Relationship between Photosensitizing and Emission Properties of

May 14, 2009 - These reactive species include oxidant triplet excited states (HS*), singlet ... Humic acid (HA) was extracted from an Irish Sphagnum p...
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Environ. Sci. Technol. 2009, 43, 4348–4354

Relationship between Photosensitizing and Emission Properties of Peat Humic Acid Fractions Obtained by Tangential Ultrafiltration LUCIANO CAVANI,† S A B R I N A H A L L A D J A , ‡,§ A L E X A N D R A T E R HALLE,‡ GHISLAIN GUYOT,‡ G I A M P I E T R O C O R R A D O , †,‡ CLAUDIO CIAVATTA,† A. BOULKAMH,§ A N D C L A I R E R I C H A R D * ,‡ Dipartimento di Scienze e Tecnologie Agroambientali, Alma Mater Studiorum-Universita` di Bologna, Viale Fanin 40, 40127 Bologna, Italy, Laboratoire de Photochimie Mole´culaire et Macromole´culaire, UMR no. 6505 CNRS-Universite´ Blaise Pascal, 63177 Aubie`re Cedex, France, and Laboratoire des Sciences et Techniques de l’Environnement, Universite´ Mentouri, 25000, Constantine, Alge´rie

Received October 27, 2008. Revised manuscript received February 6, 2009. Accepted March 3, 2009.

Peat humic acid was fractionated by tangential ultrafiltration into six nominal molecular weight (NMW) fractions, HA5-10, HA10-20, HA20-50, HA50-100, HA100-300 and HA>300, which were purified by dialysis using a 0.5 kDa membrane. The absorbing and emission properties of the separated fractions were compared and their ability to generate singlet oxygen under light excitation was evaluated, using furfuryl alcohol (FFA) as a singlet oxygen scavenger. The absorbance, the emission intensity, and the apparent first order rate constants of FFA loss were normalized per mole of organic carbon (a*, IF*, and k*, respectively). The fraction absorbance decreased with NMW, except for HA>300 which was less absorbing than HA100-300. The low NMW fractions and the HA>300 fraction generally showed lower k* and IF* values compared to the HA50-100 and HA100-300 fractions. A plot of k* versus IF* indicates that the first order rate constant of FFA photo-oxygenation increased with the intensity of fluorescence at 380, 430, and 500 nm (R2 ) 0.77-0.84). This shows that the distribution of fluorescent centers among fractions paralleled that of photosensitizing centers. Plotting k* or IF* versus a* at 365 nm reveals the apparent relative quantum efficiency of the different fractions. Higher values for low NMW fractions and HA50-100 are either due higher percentages of absorbing centers able to produce singlet oxygen or exhibit fluorescence or to lower quenching processes.

Introduction Humic substances (HS) constitute a large portion of the total organic carbon (TOC) pool in terrestrial environments. They are mainly produced through vegetation decomposition. * Corresponding author e-mail: [email protected]; tel: +33473407142; fax:+33473407700. † Dipartimento di Scienze e Tecnologie Agroambientali. ‡ Laboratoire de Photochimie Mole´culaire et Macromole´culaire. § Laboratoire des Sciences et Techniques de l’Environnement. 4348

