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Chemosphere 75 (2009) 1082–1088
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Compositional differences between soil humic acids extracted by various methods as evidenced by photosensitizing and electrophoretic properties E. Gieguzynska a, A. Amine-Khodja b, O.A. Trubetskoj c, O.E. Trubetskaya d, G. Guyot b, A. ter Halle b, D. Golebiowska a, C. Richard b,* a
Department of Physics, University of Agriculture, ul. Janosika 8, 71-424 Szczecin, Poland Université Blaise Pascal, Laboratoire de Photochimie Moléculaire et Macromoléculaire, UMR CNRS 6505, 63177 Aubière Cedex, France c Institute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia d Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia b
a r t i c l e
i n f o
Article history: Received 26 September 2008 Received in revised form 7 January 2009 Accepted 8 January 2009 Available online 15 February 2009 Keywords: Humic substances Photochemistry Spectroscopy Electrophoresis Extraction
a b s t r a c t Humic acids (HAs) were isolated from Elliott soil provided by the International Humic Substances Society (1BS102M) by three commonly used methods: (i) 0.1 M NaOH (EI-HA), (ii) neutral 0.1 M Na4P2O7 (L-HA) and subsequent 0.1 M NaOH (S-HA), and (iii) 0.1 M NaOH + 0.1 M Na4P2O7 (NP-HA). The objective was to evaluate the impact of these extractants on the photosensitizing properties of the isolated HAs. HAs were analyzed for their elemental composition, functional acid groups content, absorption and emission properties, electrophoretic characteristics and ability to produce singlet oxygen using furfuryl alcohol (FFA) as a scavenger. L-HA was slightly more aromatic and oxygenated than the other HAs and contained a higher portion of long-wavelength fluorophores and macromolecules showing low molecular size (MS) and high electrophoretic mobility. L-HA also gave a rate of FFA photooxygenation between 1.25- and 1.6-fold higher than the other HAs. This suggests that the free humic macromolecules ionized at pH 7 and/or weakly bounded on mineral surfaces via cation bridges are of relatively low MS and contribute significantly to the photosensitizing and long-wavelength emitting properties. Differences among the other HAs were more subtle, but the parallel evolution of the reactivity and electrophoretic characteristics was observed. Photochemical and electrophoretic measurements seem to be sensitive indicators to evaluate differences among the extraction procedures of HAs. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Soil humic substances (HS) constitute an important part of the soil organic matter, and over the years, their isolation has been one of the most important tasks for soil scientists and researchers from other disciplines. The objectives of the extraction are to isolate organic matter without altering it, to obtain HS that is free of clays and polyvalent cations and to obtain an organic matter representative of that existing in soils in terms of molecular size (MS) range. The occurrence of a great part of soil organic matter under insoluble macromolecular complexes or in association with minerals (cations and/or clays) makes this isolation difficult. Achard first used alkalis to extract from peat material what we now call humic (HAs) and fulvic (FAs) acids (Achard, 1786). Interestingly, Achard’s procedure is still the basis of the most common isolation methods of HS from soil. Although other procedures have been proposed for HS extraction using chelating agents, cation exchange resins, organic solvents and aqueous saline solution (see * Corresponding author. Tel.: +33 4 73 40 71 42; fax: +33 4 73 40 77 00. E-mail address:
[email protected] (C. Richard). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.01.047
the review of Hayes, 2006), alkaline solvents remain the most efficient and widely used for the isolation of soil HAs. Indeed, alkalis at pH > 12 are able to dissociate all acidic functional groups and dissolve macromolecular structures, which are either free or bound with soil minerals. However, extraction using alkalis needs to be performed under a N2 atmosphere to be the least damaging for organic matter since oxygen can oxidize compounds in anionic forms, such as phenolic functionalities. Sodium pyrophosphate (Na4P2O7) was also proposed to extract HS (Bremner and Lees, 1949; Choudhri and Stevenson, 1957; Alexandrova, 1960; Boratynski and Wilk 1965; Kononova, 1966). It is a milder extractant. Neutral pyrophosphate is able to dissolve the free macromolecules that are ionized at pH 7, and thus are the more highly oxidized ones. Pyrophosphate is also able to release the carboxylated macromolecules bound to the mineral part of soils by complexing polyvalent cations, especially Fe and Al, which form bridges between organic matter and the mineral matrix. Na4P2O7 and NaOH were also used as extractants in mixture or sequentially. Several authors have compared the extraction yields and the structural characteristics (elemental analysis, absorption ratios, FTIR or fluorescence) of HAs extracted by NaOH or Na4P2O7
