Binding of Phenol and Differently Halogenated Phenols to Dissolved

Jun 17, 2009 - nonsubstituted (phenol (P)) and halogen-substituted (2,4- dichlorophenol (DCP); 2,4,6-trichlorophenol (TCP), and 2,4,6- trifluorophenol...
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Environ. Sci. Technol. 2009, 43, 5377–5382

Binding of Phenol and Differently Halogenated Phenols to Dissolved Humic Matter As Measured by NMR Spectroscopy ´, D A N I E L A Sˇ M E J K A L O V A RICCARDO SPACCINI, BARBARA FONTAINE, AND ALESSANDRO PICCOLO* Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali Universita` di Napoli Federico II, Via Universita` 100, 80055 Portici Italy, and Centro Interdipartimentale di Ricerca sulla Spettroscopia di Risonanza Magnetica Nucleare (CERMANU), Via Universita` 100, 80055 Portici, Italy

Received February 20, 2009. Revised manuscript received May 28, 2009. Accepted June 3, 2009.

1 H- and 19F-NMR measurements of spin-lattice (T1) and spin-spin (T2) relaxation times and diffusion ordered spectroscopy (DOSY) were applied to investigate the association of nonsubstituted (phenol (P)) and halogen-substituted (2,4dichlorophenol (DCP); 2,4,6-trichlorophenol (TCP), and 2,4,6trifluorophenol (TFP)) phenols with a dissolved humic acid (HA). T1 and T2 values for both 1H and 19F in phenols decreased withenhancingHAconcentration,indicatingreductioninmolecular mobility due to formation of noncovalent interactions. Moreover, correlation times (τC) for different hydrogen and fluorine atoms in phenols showed that anisotropic mobility turned into isotropic motion with HA additions. Changes in relaxation times suggested that DCP and TCP were more extensively bound to HA than P and TFP. This was confirmed by diffusion measurements which showed full association of DCP and TCP to a less amount of HA than that required for entire complexation of P and TFP. Calculated values of binding constants (Ka) reflected the overall NMR behavior, being significantly larger for DCP- and TCP-HA (10.04 ( 1.32 and 4.47 ( 0.35 M-1, respectively) than for P- and TFP-HA complexes (0.57 ( 0.03 and 0.28 ( 0.01 M-1, respectively). Binding increased with decreasing solution pH, thus indicating a dependence on the fraction of protonated form (R) of phenols in solution. However, it was found that the hydrophobicity conferred to phenols by chlorine atoms on aromatic rings is a stronger drive than R for the phenols repartition within the HA hydrophobic domains.

Introduction Involvement of humic acids (HAs) in the transport and mobilization of aromatic contaminants has been since long recognized (1). The association of aromatic hydrocarbons with HA can significantly increase their apparent solubility, that in turn may greatly influence their bioavailability, toxicity, and biodegradation. Therefore, understanding how aromatic contaminants interact with dissolved HAs is fundamental * Corresponding author phone: +39 081 253 91 60; fax: +39 081 253 91 86; e-mail: [email protected]. 10.1021/es900559b CCC: $40.75

