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Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis
Organic Geochemistry 33 (2002) 213–220 www.elsevier.com/locate/orggeochem
Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography– polyacrylamide gel electrophoresis Olga Trubetskayaa, Oleg Trubetskojb, Ghislain Guyotc, Francis Andreuxd, Claire Richardc,* a
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia b Institute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia c Laboratoire de Photochimie Mole´culaire et Macromole´culaire UMR CNRS 6505, 63177 Aubie`re Cedex, France d Centre des Sciences de la Terre, 6bd Gabriel, F-21000 Dijon, France
Abstract Humic acids (HAs) extracted from soils of different origin (chernozem, ferralsol and ranker) and their fractions (A, B and C+D) obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis were investigated by steady-state fluorescence spectroscopy in the emission mode. Independently of HA source, high molecular size fractions A and B are shown to be weakly fluorescent. The main fluorophores, especially those emitting at long wavelength (around 500–510 nm), are contained in the polar and low molecular size fractions C+D. As indicated by the observed pH effect, aromatic structures bearing carboxylate and OH substituents may be involved in these longer wavelength emissions. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction Humic substances (HSs), usually operationally divided into humic acids (HAs, insoluble in acid) and fulvic acids (FAs, soluble in acid), constitute a large portion of the total organic pool in terrestial and aquatic environments. They play an important role in the regulation of the mobility and fate of plant nutrients and environmental contaminants (Stevenson, 1994). Despite intensive research on HSs during last past decades, the chemical structure of HA and FA is still unclear. Fluorescence spectroscopy has been often applied for investigating structural characteristics of HSs and their interactions with xenobiotics. Steady-state fluorescence
* Corresponding author. Tel.: +33-4-73-40-71-42; fax: +33-4-73-40-77-00. E-mail address: [email protected] (C. Richard).
techniques, i.e. emission, excitation and synchronous scan fluorescence spectroscopy, have been successfully used to characterize and/or to discriminate HSs of different origins (Senesi et al., 1991; Miano et al., 1988; Miano and Senesi, 1992; Pullin and Cabaniss, 1995; Mobed et al., 1996), naturally occurring organic matter (Coble et al., 1990; Belin et al., 1993; Seriti et al., 1994) and humic acids extracted from composts (Miiki et al., 1997). More recently, fluorescence spectroscopy was applied to monitor structural modifications of HSs after chlorination (Korshin et al., 1999). Even though much progress has been made in the differentiation of humic materials using fluorescence techniques, the identification of fluorophores remains difficult owing to the complexe structure of these macromolecules [i.e. molecular size (MS) range from several hundreds to several hundred thousands daltons] and to the overlapping of different fluorophores emission bands. In order to get a better insight into these emitting properties HSs were fractionated. Size exclusion
0146-6380/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(01)00153-X
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chromatography (SEC) is commonly used to fractionate HAs on the basis of MS differences (Swift, 1996). Trubetskoj et al. (1997) have developed an effective method for fractionation of HSs based on combination of SEC with polyacrylamide gel electrophoresis (PAGE) (tandem SEC–PAGE). Optimal chromatographic conditions were established for obtaining preparative quantities of soil humic acid fractions, named A, B and C+D with exactly defined electrophoretic mobility (EM) and MS (Trubetskoj et al., 1998). MS of the fractions decreased in the sequences A >B >C+D, independently of the source of the HA. Based on the protein standards the weight 3.105–105, 105–3.104 and 3.104–5.103 daltons were found for fractions A, B and C+D, respectively. A series of investigations into the physico-chemical properties of fractions obtained by coupling SEC–PAGE from soils of different origins were undertaken (Trubetskoj et al., 1999). Molar absorption coefficients at 465 nm and E4/E6 ratios (i.e. ratios of absorbances at 465 and 665 nm) were shown to increase as MS decreased. In addition, three HAs and their fractions were compared for their ability to photoinduce the transformation of 1,1-dimethylphenylurea on irradiation at 365 nm (Aguer et al., 2001) and thus shown to exhibit distinct photoinductive properties. If high MS fractions A and B appeared to be poor photoinductors compared to the whole HA, fraction C+D, in contrast, was as efficient or even more so. These results indicated that the photoinductive chromophores mainly occurred in the low MS fractions. The purpose of the present work is to compare the fluorescence properties of soil HAs and of their fractions obtained by tandem SEC–PAGE.
2. Materials and methods 2.1. Materials The HAs were extracted from A horizons of soils of different genesis: chernozem, Kursk region, Russia; ferralsol, Georgia, former USSR; ranker mountain soil under grassland, Sierra de la Demanda, Spain. HAs were extracted using the IHSS extraction procedure. The basic characteristics of these HAs are reported elsewhere (Trubetskoj et al., 1999, Aguer et al., 1997, 2001). The C/H ratio was 22.4, 14.2 and 10.0 for chernozem, ferralsol and ranker HA, respectively. 2.2. Fractionation of HAs The fractionation of soil HAs by tandem SEC–PAGE was previously reported (Trubetskoj et al., 1997, 1998). Briefly: HAs (5–10 mg) were dissolved in 7 M urea and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5100 cm), equilibrated with the same solution.