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(Schnitzer and Schuppli, 1989a; Rocha et al., 1998; Dick and Burba, 1999; Fujitake et al., 2003; Tatzer et al., 2007). HS compositions often showed several differences; however, conclusions are difficult to draw because the extraction results not only depend on the extractant used, but also on the soil structure. It should still be noted that the HAs extracted by pyrophosphate were reported to be more aromatic than those extracted by NaOH from the same soil (Piccolo and Mirabella, 1987; Schnitzer and Schuppli, 1989b; Garcia et al., 1993; Zaccone et al., 2007). Increasing interest has been given to HS in the past 30 years for their role in the fate of organic micropollutants in the environment. Due to their ability to absorb solar radiation, HS initiate a number of photochemical processes, producing radicals and/or other chemical species able to promote the chemical transformation of organic chemicals in the environment (Zepp et al., 1981; Aguer et al., 1999; Malouki et al., 2004). Upon irradiation, HS were demonstrated to produce singlet oxygen, hydroxyl radicals and oxidant triplet excited states, which further oxidize organic pollutants (Haag and Hoigné, 1986; Canonica et al., 1995; Vaughan and Blough, 1998; Richard et al., 2004; Halladja et al., 2007). The chromophoric constituents generating these species are still unknown, and their concentration in isolated HAs might depend on the extractant nature. To our knowledge, this question has never been investigated. Our purpose here was to investigate the influence of the HA extraction procedure on the ability to induce the degradation of organic chemicals under light excitation. For the sake of comparison, we characterized the different extracted HAs by standard techniques (elemental composition, functional groups analysis, UV– visible spectroscopy and fluorescence) and by a sophisticated method using polyacrylamide gel electrophoresis (PAGE). In addition, we compared extracts for their ability to sensitize the photo-oxidation of furfuryl alcohol used as a singlet oxygen quencher.
2. Experimental procedures 2.1. Materials Elliott soil I was obtained from the International Humic Substances Society (IHSS) (collection of bulk source materials, 1BS102M) (www.ihss.gatech.edu). Disodium hydrogenophosphate and potassium dihydrogenophosphate were 99% purity grade and were supplied by Prolabo (France). Furfuryl alcohol (FFA) (99% purity grade) was purchased from Aldrich. Water was purified using a Milli-Q (Millipore) device. All other reactants were of the highest purity grade. 2.2. Extraction of HAs 2.2.1. ‘‘IHSS” method (www.ihss.gatech.edu) Portions of 40 g of Elliott soil were weighed in six Erlenmeyer flasks (two flasks for a repetition). Each sample of the soil was equilibrated to pH 1–2 with 40 mL 1 M HCl at room temperature. The volume of each solution was adjusted with 360 mL of 0.1 M HCl to achieve a final ratio of 10 mL solution/1 g dry sample. The suspensions were shaken for 1 h and then allowed to settle. The supernatant was then separated from the residue by decantation. The soil residues in the flasks were neutralized with 1 M NaOH to pH 7, and 360 mL of 0.1 M NaOH solution previously bubbled with N2 for 15 min was added into each flask under a N2 atmosphere. The final ratio of extractant to soil was 1:10. The three flask pairs were placed on the mechanical plane shaker and shaken for 4.5 h under continuous N2 bubbling. After extraction, all flasks were capped and left standing overnight. On the next day, all solutions were decanted; the supernatants from each pair were com-
