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Interaction of lead (II) chloride with hydroxyflavones in methanol: A spectroscopic study
Polyhedron 24 (2005) 1593–1598 www.elsevier.com/locate/poly
Interaction of lead (II) chloride with hydroxyflavones in methanol: A spectroscopic study Lae¨titia Dangleterre, Jean-Paul Cornard
*
LASIR, CNRS UMR 8516, Universite´ des Sciences et Technologies de Lille Baˆt C5, 59655 Villeneuve d ÕAscq Cedex, France Received 18 March 2005; accepted 28 April 2005 Available online 13 June 2005
Abstract Model compounds, of reasonable size, which have chelating chemical functions (carboxylic, catechol, hydroxy-carbonyl) analogues with those met in the biopolymer of the organic matter of the soils, were studied so as to better understand the complexation processes of metal ions. The present paper reports on spectroscopic investigation of the complex formation of lead (II) with some hydroxyflavones. The purpose of this study is to determine the stoichiometric composition and stability constants of the complexes formed between Pb(II) and 3-hydroxyflavone, 5-hydroxyflavone and 3 0 ,4 0 -dihydroxyflavone, respectively, in order to propose a classification of potential sites according to their chelating capacity. The combined use of electronic absorption spectroscopy and chemometrics methods allows reaching this objective. We have shown, that in methanol solution, the catechol function presents the greatest affinity for Pb(II) and leads to complex formation of higher stoichiometry than the other sites. The comparison of the results obtained for the complexation of Pb(II) and Al(III) shows a very different behaviour of the studied sites with respect to these two metals. 2005 Elsevier Ltd. All rights reserved. Keywords: Hydroxyf1avones; Pb(II) complexation; Stability constants; UV–Vis spectroscopy; Chemometrics methods
1. Introduction The largest fraction of the natural organic matter (NOM) is composed by the humic substances. They are aromatic and aliphatic macromolecules constituting a heterogeneous and complex system which evolves in the course of time. These humic substances are subdivided in three groups: acids fulvic, humic and humin. One differentiates the humic acids from the acids fulvic by their conditions of extraction. The molecular weights lie between 500 and 2000 g mol1 for the fulvic acids and between 2000 and 5000 g mol1 for the humic acids. The humic sub*
Corresponding author. Tel.: +33 3 20 43 69 26; fax: +33 3 20 43 67
55. E-mail address: [email protected] (J.-P. Cornard). 0277-5387/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2005.04.019
stances are formed by transformation of the biogene organic matter, and constitute a complex mixture of molecules. Consequently, their structures cannot be defined by an ordinary formulation. These macromolecules present many potential sites of fixing metals and thus contribute to the retention of these last one in the soils. These macromolecules can trap metals by interactions of van der Waals type or by complexation with different potential groups such as the functions carboxylic, carboxylate, catechol, hydroxy-carbonyl [1]. The study of the interactions between a metal and humic substance taken as a whole is very delicate because of the poly-functionality of this chemical system. However, a better comprehension of the complexation mechanisms can be obtained by studying model systems, molecules precursors or fragments of the NOM, which presents functional groups identical to
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L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
those observed in the humic substances. Previous studies have shown that polyphenols and notably flavonoids are non-negligible components of organic matter [2–5]. For this reason, we have considered the flavonoidsÕ family, belonging to the polyphenolic compounds, which are molecules that occur ubiquitously in plant kingdom. The flavonoids are characterized by a common structure, in C6–C3–C6, in which two benzene rings are connected by a carbonaceous chain C3, different according to nature from the compounds. Thus, the family of flavonoids is subdivided in various groups among which one can quote: flavones and derived, anthocyanes, chalcones, aurones, etc. [6–8]. The basic skeleton common to the elements of the flavones group is constituted of two benzene rings A and B connected to each other by an oxygenated heterocycle: the c-pyrone ring (C ring). The simplest compound of the group is the flavone (Fig. 1). These omnipresent molecules in the natural environment are found partially in the beginning of the organic matter of the soil and thus constitute perfect model molecules for the study and the comparison of the different metal complexation sites. The complexes composition obtained from these compounds with a large number of metal ions are reported in the literature [9–18], but so far no information was published concerning the chelation of lead (II). The main chelating sites involving in these compounds are the catechol, a-hydroxy-carbonyl and b-hydroxy-carbonyl functions. In this work, we report the lead (II) complexation study with three ligands presenting only one binding site: 3-hydroxyflavone (3HF), 5-hydroxyflavone (5HF)
and 3 0 ,4 0 -dihydroxyflavone (3 0 4 0 diHF) (Table 1). The goals of this study are: (i) to determine the stoichiometric composition and estimate the stability of each complex obtained with these three ligands; (ii) to class the different sites according to their ability to form chelate with lead (II) in order to obtain complementary information concerning the potential chelating sites of Pb(II) in humic substances; and (iii) to compare the results with those obtained in ours previous studies concerning the chelation of Al(III) [19–23].