The void column volume (V0) and the total column volume (Vt) were 47 and 160 ml, respectively, and all humic material was eluted within the total column volume. Flow rate was 15 ml h 1. Column effluent was collected as 2 ml aliquots and each three aliquots were assayed by PAGE in the presence of denaturing agents according to Trubetskoj et al. (1991). The aliquots that consisted of individual electrophoretic zones named A, B and C+D in the PAG matrix with a similar EM in full, were combined into pools, dialysed against distilled water, lyophilized, and used for further physico-chemical analysis. The weight distribution of the HA fractions calculated using the ratio Wi/Wk, where Wi is the weight of fraction and Wk the weight of all HA fractions obtained after SEC fractionation of HA is given in Table 1. 2.3. Preparation of HAs and fractions solutions When added at a level of 5 mg in 100 ml of water purified using a Milli-Q Millipore device, fractions C+D were fully soluble while total HAs and fractions B were only partly soluble and fractions A poorly soluble. In order to solubilize all the humic materials, a pH 6.5 buffer was used. Fluorescence measurements were therefore performed at this pH using a mixture of disodium hydrogenophosphate and potassium dihydrogenophosphate (10 3 M) as a buffer. Emission spectra were recorded on solutions having the same absorbances at the selected wavelength: 0.30 0.03 at 300 nm, 0.22 0.02 at 340 nm, 0.15 0.02 at 380 nm, 0.12 0.02 at 420 nm and 0.09 0.01 at 460 nm (1 cm path-length quartz cell). The very low solubility of fractions A restricted these small absorbance values. All the solutions were filtered on 0.45 mm Sartorius filters (cellulose acetate) prior to analysis. Fluorescence measurements were also performed in acidic medium using solutions of fractions C+D. These fractions were first solubilized in pure water at a level of 1, 2 and 3 mg in 100 ml for chernozem, ranker and ferralsol HAs, respectively; then, the pH was adjusted to 2 using dilute HClO4. The solutions were stirred with a magnetic stirrer overnight and then filtered. The following absorbances were obtained: 0.170.02 at 340 nm and 0.05 0.01 at 460 nm. For studies at pH 13, HAs as well as fractions were simply dissolved in water containing 0.1 M KOH. Table 1 Weight distribution (%) of the HA fractions, obtained by tandem SEC–PAGE HA origin
Fraction A
Fraction B
Fraction BCD
Fraction C+D
Chernozem Ferralsol Ranker
243 435 212
192 192 263
21 2 15 2 23 2
364 232 303
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2.4. Analytical methods UV–visible absorption spectra were recorded on a Cary 3 spectrophotometer. Fluorescence spectra were recorded at room temperature on a Perkin-Elmer MPF3L spectrophotometer. The emission and excitation slits were set at 8 and 6 nm, respectively. A 50 nmmin 1 scan speed was used. The fluorescence emission spectra were recorded using five excitation wavelengths: 300, 340, 380, 420 and 460 nm (Raman emission bands appeared at 335, 385, 435 and 490 nm for the above excitations, respectively). The emission spectra were not corrected for the spectral sensitivity factor of the photomultiplier (Hamamatsu R106)-emission monochromator combination. They were recorded first on a X–t plotter and then replotted using Origin program collecting the fluorescence intensity (IF) each five nanometers.
3. Results and discussion 3.1. Absorption spectra Fig. 1 shows the absorption spectra of chernozem HA solutions and its fractions at a concentration of 20 mg l 1 and at pH 6.5. All the spectra exhibited a similar shape. However, absorption coefficients at a given wavelength differed from one fraction to the other. Within the wave-
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length range 250–500 nm, they decreased significantly in the order C+D>B> A. In contrast, fraction B appeared to be slightly more absorbant than fraction C+D above 525 nm. Similar results were obtained with ferralsol and ranker fractions that were, however, less dark than chernozem fractions. 3.2. Fluorescence of chernozem HA and its fractions Fluorescence emission spectra of the chernozem HA are given in Fig. 2. The emission wavelength range was large, extending from 350 nm to more than 600 nm. The change in excitation wavelength caused modifications in the shapes of the spectra. Excitations at 300, 340, 380 and 420 nm yielded featureless emission spectra as generally observed with humic material. Careful examination, however, revealed the presence of shoulders at 440–450, 480–490 and 510 nm. On the other hand, excitation at 460 nm clearly gave the emission band with maximum at 510 nm. In a general way, increase of the excitation wavelength also resulted in increase of the emission intensity. It means that excitation at longer wavelength involved chromophores either more fluorescent or more absorbant than excitation at shorter wavelength. As shown in Fig. 1, the absorbance of the solutions decreased as the wavelength increased. The latter hypothesis implies therefore that the absorbance of the solutions does not only result from the absorption of the fluorophores.
Fig. 1. UV–visible absorption spectra of solution of chernozem sample: whole HA (—), fraction A (- - - -), fraction B (. . . .) and fraction C+D (–.–.–) at a concentration of 20 mg l 1 in water buffered at pH=6.5 with phosphate buffer.
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Fig. 2. Emission spectra from chernozem HA; excitation wavelength set at 300 nm (!), 340 nm (~), 380 nm (~), 420 nm (*) and 460 nm (*). pH=6.5.
