Diffusion ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) was applied to a number of fulvic (FA) and humic (HA) acids of different origin. Spectral separation achieved by DOSY based on diffusion coefficients (D), and correlated to molecular sizes by calibration standards, showed that carbohydrates had the largest molecular size in FA, whereas alkyl or aromatic components were the most slowly diffusing moieties in HA. At increasing concentrations, these components had invariably lower D values in DOSY spectra for all humic samples,thereby indicating an aggregation into apparently larger associations, whose increased hydrodynamic radius was confirmed by viscosity measurements. When humic solutions were broughtfrom alkaline to acidic pH (3.6), components diffusivity detected by DOSY increased significantly, suggesting a decrease of aggregation and molecular size. A general comparison of HA and FA molecular sizes was achieved by multivariate statistical analysis. While a larger extent of aggregation and disaggregation was observed for HA than for FA, no aggregation was detected, under similar conditions, for a true macropolymeric standard. Such difference in diffusion between a polymeric molecule and humic samples, is in line with the supramolecular nature of humic matter. The possible formation of humic micelles was also investigated by both changes of diffusivity in DOSY spectra and shift of 1H NMR signals. Except for HA of peat and soil origin, revealing a self-assembling in micelle-like structures at the 4 mg mL(-1) concentration, no other humic sample showed evidence of critical micelle concentration (cmc) up to 20 mg mL(-1). These results indicated that DOSY-NMR spectroscopy is a useful technique to evaluate components of different molecular size in natural humic superstructures.

1H- and 19F-NMR measurements of spin-lattice (T1) and spin-spin (T2) relaxationtimes and diffusion ordered spectroscopy (DOSY) were applied to investigate the association of nonsubstituted (phenol (P)) and halogen-substituted (2,4-dichlorophenol (DCP); 2,4,6-trichlorophenol (TCP), and 2,4,6-trifluorophenol (TFP) phenols with a dissolved humic acid (HA). T1 and T2 values for both 1H and 19F in phenols decreased with enhancing HA concentration, indicating reduction in molecular mobility due to formation of noncovalent interactions. Moreover, correlation times (tau c) 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 (alpha) 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 alpha for the phenols repartition within the HA hydrophobic domains.

1H NMR measurements of spin-lattice (T1) and spin-spin (T2) relaxation times and diffusion ordered spectroscopy (DOSY) were applied to investigate the association of 2,4-dichlorophenol (DCP) with a soil fulvic (FA-VICO) and humic acid (HA(-)-VICO), and a lignite humic acid (HA-LIG). The 1H T1 and T2 values of DCP were found to decrease with increasing humic concentration, indicating reduction in molecular mobility due to formation of noncovalent interactions. The increased shortening of relaxation times observed upon addition of HA suggested more extensive association of DCP with HA than with FA. The extent of binding was inferred from diffusion coefficients (D) which showed slower diffusion for bound DCP. At 1 mg mL(-1) DCP was completely bound by 4.1 and 5.8 mg mL(-1) of HA-VICO and HA-LIG, respectively, while full DCP association was not observed even up to 20 mg mL(-1) of FA. This was reflected by association constants (Ka): 3.1 +/- 0.3 M(-1) for FA-DCP, and 15.5 +/- 3.1 M(-1) and 11.0 +/- 1.2 M(-1) for HA-VICO and HA-LIG DCP complexes, respectively. The stronger binding to HA is attributed to their larger hydrophobic character enabling formation of stable hydrophobic domains to which DCP becomes associated in host-guest complexes. DCP complexation within humic hydrophobic domains was confirmed by upfield chemical shifts and signal line broadenings observed in 1H NMR spectra. Similar chemical shift variations for the three DCP aromatic protons further indicated pi-pi interactions, rather than H-bonding, as the main driving force for noncovalent association between DCP and dissolved humic substances. Relaxation and diffusion 1H NMR techniques provide rapid and accurate measurements of binding constants and thermodynamic parameters for host-guest complexes between environmental contaminants and natural organic matter.

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