2. Experimental 2.1. Reagents and methods Flavonoids (F) were purchased from Extrasynthese (France). Anhydrous lead chloride was used without purification. Because of the very low solubility of flavonoids in water, spectroscopic-grade methanol was used. The molar ratio method has allowed us to determine the complexes composition in solution from spectrophotometric spectra. For this method, solutions containing a constant concentration (4 · 105 M) of F in methanol and variable concentration of PbCl2 (from 4 · 107 to 4 · 102 M) were prepared. The complexes stoichiometry has also been verified by the method of continuous variation (JobÕs method). 2.2. Instrumentation The UV–Vis spectra were recorded on a Cary-l (Varian) spectrophotometer with cells of 1 cm path length, at room temperature. A flow cell was used to allow successive additions of small amounts of lead (II) chloride directly in the flavonoid solution. 2.3. Calculations
Fig. 1. Structure and atomic numbering (IUPAC nomenclature) of flavonoid.
Table 1 Substitution pattern of studied flavonoids Compound
Abbreviation
R1
R2
R3
R4
R5
3-Hydroxyflavone 5-Hydroxyflavone 3 0 ,4 0 -Dihydroxyflavone Quercetin
3HF 5HF 3 0 4 0 diHF Q
OH H H OH
H OH H OH
H H OH OH
H H OH OH
H H H OH
The formation constants of the complexes were estimated by using a multivariate data analysis program for modelling and fitting equilibrium titration 3D data sets obtained from the spectrophotometric measurements. The UV–Vis spectra were refined using the Specfit software (version 3.0.32). The set of spectra obtained at variable Pb(II) concentration were treated by evolving factor analysis (EFA) method in order to determine the number of absorbing species in the system and the pure electronic spectrum of each complex [24]. The Specfit software [25] has also been used to estimate the stability constants (b). In order to obtain the best fit between the complexation model and the experimental data, several models of complexes with different stoichiometries have been envisaged for the refinement of the stability constants.
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2.4. Methodology
3. Results and discussion
The same methodology which was applied for the study of the complexation of the three ligands is as follows: (i) The study by UV–Vis spectroscopy of the lead complexation with a ligand (F) in methanol by varying the [PbCl2]/[F] molar ratio, gives us a set of spectra. The stoichiometry of the formed complexes is determined by the molar ratio method. The curve representing the variation of the absorbance according to the ratio R = [Pb(II)]/[F] at absorption maxima of the complex and the free molecule is representative of the complex composition [26]. (ii) The JobÕs method was used to validate the molar ratio method. In the JobÕs method, for a constant volume, one mixes in different proportions equimolar solutions of ligand and lead (II). The sum of the concentrations remains constant, while the molar fraction of the ligand varies. The results obtained from this method provide us one second set of spectra. Here, the stoichiometry corresponds to the change of slope of the variation curve representative to the molar fraction of ligand xF or the JobÕs function [A(kmax)complexed solution A(kmax)F] [27]. (iii) From electronic spectra obtained by varying the molar ratio R, the determination of the spectroscopically distinct species is obtained by an experimental data processing by using the EFA method. The result of this calculation allows us to find the pure absorption spectrum of the molecule alone and the complexes. In order to determine the stability constants (b) of successively formed complexes, a numerical processing was carried out on a simple model containing Pb(II) metal, the ligand and the complexes. The stability constant estimation comes from the following reaction equation:
3.1. Chelation of lead (II) with hydroxyflavones Typically, the electronic spectra of flavonoids are characterized by two strong absorption bands: the band I in the long wavelength (320–380 nm) and the band II (240–270 nm) [28]. 3-Hydroxyflavone. Fig. 2 illustrates the evolution of the UV–Vis spectra of the 3HF–Pb(II) system for different [Pb2+]/[3HF] molar ratios. The intensity of band I, located at 344 nm, of free 3HF decreases with the amount of PbCl2 added, whereas a new band appears at 405 nm. In the same time, the absorbance at 243 nm increases. The presence of an isosbestic point at 365 nm proves the existence of only two absorbing species in equilibrium. The curve of absorbance versus [Pb2+]/[3HF] molar ratio plotted at 344 nm (kmax of free ligand) and 405 nm (kmax of complex) allow us to find a stoichiometry 1:1 for the complex (Fig. 3). The JobÕs method of continuous variations has confirmed this result (not shown). To estimate the stability constant of the 1:1 complex, a numerical treatment of the electronic spectra has been carried out. The model best fitting the 2
Absorbance
1.5
1
0.5
a Pb þ b F () Pba Fb
0 200
with the corresponding stability constant ½Pba Fb a
b
½Pb ½F
.
Whatever the site implied in the complexation process, a deprotonation of hydroxyl groups occurs in order to allow the coordination to Pb(II). However, this deprotonation has not been taking into account in the constant calculation insofar as the pH notion has no much sense in methanoic medium. (iv) From these constants and extracted spectra, we simulate a theoretical spectroscopic assay and compare this one with the experimental spectra, so as to validate the initial model. Moreover, if several species are formed, the curves of the concentrations evolution can show if the complexes are formed simultaneously or successively.
350
400
450
500 0
Wavelength (nm)
Fig. 2. UV–Vis absorption spectra of 3-hydroxyflavone in methanol in the absence and in the presence of PbCl2. [Pb2+]/[3HF] molar ratios vary from 0 to 3.
.8
Absorbance
bF ¼
300
250
405 nm
.6 .4 .2
344 nm
0 0
1
2
3
[Pb2+]/[3HF]
Fig. 3. Absorbance vs. [PbCl2]/[3HF] molar ratio plots at kmax (3HF) = 344 nm and kmax (complex) = 405 nm.
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
2
Absorbance
1.5
1
0.5
0 200
25 0
300
35 0
400
450
500
Wavelength (nm) Fig. 4. UV–Vis absorption spectra of 5-hydroxyflavone in methanol in absence and in presence of PbCl2. [Pb2+]/[5HF] molar ratios vary from 0 to 5.