Excitation spectra showed that the emission band at 440 nm arose from excitation in a band with maxima at 380–390 nm, whereas fluorescence emission at 460 nm arose from excitation in a band red-shifted by 10–20 nm. The band responsible for the long wavelength emission (510 nm) had a maximum at l > 440 nm. The 467 nm maximum emission of the xenon lamp prevented us to observe the authentic maximum of this excitation band.
The emission spectra of fractions measured upon excitation at 340 and 460 nm are given in Fig. 3. Fractions A and B appeared to be poorly emissive compared to whole HA; only a weak emission with maximum around 410 nm was observed. The intensity of fluorescence at 410 nm arising from fractions A and B was only one-third and two-third of that of HA, respectively. In contrast, the emission spectra of fraction C+D showed a broad and
Fig. 3. Emission spectra from chernozem HA and fractions A, B and C+D. pH=6.5: ~, excitation wavelength set at 340 nm; *, excitation wavelength set at 460 nm.
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intense band with a maximum at 500 nm for excitation at 340 nm and 515 nm for excitation at 460 nm. When excited at 340 nm, fraction C+D showed a more intense emission at wavelengths above 450 nm but less intense at shorter wavelengths compared to the whole HA. The
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relative intensities at 410 and 500 nm as given by the 500 I410 F /IF ratio were equal to 0.25 and 0.45 for the fraction C+D and the whole HA, respectively. Accordingly, fraction C+D contains almost all the components responsible for the emission at long wave-
Fig. 4. Emission spectra from chernozem HA (a) and fraction C+D (b): ~, excitation wavelength set at 340 nm; pH=6.5 or 13; *, excitation wavelength set at 460 nm; pH=6.5 or 13.
Fig. 5. Emission spectra from fraction C+D of chernozem HA: ~, excitation wavelength set at 340 nm; pH=6.5 or 2; *, excitation wavelength set at 460 nm; pH=6.5 or 2.
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Fig. 6. Emission spectra from fraction C+D of ferralsol HA; excitation wavelength set at 300 nm (!), 340 nm (~), 380 nm (~), 420 nm (*) and 460 nm (*). pH=6.5. Inset: ~, excitation wavelength set at 340 nm; pH=6.5 or 2; *, excitation wavelength set at 460 nm; pH=6.5 or 2.
length (480–490 and 510 nm) while it only contains a part of the fluorophores emitting at shorter wavelengths. On the other hand, fractions A and B mainly contain fluorophores emitting below 450 nm. The pH effect on fluorescence emission is shown in Figs. 4 and 5. pH increase from 6.5 to 13 yielded the same modifications on the whole HA (Fig. 4a) as on fraction C+D (Fig. 4b); that is the fluorescence intensity decreased in the 450–600 nm range, especially at 510 and 515 nm, and these latter emissions were slightly red-shifted by 7 and 6 nm, respectively. These similar behaviours confirm that the main fluorophores of HA have been concentrated in fraction C+D. Measurements at pH 2 were performed on fraction C+D exclusively since this fraction was the only one to show enough solubility in acidic medium (see Section 2.3). A drastic decrease of the emission intensity was observed and the emission at 515 nm was blue-shifted by 5 nm (see Fig. 5). This pH effect (shift of maximum emission in basic and acidic media and decrease of fluorescence emission upon acidification from pH 6.5 to pH 2) suggests that the fluorescence process is strongly dependent on ionisation state of macromolecules or of fluorophores.
3.3. Fluorescence of ferralsol and ranker humic acids The emission spectra of the ferralsol HA were very similar to that of chernozem HA. Again, fractions A and B exhibited a very weak fluorescence at l < 450 nm whereas the fraction C+D was more fluorescent with emission spectra similar to those of the whole HA. Broad bands were obtained upon excitation at 300, 340, 380 and 420 nm and shoulders appeared at 420, 450, 490 and 505 nm (see Fig. 6). The excitation at 460 nm yielded an emission band with a maximum at 505 nm. As previously observed in the case of chernozem sample, acidification of fraction C+D, pH 2, strongly reduced the emission at l > 460 nm (inset of Fig. 6). The ranker samples showed the same general trends as the other HA samples. It appeared, however, that the whole HA and the fraction C+D were about four times more emissive than chernozem and ferralsol counterparts (Fig. 7). The pH effect on fraction C+D emission excited at 340 nm is shown in inset of Fig. 7. Acidification to pH 2 reduced the fluorescence intensity and blue-shifted the maximum of emission by 20 nm. The increase of pH to 13 reduced slightly the emission intensity and induced a redshift by 7 nm of the shoulder at 510 nm.
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Fig. 7. Emission spectra from fraction C+D of ranker HA; excitation wavelength set at 300 nm (!), 340 nm (~), 380 nm (~), 420 nm (*) and 460 nm (*). pH=6.5. Inset: ~, excitation wavelength set at 340 nm; pH=6.5 or 2 or 13.