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They play an important role in soil fertility and in the transport and reactivity of environmental contaminants (1). In particular, their properties arising from light absorption are essential as far as the capacity of the environment to degrade organic pollutants is concerned. HS are brown-colored compounds and show interesting properties when they interact with solar radiation. Under UV-visible light excitation, HS generate reactive species which are able to degrade organic chemicals (2-5). These reactive species include oxidant triplet excited states (HS*), singlet oxygen (1O2), hydroxyl radicals (OH · ), and peroxyl radicals (RO2 · ) ((6) and references therein). The nature of the photosensitizing chromophores from which reactive species are produced remains unknown so far. Elucidating the structure of sensitizing humic constituents would be of real interest from a fundamental and a practical point of view. The isolation of HS via a simple acid/base extraction procedure yields base-soluble and acid-insoluble humic acids (HA) and soluble fulvic acids (FA) (7). These two soluble fractions are a noncharacterized complex mixture of molecules whose molecular weight (Mw or size) varies over a wide range. Using fractionation, HA and FA can be further divided into subfractions or pools of molecules having smaller weight or size ranges than the bulk HSs (8). Fractionation, which provides more homogeneous fractions, might help to elucidate the photochemical properties of HS. Fractionation of HAs can be achieved using different techniques. Ultrafiltration and size-exclusion chromatography are the most frequently used fractionation methods for preparative purposes. It has generally been found that structural and spectral properties varied among fractions, regardless of the fractionation technique employed (9-16). Francioso, 2002 (14)and Li, 2004 (12)characterized the chemical, structural, and molecular properties of the HAs fractions obtained using ultrafiltration. As the molecular cutoff of the ultrafiltration HA fractions was increased, the apparent Mw increased accordingly. The chemical, NMR, and Py-GC-MS analyses showed that the alkyl hydrophobic components were mainly distributed in the largest molecular-size fractions, while phenolic and oxygen-containing groups were predominant in the low nominal molecular weight fractions. In this study, we fractionated a peat-extracted HA by ultrafiltration and investigated the fractions using excitation emission matrix (EEM). We also determined the ability of the fractions to photosensitize the photo-oxygenation of a probe molecule. Our goal was to determine the distribution of fluorophores and photosensitizing components among the fractions and to determine whether these two properties are connected to each other.

Experimental Section Material. Humic acid (HA) was extracted from an Irish Sphagnum peat by the protocol described in the Supporting Information and ref 17. The HA was dissolved with 0.5 M NaOH and fractionated using the tangential ultrafiltration (UF) technique (14). Six fractions with different NMW were obtained using a UF device with cellulose regenerated filters. Finally, each fraction was dialyzed (0.5 kDa cutoff) against deionized water and then freeze-dried. We used the following NMW cut-offs: 5.0-10 kDa (HA5-10); 10-20 kDa (HA10-20); 20-50 kDa (HA20-50); 50-100 kDa (HA50-100); 100-300 kDa (HA100-300); > 300 kDa (HA>300). Furfuryl alcohol (FFA) was obtained from Aldrich (99%). The stock solutions (10-3 M) were stored at 4 °C and the furaldehyde level was checked daily by UV absorption. Water was purified using a Millipore Milli-Q system (Millipore RQ, resistivity 18 MΩcm, DOC < 10.1021/es802964m CCC: $40.75

 2009 American Chemical Society

Published on Web 05/14/2009

TABLE 1. Recovery of Peat Fractions and Spectroscopic Data and Kinetic Parameters for the Photosensitized Photooxygenation of Furfuryl Alcohol by Various Humic Substances sample

recovery /%

Peat HA Elliott HA Peat FA HA>300 HA100-300 HA50-100 HA20-50 HA10-20 HA5-10

54 20 14 12 0.1 0.07

a*280/L (mol OC)-1 cm-1

a*365/L (mol OC)-1cm-1

A2/A3

IF* at 500 nm a.u.

k*/10-2 s-1 (R2) 4.17 (0.998) 10.8 (0.995) 3.39 (0.996)

