Environ. Sci. Technol. 1997, 31, 530-534
Noncovalent Interactions between Acenaphthenone and Dissolved Fulvic Acid As Determined by 13C NMR T1 Relaxation Measurements 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
Non-covalent interactions between 13C-labeled acenaphthenone (13C-labeled in the carbonyl position) and Suwannee River fulvic acid in a methanol/D2O solvent have been examined using 13C NMR T1 relaxation measurements. The influence of solvents upon the non-covalent interactions were assessed by examining acenaphthenone in pure solvents of varying solvation capacity (chloroform, methanol, methanol/D2O). Interactions with fulvic acid were examined as a function of acenaphthenone and fulvic acid concentrations, fulvic acid counter-cation (H+ or Na+), and pH. In the presence of fulvic acid in a methanol/D2O solvent, three non-covalent interactions were identified: a weak sorption interaction between acenaphthenone and fulvic acid, an enhanced solubilization of acenaphthenone by fulvic acid, and an interaction between just the solvent and acenaphthenone. The enhanced solubilization is hypothesized to arise from fulvic acid forming hydrophobic regions that are predominantly solvated with methanol and have excluded water. Acenaphthenone in these hydrophobic regions displays similar behavior to when it is dissolved in pure methanol. The ability of fulvic acid to form hydrophobic regions was found to be dependent upon the identity of the fulvic acid counter-cation and upon pH.
Introduction It is well known that the apparent solubility of many hydrophobic compounds is enhanced by soluble organic matter such as dissolved humic and fulvic acids (1-5). This impacts the transport properties of many chemicals in the environment (6), which in turn influences biodegradation, and remediation strategies of these compounds. Accordingly, extensive efforts have been made to examine the chemistry of non-covalent interactions between dissolved humic substances and hydrophobic compounds (5, 7-11). Despite this, the chemistry of non-covalent interactions is not well understood on a microscopic scale. Nuclear magnetic resonance spectroscopy (NMR) has emerged as a technique that is able to monitor weak and non-covalent interactions among molecules by measuring changes in spin-lattice relaxation time (T1). Such measurements detect changes in the overall molecular tumbling of * Corresponding author telephone: 814-865-7838; fax: 814-8653075; e-mail address:
[email protected]. † Present address: School of Civil Engineering and Environmental Science, University of Oklahoma, 202 W. Boyd, Rm. 334, Norman, OK 73019.
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a small probe molecule as it interacts with other molecules (12-14). This tumbling motion includes all modes of molecular motion: translational, rotational, and vibrational. In an NMR experiment, T1 is the time required for an excited atom to lose its excitation energy to the surrounding lattice and is directly related to the rate of molecular motion. The T1 of a probe molecule usually decreases as it non-covalently interacts with other molecules, especially large, slower moving molecules. The inter- and intramolecular behavior of molecules in solution, as they participate in various types of non-covalent bonding, has been examined by 13C and 15N NMR. Using T1 measurements, non-covalent interactions, such as hydrogen bonding of carboxylic acids (15, 16), cyclic alcohols, methoxy derivatives, cycloalkanones (17, 18), phenols (19), anilines (19), aminobenzoic acids and substituted anilines (20), and aromatic and substituted aromatic compounds (21), with various solvents and solutes have been examined as a function of pH, ionic strength, and concentration. Likewise, 7Li and 13C NMR T measurements have been employed to charac1 terize and examine the structure of lithoisobutyrophenone aggregates in ether solvents (22). Molecular anisotropic motion of pyridine (23) and phenol and aniline (19) has been examined by measuring differences in the T1 values of the individual atoms, providing detailed information regarding non-covalent interactions. Measuring T1 values of probe molecules at environmentally pertinent concentrations is typically not feasible since these concentrations are usually below NMR detection limits. Bortiatynski and Hatcher (24) overcame this problem by using phenol 13C-labeled at the C-1 position. By measuring the T1 of the labeled carbon, the interaction of phenol with a humic acid was monitored. The concentration of humic-associated 13C-labeled phenol in solution demonstrated a linear relationship with the observed T1 relaxation time. Using a similar strategy, we report here a study of the non-covalent interactions between 13C-labeled acenaphthenone (13C-labeled at the carbonyl position) and Suwannee River fulvic acid. Acenaphthenone was examined because it is a microbial metabolic byproduct of acenaphthene (25). Acenaphthene is a polycyclic aromatic hydrocarbon (PAH) typically found in coal tars and is often present in hydrocarbon-contaminated sediments and soils (26). Acenaphthenone was also used because it has a higher aqueous solubility relative to acenaphthene. Therefore, it will be present in water at a greater concentration than acenaphthene as biodegradation proceeds. In solution, the fate and transport of acenaphthenone undoubtedly will be influenced by non-covalent interactions with dissolved humic and fulvic acids.