Published on Web 06/17/2009

 2009 American Chemical Society

for the development and application of remediation strategies in aquatic and terrestrial environments (2-4). Humic substances are supramolecular systems (5), consisting of self-assembled relatively small and heterogeneous molecules of both aromatic and aliphatic origin, possessing a variety of functional groups, including COOH, phenolic, alcoholic, and enolic OH, quinone, hydroxyquinone, lactone, ether, alkane, and alkene (6). Depending on chemical functionalities of contaminants, the noncovalent binding of aromatic contaminants to HA involve hydrophobic interactions, hydrogen bonds, and pH-dependent electrostatic interactions (7). In addition, molecular recognition of aromatic hydrocarbons will also rely on the complementarity of their size and shape with the HAs assemblies (8). Since individual nuclei are very sensitive to their chemical surroundings, direct observation of interactions at molecular level can be obtained by NMR spectroscopy (9, 10). Among the limited number of NMR studies devoted to the association of aromatic hydrocarbons with HAs, most of them exploited the changes in the contaminant relaxation properties. In fact, lower molecular weight compounds, such as aromatic hydrocarbons, will have longer spin-lattice (T1) and spin-spin (T2) relaxation time as compared to HA. Therefore, in the absence of paramagnetic species, shortening of T1 and T2 can be attributed to the association of contaminants with HAs. For example, 2H T1 measurements of noncovalent interactions of HAs with phenol-d5, pyridine-d5, and benzened6 were related to the formation of π-π interactions between the monoaromatic ring of substrates and the HAs aromatic components, and the binding was found to be favored with increasing HAs aromaticity and decreasing solution pH (11). 1 H and 13C T1 measurements together with changes in line broadening were used to study the interactions of 1-naphtol and quinoline with HAs (12). This work indicated formation of strong and non specific associations of these aromatic compounds with HAs, and pointed out how other methods, such as dialysis or fluorescence quenching, greatly underestimated the binding to HAs. Upfield chemical shifts in 1H NMR spectra, together with shortened relaxation times and decreased diffusion coefficient were found to accompany the occurrence of π-π interactions between 2,4-dichlorophenol and HAs (13). Moreover, saturation-transfer doubledifference NMR spectra, that observe interactions in the form of an epitope map, indicated F and Cl halogen atoms substituted to an aromatic ring to play a dominant role in bindingwithHAsthroughhydrogenbondinganddipole-dipole interactions (14). The aim of this work was to investigate the interactions of HAs with nonsubstituted and halogen-substituted phenols, namely 2,4-dichlorophenol; 2,4,6-trichlorophenol and 2,4,6trifluorophenol, which are biotoxic chemicals of highly environmental concern (15). Due to their different substitution of halogens in the phenolic ring, they are recurrently reported to have different sorption to HAs (16, 17). The extent of binding was studied by both 1H and 19F NMR spectroscopy and evaluated by measuring chemical shift, T1 and T2 relaxation times, and diffusion coefficients. All NMR parameters were followed as a function of humic concentration. The affinity of variously substituted phenols toward HAs was expressed in the terms of binding constant.

Materials and Methods Humic Acid. A humic acid (HA) was isolated, as previously described (18) from a North Dakota leonardite (Mammoth Int. Chem. Co.). Briefly, 200 g of the parent material was VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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shaken overnight with 500 mL of a 1 M NaOH and 0.1 M Na4P2O7 solution. The HA was precipitated from the extracting solution by adding 6 M HCl until pH 1. Ashes were removed first by three cycles of dissolution in 0.5 M NaOH followed by flocculation in 6 M HCl and then by shaking HA twice in a 0.25 M HF/HCl solution for 24 h. HA was redissolved in 0.5 M NaOH and passed through a strong cation-exchange resin (Dowex 50) to eliminate remaining di- and trivalent metals. The eluate was precipitated at pH 1, dialyzed, and freezedried. After homogenization, 30 mg of HA were suspended in H2O, titrated to pH 7, and freeze-dried again. The 13C NMR CPMAS spectra of this HA indicated the presence of 8.3% of carboxyl C (190-165 ppm), 10.0% of phenolic C (165-140 ppm), 39.3% of aromatic/double bond C (140-107 ppm), 1.1% of di-O-alkyl C (107-90 ppm), 3.5% of O-alkyl C (90-64 ppm), 5.7% of methoxyl and N-alkyl C (64-45 ppm), and 32.0% of alkyl C (45-0 ppm) (13). Phenols. Phenol (P, CAS no. 108952); 2,4-dichlorophenol (DCP, CAS no. 120832); 2,4,6-trichlorophenol (TCP, CAS no. 88062), and 2,4,6-trifluorophenol (TFP, CAS no. 345806) were purchased, with purity ranging between 98 and 99%, from Sigma Aldrich (Germany) and were used without further purification. NMR Spectroscopy. NMR spectroscopy was conducted on a Bruker Avance 400 MHz instrument operating at a proton frequency of 400.13 MHz and equipped with a 5 mm Bruker multinuclear broadband observe (BBO) probe. Unless stated otherwise, phenol standards (1 mg) together with progressively larger amount (0-20 mg) of freeze-dried humic material were first added with 10 µL of CD3OD to ensure complete dissolution of phenols and then further dissolved in 1 mL of phosphate buffer at (pH7, 0.1 M NaOD + 0.1 M NaH2PO4), and transferred to NMR tubes. Since NMR spectra of the prepared phenols-HA solutions did not change after standing for several hours or several days, it was assumed that binding equilibrium was rapidly reached, and all samples were analyzed within 2 h after preparation. All NMR experiments were performed at 25.0 ( 0.1 °C. 1H NMR spectra were referenced to the chemical shift of solvent, that resonated at 4.7 ppm in all experiments, and were performed under saturation of the HOD signal, achieved with 54 dB attenuation of a 60 W amplifier for a period of 2 s. The 19F-NMR chemical shift scale was calibrated using 0.05% trifluorotoluene in CDCl3, resonating at -63.72 ppm. Relaxation NMR Spectroscopy. Prior to relaxation measurements, paramagnetic oxygen was removed by bubbling with nitrogen gas for 5 min, followed by a sonication for 15 min. Longitudinal (spin-lattice) relaxation time constants (T1) were obtained by using the inversion-recovery pulse sequence (180°-τ-90°-t)n, where n is the number of scans. A recycle time t of 200 s, and a set of τ delays ranging from 100 to 0.01 s were used to acquire T1 of phenol standards, while t was 2-50 s and τ delays ranged from 5 to 0.001 s for phenols added with HA. Transverse (spin-spin) relaxation time constants (T2) were measured using a Carr-PurcellMeiboom-Gill (CPMG) pulse sequence. A set of echo delays varying from 0.04 to 32 s were used for phenol standards, while phenols added with HA were acquired with echo delays from 0.001 to 5 s. Both T1 and T2 relaxation time constants were obtained using a nonlinear single exponential leastsquares fit to the resonance intensity. The fittings were calculated by Origin (MA, U.S.) version 6.1 software. Diffusion NMR Spectroscopy. 1H NMR diffusion-ordered (DOSY) spectra were obtained using a stimulated echo pulse sequence with bipolar gradients (STEBPGP), and a water gate 3-9-19 pulse train for water suppression. Scans (24-160) were collected using 2.0-2.5 ms sine-shaped pulses (4-5 ms bipolar pulse pair) ranging from 0.674 to 32.030 G cm-1 in 32 increments, with a diffusion time of 60-150 ms, and 8 K time domain data points. 19F DOSY spectra were acquired 5378