4. Discussion and conclusion The above results show that independent of the soil genesis, the main part of fluorophores initially contained in the HA are present in fractions C+D. This fraction contains lower MS macromolecules and exhibits a lower mass over charge ratio as shown by higher mobility in electrophoresis. Swift et al. (1992) divided soil HAs into four subfractions by SEC on Sephadex and found using NMR that the content of carboxylic groups significantly increased with a decrease of subfraction MS. Using IR and 13C NMR spectroscopy for studying HA fractions of different MS obtained by ultrafiltration, Shin et al. (1999) found that the molecules of fraction having a MS higher than 105 daltons were primarily aliphatic while the molecules of the fractions (5.104–104 daltons) contained an equal quantity of aromatic and aliphatic carbons. Moreover, titration data were consistent with an increase in the number of carboxylate groups per unit mass as MS became smaller. Based on these observations, low MS fractions C+D probably have a high content in aromatic carbons and the highest content in carboxylate functional groups. The fluorescence properties of fractions C+D are therefore in good agreement with suggestions that benzoic derivatives could be important contributors to fluorescence of humic and fulvic acids (Senesi et al., 1991).
By varying the pH of the solutions over a wide range, information on the fluorophores could be obtained. In particular, taking advantage of the fact that fluorescent chromophores were contained in the polar and therefore soluble low MS fraction, we could study the influence of acidification on emissive properties. In general, the emission band with longer wavelength appeared to be affected by changes of pH. A strong decrease of the band intensity and a slight blue-shift was observed when the pH was decreased from 6.5 to 2.0. A decrease of band intensity was also observed when the pH was raised to 13 and the maximum of emission was slightly red-shifted. Conclusions from these observations must be drawn with caution because changes of pH influence not only the ionization state of fluorophores but also molecular conformation (Ghosh and Schnitzer, 1980, Miano et al., 1988). Moreover, even though we admit that the main changes are related to fluorophores themselves, it must be kept in mind that singlet excited states from which fluorescence arises show pKa that may be different from those of ground states. Anyway, the decrease in fluorescence intensity and the blue-shift observed upon acidification strongly suggest the involvement of aromatic carboxylate functional groups. Indeed, it is known that these types of fluorophores are less emissive in molecular than in anionic form (Guilbault, 1973). On the other hand, the decrease of the 500–510 nm band and the red-shift in basic
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medium is in agreement with the involvement of phenolic functions (Bowen, 1967). All these results indicate that structures such as salicylate could be involved in the emission as already proposed by Senesi et al. (1991). An important question still remains: why do they absorb at so long a wavelength (460 nm)? The fact that fluorophores were mainly found in fraction C+D shows that they were not equally distributed in all the macromolecules but concentrated in the polar and low MS fractions. It appears that fractions A, B and C+D were not only different in sizes but also contained different chromophores, thus showing that the method of fractionation was efficient. Recently, fractions C+D were shown to contain most of the photoinductive components of HSs whereas fractions A and B did not (Aguer et al., 2001). Chromophores responsible for photoinduction are not necessarily those emitting fluorescence; however, both types of chromophores have, in common, to be contained in the low MS polar fractions.
Acknowledgements The authors wish to thank the CNRS and Russian Academy of Sciences for financial support of the investigation (project 4992). A part of this research has been supported by Russian Foundation for Basic Research (project 01-05-64666-a).
References Aguer, J.-P., Richard, C., Andreux, F., 1997. Comparison of the photoinductive properties of commercial, synthetic and soil-extracted humic substances. Journal of Photochemistry and Photobiology A: Chemistry 103, 163–168. Aguer, J.-P., Trubetskoj, O.A., Trubetskaya, O.E., Richard, C., 2001. Photoinductive properties of soil humic acids and their fractions obtained by tandem size exclusion chromatographypolyamide gel electrophoresis. Chemosphere 44, 77–81. Belin, C., Quellec, C., Lamotte, M., Ewald, M., Simon, P., 1993. Characterization by fluorescence of the dissolved organic matter in natural water. Application to fractions obtained by tangential ultrafiltration and XAD resin isolation. Environmental Technology 14, 1131–1144. Bowen, E.J., 1968. Luminescence in Chemistry. D. Van Nostram Company Ltd, London. Coble, P.G., Green, S.A., Blough, N.V., Gagosian, R.B., 1990. Characterization of dissolved organic matter in the Black Sea by fluorescence spectroscopy. Nature 348, 432–435. Ghosh, K., Schnitzer, M., 1980. Fluorescence excitation spectra of humic substances. Canadian Journal of Soil Science 60, 373–379. Guilbault, G.G., 1973. Practical Fluorescence. Theory, Methods and Techniques. Marcel Dekker, Inc, New York.
