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Alkaline hydrolysis of humic substances – spectroscopic and chromatographic investigations
Chemosphere 45 (2001) 1023±1031
www.elsevier.com/locate/chemosphere
Alkaline hydrolysis of humic substances ± spectroscopic and chromatographic investigations Michael U. Kumke, Christian H. Specht, Thomas Brinkmann, Fritz H. Frimmel
*
Division of Water Chemistry, Engler-Bunte-Institut, University of Karlsruhe, Engler-Bunte-Ring 1, D-76131 Karlsruhe, Germany Received 26 July 2000; accepted 5 December 2000
Abstract To ®nd out more on the structure of humic substances (HS), isolated dissolved organic carbon (DOC) samples from a brown water lake and a wastewater euent were fractionated and subjected to alkaline hydrolysis. UV/Vis and ¯uorescence spectroscopy, as well as size-exclusion chromatography with on-line detection of UV absorption, ¯uorescence and DOC concentration were used to investigate the structural changes caused by the hydrolysis reaction. Following hydrolysis, the ¯uorescence intensity increased considerably despite a decrease in the UV absorption. The UV absorption and the DOC data from the SEC experiments revealed a strong shift to smaller molecular sizes after hydrolysis. The spectra of the hydrolysed samples, as well as the size-exclusion chromatograms, were compared to spectra of hydroxybenzoic acids and hydroxycinnamic acids. From this comparison, it can be concluded that the hydrolysis products have a structure similar to these organic acids. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Humic substances; Alkaline hydrolysis; Fluorescence; Size-exclusion chromatography; Model compounds
1. Introduction Humic substances (HS) account for a major part of the dissolved organic carbon (DOC) in all aquatic ecosystems. Although it is impossible to describe an exact formation pathway, two general conceptual models have been discussed in the literature. The ®rst one assumes HS to be formed from the tissues of plant material by their partial breakdown and oxidation due to extracellular enzymes and abiotic processes. The second concept states a polymerization of simple compounds like quinones that are derived from degraded plant material. Aquatic HS can be derived from both external sources like plants/soils (allochthonous) and from sources pro-
* Corresponding author. Tel.: +49-721-608-2580; fax +49721-699-154. E-mail address: [email protected] (F.H. Frimmel).
duced within the aquatic ecosystem like algae/bacteria (autochthonous) (Mc Knight and Aiken, 1998). Due to the enormous number of dierent precursor materials, an exact structure of HS is impossible to de®ne. More useful is the identi®cation of structural building blocks and their relation to the reactivity of HS. Due to the vast heterogeneity of HS, an in situ characterization of structural elements is very dicult. A promising experimental approach is to reverse the formation of HS and to release structural sub-units under de®ned experimental conditions. Hydrolysis reactions have been successfully applied in the past to release amino acids, carbohydrates, aliphatic and aromatic carboxylic acids, as well as phenols from HS (Liao et al., 1982; Parsons, 1989; Hautala et al., 1997; Jahnel et al., 1998; Frimmel et al., 1998; Hautala et al., 1998). The objectives of the presented work were to investigate the eect of alkaline hydrolysis on the properties of HS, especially on their size and their spectroscopic properties. Dierences and similarities were evaluated for HS of dierent origin. The ultimate goal is to identify
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 0 6 - 6
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substructures in HS that de®ne their spectroscopic and chromatographic properties and to use them for a classi®cation of HS in terms of their reactivity with xenobiotics.
2. Experimental details 2.1. Materials, samples and sample treatment Ultrapure water (18:2 MX cm, Milli-Q P L U S , Millipore) was used in all experiments. A sodium hydroxide solution (c 1 mol/l) was prepared by diluting a commercially available concentrate (50±52% (w/w), Fluka, puriss. p. a.). The HS samples investigated were collected from a brown water lake (Hohlohsee, Northern Black Forest, Germany; HO14) and wastewater euent (water treatment plant, Neureut, Germany; ABV3). While the brown water was of natural origin and its precursors can be considered to be mainly plant remains, the wastewater euent had a strong contribution from anthropogenic activities. The original samples were freeze-dried (Orig) or separated into fulvic acids (FA), humic acids (HA) and non-humic substances (NHS) by using an XAD-8 (polymethylmethacrylate) resin according to a modi®ed method of Mantoura and Riley (Mantoura and Riley, 1975; Abbt-Braun et al., 1991). Basic sample parameters are described in detail elsewhere (Frimmel and AbbtBraun, 1999). The concentration of dissolved organic carbon was measured using a Shimadzu 5000 TOC analyzer, calibrated with potassium hydrogenphthalate standards. Sample solutions were prepared by adding 10 ml of water to approximately 2.5 mg of freeze-dried material. The resulting mixtures were soni®ed for 15 min and subsequently ®ltered (0:45 lm, polyvinylidene¯uoride). For the hydrolysis 0.75 ml of sodium hydroxide (c 1 mol/l) solution was added to 2.25 ml of sample solution.
The resulting solution was heated under nitrogen in a sealed glass vial for 48 h at 100°C. Subsequently, 25 ml of water was added, then the solution was ®ltered using a 0:45 lm polyvinylidene¯uoride ®lter. A styrene-based cation exchange resin cartridge (OnGuard-H, Dionex) was used to neutralize the solutions. After discarding the ®rst 4 ml, the solutions obtained were used in the further spectroscopic and chromatographic measurements. Dierent benzene carboxylic acids were investigated for their spectroscopic properties. The compounds are listed in Table 1. All compounds were purchased from Aldrich (grade > 97%) and used as received. The UV/Vis absorption measurements and the ¯uorescence measurements were carried out at pH values of 2, 7, and 11, respectively. To adjust the pH, aqueous HCl and NaOH were added to the solutions. In case of pH 7, a phosphate buer was used. For the ¯uorescence measurements, the optical density of the samples was adjusted to 0.1 at 265 nm in order to measure all samples under the same excitation conditions. 2.2. UV/Vis and ¯uorescence For the UV/Vis measurements, a Cary50 spectrometer (Varian) was used. The absorption spectra were recorded in the range of 200 nm < kabs < 500 nm. All spectra were background corrected. The ¯uorescence spectra were recorded using a CDT900 ¯uorescence spectrometer (Edinburgh Analytical Instruments). The instrument was equipped with a 450 W Xenon lamp and a red-sensitive photomultiplier tube, which was operated in the single photon counting mode. The instrument is described in detail elsewhere (Kumke et al., 1998; Frimmel and Kumke, 1999). Fluorescence emission spectra and ¯uorescence excitation spectra were recorded. The ¯uorescence emission spectra were measured for three dierent excitation wavelengths of kex 260, 270, and 330 nm, in the wavelength range of 265 nm < kem < 650 nm, respectively. For the ¯uores-
Table 1 Spectroscopic properties of the benzoic acids investigateda
a
UV absorption maximumb (in nm)
Fluorescence maximumc (in nm)
Gallic acid 4-Hydroxycinnamic acid 2-Hydroxycinnamic acid 3,4-Dihydroxycinnamic acid 4-Hydroxybenzoic acid Acetylsalicylic acid Coniferyl alcohol Ferulic acid
262 288 271 (314) 288 (314) 248 (266) 263 (300shoulder ) 265 (310)
349 401 498 (408shoulder ) 426 ± 411 340 442
7-hydroxycoumarin
325
453
The data were collected from solution at pH 7. b Given are the wavelengths for the absorption maximum. In case of two maxima, the second is given in parentheses. c The excitation wavelengths were chosen according to the UV absorption maxima.