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using bipolar longitudinal eddy current delay with gradients (LEDBPGP) applying 24 scans, 2.0-2.1 ms sine-shaped pulses (4.0-4.2 ms bipolar pulse pair) ranging from 0.674 to 32.030 G cm-1 in 32 increments, diffusion time of 60-80 ms, and 16 K time domain data points. Diffusion coefficients (D) were calculated from monoexponential decays using Bruker Topspin 1.3 software. All D values were corrected for changes in solution viscosity (19).

Results and Discussion Chemical Shift. The 1H NMR signals of phenol (P), 2,4dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), and 2,4,6trifluorophenol (TFP) either with or without HA are shown in Figure 1. Upon HA addition, all detected proton signals were broadened and shifted upfield. As HA concentration increased, the extent of shifting and broadening was further enhanced, thereby indicating formation of noncovalent interactions between HA and phenols (13). Since upfield shifts result from an increased electronic shielding on H nuclei, these aromatic protons must have been included into the HA hydrophobic domains. Due to the high aromatic character of this HA (49.3%), the hydrophobic domain consists mainly of aromatic rings and thus it is likely that π-π interactions are the predominant noncovalent linkages involved in binding. The extent of upfield chemical shift varied among phenols (Figure 1). The largest chemical shift variation was noted for DCP (Figure 1). It is interesting to note that H3 and H5 protons showed slightly larger upfield shifts as compared to the H6 proton. This may be attributed to the deeper insertion of H3 and H5 in hydrophobic HA cavities, while H6, being spatially close to the phenol hydrophilic OH group, may be more exposed to hydration by surrounding water molecules or hydrophilic HA constituents. No such observation was made for the DCP + HA mixtures dissolved in nonbuffered aqueous solution (13), for which H3, H5, and H6 protons had similar chemical shifts. Significant upfield shifts were also observed for TCP (Figure 1), but to a lesser extent than for DCP. Smaller but similar upfield shifts were further noted for H2, H3, and H4 of P (Figure 1), followed by those of protons in TFP (Figure 1). Since the magnitude of chemical shift variation is indicative of the interaction strength, weaker binding to HA can be inferred for TFP and P as compared to TCP and DCP. Loose interactions between TFP and HA were further confirmed by the small chemical shifts variation noted in 19F NMR spectra (Figure 2). Similarly to proton signals, fluorine resonances became broadened, but were shifted downfield. A downfield chemical shift for 19F signals is in agreement with a previous study on inclusion of fluoromethylene into hydrophobic cavities of β-cyclodextrin (20). Such downfield shift may be explained by the relative decrease of H-bonds between TFP and water molecules that accompanies the disappearance of free TFP, as the phenol is progressively incorporated into HA hydrophobic cavities and π-π interactions are concomitantly formed. In fact, formation of H-bond is known to increase electron density around fluorine atoms, thus enhancing shielding and upfield shift for F nuclei. Although entrapment of TFP in HA hydrophobic domains should also lead to a shielding effect, the resulting upfield shift is less pronounced than that given by H-bonds. Thus, the overall effect is rather a downfield shift of bound TFP as compared to its free state. Nevertheless, the chemical shift change observed for F nuclei (Figure 2), indicates that binding of TFP to HA is rather an unfavorable process. Relaxation. Formation of noncovalent interactions restricts the molecular motion of bound species and is reflected by shorter relaxation times of the involved nuclei. Therefore, the comparison of T1 (spin-lattice) and T2 (spin-spin) relaxation times of individual nuclei in phenolic molecules,