Korshin, G.V., Kumke, M.U., Li, C.W., Frimmel, F.H., 1999. Influence of chlorination on chromophores and fluorophores in humic substances. Environmental Science and Technology 33, 1207–1212. Miano, T.M., Sposito, G., Martin, J.P., 1988. Fluorescence spectroscopy of humic substances. Soil Science Society of America Journal 52, 1016–1019. Miano, T.M., Senesi, N., 1992. Synchronous excitation fluorescence spectroscopy applied to soil humic substances chemistry. Science of the Total Environment 117- 118, 41–51. Miiki, V., Senesi, N., Ha¨nninen, K., 1997. Characterization of humic material formed by composting of domestic and industrial biowastes. Chemosphere 34, 1639–1651. Mobed, J.J., Hemmingsen, S.L., Autry, J.L., McGown, L.B., 1996. Fluorescence characterization of IHSS humic substances : total luminescence spectra with absorbance correction. Environmental Science and Technology 30, 3061–6065. Pullin, M.J., Cabaniss, S.E., 1995. Rank analysis of the pHdependent synchronous fluorescence spectra of six standard humic substances. Environmental Science and Technology 29, 1460–1467. Senesi, N., Miano, T.M., Provenzano, M.R., Brunetti, G., 1991. Characterization, differentiation, and classification of humic substances by fluorescence spectroscopy. Soil Science 152, 259–271. Seriti, A., Morelli, E., Nannicini, L., Giambelluca, A., Scarano, G., 1994. Fluorescence emission characteristics of naturally occurring organic matter in relation to metal complexation studies. Science of the Total Environment 148, 73–81. Shin, H.S., Monsallier, J.-M., Choppin, G.R., 1999. Spectroscopic and chemical characterizations of molecular size fractionated humic acid. Talanta 50, 641–647. Stevenson, F.J. (Ed.), 1994. Humus Chemistry: Genesis, Composition, Reactions, 2nd Edition. Wiley, New York. Swift, R.S., Leonard, R.L., Newman, R.H., Theng, B.K.G., 1992. Changes in humic acid composition with molecular weight as detected by C-13 nuclear magnetic-resonance spectroscopy. Science of the Total Environment 118, 53–61. Swift, R., 1996. In: Methods of Soil Analysis: Part 3. Chemical Methods-SSSA Book Series No 5, pp. 1028–1031. Trubetskoj, O.A., Kudryavceva, L.Y., Shirshova, L.T., 1991. Characterization of soil humic matter by polyacrylamide gel electrophoresis in the presence of denaturing agents. Soil Biology and Biochemistry 23, 1179–1181. Trubetskoj, O.A., Trubetskaya, O.E., Afanas’eva, G.V., Reznikova, O.I., Saiz-Jimenez, C., 1997. Polyamide gel electrophoresis of soil humic acid fractionated by size exclusion chromatography and ultrafiltration. Journal of Chromatography A 767, 285–292. Trubetskoj, O.A., Trubetskaya, O.E., Afanas’eva, G.V., Reznikova, O.I., Hermosin, B., Saiz-Jimenez, C., 1998. Tandem size exclusion chromatography-polyacrylamide gel electrophoresis of humic acids. Zeitschrift fu¨r Pflanzenerna¨hrung und Bodenunde 161, 619–625. Trubetskoj, O.A., Trubetskaya, O.E., Reznikova, O.I., Afanas’eva, G.V., 1999. Weight and optical differences between soil humic acids fractions obtained by coupling SEC-PAGE. Geoderma 93, 277–287.
Fluorescence of soil humic acids and their fractions obtained by tandem size exclusion chromatography– polyacrylamide gel electrophoresis Olga Trubetskayaa, Oleg Trubetskojb, Ghislain Guyotc, Francis Andreuxd, Claire Richardc,* a
Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia b Institute of Basic Biological Problems, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia c Laboratoire de Photochimie Mole´culaire et Macromole´culaire UMR CNRS 6505, 63177 Aubie`re Cedex, France d Centre des Sciences de la Terre, 6bd Gabriel, F-21000 Dijon, France
Abstract Humic acids (HAs) extracted from soils of different origin (chernozem, ferralsol and ranker) and their fractions (A, B and C+D) obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis were investigated by steady-state fluorescence spectroscopy in the emission mode. Independently of HA source, high molecular size fractions A and B are shown to be weakly fluorescent. The main fluorophores, especially those emitting at long wavelength (around 500–510 nm), are contained in the polar and low molecular size fractions C+D. As indicated by the observed pH effect, aromatic structures bearing carboxylate and OH substituents may be involved in these longer wavelength emissions. # 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction Humic substances (HSs), usually operationally divided into humic acids (HAs, insoluble in acid) and fulvic acids (FAs, soluble in acid), constitute a large portion of the total organic pool in terrestial and aquatic environments. They play an important role in the regulation of the mobility and fate of plant nutrients and environmental contaminants (Stevenson, 1994). Despite intensive research on HSs during last past decades, the chemical structure of HA and FA is still unclear. Fluorescence spectroscopy has been often applied for investigating structural characteristics of HSs and their interactions with xenobiotics. Steady-state fluorescence
* Corresponding author. Tel.: +33-4-73-40-71-42; fax: +33-4-73-40-77-00. E-mail address: [email protected] (C. Richard).