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cence excitation spectra, two dierent detection wavelengths kem 310 and 450 nm) were chosen, and the spectra were measured in the wavelength range of 225 nm < kex < 440 nm. The spectra were recorded with a spectral bandpass of 1.8 nm in the excitation as well as in the emission monochromator. The dwell time of the measurements was set to 0.5 s and data points were taken every nanometer. 2.3. Size-exclusion chromatography The experimental set-up for the size-exclusion chromatography (SEC) measurements is described in detail elsewhere (Specht et al., 2000). Three dierent on-line detection methods were used: UV-absorption at kabs 254 nm, ¯uorescence at kex;1 260 nm, kem;1 310 nm and at kex;2 330 nm, kem;2 450 nm and DOC. A TSK-50 HW (S) column was used with a phosphate buer as the mobile phase (28 mmol/l, pH 6.8). The exclusion volume (19 ml) and the permeation volume (47 ml) were determined using dextrane blue and water, respectively. The injection volume of the samples was 2 ml and the DOC concentration of the samples was in the range of 2±10 mg/l.
3. Results and discussion 3.1. UV/Vis spectroscopy The UV/Vis spectra were recorded in the range of 200 nm < kabs < 500 nm. For the humic substances ABV3 isolated from the wastewater euent, a decrease in the UV/Vis absorption was observed after hydrolysis only for the HA fraction. For the absorption spectra of ABV3 FA and ABV3 NHS no signi®cant changes were found. More conclusive results were obtained for the brown water HO14 and its isolated fractions. Here, a decrease in the absorption was found for all fractions investigated. While the brown water samples HO14 before hydrolysis showed featureless absorption spectra, the hydrolysis caused a signi®cant change in the related absorption spectra (e.g., Fig. 1 for HO14 FA before and after hydrolysis). This was observed for the HA fraction of the wastewater euent ABV3 as well. Due to hydrolysis, a well-pronounced absorption band around kabs 265 nm appeared in the spectra (Fig. 1). It was strongest for the FA and HA fraction and least pronounced for the NHS fraction of the brown water HO14. To compare the in¯uence of the hydrolysis on the dierent HS fractions investigated, the UV/Vis spectral data were normalized to the concentration of DOC. The absorption kabs 254 nm and kabs 436 nm is frequently used to characterize the properties of HS solu-
Fig. 1. UV/Vis spectra (not normalized) of HO14 FA before and after hydrolysis. The ratio of the two spectra is shown as well as the spectra of the pure medium before (a) and after hydrolysis (b).
tions. These wavelengths were formerly used to estimate the aromaticity and the content of quinones in HS samples (Stevenson, 1982). Korshin et al. (1997) suggested the use of the absorption at kabs 203 nm and at kabs 253 nm for an estimation of the degree of functionality of the aromatic ring. Benzenoid compounds usually show an absorption band at 203 nm, which is often referred to as the benzenoid band (Bz). An absorption around 253 nm can be attributed to a chargetransfer transition (CT). While the strength of the Bz band stays relatively unchanged upon changes in the functionality of the aromatic ring, the CT band is strongly altered. Therefore, a change in the ratio of these wavelengths should be indicative of alterations in the functionality of the aromatic system. In Fig. 2 the ratio of the UV/Vis absorption at 254 nm to that at 203 nm is shown (later referred to as
Fig. 2. Comparison of the absorption ratio k254 nm =k203 nm for the HS samples before and after alkaline hydrolysis. The pH was adjusted to 4.
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ratio I). For the HS fractions investigated, an increase in ratio I was observed after hydrolysis, which indicates an increase in the degree of eective functionality of the benzoic rings. Because a direct attack of hydroxide ions on the aromatic moieties is improbable under the reaction conditions applied (Fyfe, 1971), side chain alterations like ester and amide hydrolyses, retro-aldol reactions, benzil-benzilic rearrangements, a and b ether cleavages as well as b±c±C±C cleavages may be responsible for the increase (Wallis, 1971; Parsons, 1989; Beyer, 1998). 3.2. Fluorescence spectroscopy In Fig. 3, the in¯uence of hydrolysis on the steadystate ¯uorescence of the brown water samples HO14 is shown. For comparison, the ¯uorescence intensities were normalized to the concentration of dissolved organic carbon. As a general trend , the ¯uorescence of the NOM samples investigated was increased upon hydrolysis. However, the observed increase was much stronger for the brown water samples and within the fractions decreasing in the order HO14 Orig > HO14 FA > HO14 HA > HO14 NHS. For the wastewater euent, ABV3 FA and ABV3 NHS showed a small increase. To quantify the alteration in the ¯uorescence intensity, the ¯uorescence spectra recorded at dierent excitation wavelengths were integrated and the relative change upon hydrolysis was calculated. The calculated overall change in the ¯uorescence intensity of the HS samples is shown in Fig. 4 for two dierent excitation wavelengths. Independent of the excitation wavelength, the ¯uorescence intensity of the wastewater euent fractions was only slightly changed upon hydrolysis, indicated by a relative change in ¯uorescence intensity close to one (see Fig. 4(b)). On the other hand, for the
Fig. 3. Steady-state ¯uorescence spectra kex 330 nm; pH 4 of brown water HO14 Orig before and after alkaline hydrolysis of the sample.
Fig. 4. Relative change of the overall ¯uorescence intensity of the NOM samples due to hydrolysis at dierent excitation wavelengths. Shown are the ratios of the integrated ¯uorescence intensity before hydrolysis and after hydrolysis ((a) brown water HS and (b) wastewater euent HS).