Experimental Section 13C-labeled
acenaphthenone (13C-labeled carbonyl) was synthesized by Dr. Sergey Selifonov (University of Minnesota). Fulvic acid was extracted from the Suwannee River by ultrafiltration and reverse osmosis methods (27) and prepared in the protonated form by Dr. E. M. Perdue (Georgia Institute of Technology). Methanol/D2O (55/45 vol) samples were prepared by adding milligram quantities of solid acenaphthenone (weighed with a Cahn 21 automatic electrobalance) and appropriate amounts of protonated fulvic acid stock solution to an exact volume of methanol in a 5-mm NMR tube. Following dissolution, an exact amount of D2O was added. Degassed samples were prepared in the same manner, except that methanol and D2O were bubbled with N2 gas before accurate amounts of acenaphthenone and fulvic acid were added to
S0013-936X(96)00391-4 CCC: $14.00
1997 American Chemical Society
TABLE 1. Values and Standard Deviations of Replicate T1 Relaxation Time Measurements samplea
T1 relaxation time (s)
acenaphthenone (2.550 mg/mL) (500 MHz NMR) acenaphthenone (2.500 mg/mL)b acenaphthenone (1.500 mg//mL)b fulvic acid (H+) (0.006 mg/mL)
15.605 ( 0.015 29.190 ( 0.313 39.424 ( 0.070
a Unless otherwise indicated, all samples were dissolved in a MeOH/ D2O solvent (55/45 by vol), and T1 relaxation time values were obtained with a 360-MHz NMR. b Each measurement was obtained with a different, re-made sample of the same composition.
the NMR tube. The lower limit of acenaphthenone detectability (0.5 mg/mL) was determined by the NMR spectrometer sensitivity while the upper limit (3.0 mg/mL) was determined by the solubility of acenaphthenone in a methanol/D2O (55/ 45 vol) solvent. Protonated fulvic acid was converted to the sodium form by treating with Amberlite IR-120 ion-exchange resin (Aldrich). Solution pH was measured directly in 5-mm NMR tubes with an Accumet pH meter 925 (Fisher Scientific) and a microcombination pH electrode (Lazar Research Laboratories, Inc.). Viscosity was measured at 28.5 °C with a CannonFenske viscometer, size 75. Deionized water was used for viscometer calibration. Spin-lattice relaxation times were measured using a standard inversion recovery pulse sequence with inverse gated decoupling at 303 K on a Bruker AMX 360-MHz or a Bruker AMX 500-MHz spectrometer. A recycle delay of 220 s and 42 scans per each of 10 variable delay times was used. The data were processed with no line broadening, and the resulting T1 values were obtained using a three parameter fit. Standard deviation of the fit never exceeded (0.03 s.