techniques, i.e. emission, excitation and synchronous scan fluorescence spectroscopy, have been successfully used to characterize and/or to discriminate HSs of different origins (Senesi et al., 1991; Miano et al., 1988; Miano and Senesi, 1992; Pullin and Cabaniss, 1995; Mobed et al., 1996), naturally occurring organic matter (Coble et al., 1990; Belin et al., 1993; Seriti et al., 1994) and humic acids extracted from composts (Miiki et al., 1997). More recently, fluorescence spectroscopy was applied to monitor structural modifications of HSs after chlorination (Korshin et al., 1999). Even though much progress has been made in the differentiation of humic materials using fluorescence techniques, the identification of fluorophores remains difficult owing to the complexe structure of these macromolecules [i.e. molecular size (MS) range from several hundreds to several hundred thousands daltons] and to the overlapping of different fluorophores emission bands. In order to get a better insight into these emitting properties HSs were fractionated. Size exclusion
0146-6380/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(01)00153-X
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chromatography (SEC) is commonly used to fractionate HAs on the basis of MS differences (Swift, 1996). Trubetskoj et al. (1997) have developed an effective method for fractionation of HSs based on combination of SEC with polyacrylamide gel electrophoresis (PAGE) (tandem SEC–PAGE). Optimal chromatographic conditions were established for obtaining preparative quantities of soil humic acid fractions, named A, B and C+D with exactly defined electrophoretic mobility (EM) and MS (Trubetskoj et al., 1998). MS of the fractions decreased in the sequences A >B >C+D, independently of the source of the HA. Based on the protein standards the weight 3.105–105, 105–3.104 and 3.104–5.103 daltons were found for fractions A, B and C+D, respectively. A series of investigations into the physico-chemical properties of fractions obtained by coupling SEC–PAGE from soils of different origins were undertaken (Trubetskoj et al., 1999). Molar absorption coefficients at 465 nm and E4/E6 ratios (i.e. ratios of absorbances at 465 and 665 nm) were shown to increase as MS decreased. In addition, three HAs and their fractions were compared for their ability to photoinduce the transformation of 1,1-dimethylphenylurea on irradiation at 365 nm (Aguer et al., 2001) and thus shown to exhibit distinct photoinductive properties. If high MS fractions A and B appeared to be poor photoinductors compared to the whole HA, fraction C+D, in contrast, was as efficient or even more so. These results indicated that the photoinductive chromophores mainly occurred in the low MS fractions. The purpose of the present work is to compare the fluorescence properties of soil HAs and of their fractions obtained by tandem SEC–PAGE.
2. Materials and methods 2.1. Materials The HAs were extracted from A horizons of soils of different genesis: chernozem, Kursk region, Russia; ferralsol, Georgia, former USSR; ranker mountain soil under grassland, Sierra de la Demanda, Spain. HAs were extracted using the IHSS extraction procedure. The basic characteristics of these HAs are reported elsewhere (Trubetskoj et al., 1999, Aguer et al., 1997, 2001). The C/H ratio was 22.4, 14.2 and 10.0 for chernozem, ferralsol and ranker HA, respectively. 2.2. Fractionation of HAs The fractionation of soil HAs by tandem SEC–PAGE was previously reported (Trubetskoj et al., 1997, 1998). Briefly: HAs (5–10 mg) were dissolved in 7 M urea and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5100 cm), equilibrated with the same solution.
The void column volume (V0) and the total column volume (Vt) were 47 and 160 ml, respectively, and all humic material was eluted within the total column volume. Flow rate was 15 ml h 1. Column effluent was collected as 2 ml aliquots and each three aliquots were assayed by PAGE in the presence of denaturing agents according to Trubetskoj et al. (1991). The aliquots that consisted of individual electrophoretic zones named A, B and C+D in the PAG matrix with a similar EM in full, were combined into pools, dialysed against distilled water, lyophilized, and used for further physico-chemical analysis. The weight distribution of the HA fractions calculated using the ratio Wi/Wk, where Wi is the weight of fraction and Wk the weight of all HA fractions obtained after SEC fractionation of HA is given in Table 1. 2.3. Preparation of HAs and fractions solutions When added at a level of 5 mg in 100 ml of water purified using a Milli-Q Millipore device, fractions C+D were fully soluble while total HAs and fractions B were only partly soluble and fractions A poorly soluble. In order to solubilize all the humic materials, a pH 6.5 buffer was used. Fluorescence measurements were therefore performed at this pH using a mixture of disodium hydrogenophosphate and potassium dihydrogenophosphate (10 3 M) as a buffer. Emission spectra were recorded on solutions having the same absorbances at the selected wavelength: 0.30 0.03 at 300 nm, 0.22 0.02 at 340 nm, 0.15 0.02 at 380 nm, 0.12 0.02 at 420 nm and 0.09 0.01 at 460 nm (1 cm path-length quartz cell). The very low solubility of fractions A restricted these small absorbance values. All the solutions were filtered on 0.45 mm Sartorius filters (cellulose acetate) prior to analysis. Fluorescence measurements were also performed in acidic medium using solutions of fractions C+D. These fractions were first solubilized in pure water at a level of 1, 2 and 3 mg in 100 ml for chernozem, ranker and ferralsol HAs, respectively; then, the pH was adjusted to 2 using dilute HClO4. The solutions were stirred with a magnetic stirrer overnight and then filtered. The following absorbances were obtained: 0.170.02 at 340 nm and 0.05 0.01 at 460 nm. For studies at pH 13, HAs as well as fractions were simply dissolved in water containing 0.1 M KOH. Table 1 Weight distribution (%) of the HA fractions, obtained by tandem SEC–PAGE HA origin
Fraction A
Fraction B
Fraction BCD
Fraction C+D
Chernozem Ferralsol Ranker
243 435 212
192 192 263
21 2 15 2 23 2
364 232 303
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2.4. Analytical methods UV–visible absorption spectra were recorded on a Cary 3 spectrophotometer. Fluorescence spectra were recorded at room temperature on a Perkin-Elmer MPF3L spectrophotometer. The emission and excitation slits were set at 8 and 6 nm, respectively. A 50 nmmin 1 scan speed was used. The fluorescence emission spectra were recorded using five excitation wavelengths: 300, 340, 380, 420 and 460 nm (Raman emission bands appeared at 335, 385, 435 and 490 nm for the above excitations, respectively). The emission spectra were not corrected for the spectral sensitivity factor of the photomultiplier (Hamamatsu R106)-emission monochromator combination. They were recorded first on a X–t plotter and then replotted using Origin program collecting the fluorescence intensity (IF) each five nanometers.