brown water and its fractions, a strong increase in the overall ¯uorescence intensity was observed (for HO14 Orig a 2.5-fold increase was observed for kex 260 nm; see Fig. 4(a)). Moreover, the observed increase caused by hydrolysis was dependent on the excitation wavelength. For kex 330 nm, the increase of the overall ¯uorescence intensity was smaller. Here, an increase less than 2-fold was measured for all brown water samples HO14. The completely dierent behaviour of the wastewater samples ABV3 might be attributed to their young age. On the other hand, the brown water samples HO14 are much older which means, that they have been subjected to numerous alterations like enzymatic or light-induced oxidations. These alterations seem to favour the spectroscopic changes induced by alkaline treatment. As the hydroxide ions preferently attack aliphatic moieties with oxygen-containing functional groups (Wallis, 1971; Parsons, 1989; Beyer, 1998), the results show the importance of these structural parts for the spectroscopic properties of HS. The eect of increasing ¯uorescence intensity has also been observed after chlorination, ozonation and UV
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irradiation of HS (Korshin et al., 1999; Win et al., 2000). Under these dierent conditions HS are supposed to be cleaved to smaller molecules. These might have higher ¯uorescence quantum yields than the original macromolecular substances. Another possibility is, that upon degradation, ¯uorescence quenching of small molecules by their interaction with HS (Morra et al., 1990; Doll et al., 1999) is reduced. The two explanations were further examined by size-exclusion chromatography and are discussed later in the paper. It is interesting to note that the observed increase in the ¯uorescence intensity was not uniform in the spectral range of the ¯uorescence spectrum which becomes obvious from the calculation of the dierence of ¯uorescence intensity before and after hydrolysis (see Fig. 5). The maximum increase of the ¯uorescence intensity was found in the wavelength range between 415 nm < kem < 440 nm: In Fig. 5, the calculated spectra (for kex 330 nm of the brown water samples are shown. In Fig. 5(b), the ¯uorescence excitation spectra of the brown water HO14 FA fraction before and after hydrolysis are compared. The ratio of the two spectra is
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shown as well. For a simple homogeneous solution of a ¯uorescent compound, it can be expected that the observed ¯uorescence excitation spectrum and the corresponding UV/Vis absorption spectrum are identical (assuming that the intensity distribution of the excitation lamp was considered properly). In general, for HS the measured ¯uorescence excitation spectrum and the UV/Vis absorption spectrum are not identical. While the absorption spectrum is (almost) featureless with an increasing absorption toward the UV region, in the ¯uorescence excitation spectrum, a relatively well-resolved band is observed in the spectral region around 310 nm < kex < 370 nm. These dierences indicate that only a part of the light-absorbing chromophores present in HS is ¯uorescent. Based on the ¯uorescence excitation spectra, a connection between the observed ¯uorescence increase and the related change in the absorption spectra can be drawn. The comparison of the ratio of the ¯uorescence excitation spectra and the dierence in the absorption spectra (see Fig. 1) reveals striking similarities and it also gives one explanation for the observed dependence on the ¯uorescence excitation wavelength of the ¯uorescence increase upon hydrolysis. The compounds built (or released) by the hydrolysis of the HS showed a strong absorption around kabs 265 nm. The larger increase in the ¯uorescence intensity, with kex 260 nm, can be attributed to that fact. The number of compounds (or subunits) released from HS participating in light absorption was found to be larger for k 260 nm, and therefore, the observed overall increase in the ¯uorescence intensity with that particular excitation wavelength can also be expected to be higher when compared to an excitation wavelength of lower energy (e.g., kex 330 nm). 3.3. Comparison of the spectral character of benzoic acid and the changes induced by hydrolysis in ¯uorescence and UV/Vis absorption
Fig. 5. (a) Calculated dierences of the steady-state ¯uorescence spectra of the brown water and its HS fractions upon alkaline hydrolysis kex 330 nm. (b) Comparison of the ¯uorescence excitation spectra of HO14 FA before and after hydrolysis (not corrected for lamp pro®le). The ratio of both spectra is shown as well kem 450 nm.
In a comparative approach, the UV/Vis absorption spectra and the ¯uorescence spectra of dierent benzoic acids and related compounds were measured and compared with spectra of the HS and the calculated dierences observed upon hydrolysis. Table 1 summarizes the relevant spectroscopic properties of the compounds investigated. All benzoic acid derivatives show absorption maxima between 250 nm < kabs;max < 330 nm. A strong overlap was found compared to the UV/Vis absorption spectra calculated from the measurements before and after hydrolysis of HS. This was also valid for the steady-state ¯uorescence excitation spectra of the benzoic acids. In Fig. 6, the calculated ¯uorescence spectra of the hydrolysis products of the brown water HO14 Orig is compared with the ¯uorescence spectra of various benzene carboxylic acids.
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Fig. 6. Comparison of the ¯uorescence spectra of several benzoic acids with the calculated dierence spectrum of HO14 Orig upon hydrolysis kex 260 nm; pH 7.
Assuming that some of the building processes of HS are reversed due to the alkaline treatment, a release of lignin-derived moieties like p-coumaryl, sinapyl and coniferyl alcohol-like structures can be expected. This is strongly supported by the striking agreement between the UV/Vis absorption spectra, as well as the steadystate ¯uorescence spectra of those simple model compounds and the calculated changes upon hydrolysis in the spectra of the HS. Furthermore, the results are in qualitative accordance with analytical pyrolysis data of lake Hohloh samples, where 16±22% of the volatile matter were assigned to phenols, lignin monomers and dimers (Schulten and Gleixner, 1999). A signi®cant contribution of allochthonous, soil-derived precursors to lake Hohloh HS seems to be reasonable, since it is a nutrient-poor bog lake, that will slowly become overgrown by detrital plant material. 3.4. Size-exclusion chromatography The data from the size-exclusion chromatography con®rmed the ®ndings from the UV/Vis and ¯uorescence measurements. The chromatograms of the wastewater samples were only slightly altered by hydrolysis. Whereas the DOC and UV-absorption traces of the FA and NHS sample showed virtually no changes, the ¯uorescence trace of the FA sample revealed an increase of ¯uorescence intensity. This increase was not attributable to single fractions and occurred at both sets of excitation and emission wavelengths. Both, the DOC and the UV absorption trace of the HA sample showed a shift to higher elution volumes. In size-exclusion chromatography, several non-ideal interactions between the gel and sample such as ion exclusion, ion exchange and adsorption can occur (Johansson and Gustavsson, 1988;
Potschka, 1988). These interactions are superimposed to the separation of the molecules due to their size. Under the reaction conditions applied, the most likely reactions are ester and ether cleavages (Wallis, 1971), which lead to an increase in carboxyl and hydroxyl group content. As a result, the gel±sample interactions, which may lead to a retardation, are decreased (Specht and Frimmel, 2000). Because of this, the shift to higher elution volumes can most likely be attributed to smaller molecules rather than to an increase in the attractive interactions between gel and sample. The in¯uence of hydrolysis on the brown water sample was much more pronounced in comparison to the wastewater samples. For all brown water samples (original, FA, HA, and NHS) the ¯uorescence intensity increased whereas the UV absorption decreased. In connection with a shift to higher elution volumes, this is another indication for breakdown of larger molecules due to hydrolysis. Large molecules have the ability to release the energy from an absorbed photon via processes which do not involve the emission of light. For smaller molecules, this is often not the case. Because of this, the ¯uorescence increases even if the UV absorption decreases, i.e., the ¯uorescence yield increases. Due to the chromatographic separation quenching of the smaller ¯uorescent entities by larger NOM molecules is suppressed even for the non-hydrolysed sample. Therefore, the increase of ¯uorescence is more likely caused by an increase of the ¯uorescence yield and the number of ¯uorescent entities rather than by a decrease of the quenching eect of NOM. In contrast to the wastewater sample, the increase of ¯uorescence intensity can be attributed to several fractions. Fig. 7 depicts the chromatograms obtained with DOC-, UV absorption-, and the ¯uorescence detection of the brown water sample HO14 Orig before and after hydrolysis. The DOC and UV absorption chromatograms show a considerable shift to higher elution volumes, i.e., lower molecular mass. This shift cannot be found in the ¯uorescence trace. From these results one can conclude, that the higher molecular mass fractions of natural organic matter contain structures which are potentially capable of ¯uorescence. These structures are unveiled by the hydrolysis reaction and lead to an increase in ¯uorescence intensity. This holds for all brown water samples investigated in this work. The highest increase in ¯uorescence intensity (by a factor of 3.8) was found for kex;1 260 nm and kem;1 310 nm. At kex;2 330 nm; kem;2 450 nm, ¯uorescence intensity increased by a factor of 2.7. This increase is higher than the increase in overall ¯uorescence intensity, which was found for the non-separated sample. In the non-separated sample, ¯uorescence quenching can occur through intermolecular energy transfer from smaller to larger molecules, which is not possible after separation in the SEC column. As the total area of the DOC chromato-
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Fig. 7. SEC chromatograms of brown water HO14 Orig before and after hydrolysis obtained with DOC, UV absorbance, and ¯uorescence detection.