778 850 322

humic substances 305 410 98

3.09 2.25 4.13

75.3 114 69

777 967 507 263 234 200

fractions 305 366 171 86 70 60

2.98 3.07 3.38 3.13 3.20 3.20

28.4 53.2 65.6 20.6 21.1 20.8

0.1 mg/L). The reference Elliott soil HA was purchased from IHSS (http://www.ihss.gatech.edu/). Preparation of Solutions and Methodologies. The stock solutions of FA, HAs, and fractions were prepared by precisely dissolving weighed amounts of humic material in Milli-Q water overnight at 20 °C under an air atmosphere (20 mg/L for the peat HA, Elliott HA, HA50-100, HA100-300, and HA>300 fractions and 40.0 mg/L for the HA5-10, HA10-20, and HA20-50 fractions and peat FA). The stock solutions were adjusted to pH 6.5 using a phosphate buffer (10-3 M). The solutions were then filtered on 0.45 µm carbonate filters. Portions of these filtered solutions were directly used for the organic carbon (OC) measurement and for UV-visible analysis. The other part was diluted for excitation emission matrix (EEM) analyses and photochemical experiments. EEMs were recorded for solutions adjusted by dilution to an absorbance of 0.1 at 300 nm (1-cm path length). The corresponding organic carbon concentrations were 7.5, 6.4, 5.2, 2.9, 1.5, 1.9, 1.9, 4.8, and 1.6 mg L-1 ((5%) for HA5-10, HA10-20, HA20-50, HA50-100, HA100-300, HA>300, peat HA, peat FA, and Elliott HA, respectively. For the photochemical experiments, the filtered solutions were diluted to an absorbance of 0.2 at 300 nm and were added to a 2 × 10-4 M FFA stock solution (in ultrapure water) to create a final FFA concentration of 10-4 M and an absorbance of 0.1 at 300 nm. The final solutions were used for analyses or photochemical experiments right after the preparation. Methods. The elemental analyses on solid samples were performed using a Carlo Erba model EA 1108 elemental analyzer apparatus. The organic carbon measurements on the aqueous solutions were performed using a Shimadzu 500 TOC analyzer. Calibration UV-visible absorption spectra were recorded on a Cary 3 (Varian) spectrophotometer. Milli-Q purified water was always used as the reference. The molar absorption coefficient, a*, was obtained by dividing the absorbance by the path length and the organic carbon concentration. EEMs were recorded on a Perkin-Elmer LS55 luminescence spectrometer that was equipped with a xenon excitation source. Details are given in the Supporting Information. EEMs were normalized to the organic carbon concentration. Excitation at 300 nm was chosen for fluorescence spectra because this wavelength corresponded to the emission spectrum of the lamps used for the photosensitizing experiments and also produced workable data. Excitation at 365 nm would have been better for the comparison of emission and photosensitizing properties of the fractions, but, unfortunately, the intensities of fluorescence were too low under that condition. The EEMs of irradiated solutions of peat HA and fractions were also recorded. The absorbance of solutions was initially set at 0.1 at 300 nm for those experiments; the solutions were directly analyzed after irradiation without any dilution.

3.01 4.46 3.85 1.36 1.30 1.30

Irradiation Experiments. The air-saturated solutions containing FFA and HS were irradiated in a device equipped with six polychromatic tubes that emit within the wavelength range 300-450 nm (maximum emission at 365 nm). The reactor was in Pyrex glass. Fifteen mL of these solutions were used in the experiments. Aliquots (500 µL) were removed at selected intervals and immediately analyzed by HPLC. Irradiation proceeded until the FFA reached a conversion extent between 10 and 20%. During the irradiation, solutions were re-equilibrated in air periodically in order to maintain a constant oxygen (O2) concentration. The FFA losses were monitored by HPLC using a Waters apparatus equipped with a 717 autosampler, two 515 pumps, and a 996 photodiode array detector. The reverse phase column was a C18 of dimensions 5 µm, 250 mm × 4.6 mm (Nucleodur, MachereyNagel). A flow rate of 1 mL/min was used for all analyses and the eluent was a mixture of 15% methanol and 85% water acidified with orthophosphoric acid (0.1%). The experiments and the HPLC analyses were carried out in duplicates. The irradiated solutions were also analyzed by UV-vis and fluorescence spectroscopies.

Results and Discussion Elemental Analyses. The fraction of recovery drastically decreased with NMW, ranging from 54% for HA>300 to 12% for HA20-50, down to less than 0.1% for HA5-10 (see Table 1). Results of elemental analyses are given in Supporting Information. Fractions composition in C, N, and O fell within the ranges 41-50%, 0.8-2.2%, and 29-36%, respectively. The OC measurements on the filtered aqueous solutions were in good agreement with the data measured on solid samples, taking into account that samples contained about 10% water. Variations among the fractions were generally quite small except for the H/C ratios with NMW as observed by ref 14, who found a higher concentration of methyl and methylene group in high than in low NMW fractions. UV-Visible Spectroscopy. The absorption spectra are given in Supporting Information. The absorbances drastically decreased with NMW except for HA>300, which exhibited absorbances between those of HA100-300 and HA50-100. Table 1 displays the molar absorption coefficients at 280 nm. HA100-300 and HA>300 gave values within the range of HAs (920 ( 80 L (mol of OC)-1 cm-1 for Aldrich HA and 850 ( 80 (mol of OC)-1 cm-1 for IHSS Elliott HA, for instance). HA20-50, HA10-20, and HA5-10 gave values lower than 310 ( 30 L (mol of OC)-1 cm-1 and were thus closer to the values of river fulvic acids (18) than to those of HAs. HA50-100 had an intermediate value. The shape of the absorption spectra, which can be evaluated based on the absorbance ratios, is another important metric for comparison. Table 1 lists the A2/A3 ratio, corresponding to the A254 to A365 ratio. The value of the ratio of the peat fractions varied within a narrow VOL. 43, NO. 12, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. EEM of peat HA, fractions HA>300, HA100-300, HA50-100, HA20-50, HA10-20, and HA5-10, normalized to per mole of organic carbon. range (2.98-3.38) and even though they were higher than the value of IHSS Elliott HA (2.25), they were much lower than the corresponding value of peat FA (4.13). The similarity of A2/A3 suggests that all the fractions contained the same type of absorbing centers. The absorbance differences can be thus explained by differences in the concentrations of the absorbing centers. Recent literature reports propose that the coloration of humic substances is partly due to the delocalized electrons in complexes produced via the π-π interactions between electron-donating aromatic constituents and electron-attracting aromatic constituents (19, 20). To determine whether fractionation has induced modifications in the absorbance and these charge transfer complexes, we compared the weighted sum of individual fraction absorbances and fraction 4350