3. Results and discussion 3.1. Absorption spectra Fig. 1 shows the absorption spectra of chernozem HA solutions and its fractions at a concentration of 20 mg l 1 and at pH 6.5. All the spectra exhibited a similar shape. However, absorption coefficients at a given wavelength differed from one fraction to the other. Within the wave-
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length range 250–500 nm, they decreased significantly in the order C+D>B> A. In contrast, fraction B appeared to be slightly more absorbant than fraction C+D above 525 nm. Similar results were obtained with ferralsol and ranker fractions that were, however, less dark than chernozem fractions. 3.2. Fluorescence of chernozem HA and its fractions Fluorescence emission spectra of the chernozem HA are given in Fig. 2. The emission wavelength range was large, extending from 350 nm to more than 600 nm. The change in excitation wavelength caused modifications in the shapes of the spectra. Excitations at 300, 340, 380 and 420 nm yielded featureless emission spectra as generally observed with humic material. Careful examination, however, revealed the presence of shoulders at 440–450, 480–490 and 510 nm. On the other hand, excitation at 460 nm clearly gave the emission band with maximum at 510 nm. In a general way, increase of the excitation wavelength also resulted in increase of the emission intensity. It means that excitation at longer wavelength involved chromophores either more fluorescent or more absorbant than excitation at shorter wavelength. As shown in Fig. 1, the absorbance of the solutions decreased as the wavelength increased. The latter hypothesis implies therefore that the absorbance of the solutions does not only result from the absorption of the fluorophores.
Fig. 1. UV–visible absorption spectra of solution of chernozem sample: whole HA (—), fraction A (- - - -), fraction B (. . . .) and fraction C+D (–.–.–) at a concentration of 20 mg l 1 in water buffered at pH=6.5 with phosphate buffer.
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Fig. 2. Emission spectra from chernozem HA; excitation wavelength set at 300 nm (!), 340 nm (~), 380 nm (~), 420 nm (*) and 460 nm (*). pH=6.5.
Excitation spectra showed that the emission band at 440 nm arose from excitation in a band with maxima at 380–390 nm, whereas fluorescence emission at 460 nm arose from excitation in a band red-shifted by 10–20 nm. The band responsible for the long wavelength emission (510 nm) had a maximum at l > 440 nm. The 467 nm maximum emission of the xenon lamp prevented us to observe the authentic maximum of this excitation band.
The emission spectra of fractions measured upon excitation at 340 and 460 nm are given in Fig. 3. Fractions A and B appeared to be poorly emissive compared to whole HA; only a weak emission with maximum around 410 nm was observed. The intensity of fluorescence at 410 nm arising from fractions A and B was only one-third and two-third of that of HA, respectively. In contrast, the emission spectra of fraction C+D showed a broad and
Fig. 3. Emission spectra from chernozem HA and fractions A, B and C+D. pH=6.5: ~, excitation wavelength set at 340 nm; *, excitation wavelength set at 460 nm.
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intense band with a maximum at 500 nm for excitation at 340 nm and 515 nm for excitation at 460 nm. When excited at 340 nm, fraction C+D showed a more intense emission at wavelengths above 450 nm but less intense at shorter wavelengths compared to the whole HA. The
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relative intensities at 410 and 500 nm as given by the 500 I410 F /IF ratio were equal to 0.25 and 0.45 for the fraction C+D and the whole HA, respectively. Accordingly, fraction C+D contains almost all the components responsible for the emission at long wave-
Fig. 4. Emission spectra from chernozem HA (a) and fraction C+D (b): ~, excitation wavelength set at 340 nm; pH=6.5 or 13; *, excitation wavelength set at 460 nm; pH=6.5 or 13.
Fig. 5. Emission spectra from fraction C+D of chernozem HA: ~, excitation wavelength set at 340 nm; pH=6.5 or 2; *, excitation wavelength set at 460 nm; pH=6.5 or 2.
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Fig. 6. Emission spectra from fraction C+D of ferralsol HA; excitation wavelength set at 300 nm (!), 340 nm (~), 380 nm (~), 420 nm (*) and 460 nm (*). pH=6.5. Inset: ~, excitation wavelength set at 340 nm; pH=6.5 or 2; *, excitation wavelength set at 460 nm; pH=6.5 or 2.
length (480–490 and 510 nm) while it only contains a part of the fluorophores emitting at shorter wavelengths. On the other hand, fractions A and B mainly contain fluorophores emitting below 450 nm. The pH effect on fluorescence emission is shown in Figs. 4 and 5. pH increase from 6.5 to 13 yielded the same modifications on the whole HA (Fig. 4a) as on fraction C+D (Fig. 4b); that is the fluorescence intensity decreased in the 450–600 nm range, especially at 510 and 515 nm, and these latter emissions were slightly red-shifted by 7 and 6 nm, respectively. These similar behaviours confirm that the main fluorophores of HA have been concentrated in fraction C+D. Measurements at pH 2 were performed on fraction C+D exclusively since this fraction was the only one to show enough solubility in acidic medium (see Section 2.3). A drastic decrease of the emission intensity was observed and the emission at 515 nm was blue-shifted by 5 nm (see Fig. 5). This pH effect (shift of maximum emission in basic and acidic media and decrease of fluorescence emission upon acidification from pH 6.5 to pH 2) suggests that the fluorescence process is strongly dependent on ionisation state of macromolecules or of fluorophores.