grams does not decrease, no degradation of organic matter to CO2 took place due to hydrolysis. On the other hand, the total area of the UV signal decreases by 16%. Because of the cleavage of side groups, conjugated systems might be destroyed, resulting in a lower UV absorption. In addition to the shift, the chromatograms of the samples after hydrolysis show fractions which were set free from the original sample. No major differences can be found between the dierent natural organic matter fractions in the DOC and UV absorption chromatograms. In contrast, it was possible to detect signi®cant dierences between the samples in the ¯uorescence signal. Fig. 7(c) shows the chromatogram of the ¯uorescence detector with kex;1 260 nm and kem;1 310 nm. Whereas fractions 2 and 3 could be found in the original sample as well as in the FA sample, fraction 2 was absent in the HA sample and fraction 3 could not be found in the NHS sample. Also, the HA showed no ¯uorescence at the chosen set of excitation and emission wavelengths before hydrolysis. The chromatograms of the ¯uorescence signal at kex;2 330 nm; kem;2 450 nm showed only minor changes in shape. Since humic substances show a strong ¯uorescence signal at this set of
excitation and emission wavelengths, the increase in intensity is probably caused by a decrease in the selfquenching eect of humic substances. To determine the structures of the substances which make up fractions 2 and 3 found in the ¯uorescence chromatograms, SEC experiments with the compounds listed in Table 1 were carried out under the same conditions used for the natural organic matter samples, i.e., the same UV absorption and ¯uorescence excitation and emission wavelengths were used. The data showed, that benzoic acid derivatives like gallic acid and 4-hydroxybenzoic acid had a strong ¯uorescence signal at kex;1 260 nm; kem;1 310 nm, but hardly any signal at kex;2 330 nm; kem;2 450 nm. The derivatives of cinnamic acid on the other hand showed a strong signal at kex;2 330 nm; kem;2 450 nm, but only a weak signal at kex;1 260 nm; kem;1 310 nm. In addition, the elution volume of the benzoic acid derivatives coincides with the elution volume of fraction 3. From this, one may conclude, that the fractions 2 and 3 consist of substances with a structure similar to hydroxybenzoic acids rather than hydroxycinnamic acid.
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4. Conclusions Hydrolysis reactions have been used quite extensively to obtain information on the structure of the building blocks of humic matter. In combination with spectroscopy in the UV/Vis range, hydrolysis gives information not only on the aromatic and unsaturated areas of the molecules with main absorbances around k 260 nm, but also on the molecular size. Especially ¯uorescence spectra give insight into the stability of the molecular structure in aqueous solution. The signi®cant increase in the ¯uorescence after hydrolysis makes it attractive to assume the presence of quenching mechanisms in the original dissolved macromolecule which are lost in the smaller hydrolysed products. The identi®cation of the building blocks still remains troublesome. Poor resolution of chromatographic fractionation based on water as the mobile phase and lack of powerful identi®cation methods are the main reasons. A way out of this dilemma seems to be the use of reasonable model compounds. Their properties in mixtures can be compared with the integrated character of the complex samples to be investigated. The best ®t is a good basis for structural assumptions. The application of that strategy to the hydrolysis products of HS makes the substituted representatives of salicylic acid, cinnamic acid, and 7hydroxy-coumarin to appear as attractive building blocks of the complex organic matter in brown water. Acknowledgements The work was funded by the Deutsche Forschungsgemeinschaft in the ROSIG research program and in the research project MetalOM (Grant Fr 536/21-1). The authors wish to thank Dr. Gudrun Abbt-Braun and Axel Heidt for preparing and supplying the HS samples. References Abbt-Braun, G., Frimmel, F.H., Lipp, P., 1991. Isolation of organic substances from aquatic and terrestrial systems ± comparison of some methods. Z. Wasser-Abwasser-Forsch 24, 285±292. Beyer, H., 1998. Lehrbuch der Organischen Chemie. Hirzel, Stuttgart. Doll, T.E., Frimmel, F.H., Kumke, M.U., Ohlenbusch, G., 1999. Interaction between natural organic matter (NOM) and polycyclic aromatic compounds (PAC) ± comparison of ¯uorescence quenching and solid phase micro extraction (SPME). Fresenius J. Anal. Chem. 364, 313±319. Frimmel, F.H., Jahnel, J., Hesse, S., 1998. Characterization of biogenic organic matter (BOM). Water Sci. Technol. 37, 97± 103.
Frimmel, F.H., Abbt-Braun, G., 1999. Basic characterization of reference NOM from central Europe ± similarities and dierences. Environ. Int. 25, 191±207. Frimmel, F.H., Kumke, M.U., 1999. Optische Parameter zur Stocharakterisierung vom Trinkwasser bis zum Abwasser. Wien. Mitt. 156, 1±24. Fyfe, C.A., 1971. Nucleophilic attack by hydroxide and alkoxide ions. In: Patai, S. (Ed.). The Chemistry of the Hydroxyl Group ± Part 1. The Chemistry of Functional Groups. Interscience, London, pp. 52±131. Hautala, K., Peuravuori, J., Pihlaja, K., 1997. Estimation of origin of lignin in humic DOM by CuO-oxidation. Chemosphere 35, 809±817. Hautala, K., Peuravuori, J., Pihlaja, K., 1998. Organic compounds formed by chemical degradation of lake aquatic humic matter. Environ. Int. 24, 527±536. Jahnel, J.B., Ilieva, P., Abbt-Braun, G., Frimmel, F.H., 1998. Aminosauren und Kohlenhydrate als Strukturbestandteile von refraktaren organischen Sauren. Vom Wasser 90, 205± 216. Johansson, B.-L., Gustavsson, J., 1988. Elution behavior of some proteins on fresh, acid- or base-treated sephacryl S200 HR. J. Chromatogr. 457, 205±215. Korshin, G.V., Li, C.-W., Benjamin, M.M., 1997. Monitoring the properties of natural organic matter through UV spectroscopy: a consistent theory. Water Res. 31, 1787± 1795. Korshin, G.V., Kumke, M.U., Li, C.W., Frimmel, F.H., 1999. In¯uence of chlorination on chromophores and ¯uorophores in humic substances. Environ. Sci. Technol. 33, 1207±1212. Kumke, M.U., Abbt-Braun, G., Frimmel, F.H., 1998. Timeresolved ¯uorescence measurements of aquatic natural organic matter (NOM). Acta Hydrochim. Hydrobiol. 26, 73±81. Liao, W., Christman, R.F., Johnson, J.D., Millington, D.S., 1982. Structural characterization of aquatic humic material. Environ. Sci. Technol. 16, 403±410. Mantoura, R.F.C., Riley, J.P., 1975. The analytical concentration of humic substances from natural waters. Anal. Chim. Acta 76, 97±106. Mc Knight, D.M., Aiken, G.R., 1998. Sources and age of aqutic humus. In: Hessen, D.O., Tranvik, I.J. (Eds.), Aquatic Humic Substances ± Ecology and Biogeochemistry. Springer, Berlin, pp. 9±39. Morra, M.J., Corapcioglu, M.O., von Wandruszka, R.M.A., Marshall, D.B., Topper, K., 1990. Fluorescence quenching and polarization studies of naphthalene and 1-naphthol interaction with humic acid. Soil Sci. Soc. Am. J. 54, 1283± 1289. Parsons, J.W., 1989. Hydrolytic degradations of humic substances. In: Hayes, M.H.B., MacCarthy, P., Malcolm, R.L., Swift, R.S. (Eds.), Humic Substances II ± in Search of Structure. Wiley, Chichester, pp. 99±120. Potschka, M., 1988. Size-exclusion chromatography of polyelectrolytes, experimental evidence for a general mechanism. J. Chromatogr. 441, 239±260. Schulten, H.-R., Gleixner, G., 1999. Analytical pyrolysis of humic substances and dissolved organic matter in aquatic systems: structure and origin. Water Res. 33, 2489±2498.