FIGURE 1. 1H chemical shifts of 2,4-dichlorophenol (2,4-DCP); 2,4,6-trichlorophenol (2,4,6-TCP); phenol (P); and 2,4,6-trifluorophenol (2,4,6-TFP) with and without 1, 3, and 7 mg of HA. Upfield shifts are indicated with -, downfield shifts with +. before and after HA addition, provides additional information on the strength of noncovalent interactions (13). The 1H relaxation times of P, DCP, and TCP were drastically reduced in the presence of HA (Table 1). This reflects the enlarged line width observed in 1H spectra with increasing HA concentration that is accounted to both T1 and T2 relaxation processes. The shortening of T2 suggests that the energy transfer among aromatic protons became easier, and, thus, directly supports the occurrence of inclusion of these three phenols into HA hydrophobic domains. In these, the close spatial distance among protons of both phenolic and humic aromatic moieties considerably shortened relaxation times. An analogous finding, though to a lesser extent, was observed for 19F relaxation times of TFP (Table 1). However, the T2 of F4 was found to be extremely short (T2 ) 0.13 s) in respect to F2 (T2 ) 1.41 s). Such difference can be explained by the presence of scalar couplings for F4, which do not occur for F2 (21). In fact, while F2 is scalarly coupled to only one proton, F4 is coupled to two aromatic protons (H3 and H5).

Both T1 and T2 relaxation times in solution are a function of correlation time τC, that is defined as the time required for a solute molecule to return to its original state after moving through its allowed rotational and vibrational states (22). Thus, a larger correlation time corresponds to a slower motion. The molecular correlation times were calculated according to earlier works (21, 23) and are listed in Table 1. In agreement with previous indications, it was found that correlation times increased with enhancing HA concentration, indicating an entrapment of these phenols within humic domains. Morevover, the τC values observed for H3, H4, and H5 of P and for H3, H5, and H6 of DCP were slightly different without HA, but tended to become similar with progressive HA additions (Table 1). It thus appears that the complex anisotropic motion of these molecules in their free state was progressively changed into an isotropic molecular motion, when involved in noncovalent binding with HA. A correlation time increase was mostly evident for DCP and indicated that this halogenated phenol underwent the most rigid binding with HA, followed by TCP, and P. For TFP, only the F2 correlation time significantly increased with VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. 19F chemical shifts of 2,4,6-trifluorophenol with and without 1, 3, and 7 mg of HA. Upfield shifts are indicated with -, downfield shifts with +. enhancing HA concentration, thereby suggesting that this fluorine atom was more involved in binding to HA. However, the F4 correlation time was significantly affected by the very short T2 value of free TFP. Despite this, and similarly to other phenols, the τC values of F2 and F4 became progressively equivalent with HA additions (Table 1), when TFP concomitantly developed isotropic molecular motions. Diffusion. When a small-sized substituted or unsubstituted phenol binds to HA, its diffusion constant should decrease due to the much larger molecular size and consequently slower diffusion of HA. In fact, when phenols are strongly bound to HA, they should both diffuse at the same rate, as they belong to the same supramolecular aggregate. Conversely, when their association is weak or negligible, diffusion coefficients of HA and phenols remain unchanged and different from each other. Here, we followed by DOSY-NMR spectroscopy the changes in diffusion when the variously substituted phenols were associated to progressively larger amount of HA. An example of overlaid DOSY spectra acquired for TCP is shown in Supporting Information (SI) Figure S1. The self-diffusion coefficient of TCP gradually decreased (i.e., log D became more negative) as HA concentration increased until TCP diffusion was similar to that of HA, thereby indicating a complete and strong complexation of TCP with HA. It should be noted that, concomitantly, the HA self-diffusion was slightly reduced with increasing HA concentration. This phenomenon is attributed to a further self-association of HA molecules, as previously shown (19). However, the change in HA diffusion was negligible in respect to diffusion changes of interacting phenols (13). Since the diffusion constant observed for phenols in this NMR experiment is a weighted average of diffusion for both bound and free state, the fraction of bound substrate can be determined by: F)