Results and Discussion Spin-lattice relaxation for a probe molecule can occur by several mechanisms including dipole-dipole, chemical shift anisotropy, scalar coupling, spin-rotation, and paramagnetic interactions. Chemical shift anisotropy relaxation comprises 95-100% of the total T1 relaxation mechanism of the 13Clabeled carbonyl carbon in acenaphthenone. This was determined by measuring spin-lattice relaxation times of acenaphthenone in methanol/D2O at different magnetic field strengths. Not only is the chemical shift anisotropy relaxation mechanism dependent upon molecular tumbling, it is inversely and quadratically related to the magnetic field strength (12-14). For an acenaphthenone sample (2.55 mg/ mL) in a methanol/D2O solvent with no fulvic acid present, the T1 values are 28.9 and 15.6 s at 360 and 500 MHz, respectively. Standard deviations of replicate T1 measurements are presented in Table 1. Due to the time required to obtain each T1 value (24-36 h, depending upon acenaphthenone concentration), only select samples were examined in duplicate. For the samples that were remeasured, the standard deviaiton was equal to or less than 1%. T1 Relaxation of Acenaphthenone in Various Solvents. Table 2 presents T1 data for the carbonyl carbon (C-1 carbon) of acenaphthenone in various solvents. T1 values decrease from chloroform to methanol to methanol/D2O (55/45), demonstrating that the overall molecular motion of acenaphthenone decreases in this solvent series. This decrease in T1 values going from chloroform to methanol occurs without a corresponding increase in solvent viscosity, illustrating that a reduction in overall molecular motion is due to a noncovalent interaction and not viscosity. Presumably, hydrogen bonding between acenaphthenone and solvent is the noncovalent interaction occurring. Minuscule changes in T1
FIGURE 1. T1 relaxation time of acenaphthenone as a function of acenaphthenone concentration and pH in a MeOH/D2O (55/45 vol) solvent.
TABLE 2. Acenaphthenone-Solvent Interactions solvent
T1 relaxation time (s)
viscosity (cP)a
CDCl3 CDCl3 (degassed) CD3OD (degassed) MeOH/D2O pH ) 5.80 pH ) 6.83 pH ) 6.83 (degassed)
46.300 44.510 40.339
0.522 0.522 0.521
28.492 29.423 30.800
1.440 1.430 1.430
a
Viscosity data obtained with a Cannon-Fenske viscometer.
values after degassing both chloroform and methanol/D2O (55/45) solvents demonstrate that dissolved oxygen does not contribute appreciably to the T1 relaxation process. Changes in pH of the methanol/D2O (55/45) solvent produces negligible changes in solvent viscosity and T1 values. Figure 1 presents T1 values as not only a function of pH but also a function of acenaphthenone concentration and illustrates that moderate pH changes do not influence noncovalent interactions between acenaphthenone and the methanol/D2O (55/45) solvent. The T1 is invariant at approximately 30 s. The fact that T1 remains constant over the entire acenaphthenone concentration range indicates that acenaphthenone is dissolved rather than aggregating. Incorporation of acenaphthenone into aggregates would greatly reduce the overall molecular motion of acenaphthenone, which would be detected by a dramatic decrease in T1. Spin-Lattice Relaxation of Acenaphthenone in Presence of Fulvic Acid (Sodium Ion Form). Figure 2 presents T1 values for acenaphthenone in the presence of Suwannee River fulvic acid in the sodium form. The T1 of acenaphthenone at all concentrations of acenaphthenone is indirectly related to the fulvic acid concentration. We interpret this as evidence that acenaphthenone is interacting non-covalently with the fulvic acid, causing a decrease in the overall molecular motion of acenaphthenone. This association is believed to be a weak hydrophobic sorption of the acenaphthenone to the fulvic acid or hydrogen-bond formation with fulvic acid. It is important to note that the observed T1 is an average value of the weakly sorbed acenaphthenone and the unassociated acenaphthenone. Thus, as the fulvic acid concentration is increased, a greater percentage of acenaphthenone molecules non-covalently interact, reducing the average overall motion of all the acenaphthenone molecules. This in turn causes a reduction in the measured T1 values. The trend toward lower T1 values at all concentrations of acenaphthenone indicates that the interaction is one that involves partitioning of the acenaphthenone between solvent and fulvic acid.