1
Absorbance
experimental data gives a stability constant: log b3HF = 4.97 ± 4 · 106. As the chelation of lead occurs at the level of the a-hydroxy-carbonyl group with a complete deprotonation of the hydroxyl group, the formula of the complex is [(3HF)Pb]+. 5-Hydroxyflavone. With the addition of lead, the evolution of the electronic spectra of this ligand differs widely from that observed for its homolog the 3HF molecule. The general shape of the free ligand spectrum is not at all modified by the addition of PbCl2, only an increase in the absorbance, more or less important according to the wavelength, is observed (Fig. 4). However, a new band appearing at 241 nm differentiates the spectrum of the complex from that of the free ligand. Both the molar ratio and continuous variations methods give a stoichiometry 1:1 for the complex of 5HF, leading to a chelate [(5HF)Pb]+. The treatment of the spectra with Specfit program allows us the determination of the stability constant of this complex: log b5HF = 4.51 ± 3 · 102. So, the comparison of the stability constants indicates that the complex obtained with the 5HF molecule is slightly less stable than the one formed with 3HF. 3 0 ,4 0 -Dihydroxyflavone. The behaviour of 3 0 4 0 diHF in the presence of Pb(II) still differs from that found in the previous compounds. The first spectrum obtained in the absence of Pb(II) is characteristic of the ligand in methanol solution and presents some bands at 343, 306, 244 and 213 nm (Fig. 5). The addition of an increasing quantity of lead chloride to the solution of 3 0 4 0 diHF induces a decrease of the intensity of the band at 343 nm relating to the free flavonoid and an appearance of a new band located at 399 nm whose absorbance increases with the quantity of Pb(II) added. The series of spectra does not present a clearly definite isosbestic point, which would tend to mean that several species coexist in solution. The molar ratio plots at 343 and 399 nm (Fig. 6) show inflection at [Pb2+]/[3 0 4 0 diHF] = 0.5 and 1 (the break
0 200
250
300
350
400
450
500
Wavelength (nm) Fig. 5. UV–Vis absorption spectra of 3 0 ,4 0 -dihydroxyflavone in methanol in absence and in presence of PbCl2. [Pb2+]/[3 0 4 0 diHF] molar ratios vary from 0 to 4.
Absorbance
1596
343 nm
.6 .4 .2
0
399 nm
1
2 3 [Pb2+] / [3'4'diHF]
4
Fig. 6. Absorbance vs. [PbCl2]/[3 0 4 0 diHF] molar ratio plots at kmax (3 0 4 0 diHF) = 343 nm and kmax (complex) = 399 nm.
at 1 appears clearly on the plot at 399 nm), indicating the formation of two complexes of stoichiometry 1:2 and 1:1. This result is corroborated by the JobÕs method. From the UV–Vis spectra, the determination of the number of different absorbing species was estimated by the EFA method with the Spectfit software. Three distinct components were found corresponding to the free 3 0 4 0 diHF and the two complexed forms [Pb(3 0 4 0 diHF)2]2 and Pb(3 0 4 0 diHF). The electronic absorption spectra of these different pure species are presented in Fig. 7. The spectra of the two complexed species are very close, however, an additional intense band located at 237 nm characterizes the Pb(3 0 4 0 diHF) form. From these pure species spectra, the stability constants have been estimated and the model which provides the best fit to the experimental data gives: log b3 0 4 0 diHF = 8.82 ± 0.15 and log b3 0 4 0 diHF = 5.09 ± 0.10 for [Pb(3 0 4 0 diHF)2]2 and Pb(3 0 4 0 diHF), respectively. The concentration variations of the free and complexed ligands versus quantity of lead added is illustrated in Fig. 8. The curves show that two complexes are simultaneously formed from the very beginning of the lead addition, however, the Pb(3 0 4 0 diHF) species is largely predominant. If the formation of the complex 1:2 is in the minority, it is
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
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capacity in the following way: catechol > a-hydroxycarbonyl > b-hydroxy-carbonyl. 3.2. Comparison of Pb(II)- and Al(III)-hydroxyflavone systems
Fig. 7. UV–Vis spectra of the free and two complexed forms of 3 0 4 0 diHF extracted using the EFA method.
Fig. 8. Distribution curves of the concentration (M) of lead complexes with 3 0 4 0 diHF in methanol as a function of [PbCl2] added.