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Dsub,obs - Dsub,free Dcomplex - Dsub,free

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where Dsub,obs is the apparent (weighted average) diffusion constant for phenol, and Dsub,free is the diffusion constant of phenol without HA. Since Dcomplex is the diffusion of HA-phenol complex that is unknown and cannot be determined in practice, a suitable approximation is to substitute Dcomplex with DHA, because phenols have much smaller sizes than HA (24). The resulting amount of bound phenols in respect to HA concentration are plotted in Figure 3. Full complexation was observed only for DCP and TCP. However, while DCP was already completely bound to 5 mg of HA, full TCP complexation occurred only when HA was larger than 10 mg. Conversely, P and TFP binding occurred only beyond 5 mg of HA addition. The shapes of binding curves for P and TFP (Figure 3) showed a much weaker interaction, as compared to DCP and TCP. In fact, with 20 mg of HA, about 74% of P, and only 38% of TFP were bound to HA. In order to express and compare the complex stability with HA, association constants (Ka) had to be determined for each phenol. The exact derivation of Ka is reported in the SI, association values are summarized in Table 2. In agreement with previous discussions for chemical shift changes and relaxation times, the strongest complexation was observed for DCP, where Ka reached 10.04 ( 1.32 M-1. The complexation strength with HA was then decreased by 45% for TCP, 94% for P, and 97% for TFP. In fact, the derived values of Gibbs free energies of transfer (∆G°transfer ) -RTlnKa, Table 2) confirmed a spontaneous and overall energetically favorable interaction process only in case of DCP and TCP. Values of NMR-derived Ka for selected phenols at different pHs were obtained (Table 2). A similar complexation strength was found for DCP at pH 6 and 7, whereas the interaction of TFP increased slightly at the lower pH. The strongly interacting TCP at pH 7, showed no affinity to HA at pH 12. The different binding behavior of phenols can be explained by the magnitude of their fraction of protonated form (R) at different pHs (Table 2), as calculated from pKa of each phenol (25, 26). The slight different R values at pH 6 and 7 did not appear to significantly alter the DCP binding capacity. Conversely, the increasing affinity of TFP at pH 6 depends on the almost doubling of R, that implies both a larger H-donor capacity and greater hydrophobicity of TFP at this pH. The same mechanism accounts for the loss of TCP binding at pH 12, whereby this phenol is totally deprotonated (R ) 1.6 × 10-6) and fails to act as H-donor to complementary HA molecules. However, the lowest R values among phenols found for TCP at pH 7 indicates that its binding to HA is less due to its H-donor capacity than to the hydrophobicity conferred by the three chlorine atoms (25). Concomitantly, it must be noted that the low P affinity to HA at pH 7 cannot be explained only by its fraction of protonated form, that was the largest of all phenols (R ) 0.99), and it should have enhanced rather than decreased complexation with HA. Hence, it is likely that the OH group alone and its H-donor capacity is not sufficient to ensure binding of phenols to HA. Therefore, it is the substitution of chlorine atoms on aromatic ring that, by increasing phenols hydrophobicity, becomes mostly responsible for their repartition into the HA hydrophobic domains (25). This study has shown that 1H NMR relaxation and diffusion spectroscopy is a fast and versatile technique to quantitatively evaluate interactions of phenolic contaminants with humic compounds. The upfield chemical shifts of aromatic signals observed upon HA addition indicated noncovalent inclusion of phenols into HA hydrophobic domains. This incorporation was further shown by the broadening of aromatic proton signals, related to the enhanced correlation time, and thus to a restricted motion of aromatic molecules. The extent of the upfield shift and broadening was a good indication of the effective binding strength between phenols and HA. The

TABLE 1. T1 and T2 Relaxation and Correlation Time (τc) Values Calculated from 1H NMR Spectra of Phenol (P), 2,4-Dichlorophenol (DCP), and 2,4,6-Trichlorophenol (TCP), and from 19F NMR Spectra of 2,4,6-Trifluorophenol (TFP) as a Function of Humic Acid Concentration at 25°C humic concentration (mg mL-1) DCP, control DCP+HA 1.0 2.0 3.0 4.0 5.0 7.0