M.U. Kumke et al. / Chemosphere 45 (2001) 1023±1031 Specht, C.H., Frimmel, F.H., 2000. Speci®c interactions of organic substances in size-exclusion chromatography. Environ. Sci. Technol. 34, 2361±2366. Specht, C.H., Kumke, M.U., Frimmel, F.H., 2000. Characterization of NOM adsorption to clay minerals by size exclusion chromatography. Water Res. 34, 4063±4069. Stevenson, F.J., 1982. Humus Chemistry, Genesis, Composition, Reactions. Wiley, New York. Wallis, A.F.A., 1971. Solvolysis by acids and bases. In: Sarkanen, K.V., Ludwig, C.H. (Eds.), Lignins, Occurrence,
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Formation, Structure and Reactions. Wiley±Interscience, New York, pp. 345±372. Win, Y.Y., Kumke, M.U., Specht, C.H., Schindelin, A.J., Kolliopoulos, G., Ohlenbusch, G., Kleiser, G., Hesse, S., Frimmel, F.H., 2000. In¯uence of oxidation of dissolved organic matter (DOM) on subsequent water treatment processes. Water Res. 34, 2098±2104.
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Alkaline hydrolysis of humic substances ± spectroscopic and chromatographic investigations Michael U. Kumke, Christian H. Specht, Thomas Brinkmann, Fritz H. Frimmel
*
Division of Water Chemistry, Engler-Bunte-Institut, University of Karlsruhe, Engler-Bunte-Ring 1, D-76131 Karlsruhe, Germany Received 26 July 2000; accepted 5 December 2000
Abstract To ®nd out more on the structure of humic substances (HS), isolated dissolved organic carbon (DOC) samples from a brown water lake and a wastewater euent were fractionated and subjected to alkaline hydrolysis. UV/Vis and ¯uorescence spectroscopy, as well as size-exclusion chromatography with on-line detection of UV absorption, ¯uorescence and DOC concentration were used to investigate the structural changes caused by the hydrolysis reaction. Following hydrolysis, the ¯uorescence intensity increased considerably despite a decrease in the UV absorption. The UV absorption and the DOC data from the SEC experiments revealed a strong shift to smaller molecular sizes after hydrolysis. The spectra of the hydrolysed samples, as well as the size-exclusion chromatograms, were compared to spectra of hydroxybenzoic acids and hydroxycinnamic acids. From this comparison, it can be concluded that the hydrolysis products have a structure similar to these organic acids. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Humic substances; Alkaline hydrolysis; Fluorescence; Size-exclusion chromatography; Model compounds
1. Introduction Humic substances (HS) account for a major part of the dissolved organic carbon (DOC) in all aquatic ecosystems. Although it is impossible to describe an exact formation pathway, two general conceptual models have been discussed in the literature. The ®rst one assumes HS to be formed from the tissues of plant material by their partial breakdown and oxidation due to extracellular enzymes and abiotic processes. The second concept states a polymerization of simple compounds like quinones that are derived from degraded plant material. Aquatic HS can be derived from both external sources like plants/soils (allochthonous) and from sources pro-
* Corresponding author. Tel.: +49-721-608-2580; fax +49721-699-154. E-mail address: [email protected] (F.H. Frimmel).
duced within the aquatic ecosystem like algae/bacteria (autochthonous) (Mc Knight and Aiken, 1998). Due to the enormous number of dierent precursor materials, an exact structure of HS is impossible to de®ne. More useful is the identi®cation of structural building blocks and their relation to the reactivity of HS. Due to the vast heterogeneity of HS, an in situ characterization of structural elements is very dicult. A promising experimental approach is to reverse the formation of HS and to release structural sub-units under de®ned experimental conditions. Hydrolysis reactions have been successfully applied in the past to release amino acids, carbohydrates, aliphatic and aromatic carboxylic acids, as well as phenols from HS (Liao et al., 1982; Parsons, 1989; Hautala et al., 1997; Jahnel et al., 1998; Frimmel et al., 1998; Hautala et al., 1998). The objectives of the presented work were to investigate the eect of alkaline hydrolysis on the properties of HS, especially on their size and their spectroscopic properties. Dierences and similarities were evaluated for HS of dierent origin. The ultimate goal is to identify
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 0 0 6 - 6
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substructures in HS that de®ne their spectroscopic and chromatographic properties and to use them for a classi®cation of HS in terms of their reactivity with xenobiotics.
2. Experimental details 2.1. Materials, samples and sample treatment Ultrapure water (18:2 MX cm, Milli-Q P L U S , Millipore) was used in all experiments. A sodium hydroxide solution (c 1 mol/l) was prepared by diluting a commercially available concentrate (50±52% (w/w), Fluka, puriss. p. a.). The HS samples investigated were collected from a brown water lake (Hohlohsee, Northern Black Forest, Germany; HO14) and wastewater euent (water treatment plant, Neureut, Germany; ABV3). While the brown water was of natural origin and its precursors can be considered to be mainly plant remains, the wastewater euent had a strong contribution from anthropogenic activities. The original samples were freeze-dried (Orig) or separated into fulvic acids (FA), humic acids (HA) and non-humic substances (NHS) by using an XAD-8 (polymethylmethacrylate) resin according to a modi®ed method of Mantoura and Riley (Mantoura and Riley, 1975; Abbt-Braun et al., 1991). Basic sample parameters are described in detail elsewhere (Frimmel and AbbtBraun, 1999). The concentration of dissolved organic carbon was measured using a Shimadzu 5000 TOC analyzer, calibrated with potassium hydrogenphthalate standards. Sample solutions were prepared by adding 10 ml of water to approximately 2.5 mg of freeze-dried material. The resulting mixtures were soni®ed for 15 min and subsequently ®ltered (0:45 lm, polyvinylidene¯uoride). For the hydrolysis 0.75 ml of sodium hydroxide (c 1 mol/l) solution was added to 2.25 ml of sample solution.