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FIGURE 2. T1 relaxation time of acenaphthenone as a function of acenaphthenone concentration and fulvic acid concentration (Na+) in a MeOH/D2O (55/45 vol) solvent.
FIGURE 4. Model representing the three non-covalent interactions between acenaphthenone and fulvic acid (H+) in a MeOH/ D2O (55/ 45 vol) solvent.
FIGURE 3. T1 relaxation time of acenaphthenone as a function of acenaphthenone concentration and fulvic acid concentration (H+) in a MeOH/D2O (55/45 vol) solvent. Solvent viscosity did not change upon increasing fulvic acid concentration, and pH changed only slightly, ranging from 6.83 in the absence of fulvic acid to 6.54 for a fulvic acid concentration of 0.180 mg/mL. Therefore, neither of these parameters are responsible for changing the T1 values. Spin-Lattice Relaxation of Acenaphthenone in Presence of Fulvic Acid (Protonated Form). Protonation of Suwannee River fulvic acid changes dramatically its interaction with acenaphthenone. Figure 3 illustrates that at low acenaphthenone concentrations (∼0.5 mg/mL), the T1 is indirectly related to the protonated fulvic acid concentration. Although this is similar to that observed with fulvic acid in the sodium form discussed above, the reduction in T1 is greater per unit of protonated fulvic acid. Because the measured T1 represents an average value for both the associated and unassociated acenaphthenone molecules, the greater reduction in measured T1 demonstrates that protonated fulvic acid molecules can non-covalently interact with more acenaphthenone molecules than can fulvic acid molecules in the sodium form. As with fulvic acid in the sodium form, it is hypothesized that this non-covalent association is a weak hydrophobic sorption of the acenaphthenone to the fulvic acid or the formation of hydrogen bonds with the fulvic acid as depicted in Figure 4a. Viscosity does not change upon increasing fulvic acid concentration, and the solution pH decreases from 6.83 in the absence of fulvic acid to 6.04 in the presence of 0.25 mg/ mL protonated fulvic acid. Figure 3 shows that for samples with acenaphthenone concentrations of approximately 1 mg/mL or more and fulvic acid concentrations of 0.0003 and 0.006 mg/mL, the T1
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relaxation time becomes greater than that of acenaphthenone alone (i.e., no fulvic acid present) in the methanol/D2O solution. The T1 relaxation time remains greater than 29.0 s for acenaphthenone concentrations of approximately 2 and 3 mg/mL, respectively. Since the predominant T1 relaxation mechanism is chemical shift anisotropy, an increase in the T1 of acenaphthenone indicates that the environment surrounding the acenaphthenone is such that the acenaphthenone is experiencing enhanced overall molecular motion. This is unexpected because any association of acenaphthenone with fulvic acid or incorporation into an aggregate would decrease T1. It is important to note that for acenaphthenone in the presence of a protonated fulvic acid concentration of 0.006 mg/mL, the maximum T1 is 39.1 s, nearly that of acenaphthenone in pure methanol. This suggests that acenaphthenone is present in regions consisting predominantly of methanol. We propose as the only logical explanation for the increased T1 that the fulvic acid forms hydrophobic regions that exclude D2O but concentrate methanol. The acenaphthenone preferentially partitions into the hydrophobic region and acts as though it were dissolved in methanol rather than associated with fulvic acid or the methanol/D2O solvent (Figure 4b). Although an alteration in 13C chemical shift might be expected to accompany an association with pure methanol, no such change was observed in the case of these experiments. The 13C chemical shift that is observed for the C-1 carbon in methanol/D2O is approximately 209.2 ppm, while that in methanol-d3 it is approximately 207.6 ppm. We have no explanation for the apparent lack of change in chemical shift as T1 changes in the associated acenaphthenone. This hypothesis is consistent with fluorescence quenching and fluorescence anisotropy behavior of probes in the presence of dissolved humic substances (28-31). Studies of the interactions between humic acids and various environmentally pertinent compounds such as naphthalene and