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bined and subsequently centrifuged at 3600 rpm for 30 min. After centrifugation, each supernatant was acidified using 6 M HCl to pH 1 under constant stirring and left for one night, allowing the HAs to precipitate. The precipitated HAs were then separated from the supernatant by centrifugation (3600 rpm, 45 min). Small volumes of 0.1 M KOH (previously bubbled with N2) were added under N2, step by step, until all the HAs were dissolved; solid KCl was then added to reach a concentration of 0.3 M in K+. The obtained solution was centrifuged (12,000 rpm, 15 min). The HAs were re-precipitated from the supernatant by adding 6 M HCl, under constant stirring, to pH 1. The HA suspension was left again overnight, then again centrifuged at 3600 rpm for 40 min. The supernatant was discarded, and the HAs were suspended in 50 mL 0.1 M HCl/0.3 M HF solution in a plastic bottle and shaken overnight at room temperature. The suspension was then centrifuged (3600 rpm, 30 min), the supernatant discarded, and HA was once again shaken overnight with 60 mL 0.1 M HCl/0.3 M HF. After the second centrifugation (3600 rpm, 30 min), all the HAs were dialyzed against distilled water using Medicell dialysis tubing with MWCO 12–14 kDa. The HAs were then lyophilized, dried overnight at 60 °C and stored in a dessicator over P2O5. The sample was named EI-HA; it was obtained in triplicate. 2.2.2. ‘‘Two-step” method (Boratynski and Wilk,1965) 2.2.2.1. First step. Portions of 50 g of Elliott soil were weighed in six Erlenmeyer flasks (two flasks for one repetition). For each flask, 300 mL 0.1 M Na4P2O7 (pH 7.0) solution was added. The three flask pairs were placed on the mechanical plane shaker, shaken for 4 h and then allowed to stand overnight for settling of soil particles. The next day, the suspension above the soil was decanted and centrifuged 40 min at 3600 rpm. After centrifugation, the supernatants from each flask pair were combined, transferred into 1000 mL tubes and allowed to stand. The remaining soils were extracted a second time with 250 mL 0.1 M Na4P2O7 (pH 7.0) solution for 3 h, allowed to stand and centrifuged in the same manner as described previously. The supernatant obtained after the second extraction was combined with the one from the first extraction. All the combined extracts were allowed to stand for settling of the fine soil particles. The supernatant was decanted and then acidified with 6 M HCl to pH 1 with constant stirring. The suspensions were left for one night to settle the precipitated labile humic acids; they were then separated from the supernatant by centrifugation (3600 rpm, 40 min). The samples were purified as explained in the IHSS method, named L-HA and obtained in triplicate. 2.2.2.2. Second step. Three hundred milliliters of N2-purged 0.1 M NaOH solution was added to each of the remaining soil residues. The flask pairs were placed on the mechanical plane shaker and shaken for 4 h with simultaneous N2 bubbling. After the extraction, all the flasks were capped and left standing overnight. The next day, the solution above the soil was decanted, and the supernatants from each pair were combined and centrifuged at 3600 rpm for 40 min. Following centrifugation, the supernatants from each of the two flasks were combined, transferred into a 1000 mL tube and allowed to stand. The remaining soil residues were extracted a second time with 250 mL of 0.1 M NaOH solution for 3 h and then centrifuged in the same manner described previously. The supernatant obtained after the second extraction was combined with the one after the first extraction. All the combined alkaline extracts were allowed to stand for three days because the suspended soil colloids were extremely dispersed. When these suspensions settled, each supernatant was decanted and acidified with 6 M HCl to pH 1, with constant stirring. The suspensions were left for one night to settle the precipitated ‘‘stabile” humic acids; the suspensions were then separated from the supernatant by centrifugation
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(3600 rpm, 40 min). The samples obtained in triplicate were purified as explained in the IHSS method and were named S-HA. 2.2.3. ‘‘Mixed extractants” method (Kononova, 1966) Portions of 50 g of Elliott soil were weighed in six Erlenmeyer flasks (two flasks for 1 repetition). For each flask, 500 mL of 0.1 M NaOH + 0.1 M Na4P2O7 (pH 13) solution was added, which was previously bubbled for 30 min with N2. The three flask pairs were placed on the mechanical plane shaker, shaken for 6 h and then allowed to stand overnight to settle the soil particles. N2 bubbling was maintained during shaking and standing. The next day, the suspension above the soil was decanted and then centrifuged for 40 min at 3600 rpm. After centrifugation, the supernatants from each flask pair were combined and then acidified with 6 M HCl to pH 1 with constant stirring. The suspensions were left standing for one night to settle the precipitated HAs; the suspensions were then separated from the supernatant by centrifugation (3600 rpm, 40 min). The samples were obtained in triplicate and were purified as explained in the IHSS method; these samples were named NP-HA.
