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Comment on “Noncovalent Interactions between Acenaphthenone and Dissolved Fulvic Acid As Determined by 13C NMR T1 Relaxation Measurements” SIR: The recent contribution by Nanny et al. (1) presented some new data and observations regarding the use of 13C NMR T1 relaxation measurements to determine noncovalent interactions of acenaphthenone with fulvic acid (FA). While this paper provides a very good example of the use of NMR as a powerful molecular probe technique, we would like to make several comments regarding their interpretation of the results. Nanny et al. (1) have determined that chemical shift anisotropy (CSA) was the dominating relaxation mechanism for acenaphthenone in MeOH/D2O. Dipole-dipole (DD) relaxation generally dominate for 13C nuclei in organic molecules with protons and in the presence of unpaired spins, such as dissolved oxygen. In this study, by selecting only the 13C-labeled carbonyl of the acenaphthenone molecule, which has no protons attached, the DD relaxation effect is minimized, and thus CSA dominates. The minimal effects of dissolved oxygen on T1 relaxation observed can also be due to the selected labeled site in their molecule. Thus, we believe that their interpretation should be based on the labeled site rather than the molecule as a whole. In Figure 2, Nanny et al. (1) show that the T1 relaxation of acenaphthenone, at all concentrations, was reduced by approximately the same ∆T1 as the FA-Na+ concentration was increased. They concluded that partitioning of the acenaphthenone between the solvent and the FA-Na+ was occurring. A similar behavior in T1 relaxation was observed at low acenaphthenone concentrations (∼0.5 mg/mL) when FA-H+ was present and was also indirectly related to the FA concentration. However, the ∆T1 was greater than that observed in the case of FA-Na+. The authors explained this behavior as being due to more acenaphthenone molecules interacting with the FA-H+. This can also be explained using the following model presentation: FA-H+ is more hydrophilic in nature than FA-Na+ and may cause the orientation of acenaphthenone to change at the surface, to favor hydrophilic interactions, and in turn may induce more CSA relaxation of the carbonyl (13C-labeled position of the molecule).
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As the acenaphthenone concentration is increased (∼1 to 2.5 mg/mL) at FA-H+ concentrations of 0.0003 and 0.006 mg/mL, the T1 becomes greater than that of acenaphthenone by itself, indicating an increase in overall molecular motion (Figure 3). The explanation given by the authors for this increase in T1 is that FA-H+ forms hydrophobic regions that exclude D2O but concentrate MeOH and that acenaphthenone preferentially partitions into these hydrophobic regions and acts as dissolved in MeOH. This explanation was given based on the fact that, in the presence of 0.006 mg/mL of FA-H+, the maximum T1 of acenaphthenone was 39.1 s, which was approximately that of acenaphthenone in CD3OD (40.3 s). However, this interpretation of the behavior of acenaphthenone by Nanny et al. (1) is not completely supported by the data presented due to the following reasons: (1) The quadrapolar relaxation rate of acenaphthenone in deuterated methanol (CD3OD) is very different from dipolar rates in non-deuterated methanol (MeOH). Hydrogen bonding in methanol can be measured by changes in dipolar relaxation only if MeOH or CD3OH is present. In the solvent mixture MeOH/D2O (55/45 v/v), rapid exchange occurs between the solvents MeOH + D2O, yielding equal weights of MeOD + HOD. Frequencies and relaxation times due to individual solvent components (MeOH, HOD, and MeOD) are indistinguishable on the NMR time scale. From the results presented, we cannot assume the spartial resolution of MeOH, HOD, and/or MeOD nor that an individual solvent component is effecting the relaxation. (2) The chemical shift of the carbonyl-C in MeOH that would indicate binding (i.e., a solvent related change in the observed chemical environment of the carbonyl group) was not observed. (3) Localized hydrophobic regions would reduce molecular motion and hence reduce T1 (2-4) but not increase as proposed by the authors. (4) No supporting evidence is presented to verify that different regions of localized ordering of small molecular weight solvents (like MeOH in mixture with D2O) occurs on surface structures (like acenaphthenone) and/or can be observed on the NMR time scale. Furthermore, as the concentration of acenaphthenone increased after the T1 relaxation reached a maximum of 39.5 and 31.8 s in FA-H+ at concentrations of 0.006 and 0.0003 mg/mL, respectively, the T1 reduced and was below that of acenaphthenone in MeOH/D2O (29.0 s) beyond ∼2.7 mg/mL (acenaphthenone concentration). The authors, however, consider the T1 to be “invariable” around 29.0 s (although Figure 3 shows a reducing trend). They explain this behavior as the saturation of the MeOH in the hydrophobic regions, by acenaphthenone, and forcing out the excess acenaphthenone to interact with the MeOH/D2O solvent. This interpretation lacks support from experimental evidence. We would like to propose other possible explanations for the observed behavior of acenaphthenone in FA-H+. If the hydrophobicity of the bulk FA-H+ was low and the FA-H+ was present at low concentrations, then as the acenaphthenone concentration increases, a lesser number of interactions at the carbonyl-C will be observed, indicating an increase in the relaxation times. In the pH-dependent study of acenaphthenone (1.162 mg/mL) association with FA (0.006 mg/mL) in a MeOH/D2O solvent, it is shown that the T1 of acenaphthenone varied with pH and reached a maximum of ∼39.0 s around a pH of 6.1 and decreased to a value of ∼32 s around pH 6.85 (Figure 5). Although the pH of the FA-H+ at a concentration of 0.006 mg/mL in the acnaphthenoneMeOH/D2O system discussed in Figure 3 has not been reported, the behavior of acenaphthenone in this figure may
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1997 American Chemical Society
be similar to that observed in Figure 5. At low acenaphthenone concentrations (∼