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A2/A3 ratios to the absorbance and A2/A3 ratio of peat HA. Using the percentage of recovery for each fraction and the values of a*280 and A2/A3 for each fraction (see Table 1), the weighted sum absorbance at 280 nm was 720 L (mol OC)-1 cm-1 compared with 778 in the case of peat HA, and the weighted sum of A2/A3 ratio was 3.07 compared with 3.09 for peat HA. These results show that fractionation did not affect the possible formation of charge transfer complexes. It was recently reported that the absorbance at 350 nm of terrestrial humic substances from various sources and isolation procedures is positively correlated with the aromatic carbon (mg aromatic carbon L-1 ) 0.0710 × a350 + 0.1646 for 5 mg L-1 of HS) (20). Applied to our case, one gets that the concentration of aromatic carbon would range from 0.78 mg L-1 for HA100-300 to 0.27 mg L-1 for HA5-10.

FIGURE 2. EEM of peat HA before and after 8 h of irradiation using polychromatic light (300-450 nm) in the absence or presence of FFA (10-4 M).

FIGURE 3. Plots of k* vs a*365 and IF*500 vs a*365 for peat HA and FA, peat fractions, and Elliott HA.

Fluorescence Spectroscopy. The EEM spectra of peat HA and fractions normalized to the organic carbon are presented in Figure 1 and the emission spectra recorded upon excitation at 300 nm are presented in the Supporting Information. The spectrum of peat HA showed a broad emission band extending from the excitation wavelength (280-500 nm) to 600 nm. The maximum of emission was located around 430 nm, but several shoulders were also visible at 360, 380, 400, 500, and 550 nm. This indicates the contributions of different types of fluorophores to the global spectrum. Even though the low NMW fractions (HA5-10, HA10-20, HA20-50) emitted much less than peat HA, maxima with varying intensities appeared nonetheless. In particular, the emission band around 380 nm was more intense in HA10-20 than in the other low NMW fractions. The high NMW fractions (HA50-100, HA100-300, HA>300) emitted more intensively than the low NMW fractions. HA50-100 and HA100-300 gave similar intensities of emission around 430 nm. While the emission from HA100-300 was higher than from HA50-100 below 430 nm, the former emitted less intensely between 440 and 600 nm. HA>300 emitted much less intensely than the other two high NMW fractions. To summarize, even though the fractions seemed to contain the same fluorophores as peat HA, their emission intensities varied among fractions. The intensity variations were either due to a distinct distribution of fluorophores among the fractions or, in the case where the fluorophores were randomly distributed among fractions, to the existence of deactivation processes in only some of the fractions. The weighted sum of individual fraction fluorescence intensity can be compared to the fluorescence intensity of peat HA, as in the case for absorbance. Using the emission spectra obtained by excitation at 300 nm, we got that the weighted sum was generally significantly lower, by up to 50%. This indicates either the loss of many fluorescent centers during fractionation or that deactivation processes were favored in fractions compared to peat HA. It is quite difficult to compare our results with those in the literature because the previously reported data are not always normalized to the organic carbon. However, there is generally a good agreement between the values obtained VOL. 43, NO. 12, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Variations of IF*500 and k* for peat HA and fractions. here and those for UV absorbing and fluorescent carbon of the same size fractions in the literature (10, 11, 15, 22). Ability to Sensitize the Photo-Oxygenation of FFA. The ability of the fractions to generate singlet oxygen upon light excitation was investigated using FFA as a singlet oxygen scavenger (23). The experiments were conducted at the same absorbance (0.1 at 300 nm) for all samples. FFA (10-4 M) negligibly disappeared when irradiated alone. In contrast, when irradiated in the presence of HAs, peat FA, or peat HA fractions, 10-20% of FFA disappeared after 8 h. Singlet oxygen produced by the excitation of the humic substances either deactivates or reacts with FFA (24, 25). The concentration of