3. Analytical methods Elemental C, H, N analyses were performed on a VARIO EL III Analyzer. Before the analysis, the HA samples were air-dried at 105 °C. The atomic ratios are expressed on an ash and water-free basis. The percentage of oxygen was calculated by subtracting the sum of C, H, and N from 100%. Total and carboxylic OH acidities were determined according to the procedure described in Stevenson (1982) on 105 °C dried samples. This was based on indirect titrations with Ba(OH)2 and Ca(OAc)2. The quantity of phenolic OH was calculated as the difference between total acidity and COOH content. Electrophoresis was performed according to Trubetskoj et al. (1997). The apparatus was a vertical electrophoresis device (LKB 2001 Vertical Electrophoresis, Sweden) with gel slab (20 20 cm). As the gel buffer, 89 mM Tris–borate pH 8.3 with 1 mM EDTA and 7 M urea was used. The HA sample (0.15 mg) was dissolved in buffer (0.05 mL) containing 89 mM Tris–borate (pH 8.3), 7 M urea, 1% sodium dodecyl sulfate and 1 mM EDTA. The electrode buffer was 89 mM Tris–borate at pH 8.3 with 1 mM EDTA. Electrophoresis was carried out in 10% PAG at room temperature for 1 h at a current intensity of 25 mA. The stock HA solutions were prepared by dissolving 40 ± 2 mg L 1 of humic material in Milli-Q water buffered at pH 6.5 using phosphate buffer (3 10 3 M). The solutions were stirred overnight at 20 °C under air atmosphere until the full dissolution of the HAs was complete. The solutions were filtrated on 0.45 lm carbonate filters prior to use. UV–Visible spectra of these solutions were recorded on a Cary 3 (Varian) spectrophotometer in a 1-cm quartz cuvette against a blank solvent. Dissolved organic carbon was measured using a Shimadzu 500 TOC analyzer. Specific absorbances at 254 nm were obtained by dividing the absorbance by the concentration of organic carbon expressed in g L 1. Excitation emission matrices (EEM) spectra were recorded on a Perkin–Elmer LS-55 luminescence spectrometer equipped with a xenon excitation source. The excitation and emission slits were set to 10 and 5 nm band pass, respectively. To limit the second order Rayleigh scattering, Raman diffusion and fluorescence emission, a 290 nm cut-off filter was used. A correction for the instrumental configuration was done using the calibration data for excitation and emission factors provided by the manufacturer. To minimize the inner filter effects and re-absorption, an absorbance of 0.115 ± 0.005 at 320 nm was chosen for all the samples by dilution of the stock solutions. The excitation wavelength range was 300–500 nm, and
the emission wavelength range was 350–600 nm. Stepwise increments of 1 and 10 nm were used for the emission and excitation wavelengths, respectively. 4. Ability of HAs to sensitize the photooxygenation of FFA Air-saturated solutions of FFA and HAs were irradiated in a device equipped with six polychromatic tubes emitting within the wavelength range 300–450 nm (maximum emission at 365 nm) into a cylindrical glass reactor filled with 18 mL of solution. Aliquots (500 lL) were withdrawn each 60 min for 6–7 h after initiating the reaction and were immediately analyzed by HPLC. During irradiations, solutions were periodically re-equilibrated in air to maintain the oxygen (O2) concentration constant. Losses of FFA 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 5 lm 250 mm 4.6 mm Nucleodur, Macherey-Nagel. A flow rate of 1 mL min 1 was used for all analyses, and the eluent was a mixture of 15% methanol and 85% water acidified with orthophosphoric acid (0.1%). All experiments and HPLC analyses were carried out in duplicate. For control experiments, FFA was irradiated alone or left in the dark in the presence of HAs during the time of the photochemical experiments. No losses of FFA were measured in solution left in the dark. FFA was quite photostable in the buffered solution, the consumption being within the experimental error of the HPLC analysis. 5. Statistical treatment of data Elemental analyses and total and carboxylic OH acidities measurements were made in triplicate for each sample and for each type of HA three samples were analyzed. Finally, the standard deviations did not exceed 1% for elemental data and were up to 10% for total acidities and up to 15% for carboxylic acidities. Specific absorbances were measured for one sample of each series. The same solution was used for absorbance and dissolved organic carbon measurements. The dissolved organic carbon measurement was made in triplicate. The standard deviations for specific absorbance were around 2%. Absorbance and emission intensity ratios were estimated with uncertainties around 4% and 6%, respectively. Pseudo first order rate constants are expressed with errors representing the 95% confidence level. The comparison between rates constants was achieved by using a statistical hypothesis test with a critical value of 5%. If the rates were significantly different we reported the p-value. 6. Results and discussion 6.1. Elemental and functional group analyses The quotas of EI-HA and NP-HA in the total soil organic matter were between 15% and 17%, respectively (Table 1). These values are in the range of those generally reported. L-HA and S-HA samples were obtained in half yields, but the combined value was close to that of EI-HA and NP-HA. The elemental composition of our extracted HAs and of the Elliott standard HA is reported in Table 1. As expected, EI-HA and the Elliott standard HA showed close results in atomic composition. NP-HA showed data close to EI-HA. L-HA had a similar C content to the other HAs, a similar H content than EI-HA and NP-HA, while it also had significantly lower N contents and higher O content than other HAs and significantly lower H content than S-HA. On the contrary, S-HA showed higher H and N contents and lower O contents than other HAs. This resulted in significant