sufficient to have a significant role in the complexation mechanism of Pb(II) by 3 0 4 0 diHF, in particular in the presence of small amounts of lead. The graph also indicates that it remains 32% of free molecules in solution for a molar ratio of 1. Among the three flavonoids, 3 0 4 0 diHF is the only compound which leads to the formation of species of high stoichiometry (1:2) in methanol, and moreover, the stability of its complex 1:1 is higher than those of 3HF and 5HF. These results suggest that the catechol group presents a chelating power with respect to Pb(II) higher than that of other studied functions. Thus, it is possible to classify the three sites of fixation of Pb(II) according to their chelating
In a similar way, our preceding works [29] had made it possible to obtain a classification of these three sites with respect to their power of complexation of Al(III), under the same physico-chemical conditions. The comparative results of the two systems are reported in Table 2. It is obviously observed that the behaviour of the three studied sites is very different according to the metal ion implied in the complexation process. With regard to Al(III)-hydroxyflavone systems, it is the a-hydroxycarbonyl site which presents the greatest affinity for aluminium ion and gives a complex of stoichiometry (metal:ligand) 1:2. The catechol function leads to the formation of a complex 1:1 with Al(III) which has a greater stability than that obtained with the b-hydroxy-carbonyl function. Thus, it is observed that the change of Al(III) by Pb(II) in the system provokes an inversion of the chelating power of the catechol and a-hydroxy-carbonyl sites. The electronic spectra of the predominant complexes obtained for the three hydroxyflavones with both Al(III) and Pb(II) are presented in Fig. 9. Except for the [Pb(5HF)]+ complex, the electronic spectra of the different complexed species obtained with both Al(III) and Pb(II) exhibit a bathochromic shift of the band I compared to the free molecule spectra. This observation seems to indicate that the complexation mechanism of lead (II) by the b-hydroxy-carbonyl group is very different from that observed with aluminium (III). The hyperchromic effect on the absorption bands of the spectrum of the 5HF solution occurring with the addition of PbCl2 tends to show that low electronic changes at the level of the ligand are generated by complexation. It is interesting to notice that the electronic spectra of the complexes obtained with Al(III) or Pb(II) and the 3HF molecule (which present different stoichiometries but the same charge) are almost similar. That would like to say that the absorption bands of the complexes come from electronic transitions involving only molecular orbitals localized on the ligand, and that no metal– ligand charge transfer is observed.
Table 2 Comparison of the stoichiometry and stability constants of the complexes of hydroxyflavones obtained with Pb(II) and Al(III) Compounds
kmax (nm)
Pb(II) complexes Pb:L
kmax (nm)
log (b)
Al:L
kmax (nm)
log (b)
3HF 5HF 3 0 4 0 diHF
344 270 342
1:1 1:1 1:2 1:1
405 268/241 399
4.97 4.51 8.82 5.09
1:2 1:1 1:1
402 397 384
12.3 6.5 6.7
Al(III) complexes
The wavelengths of the band I (in the long wavelength) are given for the free ligand and for all the complexes.
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
Absorbance
1598
the influence of the pH on the capacity of complexation of Pb(II) by these functions. [Pb(3HF)]+ + [Al(3HF) [Al(3HF) ] 2] +
2
Acknowledgements This work is part of the ‘‘Programme de Recherche Concerte´e: Sites et Sols Pollue´s’’ supported by the ‘‘Re´gion Nord-Pas de Calais’’ and the ‘‘Fonds Europe´en de De´veloppement Economique des Re´gions’’ (FEDER).
+
[Pb(3HF)]
[Al(3'4'diHF)]+ +
[Al(3'4'diHF)]
References
[Pb(3'4'diHF)]
[Pb(3'4'diHF)]
[Al(5HF)]2+ [Pb(5HF)]+ 200
300
400
500
Wavelength (nm)
Fig. 9. Comparison of the spectra of the major complexes obtained with Al(III) and lead (II) and hydroxyflavones in methanol.
4. Conclusion This study made it possible to establish a classification of various chelating sites of humic substances with respect to lead (II) in methanol solution. The interactions between lead (II) and hydroxyflavones had never yet been studied, however, this paper reports the first step of our work. In a second time, we will focus our attention on the complexation mechanism of a molecule presenting simultaneously the functions catechol, a-hydroxy-carbonyl and b-hydroxy-carbonyl: the quercetin molecule (Table 1). That will make it possible to observe if classification obtained with these results remains valid when the three sites are in competition within the same molecule. Moreover, for reasons of solubility this study was carried out in methanol solution. If our preceding studies concerning Al(III) showed that the results obtained in water are comparable with those obtained in methanol, we will study these three molecules in a mixed solvent water–methanol in order to be able to observe
[1] F.J. Stevenson, Humus Chemistry: Genesis, Composition, Reactions, Wiley, New York, 1982. [2] C.B. Coulson, R.I. Davies, D.A. Lewis, J. Soil Sci. 11 (1960) 20. [3] M. Remko, J. Polcin, Collect. Czech. Chem. Commun. 45 (1980) 201. [4] D.S. Smith, J.R. Kramer, Environ. Int. 25 (1999) 295. [5] D.S. Smith, J.R. Kramer, Anal. Chim. Acta 416 (2000) 211. [6] E. Middelton, C. Kandaswami, in: J.B. Harborne (Ed.), The Flavonoids: Advances in Research Since 1986, Chapman & Hall, London, 1994. [7] L. Bravo, Nutr. Rev. 56 (1998) 317. [8] E. Middelton, C. Kandaswami, T.C. Theoharides, Pharmacol. Rev. 52 (2000) 673. [9] G. Speier, V. Fu¨lo¨p, E. Balogh-Hergovich, Z. Tyeklar, J. Inorg. Biochem. 36 (1989) 329. [10] G.J. Smith, S.J. Thomsen, K. Markham, C. Andary, D. Cardon, J. Photochem. Photobiol. A 136 (2000) 87. [11] M. Hauteville, M. Rakotovao, M.C. Duclos, B. Voirin, Phytochemistry 48 (1998) 547. [12] J. Zhou, L. Wang, J. Wang, N. Tang, J. Inorg. Biochem. 83 (2001) 41. [13] H. Deng, G.J. Van Berkel, J. Mass Spectrom. 33 (1998) 1080. [14] J.E. Brown, H. Khodr, R.C. Hider, C.A. Rice-Evans, Biochem. J. 330 (1998) 1173. [15] P. Hollman, J. Van Trijp, M. Buysman, Anal. Chem. 68 (1996) 3511. [16] M. Yoshino, K. Murakami, Anal. Biochem. 257 (1998) 40. [17] M. Katyal, S. Prakash, Talanta 24 (1977) 367. [18] G. Le Nest, O. Caille, M. Woudstra, S. Roche, F. Guerlesquin, D. Lexa, Inorg. Chim. Acta 357 (2004) 775. [19] A.C. Boudet, J.P. Cornard, J.C. Merlin, Spectrochim. Acta 56 (2000) 829. [20] J.P. Cornard, A.C. Boudet, J.C. Merlin, Spectrochim. Acta 57 (2001) 591. [21] J.P. Cornard, J.C. Merlin, J. Mol. Struct. 569 (2001) 129. [22] J.P. Cornard, J.C. Merlin, J. Inorg. Biochem. 92 (2002) 19. [23] J.P. Cornard, J.C. Merlin, Polyhedron 21 (2002) 2801. [24] H. Gampp, M. Maeder, C.J. Meyer, A. Zuberbu¨hler, Talanta 33 (1986) 943. [25] Specfit Global Analysis System, Spectrum software Associates, Marlborough, MA, USA. [26] J.H. Yoe, L. Jones, Ind. Eng. Chem. Anal. Ed. 16 (1944) 11. [27] P. Job, C. R. Acad. Sci. 180 (1925) 928. [28] L.J. Porter, K.R. Markham, Phytochemistry 24 (1970) 835. [29] J.P. Cornard, J.C. Merlin, J. Mol. Struct. 651–653 (2003) 381.
