone concentration, the fulvic acid concentration, the fulvic acid countercation (Na+ or H+), and pH. The three noncovalent interactions of acenaphthenone are a weak sorption interaction with fulvic acid, an enhanced solubilization of acenaphthenone by fulvic acid, and a noncovalent interaction with solvent. We hypothesized that the enhanced solubilization of acenaphthenone arose from fulvic acid forming hydrophobic regions predominantly solvated with methanol. Acenaphthenone encapsulated in the hydrophobic region displayed similar behavior to when it was dissolved in pure methanol. At low acenaphthenone concentrations (∼0.5 mg/mL), an inverse relationship exists between the fulvic acid concentration and the T1 relaxation time of acenaphthenone, indicating that the averaged molecular tumbling motion of sorbed and dissolved is decreasing due to increasing sorption of acenaphthenone to fulvic acid. Protonated fulvic acid (FAH) causes a greater reduction in T1 relaxation time per mass of fulvic acid than does the fulvic acid with sodium as the countercation (FA-Na). We proposed that this difference is due to the fact that FA-H is more hydrophobic than FA-Na; therefore, there is greater hydrophobic sorption of acenaphthenone to FA-H. Jayasundera et al. propose another interpretation in which the orientation of the sorbed acenaphthenone is a function of fulvic acid countercation. They claim incorrectly that the FA-H is more hydrophilic than FA-Na and therefore favors hydrophilic interactions with acenaphthenone, which in turn enhances the T1 relaxation rate. It may be possible that hydrogen bonding (which Jayasundera et al. do not mention) between the acenaphthenone carbonyl group and FA-H is occurring; however, the highly hydrophobic nature of FA-H would favor hydrophobic sorption of the aromatic portion of acenaphthenone. As the concentration of acenaphthenone was increased in the presence of FA-H (0.0003 to 0.006 mg of FA-H/mL), the T1 relaxation time of acenaphthenone increased to a value greater than that of acenaphthenone in pure methanol/D2O solvent. This increase demonstrates that the acenaphthenone molecule is experiencing increased overall molecular motion, which is more than it would experience in its interactions with just the methanol/D2O solvent. As the acenaphthenone concentration was increased in solution containing FA-H at a concentration of 0.006 mg/mL, the T1 relaxation time reached a maximum of ∼39 s before returning to ∼30 s, the T1 relaxation time of acenaphthenone in pure methanol/ D2O. We proposed that the T1 relaxation time increase of acenaphthenone is due to FA-H forming hydrophobic regions that preferentially encapsulate methanol molecules and exclude D2O molecules. Acenaphthenone molecules also become encapsulated into these hydrophobic pockets and behave as if they were dissolved in methanol and therefore experience a greater molecular tumbling motion than when present in a MeOH/D2O solvent.
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Jayasundera et al. claim that this interpretation is not supported by the data for several reasons. First, they state that “from the results presented we cannot assume spartial [sic] resolution of MeOH, HOD, and/or MeOD nor that an individual solvent component is effecting the relaxation”. We agree with this statement and note that the T1 relaxation time observed in the methanol/D2O solvent is an averaged value of the T1 relaxation times of acenaphthenone with methanol, deuterated methanol, and D2O. However, as mentioned in our paper (1), a decrease in the acenaphthenone T1 relaxation time is to be expected when going from pure deuterated methanol to a methanol/D2O mixture because of the increased hydrogen-bond forming propensity of D2O as compared to deuterated methanol. However, the hydrophobic region created by the fulvic acid is predominantly methanol and/or deuterated methanol; therefore, the acenaphthenone present in the hydrophobic region is essentially dissolved in methanol. The concern that the quadrupolar relaxation of acenaphthenone in deuterated prevents us from comparing the relaxation of the carbonyl carbon of acenapthenone in MeOD with that of MeOH is not clear, since we have shown chemical shift anisotropy to be the predominant relaxation pathway for the carbonyl carbon. In this case, changes in quadrupolar relaxation should be negligible. The second criticism of our interpretation is based on lack of observed 13C chemical shift changes for the carbonyl carbon of the acenaphthenone. A change in the 13C chemical shift of the carbonyl carbon was observed when acenaphthenone was examined in pure MeOD versus MeOH/D2O (209.2 ppm and 207.6 ppm, respectively). Once fulvic acid, which is very low