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Table 1 Quota, elemental composition (in atomic%), atomic ratio, ash content and functional group analyses of HA samples, isolated from Elliott soil by different methods. Elemental compositions (C, H, N and O) are given on an ash and water-free basis. Given data were performed in triplicate and are averages of those results. Name of samples a
Quota (%) in total soil organic matter C H N O H/C O/C N/C Ash content (%) Total acidity (mol/kg C) Carboxylic groups content (mol/kg C) Phenolic groups content (mol/kg C) a
EI-HA
L-HA
S-HA
NP-HA
Standard IHSS Elliott soil HA (1S102H)
15 44.1 ± 0.4 34.3 ± 0.5 2.79 ± 0.01 18.8 ± 0.1 0.78 ± 0.02 0.427 ± 0.002 0.0633 ± 0.0003 2.1 12.2 ± 0.8 7.4 ± 0.9 4.8 ± 0.8
8.5 44.6 ± 0.3 32.8 ± 0.3 2.38 ± 0.03 20.1 ± 0.2 0.74 ± 0.01 0.451 ± 0.006 0.0533 ± 0.0003 1.4 13.6 ± 0.8 8.2 ± 0.9 5.4 ± 0.8
8.5 42.7 ± 0.4 37.2 ± 0.2 3.08 ± 0.02 17.1 ± 0.3 0.87 ± 0.01 0.399 ± 0.008 0.0722 ± 0.0009 0.80 10.8 ± 0.8 6.4 ± 0.9 4.4 ± 0.8
17 45.0 ± 0.2 34.1 ± 0.3 2.77 ± 0.03 18.2 ± 0.2 0.76 ± 0.01 0.405 ± 0.003 0.0615 ± 0.0004 0.79 11.6 ± 0.8 7.6 ± 0.9 4.0 ± 0.8
44.2 33.6 2.70 19.5 0.76 0.44 0.06 0.88 10.15 8.28 1.87
Extracting yields were computed taking into account that Elliott soil contains 2.9% of organic carbon.
lower H/C and N/C ratios for L-HA than for S-HA. The O/C ratio was higher for L-HA than for the other samples, especially S-HA and NP-HA. These results suggest that neutral pyrophosphate released less aliphatic (or more aromatic compounds) and/or more highly oxidized molecules than the alkali extractant, which is in agreement with the literature (Piccolo and Mirabella, 1987; Schnitzer and Schuppli, 1989b; Garcia et al., 1993; Zaccone et al., 2007). Differences between the acidity (especially phenolic OH acidity) of standard IHSS HA and EI-HA were important (Table 1). One must take into account that the acidity of these samples was measured by quite different methods. The acidity of standard IHSS HA has been determined by direct titrations, while we used the method based on indirect titrations with Ba(OH)2 and Ca(OAc)2. As Ritchie and Perdue (2003) indicated, the carboxyl contents for the IHSS samples are obviously in agreement with both methods of titration, whereas the phenolic contents based on indirect titrations are significantly greater than those estimated from direct titrations. The main reason for this difference is the absorption of CO2 in the Ba(OH)2 solutions occurring during filtration, washing and titration, although we worked only under N2 atmosphere. Therefore, the results from indirect titrations can be only used for comparing the results of different HA preparations obtained in the same laboratory. Data for carboxylic and phenolic group contents were not significantly different from a HA to the other considering the important values of standard deviations.
were too low. A*254 was significantly higher for L-HA with a value of 102 (g of OC) 1 L cm 1 than for the other samples with values of 85–89 (g of OC) 1 L cm 1. In general, the absorbance between 250 and 280 nm normalized to the organic carbon is proportional to the aromatic content for any source of HS (Peuravuori and Pihlaja, 1997). Applied to our case, this would imply that L-HA is the sample containing the highest concentration of aromatic moieties; this also confirms the elemental composition data. The ratio A350/A550 varied only slightly from a HA to the other, contrary to reported data (Dick and Burba, 1999). The EEM were normalized per mol of OC. All the EEM presented a broad emission band centered between 500 and 550 nm. A twoband feature with maximum at 515 and 545 nm was generally observed as reported in the literature (Jones and Indig, 1996; Alberts and Takacs, 2004). L-HA gave a more intense emission in the 480– 580 nm wavelength region than that of the other samples and a clear double maximum. In S-HA, the 545 nm maximum was much less intense than that 515 nm suggesting the absence of some emitting components compared to the other samples. For easier visualization of the shape differences among samples, we present in Fig. 1 extracted emission spectra (320 nm for the excitation) normalized at 510 nm. Between 380 and 510 nm, the emission intensity varied in the following order: L-HA < EI-HA < NP-HA < SHA; meanwhile, between 510 and 600 nm, it varied in an almost opposite order: S-HA < EI-HA < NP-HA < L-HA. Using the ratio of
6.2. Spectral characteristics 120
Table 2 Specific absorbance at 254 nm, absorbance ratio and emission ratio of different HA extracts.