FFA used here was low enough for deactivation to be the main reaction pathway of the singlet oxygen. Thus, the rate of FFA loss must be proportional to [FFA]. The pseudo first order rate constant normalized per mole of organic carbon contained in HA samples (k*) was obtained by plotting ln [FFA]/[FFA]0 against the irradiation time. The values of k* and R2 (7 points) are given in Table 1. The low NMW fractions gave k* values of 1.3-1.4 × 10-2 s-1, while the high NMW fractions and peat HA and FA gave k* values in the range 3.0-4.5 × 10-2 s-1. Photosensitizing properties of fractions were previously studied by ref 25 on soil and aquatic HAs fractionated by gel permeation chromatography and by ref 13 on soil humic acids fractionated by SEC-PAGE in the presence of urea. In the former case, the rate of singlet oxygen production normalized to the OC did not vary significantly among fractions, while in the latter case, significant differences were found between fractions. In particular, low molecular weight fractions surpassed the others in their ability to photosensitize the photo-oxidation of a phenolic probe (13). Again, we compared the weighted sum of k* to the k* of peat HA. One found 3.4 × 10-2 s-1, against 4.2 × 10-2 s-1 for peat HA. This means that 20% of photosensitizing properties were lost by fractionation. As in the case of fluorescence, this result could be rationalized by an enhancement of deactivation processes in fractions. Effect of Irradiation on the Properties of Fractions. The irradiation of peat HA and the fractions over 8 h in the polychromatic device induced only a modest decrease in absorbance of between 5 and 10%. Nonetheless, the fluorescence was greatly affected by irradiation and all the samples showed similar behavior. Figure 2 displays the results obtained from peat HA. After 8 h of irradiation, the intensities of fluorescence decreased drastically and uniformly over the whole wavelength range. The loss was about 50%. 4352

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In contrast, the consumption profiles of FFA against the irradiation time did not show any visible variations, indicating that chromophores producing singlet oxygen are not affected by the irradiation. Examination of the EEM in Figure 2 reveals the presence of new emission bands upon irradiation: one located at 250/ 360 nm and another at 280/350 nm reminiscent to that previously observed by ref 26. HA was irradiated in the presence of FFA (10-4 M) in another experiment, and a decrease in fluorescence was observed in the absence of any new emission band. This suggests that FFA inhibited the formation of the new emission band.

Discussion The data presented here demonstrate that the fractions obtained by ultrafiltration behaved differently in terms of emission properties and of capacity to generate singlet oxygen under irradiation. Assuming that k*, the first order rate constant normalized to the organic carbon, and IF*, the fluorescence intensy normalized to the organic carbon, are equal to: SO* k* ) ISO* ΦSOfFFA a ΦSOfFFA ) 2.3I0A F* IF* ) cteIF* a ΦF ) 2.3cteI0A ΦF

where IaSO* and IaF* are the photon fluence rate absorbed by the sensitizing and fluorescent chromophores per organic carbon, ΦSO and ΦF are the quantum yields of singlet oxygen formation and of fluorescence, fFFA is the fraction of singlet oxygen trapped by FFA at the chosen concentration, I0 is the photon fluence rate arriving on the reactor, and ASO* and AF* are the absorbances of sensitizing and fluorescent chromophores per organic carbon. As experiments were performed at low absorbance, one can write IaSO* ≈ 2.3 I0 ASO* and IaF* ≈ 2.3 I0 AF*. In a first step, we investigated the variations of k* and IF* with a*, ASO* and AF* being related to a*. IF* was measured at three wavelengths: 380, 430, and 500 nm. Plots of k* and IF* at 500 nm vs a* at 365 nm (a365*) are given in Figure 3, and plots of IF* at 380 and 430 nm vs a365* are shown in Supporting Information. The 365 nm wavelength was chosen because it corresponds to the maximum of the lamp’s emission. k* showed a tendency to increase linearly with a365*. Data of the low NMW fractions and of HA50-100 lie above the straight line while data of HA>300 lie below. Either the low NMW fractions and HA50-100 are enriched with