The resulting solution was heated under nitrogen in a sealed glass vial for 48 h at 100°C. Subsequently, 25 ml of water was added, then the solution was ®ltered using a 0:45 lm polyvinylidene¯uoride ®lter. A styrene-based cation exchange resin cartridge (OnGuard-H, Dionex) was used to neutralize the solutions. After discarding the ®rst 4 ml, the solutions obtained were used in the further spectroscopic and chromatographic measurements. Dierent benzene carboxylic acids were investigated for their spectroscopic properties. The compounds are listed in Table 1. All compounds were purchased from Aldrich (grade > 97%) and used as received. The UV/Vis absorption measurements and the ¯uorescence measurements were carried out at pH values of 2, 7, and 11, respectively. To adjust the pH, aqueous HCl and NaOH were added to the solutions. In case of pH 7, a phosphate buer was used. For the ¯uorescence measurements, the optical density of the samples was adjusted to 0.1 at 265 nm in order to measure all samples under the same excitation conditions. 2.2. UV/Vis and ¯uorescence For the UV/Vis measurements, a Cary50 spectrometer (Varian) was used. The absorption spectra were recorded in the range of 200 nm < kabs < 500 nm. All spectra were background corrected. The ¯uorescence spectra were recorded using a CDT900 ¯uorescence spectrometer (Edinburgh Analytical Instruments). The instrument was equipped with a 450 W Xenon lamp and a red-sensitive photomultiplier tube, which was operated in the single photon counting mode. The instrument is described in detail elsewhere (Kumke et al., 1998; Frimmel and Kumke, 1999). Fluorescence emission spectra and ¯uorescence excitation spectra were recorded. The ¯uorescence emission spectra were measured for three dierent excitation wavelengths of kex 260, 270, and 330 nm, in the wavelength range of 265 nm < kem < 650 nm, respectively. For the ¯uores-
Table 1 Spectroscopic properties of the benzoic acids investigateda
a
UV absorption maximumb (in nm)
Fluorescence maximumc (in nm)
Gallic acid 4-Hydroxycinnamic acid 2-Hydroxycinnamic acid 3,4-Dihydroxycinnamic acid 4-Hydroxybenzoic acid Acetylsalicylic acid Coniferyl alcohol Ferulic acid
262 288 271 (314) 288 (314) 248 (266) 263 (300shoulder ) 265 (310)
349 401 498 (408shoulder ) 426 ± 411 340 442
7-hydroxycoumarin
325
453
The data were collected from solution at pH 7. b Given are the wavelengths for the absorption maximum. In case of two maxima, the second is given in parentheses. c The excitation wavelengths were chosen according to the UV absorption maxima.
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cence excitation spectra, two dierent detection wavelengths kem 310 and 450 nm) were chosen, and the spectra were measured in the wavelength range of 225 nm < kex < 440 nm. The spectra were recorded with a spectral bandpass of 1.8 nm in the excitation as well as in the emission monochromator. The dwell time of the measurements was set to 0.5 s and data points were taken every nanometer. 2.3. Size-exclusion chromatography The experimental set-up for the size-exclusion chromatography (SEC) measurements is described in detail elsewhere (Specht et al., 2000). Three dierent on-line detection methods were used: UV-absorption at kabs 254 nm, ¯uorescence at kex;1 260 nm, kem;1 310 nm and at kex;2 330 nm, kem;2 450 nm and DOC. A TSK-50 HW (S) column was used with a phosphate buer as the mobile phase (28 mmol/l, pH 6.8). The exclusion volume (19 ml) and the permeation volume (47 ml) were determined using dextrane blue and water, respectively. The injection volume of the samples was 2 ml and the DOC concentration of the samples was in the range of 2±10 mg/l.
3. Results and discussion 3.1. UV/Vis spectroscopy The UV/Vis spectra were recorded in the range of 200 nm < kabs < 500 nm. For the humic substances ABV3 isolated from the wastewater euent, a decrease in the UV/Vis absorption was observed after hydrolysis only for the HA fraction. For the absorption spectra of ABV3 FA and ABV3 NHS no signi®cant changes were found. More conclusive results were obtained for the brown water HO14 and its isolated fractions. Here, a decrease in the absorption was found for all fractions investigated. While the brown water samples HO14 before hydrolysis showed featureless absorption spectra, the hydrolysis caused a signi®cant change in the related absorption spectra (e.g., Fig. 1 for HO14 FA before and after hydrolysis). This was observed for the HA fraction of the wastewater euent ABV3 as well. Due to hydrolysis, a well-pronounced absorption band around kabs 265 nm appeared in the spectra (Fig. 1). It was strongest for the FA and HA fraction and least pronounced for the NHS fraction of the brown water HO14. To compare the in¯uence of the hydrolysis on the dierent HS fractions investigated, the UV/Vis spectral data were normalized to the concentration of DOC. The absorption kabs 254 nm and kabs 436 nm is frequently used to characterize the properties of HS solu-
Fig. 1. UV/Vis spectra (not normalized) of HO14 FA before and after hydrolysis. The ratio of the two spectra is shown as well as the spectra of the pure medium before (a) and after hydrolysis (b).
tions. These wavelengths were formerly used to estimate the aromaticity and the content of quinones in HS samples (Stevenson, 1982). Korshin et al. (1997) suggested the use of the absorption at kabs 203 nm and at kabs 253 nm for an estimation of the degree of functionality of the aromatic ring. Benzenoid compounds usually show an absorption band at 203 nm, which is often referred to as the benzenoid band (Bz). An absorption around 253 nm can be attributed to a chargetransfer transition (CT). While the strength of the Bz band stays relatively unchanged upon changes in the functionality of the aromatic ring, the CT band is strongly altered. Therefore, a change in the ratio of these wavelengths should be indicative of alterations in the functionality of the aromatic system. In Fig. 2 the ratio of the UV/Vis absorption at 254 nm to that at 203 nm is shown (later referred to as
Fig. 2. Comparison of the absorption ratio k254 nm =k203 nm for the HS samples before and after alkaline hydrolysis. The pH was adjusted to 4.
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ratio I). For the HS fractions investigated, an increase in ratio I was observed after hydrolysis, which indicates an increase in the degree of eective functionality of the benzoic rings. Because a direct attack of hydroxide ions on the aromatic moieties is improbable under the reaction conditions applied (Fyfe, 1971), side chain alterations like ester and amide hydrolyses, retro-aldol reactions, benzil-benzilic rearrangements, a and b ether cleavages as well as b±c±C±C cleavages may be responsible for the increase (Wallis, 1971; Parsons, 1989; Beyer, 1998). 3.2. Fluorescence spectroscopy In Fig. 3, the in¯uence of hydrolysis on the steadystate ¯uorescence of the brown water samples HO14 is shown. For comparison, the ¯uorescence intensities were normalized to the concentration of dissolved organic carbon. As a general trend , the ¯uorescence of the NOM samples investigated was increased upon hydrolysis. However, the observed increase was much stronger for the brown water samples and within the fractions decreasing in the order HO14 Orig > HO14 FA > HO14 HA > HO14 NHS. For the wastewater euent, ABV3 FA and ABV3 NHS showed a small increase. To quantify the alteration in the ¯uorescence intensity, the ¯uorescence spectra recorded at dierent excitation wavelengths were integrated and the relative change upon hydrolysis was calculated. The calculated overall change in the ¯uorescence intensity of the HS samples is shown in Fig. 4 for two dierent excitation wavelengths. Independent of the excitation wavelength, the ¯uorescence intensity of the wastewater euent fractions was only slightly changed upon hydrolysis, indicated by a relative change in ¯uorescence intensity close to one (see Fig. 4(b)). On the other hand, for the
Fig. 3. Steady-state ¯uorescence spectra kex 330 nm; pH 4 of brown water HO14 Orig before and after alkaline hydrolysis of the sample.