100
Emission intensity (a. u.)
Samples were also compared for their spectral characteristics (absorption and fluorescence). The UV–Visible spectra of the HA samples exhibited a monotonous decrease, similar to that generally observed for HS. Differences in absorption intensities were however observed among the samples. In particular, the L-HA samples were 12–18% more absorbing between 250 and 500 nm than other HAs. A comparison of the samples can be performed on the basis of the specific spectral absorbance at 254 nm, A*254, and on that of the absorbance ratio A350/A550 (ratio of absorbance at 350 nm and to that at 550 nm) (Table 2). This absorbance ratio was previously used by other authors (Dick and Burba, 1999) instead of the classical A465/A665, since the absorbances at 665 nm
S-HA 80 NP-HA 60 EI-NP 40 L-HA 20
0 400
500
600
Wavelength (nm) Name of sample A*254 in (g of OC) A350/A550 I550/I430
1
L cm
1
EI-HA
L-HA
S-HA
NP-HA
84 ± 2 4.9 ± 0.2 1.8 ± 0.1
102 ± 2 5.4 ± 0.2 2.2 ± 0.1
84 ± 2 5.0 ± 0.2 1.5 ± 0.1
89 ± 2 5.0 ± 0.2 1.6 ± 0.1
Fig. 1. Emission spectra of neutral aqueous solutions of EI-HA, L-HA, S-HA and NPHA, isolated from Elliott soil by different methods. The excitation wavelength was set at 320 nm, with the spectra normalized at 510 nm. Spectra were recorded 1 h after the solutions were made.
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the emission intensity at 550 nm to the emission intensity at 430 nm gave the data reported in Table 2. The highest value was obtained for the L-HA sample (2.2) and the lowest for the S-HA sample (1.5). EI-HA and NP-HA gave median values of 1.8 and 1.6, respectively. In summary, the EEM spectra showed that L-HA was the more concentrated sample in long-wavelength emission fluorophores, and the normalized spectra indicated that the distribution of fluorophores among the samples, especially among L-HA and S-HA, were different. The relative amount of long-wavelength emission fluorophores to medium-wavelength fluorophores was higher in L-HA than in S-HA. If the long-emission wavelength is related to a high degree of conjugation, then one can conclude that neutral pyrophosphates extract higher amounts of unsaturated bond systems than other extractant. 6.3. Electrophoretic properties The soil HAs extracted by the different methods were fractionated by PAGE in the presence of denaturing agents (Fig. 2). This method has been developed by Trubetskoj et al. (1997), and it was successfully used previously for the comparison of (i) soil HAs and model phenolic polymers (Saiz-Jimenez et al., 1999); (ii) soil HAs before and after acid hydrolysis (Trubetskaya et al., 2001), and (iii) soil, river and lake HS (Trubetskoi and Trubetskaya, 2004). During the electrophoresis, all the samples were separated into four discrete fractions: A or the start zone, which did not move into the gel and B, C, and D, which were three narrow, intensely naturally colored zones. Zone B differed greatly from zones C and D in electrophoretic mobility (EM), and zones C and D were combined into one fraction C + D due to the relatively close electrophoretic behavior. It has been previously shown that the MS of electrophoretic fractions decreased with increasing EM (Trubetskoj et al., 1997). Fraction A had the highest MS, and fraction C + D had the smallest MS. All samples studied in the present work exhibited the four A, B, C and D zones. A careful examination of electropherograms revealed differences among the samples in the coloration intensities of bands. The color of zone A varied in the following order: L-HA < NP-HA < EI-HA < S-HA. The coloration of zone B was roughly the same for all samples. Zone C + D was slightly darker for L-HA than for EI-HA and N-HA, and it was significantly clearer for S-HA than for L-HA. These results confirm the differences in the sample composition, particularly between L-HA and S-HA. In previous works, we showed that zone A is associated with macromolecules more aliphatic and with a lower carboxylic group content than those of zones B and C + D (Trubetskaya et al., 2002; Richard