1-naphthol (28), difenzoquat and 1-naphthol (29), pyrene (30), and 2,5-diphenyloxazole (31) have demonstrated that humic substances have the ability to form hydrophobic regions and incorporate probe molecules. Also, this behavior was shown to be dependent upon pH, ionic strength, and humic acid concentration. Bertsch and Herbert (32) proposed a similar hypothesis using 19F NMR to examine changes in line width of fluorobenzene in the presence of either humic acids or surfactants. They suggested that the sorbed fluorobenzene exists within a three-dimensional cage-like environment created by the organic macromolecule. The size of the cage-like environment was suggested to be influenced by solution pH. In contrast to our results, which demonstrate enhanced molecular motion of the probe molecule while in the presumed hydrophobic region, fluorescence and NMR data presented above suggest that the overall molecular motion of the probe molecule is reduced. These contrasting observations are probably explained by the fact that the fluorescence and NMR experiments listed above were performed in water, while our experiments were performed in a methanol/ D2O solvent. The hydrophobic regions created by humic substances will, by their nature, exclude water in which any experiments performed in water as the only solvent will result in the formation of a hydrophobic region in which hydrophobic probe molecules will predominantly interact with the humic substance, presumably through hydrophobic sorption. In a methanol/D2O solvent, methanol will selectively enter into the hydrophobic region, forming a pocket of methanol in which the hydrophobic probe molecule will act as though it were dissolved, displaying enhanced molecular motion. Any additional acenaphthenone added reduces T1 values to that of acenaphthenone in a solvent without fulvic acids present. This behavior is seen for both the samples containing fulvic acid at concentrations of 0.006 and 0.0003 mg/mL. After the T1 relaxation time reaches a maximum of 39.50 and 31.80 s, respectively, the T1 relaxation time decreases to values near 29 s, the T1 relaxation time of acenaphthenone in the pure methanol/D2O solvent. We interpret this to indicate that the increased acenaphthenone concentration saturates the methanol solvent held in hydrophobic regions and excess acenaphthenone is forced to interact with the methanol/D2O solvent, giving it a lower T1 value (Figure 4c). At high fulvic acid concentrations (0.2500 mg/mL), no enhancement in T1 is observed. This suggests that the only non-covalent interaction observed is hydrophobic sorption or hydrogen bonding with the fulvic acid (Figure 3), which diminishes the T1. Presumably, there is enough fulvic acid present such that the addition of up to ∼3 mg of acenaphthenone does not saturate this interaction. From the results discussed above, three different noncovalent interactions of acenaphthenone were identified. The first is an initial interaction that reduces the overall molecular motion of the acenaphthenone, presumably by hydrophobic sorption or hydrogen bonding with fulvic acid. The second interaction enhances overall molecular motion and is hypothesized to result from the formation of solvated hydrophobic regions. Upon saturation of the second interaction, the third interaction occurs, which is one in which excess acenaphthenone interacts primarily with the solvent mixture and not the fulvic acid. Viscosity was constant for all these samples. Degassing the methanol/D2O solvent, which contained 0.006 mg/mL fulvic acid (protonated form) and either 1.162 mg or 2.098 mg of acenaphthenone, produced a negligible difference in T1 of 31.70 and 30.90 s, respectively. These results demonstrate that neither viscosity or dissolved oxygen influenced T1 of acenaphthenone. T1 Dependence upon Solvent pH. To test the dependence of T1 of acenaphthenone upon pH, a sample containing acenaphthenone and protonated fulvic acid concentrations
FIGURE 5. T1 relaxation time of acenaphthenone (1.162 mg/mL) as a function of pH in a MeOH/D2O (55/45 vol) solvent and with a fulvic acid (H+) concentration of 0.006 mg/mL.