3.3. Fluorescence of ferralsol and ranker humic acids The emission spectra of the ferralsol HA were very similar to that of chernozem HA. Again, fractions A and B exhibited a very weak fluorescence at l < 450 nm whereas the fraction C+D was more fluorescent with emission spectra similar to those of the whole HA. Broad bands were obtained upon excitation at 300, 340, 380 and 420 nm and shoulders appeared at 420, 450, 490 and 505 nm (see Fig. 6). The excitation at 460 nm yielded an emission band with a maximum at 505 nm. As previously observed in the case of chernozem sample, acidification of fraction C+D, pH 2, strongly reduced the emission at l > 460 nm (inset of Fig. 6). The ranker samples showed the same general trends as the other HA samples. It appeared, however, that the whole HA and the fraction C+D were about four times more emissive than chernozem and ferralsol counterparts (Fig. 7). The pH effect on fraction C+D emission excited at 340 nm is shown in inset of Fig. 7. Acidification to pH 2 reduced the fluorescence intensity and blue-shifted the maximum of emission by 20 nm. The increase of pH to 13 reduced slightly the emission intensity and induced a redshift by 7 nm of the shoulder at 510 nm.
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Fig. 7. Emission spectra from fraction C+D of ranker HA; excitation wavelength set at 300 nm (!), 340 nm (~), 380 nm (~), 420 nm (*) and 460 nm (*). pH=6.5. Inset: ~, excitation wavelength set at 340 nm; pH=6.5 or 2 or 13.
4. Discussion and conclusion The above results show that independent of the soil genesis, the main part of fluorophores initially contained in the HA are present in fractions C+D. This fraction contains lower MS macromolecules and exhibits a lower mass over charge ratio as shown by higher mobility in electrophoresis. Swift et al. (1992) divided soil HAs into four subfractions by SEC on Sephadex and found using NMR that the content of carboxylic groups significantly increased with a decrease of subfraction MS. Using IR and 13C NMR spectroscopy for studying HA fractions of different MS obtained by ultrafiltration, Shin et al. (1999) found that the molecules of fraction having a MS higher than 105 daltons were primarily aliphatic while the molecules of the fractions (5.104–104 daltons) contained an equal quantity of aromatic and aliphatic carbons. Moreover, titration data were consistent with an increase in the number of carboxylate groups per unit mass as MS became smaller. Based on these observations, low MS fractions C+D probably have a high content in aromatic carbons and the highest content in carboxylate functional groups. The fluorescence properties of fractions C+D are therefore in good agreement with suggestions that benzoic derivatives could be important contributors to fluorescence of humic and fulvic acids (Senesi et al., 1991).
By varying the pH of the solutions over a wide range, information on the fluorophores could be obtained. In particular, taking advantage of the fact that fluorescent chromophores were contained in the polar and therefore soluble low MS fraction, we could study the influence of acidification on emissive properties. In general, the emission band with longer wavelength appeared to be affected by changes of pH. A strong decrease of the band intensity and a slight blue-shift was observed when the pH was decreased from 6.5 to 2.0. A decrease of band intensity was also observed when the pH was raised to 13 and the maximum of emission was slightly red-shifted. Conclusions from these observations must be drawn with caution because changes of pH influence not only the ionization state of fluorophores but also molecular conformation (Ghosh and Schnitzer, 1980, Miano et al., 1988). Moreover, even though we admit that the main changes are related to fluorophores themselves, it must be kept in mind that singlet excited states from which fluorescence arises show pKa that may be different from those of ground states. Anyway, the decrease in fluorescence intensity and the blue-shift observed upon acidification strongly suggest the involvement of aromatic carboxylate functional groups. Indeed, it is known that these types of fluorophores are less emissive in molecular than in anionic form (Guilbault, 1973). On the other hand, the decrease of the 500–510 nm band and the red-shift in basic
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medium is in agreement with the involvement of phenolic functions (Bowen, 1967). All these results indicate that structures such as salicylate could be involved in the emission as already proposed by Senesi et al. (1991). An important question still remains: why do they absorb at so long a wavelength (460 nm)? The fact that fluorophores were mainly found in fraction C+D shows that they were not equally distributed in all the macromolecules but concentrated in the polar and low MS fractions. It appears that fractions A, B and C+D were not only different in sizes but also contained different chromophores, thus showing that the method of fractionation was efficient. Recently, fractions C+D were shown to contain most of the photoinductive components of HSs whereas fractions A and B did not (Aguer et al., 2001). Chromophores responsible for photoinduction are not necessarily those emitting fluorescence; however, both types of chromophores have, in common, to be contained in the low MS polar fractions.
Acknowledgements The authors wish to thank the CNRS and Russian Academy of Sciences for financial support of the investigation (project 4992). A part of this research has been supported by Russian Foundation for Basic Research (project 01-05-64666-a).
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