et al., 2007). On this basis, S-HA is more aliphatic and less carboxylated than L-HA as also indicated by the elemental and functional groups analyses. 6.4. Photosensitizing properties FFA is often used to probe and measure the production of singlet oxygen, since it readily reacts with this oxidant species (k = 1.2 108 M 1 s 1) (Haag and Hoigné, 1986). The standard conditions used to measure FFA photo-oxidation were as follows: 10 4 M FFA, pH 6.5 using phosphate buffers, and 2 mg L 1 of HA. At this concentration, the HAs showed an absorbance comprise between 0.039 ± 0.002 and 0.045 ± 0.002 at 365 nm, the emission maximum of the lamps. At this low absorbance, one can postulate that the rate of FFA loss, which is proportional to the rate of singlet oxygen formation, is also directly related to the concentration of HA constituents capable of photosensitizing the production of singlet oxygen. Among all the absorbing constituents of the HAs, only a part (and likely a small part) showed sensitizing properties, i.e., were able to transfer their energy to oxygen ground state molecules after having been promoted in their triplet excited state. The other absorbing constituents give excited states which deactivate or undergo reactions but are not involved in the process of FFA photooxygenation. At low absorbance ( NP-HA > EI-HA > S-HA; additionally, the rate of FFA loss followed a pseudo-first order kinetics, as shown by the linearity of the plots ln [FFA]0/[FFA] with the irradiation time (Fig. 3). The rate coefficients correspond to the slopes. The rate coefficient was (1.27 ± 0.03) 10 3 min 1 for EI-HA, (1.76 ± 0.07) 10 3 min 1 for L-HA, (1.08 ± 0.02) 10 3 min 1 for S-HA, and (1.47 ± 0.04) 10 3 min 1 for NP-HA. All these rates are significant different. L-HA showed the highest photosensitizing capacities and S-HA the lowest. To confirm that FFA mainly disappeared via reaction with singlet oxygen generated by the HAs, we used the scavenging technique. For that, we monitored the photooxygenation of FFA in irradiated solutions containing both HA and either isopropanol or
0.8
EI-HA
L-HA
S-HA
L-HA
0.7
NP-HA
NP-HA 0.6
A
EI-HA
ln[FFA] 0/[FFA]
0.5 0.4
S-HA
0.3 0.2
B 0.1 0.0
C+D Fig. 2. Electrophoresis of 0.15 mg EI-HA, L-HA, S-HA and NP-HA, isolated from Elliott soil by different methods, on 10% polyacrylamide gel in the presence of denaturing agents. A, B and C + D are discrete, naturally colored zones.
0
1
2
3
4
5
6
7
8
irradiation time (h) Fig. 3. Loss of furfuryl alcohol (logarithmic plots), when irradiated in the presence of EI-HA, L-HA, S-HA, and NP-HA at a concentration of 2 mg L 1. Neutral medium, [FFA]0 = 10 4 M.
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0.0015
rate coefficient/10
-4
in min
-1
0.0020
0.0010
0.0005
0.0000 L-HA
NP-HA
EI-HA
S-HA
Fig. 4. Influence of isopropanol (5 10 3 M) (dash) and azide ions (3 10 3 M) (white) on the photooxygenation of furfuryl alcohol in the presence of extracted Elliott soil HAs (2 mg/L). Black columns correspond to results in the absence of isopropanol and azide. Neutral medium, [FFA]0 = 10 4 M.
azide ions. Isopropanol was used as a hydroxyl radical scavenger (k = 1.9 1010 M 1 s 1) and azide ions as a source of singlet oxygen (k = 7.8 108 M 1 s 1). In the presence of isopropanol added at a concentration of 5 10 3 M, which was sufficient to trap at least 90% of the formed hydroxyl radicals, the loss of FFA was insignificantly affected. This confirmed the non-involvement of these species in the FFA photo-oxidation. In the case of S-HA, the addition of isopropanol slightly increased the rate of FFA loss. This result was unexpected but however reproducible. On the contrary, azide ions at a concentration of 3 10 3 M inhibited the FFA loss by 90%, as expected on the basis of the singlet oxygen scavenging rate constant of azide (Fig. 4). Since all the HAs were tested in the same conditions, the rate of singlet oxygen production by HAs varied in the same order as that of FFA loss. If one considers that the rate of singlet oxygen production is proportional to the concentration of sensitizing constituents, it follows that L-HA was enriched with sensitizing components compared to S-HA, while NP-HA and EI-HA were in the middle range. 7. Discussion Data gained in this study indicated that isolation methods employing NaOH, neutral pyrophosphate or basic pyrophosphate produced chemically different HAs. The most evident differences occurred between the pool of macromolecules extracted by neutral pyrophosphate and that extracted, subsequently, by NaOH. To summarize, L-HA was both more aromatic than S-HA, as confirmed by the H/C ratio and specific absorbance at 254 nm, and more oxygenated, as shown by the O/C ratio. These results are in line with previous reports on HAs extracted from peats (Piccolo and Mirabella, 1987; Garcia et al., 1993; Zaccone et al., 2007) or from various soils (Schnitzer and Schuppli, 1989b; Fujitake et al., 2003). Additionally, the electrophoretic characterization of L-HA and S-HA gave very distinct pictures. The zone attributed to low EM and high apparent MS macromolecules (fraction A in Fig. 2) was more pale for L-HA than for S-HA; conversely, the zone corresponding to high EM and low apparent MS macromolecules (fraction C + D in Fig. 2) was darker for L-HA than for S-HA. In accordance, Piccolo and Mirabella (1987) reported that HAs extracted from a peat by NaOH showed a higher molecular weight than HAs extracted by neutral pyrophosphate. Under fluorescence, L-HA was slightly more emitting than S-HA in the region 500–550 nm, and the distribution of fluorophores was different, with a relative enrichment of the fluo-