Fig. 4. Relative change of the overall ¯uorescence intensity of the NOM samples due to hydrolysis at dierent excitation wavelengths. Shown are the ratios of the integrated ¯uorescence intensity before hydrolysis and after hydrolysis ((a) brown water HS and (b) wastewater euent HS).
brown water and its fractions, a strong increase in the overall ¯uorescence intensity was observed (for HO14 Orig a 2.5-fold increase was observed for kex 260 nm; see Fig. 4(a)). Moreover, the observed increase caused by hydrolysis was dependent on the excitation wavelength. For kex 330 nm, the increase of the overall ¯uorescence intensity was smaller. Here, an increase less than 2-fold was measured for all brown water samples HO14. The completely dierent behaviour of the wastewater samples ABV3 might be attributed to their young age. On the other hand, the brown water samples HO14 are much older which means, that they have been subjected to numerous alterations like enzymatic or light-induced oxidations. These alterations seem to favour the spectroscopic changes induced by alkaline treatment. As the hydroxide ions preferently attack aliphatic moieties with oxygen-containing functional groups (Wallis, 1971; Parsons, 1989; Beyer, 1998), the results show the importance of these structural parts for the spectroscopic properties of HS. The eect of increasing ¯uorescence intensity has also been observed after chlorination, ozonation and UV
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irradiation of HS (Korshin et al., 1999; Win et al., 2000). Under these dierent conditions HS are supposed to be cleaved to smaller molecules. These might have higher ¯uorescence quantum yields than the original macromolecular substances. Another possibility is, that upon degradation, ¯uorescence quenching of small molecules by their interaction with HS (Morra et al., 1990; Doll et al., 1999) is reduced. The two explanations were further examined by size-exclusion chromatography and are discussed later in the paper. It is interesting to note that the observed increase in the ¯uorescence intensity was not uniform in the spectral range of the ¯uorescence spectrum which becomes obvious from the calculation of the dierence of ¯uorescence intensity before and after hydrolysis (see Fig. 5). The maximum increase of the ¯uorescence intensity was found in the wavelength range between 415 nm < kem < 440 nm: In Fig. 5, the calculated spectra (for kex 330 nm of the brown water samples are shown. In Fig. 5(b), the ¯uorescence excitation spectra of the brown water HO14 FA fraction before and after hydrolysis are compared. The ratio of the two spectra is
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shown as well. For a simple homogeneous solution of a ¯uorescent compound, it can be expected that the observed ¯uorescence excitation spectrum and the corresponding UV/Vis absorption spectrum are identical (assuming that the intensity distribution of the excitation lamp was considered properly). In general, for HS the measured ¯uorescence excitation spectrum and the UV/Vis absorption spectrum are not identical. While the absorption spectrum is (almost) featureless with an increasing absorption toward the UV region, in the ¯uorescence excitation spectrum, a relatively well-resolved band is observed in the spectral region around 310 nm < kex < 370 nm. These dierences indicate that only a part of the light-absorbing chromophores present in HS is ¯uorescent. Based on the ¯uorescence excitation spectra, a connection between the observed ¯uorescence increase and the related change in the absorption spectra can be drawn. The comparison of the ratio of the ¯uorescence excitation spectra and the dierence in the absorption spectra (see Fig. 1) reveals striking similarities and it also gives one explanation for the observed dependence on the ¯uorescence excitation wavelength of the ¯uorescence increase upon hydrolysis. The compounds built (or released) by the hydrolysis of the HS showed a strong absorption around kabs 265 nm. The larger increase in the ¯uorescence intensity, with kex 260 nm, can be attributed to that fact. The number of compounds (or subunits) released from HS participating in light absorption was found to be larger for k 260 nm, and therefore, the observed overall increase in the ¯uorescence intensity with that particular excitation wavelength can also be expected to be higher when compared to an excitation wavelength of lower energy (e.g., kex 330 nm). 3.3. Comparison of the spectral character of benzoic acid and the changes induced by hydrolysis in ¯uorescence and UV/Vis absorption
Fig. 5. (a) Calculated dierences of the steady-state ¯uorescence spectra of the brown water and its HS fractions upon alkaline hydrolysis kex 330 nm. (b) Comparison of the ¯uorescence excitation spectra of HO14 FA before and after hydrolysis (not corrected for lamp pro®le). The ratio of both spectra is shown as well kem 450 nm.
In a comparative approach, the UV/Vis absorption spectra and the ¯uorescence spectra of dierent benzoic acids and related compounds were measured and compared with spectra of the HS and the calculated dierences observed upon hydrolysis. Table 1 summarizes the relevant spectroscopic properties of the compounds investigated. All benzoic acid derivatives show absorption maxima between 250 nm < kabs;max < 330 nm. A strong overlap was found compared to the UV/Vis absorption spectra calculated from the measurements before and after hydrolysis of HS. This was also valid for the steady-state ¯uorescence excitation spectra of the benzoic acids. In Fig. 6, the calculated ¯uorescence spectra of the hydrolysis products of the brown water HO14 Orig is compared with the ¯uorescence spectra of various benzene carboxylic acids.
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Fig. 6. Comparison of the ¯uorescence spectra of several benzoic acids with the calculated dierence spectrum of HO14 Orig upon hydrolysis kex 260 nm; pH 7.