Interaction of lead (II) chloride with hydroxyflavones in methanol: A spectroscopic study Lae¨titia Dangleterre, Jean-Paul Cornard
*
LASIR, CNRS UMR 8516, Universite´ des Sciences et Technologies de Lille Baˆt C5, 59655 Villeneuve d ÕAscq Cedex, France Received 18 March 2005; accepted 28 April 2005 Available online 13 June 2005
Abstract Model compounds, of reasonable size, which have chelating chemical functions (carboxylic, catechol, hydroxy-carbonyl) analogues with those met in the biopolymer of the organic matter of the soils, were studied so as to better understand the complexation processes of metal ions. The present paper reports on spectroscopic investigation of the complex formation of lead (II) with some hydroxyflavones. The purpose of this study is to determine the stoichiometric composition and stability constants of the complexes formed between Pb(II) and 3-hydroxyflavone, 5-hydroxyflavone and 3 0 ,4 0 -dihydroxyflavone, respectively, in order to propose a classification of potential sites according to their chelating capacity. The combined use of electronic absorption spectroscopy and chemometrics methods allows reaching this objective. We have shown, that in methanol solution, the catechol function presents the greatest affinity for Pb(II) and leads to complex formation of higher stoichiometry than the other sites. The comparison of the results obtained for the complexation of Pb(II) and Al(III) shows a very different behaviour of the studied sites with respect to these two metals. 2005 Elsevier Ltd. All rights reserved. Keywords: Hydroxyf1avones; Pb(II) complexation; Stability constants; UV–Vis spectroscopy; Chemometrics methods
1. Introduction The largest fraction of the natural organic matter (NOM) is composed by the humic substances. They are aromatic and aliphatic macromolecules constituting a heterogeneous and complex system which evolves in the course of time. These humic substances are subdivided in three groups: acids fulvic, humic and humin. One differentiates the humic acids from the acids fulvic by their conditions of extraction. The molecular weights lie between 500 and 2000 g mol1 for the fulvic acids and between 2000 and 5000 g mol1 for the humic acids. The humic sub*
Corresponding author. Tel.: +33 3 20 43 69 26; fax: +33 3 20 43 67
55. E-mail address: [email protected] (J.-P. Cornard). 0277-5387/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2005.04.019
stances are formed by transformation of the biogene organic matter, and constitute a complex mixture of molecules. Consequently, their structures cannot be defined by an ordinary formulation. These macromolecules present many potential sites of fixing metals and thus contribute to the retention of these last one in the soils. These macromolecules can trap metals by interactions of van der Waals type or by complexation with different potential groups such as the functions carboxylic, carboxylate, catechol, hydroxy-carbonyl [1]. The study of the interactions between a metal and humic substance taken as a whole is very delicate because of the poly-functionality of this chemical system. However, a better comprehension of the complexation mechanisms can be obtained by studying model systems, molecules precursors or fragments of the NOM, which presents functional groups identical to
1594
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
those observed in the humic substances. Previous studies have shown that polyphenols and notably flavonoids are non-negligible components of organic matter [2–5]. For this reason, we have considered the flavonoidsÕ family, belonging to the polyphenolic compounds, which are molecules that occur ubiquitously in plant kingdom. The flavonoids are characterized by a common structure, in C6–C3–C6, in which two benzene rings are connected by a carbonaceous chain C3, different according to nature from the compounds. Thus, the family of flavonoids is subdivided in various groups among which one can quote: flavones and derived, anthocyanes, chalcones, aurones, etc. [6–8]. The basic skeleton common to the elements of the flavones group is constituted of two benzene rings A and B connected to each other by an oxygenated heterocycle: the c-pyrone ring (C ring). The simplest compound of the group is the flavone (Fig. 1). These omnipresent molecules in the natural environment are found partially in the beginning of the organic matter of the soil and thus constitute perfect model molecules for the study and the comparison of the different metal complexation sites. The complexes composition obtained from these compounds with a large number of metal ions are reported in the literature [9–18], but so far no information was published concerning the chelation of lead (II). The main chelating sites involving in these compounds are the catechol, a-hydroxy-carbonyl and b-hydroxy-carbonyl functions. In this work, we report the lead (II) complexation study with three ligands presenting only one binding site: 3-hydroxyflavone (3HF), 5-hydroxyflavone (5HF)
and 3 0 ,4 0 -dihydroxyflavone (3 0 4 0 diHF) (Table 1). The goals of this study are: (i) to determine the stoichiometric composition and estimate the stability of each complex obtained with these three ligands; (ii) to class the different sites according to their ability to form chelate with lead (II) in order to obtain complementary information concerning the potential chelating sites of Pb(II) in humic substances; and (iii) to compare the results with those obtained in ours previous studies concerning the chelation of Al(III) [19–23].