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rophores emitting in the region 500–550 nm compared to those emitting between 400 and 500 nm. Lastly, L-HA produced a greater amount of singlet oxygen upon excitation between 300 and 450 nm than S-HA. The factor was about 1.6. Combined together, these results indicated that long-wavelength emitting fluorophores and photosensitizing constituents are in the aromatic and oxidized moieties and are contained in the electrophoretic zone C + D. This is in line with our previous observations (Richard et al., 2004, 2007). Neutral pyrophosphate is known to dissolve the free macromolecules ionized at pH 7 and the macromolecules weakly bounded on mineral surfaces via cation bridges. Polyvalent cations, such as Al3+, Fe3+, Ca2+ or Mg2+, are able to form cross-linking bridges between the negatively charged surface sites and the anionic humic macromolecules. By complexing the cation, pyrophosphate can release the organic macromolecules (Kononova, 1966). This is fully consistent to observe that L-HA is enriched with oxygen compared to SHA because carboxylic and/or phenolic groups are likely to be involved in surface complexes. Our data indicate that the weakly bound macromolecules were of small or medium size with aromatic character and were one of the sources of the photosensitizing components. The subsequent isolation of S-HA by NaOH yielded macromolecules of bigger size and aliphatic character, which were less oxidized and emitted at a shorter wavelength. These macromolecules were strongly bounded to minerals, as only high pH could reveal them. On the other hand, the differences between EI-HA and NP-HA were very small. No differences on the basis of elemental analysis, acidity measurement and absorbance were found, and the ratio of fluorophores emitting within the region 500–550 nm to those emitting between 400 and 500 nm was only slightly higher in EIHA than in NP-HA. Yet, in electrophoresis, zone A was visibly more colored in EI-HA than in NP-HA, and NP-HA was more photosensitizing than EI-HA by 15%. Thus, the introduction of pyrophosphate into the extracting medium did not measurably change the global structural characteristics, but it instead redistributed the humic components to some extent, as shown by the electrophoretic and photosensitizing analyses. Thus, neutral pyrophosphate and NaOH used in the two step extraction method allowed some separation of distinct macromolecule categories. This was demonstrated on reactivity and a little on structural characteristics. On the other hand, the use of pyrophosphate in basic medium did not yield measurable differences as far as basic structural characteristics were concerned; rather, only the reactivity allowed differentiation. Acknowledgements The work was supported by INTAS Grant 06-8055 and ECONET program (Ministère des Affaires Etrangères, France). References Achard, F.K., 1786. Crell’s Chem. Ann. Chemische untersuchungen des torfs. 2, 391– 403. Aguer, J.-P., Richard, C., Andreux, F., 1999. Effect of light on the humic substances: production of reactive species. Analysis 27, 387–390. Alberts, J., Takacs, M., 2004. Total luminescence spectra of IHSS standard and Reference fulvic acids, humic acids and natural organic matter: comparison of aquatic and terrestrial sources terms. Org. Geochem. 35, 243–256. Alexandrova, L.N., 1960. The use of sodium pyrophosphate for isolating free humic substances and their organic mineral compounds from soil. Sov. Soil Sci. 2, 190– 197. Boratynski, K., Wilk, K., 1965. Fractionation of humus compounds with complexing solutions and diluted alkali solutions. Roczn. Glebozn. 15, 53–63. Bremner, J.M., Lees, H., 1949. Studies of soil organic matter: II The extraction of organic mater from soil by neutral reagents. J. Soil Sci. 1, 198–204. Canonica, S., Jans, U., Stemmler, K., Hoigné, J., 1995. Transformation kinetics of phenols in water: photosensitization by dissolved natural organic material and aromatic ketones. Environ. Sci. Technol. 29, 1822–1831.
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