FIGURE 6. Model representing the influence of pH upon the macrostructure of fulvic acid and the encapsulation capability of fulvic acid. of 1.550 and 0.006 mg/mL, respectively, was prepared. Its pH was varied from 4.31 to 7.36 with the addition of small aliquots of concentrated HCl or NaOH. Figure 5 illustrates the pH dependence of T1 for acenaphthenone. It is evident from these data that major changes in the non-covalent interactions between acenaphthenone and fulvic acid are occurring. As demonstrated previously in Figure 1, varying the pH of the methanol/D2O solvent in the absence of fulvic acid has very little, if any, effect upon the T1, and moderate pH changes do not affect the interactions between the solvent system and acenaphthenone. On the other hand, in the presence of fulvic acid, varying the pH causes dramatic changes in the measured T1, inducing a T1 maximum of 39.0 s at a pH of 6.10. We interpret this pH behavior as reflecting changes in the three-dimensional macrostructure of the
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protonated fulvic acid, which in turn changes the way in which fulvic acid interacts with acenaphthenone (Figure 6). Increasing pH deprotonates fulvic acid, ionizing it, and rendering it more soluble in the polar methanol/D2O solvent. As the fulvic acid becomes ionized, it expands and becomes unable to encapsulate hydrophobic regions (Figure 4c). Decreasing pH causes fulvic acid to become protonated, increasing its hydrophobicity, leading to the collapse of its three-dimensional order that allows the existence of hydrophobic regions. Acenaphthenone molecules that were encapsulated are now forced out to interact with the methanol/ D2O solvent and, therefore, have a slower overall molecular motion (Figure 4c). Additionally, increasing the hydrophobicity of fulvic acid probably enhances sorptive associations which, in turn, lowers the T1. It is apparent from these data that the existence of hydrophobic regions are highly dependent upon the pH of the solvent. Engebretson et al. (31) have observed similar behavior between 2,5-diphenyloxazole and humic acid as a function of pH in a pure aqueous solution. In a pH range from 6.2 to 9.8, they detected maximum association between 2,5 diphenyloxazole and humic acid at a pH of 6.2, which then decreased as pH rose. They proposed that, at a pH of 6.2, hydrophobic regions were present that contained 2,5-diphenyloxazole. Increasing pH deprotonated the humic acid macromolecule, and the repulsion of the resulting ionic charges caused it to adopt an elongated configuration, which in turn destroyed the hydrophobic region and caused a reduction in the interaction between 2,5 diphenyloxazole and humic acid. Implications. This study indicates by use of a new molecular probe technique that fulvic acids can form hydrophobic regions. These can trap an organic solvent such as methanol and then encapsulate hydrophobic compounds. Such behavior could have a direct impact upon understanding the hydrophobic pollutant transport chemistry occurring in aquatic environments influenced by contamination with organic solvents. Organic solvents (referred to as NAPLs) trapped in a hydrophobic pocket of dissolved humic substances can act as vehicles for the transport or dissemination of pollutants. The structure of the hydrophobic pocket and its ability to encapsulate solvents and hydrophobic molecules is sensitive to changes in pH values commonly found in the environment. Therefore, slight changes in groundwater or soil water pH could have dramatic effects upon the interaction of hydrophobic compounds with natural organic matter. Thus, it is apparent that fulvic acid plays an important role in noncovalent interactions and, hence, the fate and transport of many compounds in the environment.
Acknowledgments We would like to thank Dr. Sergey Selifonov (University of Minnesota) and Dr. E. M. Perdue (Georgia Institute of Technology) for the gifts of 13C-labeled acenaphthenone and Suwannee River fulvic acid, respectively. We also wish to thank Dr. Alan Benesi (Penn State University) for analyzing samples on the 500-MHz NMR and Ms. Tara Sewell for assistance in using viscometers. Likewise, we want to thank Dr. L. M. Jackman (Penn State University, Emeritus) for his helpful discussions and comments. Finally, we gratefully acknowledge the Office of Naval Research for their financial support (Grant N00014-95-1-0209).
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Received for review May 3, 1996. Revised manuscript received September 24, 1996. Accepted September 27, 1996.X ES960391A X
Abstract published in Advance ACS Abstracts, December 15, 1996.