2. Experimental 2.1. Reagents and methods Flavonoids (F) were purchased from Extrasynthese (France). Anhydrous lead chloride was used without purification. Because of the very low solubility of flavonoids in water, spectroscopic-grade methanol was used. The molar ratio method has allowed us to determine the complexes composition in solution from spectrophotometric spectra. For this method, solutions containing a constant concentration (4 · 105 M) of F in methanol and variable concentration of PbCl2 (from 4 · 107 to 4 · 102 M) were prepared. The complexes stoichiometry has also been verified by the method of continuous variation (JobÕs method). 2.2. Instrumentation The UV–Vis spectra were recorded on a Cary-l (Varian) spectrophotometer with cells of 1 cm path length, at room temperature. A flow cell was used to allow successive additions of small amounts of lead (II) chloride directly in the flavonoid solution. 2.3. Calculations
Fig. 1. Structure and atomic numbering (IUPAC nomenclature) of flavonoid.
Table 1 Substitution pattern of studied flavonoids Compound
Abbreviation
R1
R2
R3
R4
R5
3-Hydroxyflavone 5-Hydroxyflavone 3 0 ,4 0 -Dihydroxyflavone Quercetin
3HF 5HF 3 0 4 0 diHF Q
OH H H OH
H OH H OH
H H OH OH
H H OH OH
H H H OH
The formation constants of the complexes were estimated by using a multivariate data analysis program for modelling and fitting equilibrium titration 3D data sets obtained from the spectrophotometric measurements. The UV–Vis spectra were refined using the Specfit software (version 3.0.32). The set of spectra obtained at variable Pb(II) concentration were treated by evolving factor analysis (EFA) method in order to determine the number of absorbing species in the system and the pure electronic spectrum of each complex [24]. The Specfit software [25] has also been used to estimate the stability constants (b). In order to obtain the best fit between the complexation model and the experimental data, several models of complexes with different stoichiometries have been envisaged for the refinement of the stability constants.
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2.4. Methodology
3. Results and discussion
The same methodology which was applied for the study of the complexation of the three ligands is as follows: (i) The study by UV–Vis spectroscopy of the lead complexation with a ligand (F) in methanol by varying the [PbCl2]/[F] molar ratio, gives us a set of spectra. The stoichiometry of the formed complexes is determined by the molar ratio method. The curve representing the variation of the absorbance according to the ratio R = [Pb(II)]/[F] at absorption maxima of the complex and the free molecule is representative of the complex composition [26]. (ii) The JobÕs method was used to validate the molar ratio method. In the JobÕs method, for a constant volume, one mixes in different proportions equimolar solutions of ligand and lead (II). The sum of the concentrations remains constant, while the molar fraction of the ligand varies. The results obtained from this method provide us one second set of spectra. Here, the stoichiometry corresponds to the change of slope of the variation curve representative to the molar fraction of ligand xF or the JobÕs function [A(kmax)complexed solution A(kmax)F] [27]. (iii) From electronic spectra obtained by varying the molar ratio R, the determination of the spectroscopically distinct species is obtained by an experimental data processing by using the EFA method. The result of this calculation allows us to find the pure absorption spectrum of the molecule alone and the complexes. In order to determine the stability constants (b) of successively formed complexes, a numerical processing was carried out on a simple model containing Pb(II) metal, the ligand and the complexes. The stability constant estimation comes from the following reaction equation:
3.1. Chelation of lead (II) with hydroxyflavones Typically, the electronic spectra of flavonoids are characterized by two strong absorption bands: the band I in the long wavelength (320–380 nm) and the band II (240–270 nm) [28]. 3-Hydroxyflavone. Fig. 2 illustrates the evolution of the UV–Vis spectra of the 3HF–Pb(II) system for different [Pb2+]/[3HF] molar ratios. The intensity of band I, located at 344 nm, of free 3HF decreases with the amount of PbCl2 added, whereas a new band appears at 405 nm. In the same time, the absorbance at 243 nm increases. The presence of an isosbestic point at 365 nm proves the existence of only two absorbing species in equilibrium. The curve of absorbance versus [Pb2+]/[3HF] molar ratio plotted at 344 nm (kmax of free ligand) and 405 nm (kmax of complex) allow us to find a stoichiometry 1:1 for the complex (Fig. 3). The JobÕs method of continuous variations has confirmed this result (not shown). To estimate the stability constant of the 1:1 complex, a numerical treatment of the electronic spectra has been carried out. The model best fitting the 2
Absorbance
1.5
1
0.5
a Pb þ b F () Pba Fb
0 200
with the corresponding stability constant ½Pba Fb a
b
½Pb ½F
.