Assuming that some of the building processes of HS are reversed due to the alkaline treatment, a release of lignin-derived moieties like p-coumaryl, sinapyl and coniferyl alcohol-like structures can be expected. This is strongly supported by the striking agreement between the UV/Vis absorption spectra, as well as the steadystate ¯uorescence spectra of those simple model compounds and the calculated changes upon hydrolysis in the spectra of the HS. Furthermore, the results are in qualitative accordance with analytical pyrolysis data of lake Hohloh samples, where 16±22% of the volatile matter were assigned to phenols, lignin monomers and dimers (Schulten and Gleixner, 1999). A signi®cant contribution of allochthonous, soil-derived precursors to lake Hohloh HS seems to be reasonable, since it is a nutrient-poor bog lake, that will slowly become overgrown by detrital plant material. 3.4. Size-exclusion chromatography The data from the size-exclusion chromatography con®rmed the ®ndings from the UV/Vis and ¯uorescence measurements. The chromatograms of the wastewater samples were only slightly altered by hydrolysis. Whereas the DOC and UV-absorption traces of the FA and NHS sample showed virtually no changes, the ¯uorescence trace of the FA sample revealed an increase of ¯uorescence intensity. This increase was not attributable to single fractions and occurred at both sets of excitation and emission wavelengths. Both, the DOC and the UV absorption trace of the HA sample showed a shift to higher elution volumes. In size-exclusion chromatography, several non-ideal interactions between the gel and sample such as ion exclusion, ion exchange and adsorption can occur (Johansson and Gustavsson, 1988;
Potschka, 1988). These interactions are superimposed to the separation of the molecules due to their size. Under the reaction conditions applied, the most likely reactions are ester and ether cleavages (Wallis, 1971), which lead to an increase in carboxyl and hydroxyl group content. As a result, the gel±sample interactions, which may lead to a retardation, are decreased (Specht and Frimmel, 2000). Because of this, the shift to higher elution volumes can most likely be attributed to smaller molecules rather than to an increase in the attractive interactions between gel and sample. The in¯uence of hydrolysis on the brown water sample was much more pronounced in comparison to the wastewater samples. For all brown water samples (original, FA, HA, and NHS) the ¯uorescence intensity increased whereas the UV absorption decreased. In connection with a shift to higher elution volumes, this is another indication for breakdown of larger molecules due to hydrolysis. Large molecules have the ability to release the energy from an absorbed photon via processes which do not involve the emission of light. For smaller molecules, this is often not the case. Because of this, the ¯uorescence increases even if the UV absorption decreases, i.e., the ¯uorescence yield increases. Due to the chromatographic separation quenching of the smaller ¯uorescent entities by larger NOM molecules is suppressed even for the non-hydrolysed sample. Therefore, the increase of ¯uorescence is more likely caused by an increase of the ¯uorescence yield and the number of ¯uorescent entities rather than by a decrease of the quenching eect of NOM. In contrast to the wastewater sample, the increase of ¯uorescence intensity can be attributed to several fractions. Fig. 7 depicts the chromatograms obtained with DOC-, UV absorption-, and the ¯uorescence detection of the brown water sample HO14 Orig before and after hydrolysis. The DOC and UV absorption chromatograms show a considerable shift to higher elution volumes, i.e., lower molecular mass. This shift cannot be found in the ¯uorescence trace. From these results one can conclude, that the higher molecular mass fractions of natural organic matter contain structures which are potentially capable of ¯uorescence. These structures are unveiled by the hydrolysis reaction and lead to an increase in ¯uorescence intensity. This holds for all brown water samples investigated in this work. The highest increase in ¯uorescence intensity (by a factor of 3.8) was found for kex;1 260 nm and kem;1 310 nm. At kex;2 330 nm; kem;2 450 nm, ¯uorescence intensity increased by a factor of 2.7. This increase is higher than the increase in overall ¯uorescence intensity, which was found for the non-separated sample. In the non-separated sample, ¯uorescence quenching can occur through intermolecular energy transfer from smaller to larger molecules, which is not possible after separation in the SEC column. As the total area of the DOC chromato-
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Fig. 7. SEC chromatograms of brown water HO14 Orig before and after hydrolysis obtained with DOC, UV absorbance, and ¯uorescence detection.
grams does not decrease, no degradation of organic matter to CO2 took place due to hydrolysis. On the other hand, the total area of the UV signal decreases by 16%. Because of the cleavage of side groups, conjugated systems might be destroyed, resulting in a lower UV absorption. In addition to the shift, the chromatograms of the samples after hydrolysis show fractions which were set free from the original sample. No major differences can be found between the dierent natural organic matter fractions in the DOC and UV absorption chromatograms. In contrast, it was possible to detect signi®cant dierences between the samples in the ¯uorescence signal. Fig. 7(c) shows the chromatogram of the ¯uorescence detector with kex;1 260 nm and kem;1 310 nm. Whereas fractions 2 and 3 could be found in the original sample as well as in the FA sample, fraction 2 was absent in the HA sample and fraction 3 could not be found in the NHS sample. Also, the HA showed no ¯uorescence at the chosen set of excitation and emission wavelengths before hydrolysis. The chromatograms of the ¯uorescence signal at kex;2 330 nm; kem;2 450 nm showed only minor changes in shape. Since humic substances show a strong ¯uorescence signal at this set of
excitation and emission wavelengths, the increase in intensity is probably caused by a decrease in the selfquenching eect of humic substances. To determine the structures of the substances which make up fractions 2 and 3 found in the ¯uorescence chromatograms, SEC experiments with the compounds listed in Table 1 were carried out under the same conditions used for the natural organic matter samples, i.e., the same UV absorption and ¯uorescence excitation and emission wavelengths were used. The data showed, that benzoic acid derivatives like gallic acid and 4-hydroxybenzoic acid had a strong ¯uorescence signal at kex;1 260 nm; kem;1 310 nm, but hardly any signal at kex;2 330 nm; kem;2 450 nm. The derivatives of cinnamic acid on the other hand showed a strong signal at kex;2 330 nm; kem;2 450 nm, but only a weak signal at kex;1 260 nm; kem;1 310 nm. In addition, the elution volume of the benzoic acid derivatives coincides with the elution volume of fraction 3. From this, one may conclude, that the fractions 2 and 3 consist of substances with a structure similar to hydroxybenzoic acids rather than hydroxycinnamic acid.
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4. Conclusions Hydrolysis reactions have been used quite extensively to obtain information on the structure of the building blocks of humic matter. In combination with spectroscopy in the UV/Vis range, hydrolysis gives information not only on the aromatic and unsaturated areas of the molecules with main absorbances around k 260 nm, but also on the molecular size. Especially ¯uorescence spectra give insight into the stability of the molecular structure in aqueous solution. The signi®cant increase in the ¯uorescence after hydrolysis makes it attractive to assume the presence of quenching mechanisms in the original dissolved macromolecule which are lost in the smaller hydrolysed products. The identi®cation of the building blocks still remains troublesome. Poor resolution of chromatographic fractionation based on water as the mobile phase and lack of powerful identi®cation methods are the main reasons. A way out of this dilemma seems to be the use of reasonable model compounds. Their properties in mixtures can be compared with the integrated character of the complex samples to be investigated. The best ®t is a good basis for structural assumptions. The application of that strategy to the hydrolysis products of HS makes the substituted representatives of salicylic acid, cinnamic acid, and 7hydroxy-coumarin to appear as attractive building blocks of the complex organic matter in brown water. Acknowledgements The work was funded by the Deutsche Forschungsgemeinschaft in the ROSIG research program and in the research project MetalOM (Grant Fr 536/21-1). The authors wish to thank Dr. Gudrun Abbt-Braun and Axel Heidt for preparing and supplying the HS samples. References Abbt-Braun, G., Frimmel, F.H., Lipp, P., 1991. Isolation of organic substances from aquatic and terrestrial systems ± comparison of some methods. Z. Wasser-Abwasser-Forsch 24, 285±292. Beyer, H., 1998. Lehrbuch der Organischen Chemie. Hirzel, Stuttgart. Doll, T.E., Frimmel, F.H., Kumke, M.U., Ohlenbusch, G., 1999. Interaction between natural organic matter (NOM) and polycyclic aromatic compounds (PAC) ± comparison of ¯uorescence quenching and solid phase micro extraction (SPME). Fresenius J. Anal. Chem. 364, 313±319. Frimmel, F.H., Jahnel, J., Hesse, S., 1998. Characterization of biogenic organic matter (BOM). Water Sci. Technol. 37, 97± 103.
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Formation, Structure and Reactions. Wiley±Interscience, New York, pp. 345±372. Win, Y.Y., Kumke, M.U., Specht, C.H., Schindelin, A.J., Kolliopoulos, G., Ohlenbusch, G., Kleiser, G., Hesse, S., Frimmel, F.H., 2000. In¯uence of oxidation of dissolved organic matter (DOM) on subsequent water treatment processes. Water Res. 34, 2098±2104.