TCP, control TCP+HA 1.0 2.0 3.0 4.0 5.0 7.0

P, control P+HA 1.0 3.0 5.0 7.0

TFP, control TFP+HA 1.0 3.0 5.0 7.0

T1 (s)

T2 (s)

τc (ns)

H3

H5

H6

H3

H5

H6

H3

H5

H6

12.19

5.76

5.20

4.921

1.043

1.560

0.4

0.9

0.6

0.25 0.14 0.09 0.08 0.08 0.07

0.18 0.11 0.07 0.06 0.07 0.08

0.13 0.08 0.06 0.05 0.06 0.04

0.027 0.015 0.008 0.007 0.003 0.003

0.025 0.010 0.005 0.005 0.003 nd

0.024 0.009 0.005 0.004 0.003 nd

0.6 1.3 1.5 1.4 2.1 2.1

0.5 0.9 1.6 1.5 2.1 nd

0.4 0.9 1.4 1.4 2.0 nd

H3

H3

H3

17.75

11.894

0.2

0.44 0.31 0.17 0.13 0.11 0.10

0.057 0.047 0.021 0.015 0.014 0.011

1.1 1.0 1.2 1.2 1.2 1.3

H3

H4

H5

H3

H4

H5

H3

H4

H5

9.04

6.11

10.15

3.899

2.021

0.943

0.4

0.5

1.4

1.04 0.43 0.27 0.19

1.09 0.43 0.28 0.19

1.16 0.40 0.26 0.18

0.251 0.062 0.042 0.029

0.186 0.059 0.036 0.025

0.198 0.064 0.037 0.026

1.1 1.0 1.2 1.2

0.4 1.1 1.1 1.1

0.3 1.0 1.1 1.1

F4

F2

F4

F2

F4

F2

6.14

3.45

0.130

1.405

28.6

2.7

1.71 0.57 0.30 0.20

1.49 0.55 0.29 0.20

0.083 0.039 0.019 0.016

0.182 0.049 0.021 0.016

19.1 16.9 17.4 16.0

11.4 14.2 16.2 15.2

TABLE 2. Fraction of Acidic Form (r) of Phenolic Substrates, And Binding Constant (Ka), and Free Gibbs Energy (∆G°) for the Association of Phenol (P), 2,4-Dichlorophenol (DCP), 2,4,6-Trichlorophenol (TCP), and 2,4,6-Trifluorophenol (TFP) with Lignite Humic Acid at 25°C Observed in Phosphate Buffer (pH 7), Deuterated Water (∼pH 6) and 0.1M NaOD (pH 12)a pH

substrateb

rc

Ka (M-1)

∆G0(kJmol-1)

7

DCP TCP P TFP DCP TFP TCP

0.88 0.13 0.99 0.59 0.98 0.93 1.6 × 10-6

10.04 ( 1.32 4.47 ( 0.35 0.57 ( 0.03 0.28 ( 0.01 9.10 ( 1.06 0.42 ( 0.04 0.06 ( 0.00

-5.7 ( 0.8 -3.7 ( 0.3 1.4 ( 0.1 3.1 ( 0.2 -5.5 ( 0.6 2.2 ( 0.2 6.9 ( 0.3

6

FIGURE 3. Percentage of humic-associated phenols as a function of HA concentration. P is for phenol, DCP is for 2,4-dichlorophenol, TCP is for 2,4,6-trichlorophenol and TFP is for 2,4,6-trifluorophenol. different binding was further confirmed by DOSY experiments. In fact, determination of self-diffusion allowed evaluation of binding constants for phenols. While very weak interactions with HA resulted for P and TFP, formation of strong noncovalent complexes was observed for the much faster relaxing DCP and TCP. This finding was explained by

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a Ka values were calculated as indicated by eq 3 in the SI. b pKa: [P ) 9.95, DCP ) 7.85, TCP ) 6.19 (25)]; TFP ) 7.17. (26) Log Kow: P ) 1.44; DCP ) 3.09; TCP ) 3.67; TFP ) 2.24 (25). c R ) 1/(1 + 10pH-pKa) (25).

the preferential inclusion of the most hydrophobic chlorinated phenols within the HA hydrophobic domains.

Acknowledgments D.S. acknowledges a grant from the Italian Ministry of University and Research (MIUR) under the FISR programme. VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Supporting Information Available Investigation on relaxation and correlation times of P, TCP, DCP, TFP and their complexes with HA, amount of bound phenols, Job’s plot, and fitting to eq 2 and 3. This material is available free of charge via the Internet at http:// pubs.acs.org.

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