Whatever the site implied in the complexation process, a deprotonation of hydroxyl groups occurs in order to allow the coordination to Pb(II). However, this deprotonation has not been taking into account in the constant calculation insofar as the pH notion has no much sense in methanoic medium. (iv) From these constants and extracted spectra, we simulate a theoretical spectroscopic assay and compare this one with the experimental spectra, so as to validate the initial model. Moreover, if several species are formed, the curves of the concentrations evolution can show if the complexes are formed simultaneously or successively.
350
400
450
500 0
Wavelength (nm)
Fig. 2. UV–Vis absorption spectra of 3-hydroxyflavone in methanol in the absence and in the presence of PbCl2. [Pb2+]/[3HF] molar ratios vary from 0 to 3.
.8
Absorbance
bF ¼
300
250
405 nm
.6 .4 .2
344 nm
0 0
1
2
3
[Pb2+]/[3HF]
Fig. 3. Absorbance vs. [PbCl2]/[3HF] molar ratio plots at kmax (3HF) = 344 nm and kmax (complex) = 405 nm.
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
2
Absorbance
1.5
1
0.5
0 200
25 0
300
35 0
400
450
500
Wavelength (nm) Fig. 4. UV–Vis absorption spectra of 5-hydroxyflavone in methanol in absence and in presence of PbCl2. [Pb2+]/[5HF] molar ratios vary from 0 to 5.
1
Absorbance
experimental data gives a stability constant: log b3HF = 4.97 ± 4 · 106. As the chelation of lead occurs at the level of the a-hydroxy-carbonyl group with a complete deprotonation of the hydroxyl group, the formula of the complex is [(3HF)Pb]+. 5-Hydroxyflavone. With the addition of lead, the evolution of the electronic spectra of this ligand differs widely from that observed for its homolog the 3HF molecule. The general shape of the free ligand spectrum is not at all modified by the addition of PbCl2, only an increase in the absorbance, more or less important according to the wavelength, is observed (Fig. 4). However, a new band appearing at 241 nm differentiates the spectrum of the complex from that of the free ligand. Both the molar ratio and continuous variations methods give a stoichiometry 1:1 for the complex of 5HF, leading to a chelate [(5HF)Pb]+. The treatment of the spectra with Specfit program allows us the determination of the stability constant of this complex: log b5HF = 4.51 ± 3 · 102. So, the comparison of the stability constants indicates that the complex obtained with the 5HF molecule is slightly less stable than the one formed with 3HF. 3 0 ,4 0 -Dihydroxyflavone. The behaviour of 3 0 4 0 diHF in the presence of Pb(II) still differs from that found in the previous compounds. The first spectrum obtained in the absence of Pb(II) is characteristic of the ligand in methanol solution and presents some bands at 343, 306, 244 and 213 nm (Fig. 5). The addition of an increasing quantity of lead chloride to the solution of 3 0 4 0 diHF induces a decrease of the intensity of the band at 343 nm relating to the free flavonoid and an appearance of a new band located at 399 nm whose absorbance increases with the quantity of Pb(II) added. The series of spectra does not present a clearly definite isosbestic point, which would tend to mean that several species coexist in solution. The molar ratio plots at 343 and 399 nm (Fig. 6) show inflection at [Pb2+]/[3 0 4 0 diHF] = 0.5 and 1 (the break
0 200
250
300
350
400
450
500
Wavelength (nm) Fig. 5. UV–Vis absorption spectra of 3 0 ,4 0 -dihydroxyflavone in methanol in absence and in presence of PbCl2. [Pb2+]/[3 0 4 0 diHF] molar ratios vary from 0 to 4.
Absorbance
1596
343 nm
.6 .4 .2
0
399 nm
1
2 3 [Pb2+] / [3'4'diHF]
4
Fig. 6. Absorbance vs. [PbCl2]/[3 0 4 0 diHF] molar ratio plots at kmax (3 0 4 0 diHF) = 343 nm and kmax (complex) = 399 nm.
at 1 appears clearly on the plot at 399 nm), indicating the formation of two complexes of stoichiometry 1:2 and 1:1. This result is corroborated by the JobÕs method. From the UV–Vis spectra, the determination of the number of different absorbing species was estimated by the EFA method with the Spectfit software. Three distinct components were found corresponding to the free 3 0 4 0 diHF and the two complexed forms [Pb(3 0 4 0 diHF)2]2 and Pb(3 0 4 0 diHF). The electronic absorption spectra of these different pure species are presented in Fig. 7. The spectra of the two complexed species are very close, however, an additional intense band located at 237 nm characterizes the Pb(3 0 4 0 diHF) form. From these pure species spectra, the stability constants have been estimated and the model which provides the best fit to the experimental data gives: log b3 0 4 0 diHF = 8.82 ± 0.15 and log b3 0 4 0 diHF = 5.09 ± 0.10 for [Pb(3 0 4 0 diHF)2]2 and Pb(3 0 4 0 diHF), respectively. The concentration variations of the free and complexed ligands versus quantity of lead added is illustrated in Fig. 8. The curves show that two complexes are simultaneously formed from the very beginning of the lead addition, however, the Pb(3 0 4 0 diHF) species is largely predominant. If the formation of the complex 1:2 is in the minority, it is
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
1597
capacity in the following way: catechol > a-hydroxycarbonyl > b-hydroxy-carbonyl. 3.2. Comparison of Pb(II)- and Al(III)-hydroxyflavone systems
Fig. 7. UV–Vis spectra of the free and two complexed forms of 3 0 4 0 diHF extracted using the EFA method.