sensitizing constituents compared to peat HA, while HA>300 is more depleted, or all the fractions have the same proportion of sensitizing constituents and quenching processes reduces the apparent quantum efficiencies. For IF*, the data are more distributed on both sides of the straight line that joins the origin to the point corresponding to peat HA. Data of HA50-100 lie above the straight line and data of HA100-300 and HA>300 lie below. Again, these differences can be explained by differences in fluorophores concentration or in quenching efficiencies. Peat FA and Elliott HA are both well above the straight line, indicating that they contain higher percentages of absorbing centers that generate singlet oxygen and fluorophores compared to peat HA or are less subject to quenching processes. We then compared IF* and k* values. It is shown in Figure 4 and in Supporting Information that IF* are satisfactorily correlated with k*, with R2 values ranging from 0.77 to 0.84. This correlation between the two properties shows that sensitizing and fluorescent chromophores are similarly distributed among fractions. This suggests that the same absorbing centers are responsible for the two phenomena. Alternatively, these chromophores might be different, but intimately linked each to another. The contrasting effect of irradiation on the properties of the fractions (a drastic reduction in emission even though there is no effect on the singlet oxygen generation and absorption) do not offer a direct answer. Indeed, the chromophores might lose emitting properties under irradiation through conformational changes but remain absorbing and sensitizing. Also, the question regarding the contributions of the sensitizing and emitting absorbing centers to the total absorbance remains. The fact that the fractionation does not affect the quantity of absorbing centers recovered while it decreases fluorescence and singlet oxygen generation capacity might suggest that fluorescent absorbing centers and photosensitizing absorbing centers contribute for a small part to the total absorbance. In previous works, we compared the emitting properties and the ability to sensitize the transformation of a phenolic probe of soil HAs fractions obtained by tandem size exclusion chromatography and polyacrylamide gel electrophoresis (SEC-PAGE) fractionation in the presence of urea used as a hydrogen bond denaturing agent (11, 13). We found that the 5-30 kDa fractions were much more emitting, more absorbing, and more sensitizing than the high MW fractions. Here, differences among fractions are not so big, even though low MW fractions and 50-100 kDa show higher quantum efficiencies. Consequently, we think that the mode of fractionation is essential in the distribution of absorbing, emitting, and singlet oxygen producing absorbing centers, and that SEC-PAGE fractionation in the presence of a hydrogen bond denaturing agent is more selective than ultrafiltration. Finally, we compared the results obtained from peat HA and its fractions with those obtained from peat FA and Elliott HA. When the data related to peat FA and Elliott HA are added to the graphs, R2 is 0.85 for the plot IF*500 vs k*, but is very low for the IF*430 vs k* and IF*380 vs k* plots. This suggests that the correlation could be extended to other HAs only for long-wavelength fluorophores. This requires further confirmation. In conclusion, ultrafiltration yielded fractions showing distinct absorbance, emission, and capacity to generate singlet oxygen. The high NMW fractions have a higher concentration of absorbing centers compared to the low NMW fractions. Yet, the apparent relative quantum efficiencies of fractions showing NMW higher than 100 kDa are in general lower than those of lower NMW. Either the percentages of absorbing centers which are able to fluoresce and to generate singlet oxygen under irradiation are lower in the former, or the deactivation processes are more important.

The emitting absorbing centers and the absorbing centers producing singlet oxygen are distributed similarly among fractions. The two types of absorbing centers arise from the same molecule or two different but closely linked molecules. The relation between fluorescence around 500 nm and capacity to generate singlet oxygen might be extended to a variety of humic substances. Work is in progress to determine whether a generalization is possible. In the affirmative, as fluorescence is an easy to measure property, this result would be of help to assess the pollutant remediation capacity of humic solutions.

Acknowledgments G.C. acknowledges support by IHSS Training Bursary 2005.

Supporting Information Available Elemental analyses, emission spectra, experimental details, and some correlations. This material is available free of charge via the Internet at http://pubs.acs.org.

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