concentration, is added to the MeOH/D2O solvent system, an additional change in chemical shift was not detected. In a previous study with [1-13C]phenol, (2) 13C chemical shift changes were not observed in any of our solutions containing humic or fulvic acids; however, sorption studies with chlorinated phenols carried out by other investigators indicated that noncovalent binding interactions are present under similar conditions (3, 4). It is known that 13C chemical shifts are largely insensitive to medium effects and they are used as a gauge for changes in chemical structure, which is an attribute of this physical parameter. The only explanation that we can offer at this time is that the medium effects in solutions containing low concentrations of humic or fulvic acids are not severe enough to produce a measurable change in the 13C chemical shift. Thirdly, their statement that localized hydrophobic regions have been observed by other researchers to reduce molecular motion of encapsulated hydrophobic molecules (5-7) is correct. However, as explained in our paper (1), the other researchers did their experiments in pure water, while our experiments were in a methanol/D2O mixture. In the absence of organic solvent, the hydrophobic regions formed by FA-H remain “dry”, and any hydrophobic molecules entering them will sorb to the hydrophobic walls of the region or the hydrophobic regions will be collapsed in the absence of an organic solvent. We hypothesized that in our experiment the hydrophobic region was filled with solvent molecules, which kept the FA-H+ hydrophobic region swelled with solvent and the acenaphthenone dissolved. Jayasundera et al. propose another explanation for the behavior of acenaphthenone with FA-H. They claim that at low fulvic acid concentrations, as the acenaphthenone concentration increases, “lesser number of interactions at the carbonyl-C will be observed, indicating an increase in the relaxation times”. This agrees with our explanation. However, they continue to propose that as the acenaphthenone concentration increases to greater than 0.75 mg/mL and the T1 relaxation
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1997 American Chemical Society
time increases above ∼31 s that the acenaphthenone may be controlling the pH such that the pH is increased from 6.04 (the reported value of a methanol/D2O solution of 0.25 mg/ mL FA-H) to a value of approximately 6.85, causing the T1 relaxation time to change from ∼39 to ∼32 s (as indicated by Figure 5). It is unlikely, based upon the chemical structure of acenaphthenone, that such a dramatic increase in pH would occur. In addition, Jayasundera et al.’s proposal does not provide any explanation why the T1 relaxation time in the FA-H sample, at a concentration of 0.25 mg/mL and a pH of 6.04, does not show similar behavior. Based on Figure 5 and their explanation, a T1 relaxation time value of ∼39 s would be expected. Likewise, their proposal does not provide an explanation as to why the T1 relaxation time does increase to values greater than that of acenaphthenone in methanol/ D2O solutions (∼32 s) and why the T1 relaxation time eventually decreases with increasing acenaphthenone concentration. We are aware that additional information is needed to confirm the proposed association mechanisms presented in this study of the interaction of fulvic acid with 13C-labeled acenaphthenone. We appreciate the interest our work has generated in this area and would like to thank Jayasundera et al. for presenting alternative mechanisms to explain the data.
Literature Cited (1) Nanny, M. A.; Bortiatynski, J. M.; Hatcher, P. G. Environ. Sci. Technol. 1997, 31, 530-534. (2) Bortiatynski, J. M.; Hatcher, P. G.; Minard, R. D. In Nuclear Magnetic Resonance Spectroscopy in Environmental Chemistry; Nanny, M. A., Minear, R. A., Leenheer, J. A., Eds.; Oxford University Press: New York, 1997; pp 26-50. (3) Schellenberg, K.; Leuenberger, C.; Schwarzenbach, R. P. Environ. Sci. Technol. 1984, 18, 652. (4) Lee, L. S.; Suresch, P.; Rao, R. S. C.; Brusseau, M. L. Environ. Sci. Technol. 1991, 25, 722. (5) Engebretson, R. R.; von Wandruszka, R. Environ. Sci. Technol. 1994, 28, 1934-1941. (6) Engebretson, R. R.; Amos, T.; von Wandruszka, R. Environ. Sci. Technol. 1996, 30, 990-997. (7) Herbert, B. E.; Bertsch, P. M. In Nuclear Magnetic Resonance Spectroscopy in Environmental Chemistry; Nanny, M. A., Minear, R. A., Leenheer, J. A., Eds.; Oxford University Press: New York, 1997; pp 73-90.
Mark A. Nanny, Jacqueline M. Bortiatynski, and Patrick G. Hatcher* Fuel Science Program 209 Academic Projects Building The Pennsylvania State University University Park, Pennsylvania 16802 ES972017W
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