Fig. 8. Distribution curves of the concentration (M) of lead complexes with 3 0 4 0 diHF in methanol as a function of [PbCl2] added.
sufficient to have a significant role in the complexation mechanism of Pb(II) by 3 0 4 0 diHF, in particular in the presence of small amounts of lead. The graph also indicates that it remains 32% of free molecules in solution for a molar ratio of 1. Among the three flavonoids, 3 0 4 0 diHF is the only compound which leads to the formation of species of high stoichiometry (1:2) in methanol, and moreover, the stability of its complex 1:1 is higher than those of 3HF and 5HF. These results suggest that the catechol group presents a chelating power with respect to Pb(II) higher than that of other studied functions. Thus, it is possible to classify the three sites of fixation of Pb(II) according to their chelating
In a similar way, our preceding works [29] had made it possible to obtain a classification of these three sites with respect to their power of complexation of Al(III), under the same physico-chemical conditions. The comparative results of the two systems are reported in Table 2. It is obviously observed that the behaviour of the three studied sites is very different according to the metal ion implied in the complexation process. With regard to Al(III)-hydroxyflavone systems, it is the a-hydroxycarbonyl site which presents the greatest affinity for aluminium ion and gives a complex of stoichiometry (metal:ligand) 1:2. The catechol function leads to the formation of a complex 1:1 with Al(III) which has a greater stability than that obtained with the b-hydroxy-carbonyl function. Thus, it is observed that the change of Al(III) by Pb(II) in the system provokes an inversion of the chelating power of the catechol and a-hydroxy-carbonyl sites. The electronic spectra of the predominant complexes obtained for the three hydroxyflavones with both Al(III) and Pb(II) are presented in Fig. 9. Except for the [Pb(5HF)]+ complex, the electronic spectra of the different complexed species obtained with both Al(III) and Pb(II) exhibit a bathochromic shift of the band I compared to the free molecule spectra. This observation seems to indicate that the complexation mechanism of lead (II) by the b-hydroxy-carbonyl group is very different from that observed with aluminium (III). The hyperchromic effect on the absorption bands of the spectrum of the 5HF solution occurring with the addition of PbCl2 tends to show that low electronic changes at the level of the ligand are generated by complexation. It is interesting to notice that the electronic spectra of the complexes obtained with Al(III) or Pb(II) and the 3HF molecule (which present different stoichiometries but the same charge) are almost similar. That would like to say that the absorption bands of the complexes come from electronic transitions involving only molecular orbitals localized on the ligand, and that no metal– ligand charge transfer is observed.
Table 2 Comparison of the stoichiometry and stability constants of the complexes of hydroxyflavones obtained with Pb(II) and Al(III) Compounds
kmax (nm)
Pb(II) complexes Pb:L
kmax (nm)
log (b)
Al:L
kmax (nm)
log (b)
3HF 5HF 3 0 4 0 diHF
344 270 342
1:1 1:1 1:2 1:1
405 268/241 399
4.97 4.51 8.82 5.09
1:2 1:1 1:1
402 397 384
12.3 6.5 6.7
Al(III) complexes
The wavelengths of the band I (in the long wavelength) are given for the free ligand and for all the complexes.
L. Dangleterre, J.-P. Cornard / Polyhedron 24 (2005) 1593–1598
Absorbance
1598
the influence of the pH on the capacity of complexation of Pb(II) by these functions. [Pb(3HF)]+ + [Al(3HF) [Al(3HF) ] 2] +
2
Acknowledgements This work is part of the ‘‘Programme de Recherche Concerte´e: Sites et Sols Pollue´s’’ supported by the ‘‘Re´gion Nord-Pas de Calais’’ and the ‘‘Fonds Europe´en de De´veloppement Economique des Re´gions’’ (FEDER).
+
[Pb(3HF)]
[Al(3'4'diHF)]+ +
[Al(3'4'diHF)]
References
[Pb(3'4'diHF)]
[Pb(3'4'diHF)]
[Al(5HF)]2+ [Pb(5HF)]+ 200
300
400
500
Wavelength (nm)
Fig. 9. Comparison of the spectra of the major complexes obtained with Al(III) and lead (II) and hydroxyflavones in methanol.
4. Conclusion This study made it possible to establish a classification of various chelating sites of humic substances with respect to lead (II) in methanol solution. The interactions between lead (II) and hydroxyflavones had never yet been studied, however, this paper reports the first step of our work. In a second time, we will focus our attention on the complexation mechanism of a molecule presenting simultaneously the functions catechol, a-hydroxy-carbonyl and b-hydroxy-carbonyl: the quercetin molecule (Table 1). That will make it possible to observe if classification obtained with these results remains valid when the three sites are in competition within the same molecule. Moreover, for reasons of solubility this study was carried out in methanol solution. If our preceding studies concerning Al(III) showed that the results obtained in water are comparable with those obtained in methanol, we will study these three molecules in a mixed solvent water–methanol in order to be able to observe
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