Kinetic analysis of aluminum complex formation with different soil fulvic acids

A fluorescence technique was used to investigate the complex formation kinetics of aluminum with fulvic acids isolated from different forest soil environments. In the pH range of 2.4–3.6, all of the fulvic acids were found to contain two kinetically distinguishable components, which define two types of average aluminum binding sites. Both of these average sites on all of the fulvic acids conformed to a bidentate chelating binding site kinetic analysis, from which average rate and equilibrium parameters were obtained. Evidence indicated that the difference in reaction rate between the two types of aluminum binding sites on the fulvic acids was due to a steric strain, whereby aluminum was repelled from the slower reacting sites. In comparing this study with a similar kinetic study carried out in acetate buffered solutions, it was found that the presence of buffer changed both the overall mechanism by which aluminum reacted with fulvic acid, and the nature of the sites on fulvic acid that bind aluminum.     

Stability constants for the complexes of transition-metal ions with fulvic and humic acids in sediments measured by gel filtration

The molecular-weight distributions of fulvic and humic acids in sediments and their complexes with metal ions (Cu(2+), Zn(2+), Mn(2+)) were investigated by gel filtration. In all cases, metal complexes were found in the fulvic and humic acids. In the complexes the metals were bound to the high molecular-weight fulvic and humic polymers. By use of gel filtration, stability constants for the complexes of copper, zinc and manganese with fulvic acids have been measured. Scatchard plots indicate the presence of two classes of binding site with stability constants of 2.3 x 10(7) and 1.4 x 10(6) for copper, 2.1 x 10(6) and 6.6 x 10(4) for zinc and 1.8 x 10(5) and 7.3 x 10(3) for manganese, respectively.     

Flocculation of humic substances with metal ions as followed by capillary zone electrophoresis

Humic and fulvic acids from various sources have been shown to give different electropherograms by capillary zone electrophoresis (CZE), depending on the pH of the electrolyte. This CZE work is extended here through investigations involving the titration of humic and fulvic acids with Fe(III) and Cu(II) cations. As increasing amounts of these cations were added to the humic substances (HUS), flocculation of metal-humic complexes occurred. This is believed to be caused by binding of the metal cations with negative carboxyl and phenolic sites on the HUS, resulting in a decrease of the repulsive forces that keep the HUS in a conformation more suitable for water solubility. The flocculated complexes were separated from the supernatant by centrifugation, and the supernatants were characterized as to total organic carbon (TOC) content, molecular weight (MW) using gel permeation chromatography, and average electrophoretic mobility (AEM) using CZE. The extent of flocculation correlated with both TOC and quantitative CZE measurements. The MW of the HUS remaining in solution actually decreased, presumably because of precipitation of larger molecules as they became insoluble because of reactions with the metals. Humic acids showed total precipitation of TOC with both metals at a concentration equivalent to their measured acidity. CZE demonstrated that certain fulvic acid fractions (low molecular weight phenolic acids) remained in solution even at high metal concentrations. In summary, changes in electrophoretic behavior of the soluble HUS could be related to changes in charge-to-mass ratios (charge densities) of both humic and fulvic acids with increasing metal cation concentration (neutralization). The copper treated HUS showed changes in their electrophoretic behavior even at low metal concentrations before flocculation, whereas the iron treated HUS flocculated uniformally over the range of added iron without significant changes in AEM. Thus these changes in CZE patterns illustrate different specific binding sites of the HUS for each metal.     

Flocculation of humic substances with metal ions as followed by capillary zone electrophoresis

Humic and fulvic acids from various sources have been shown to give different electropherograms by capillary zone electrophoresis (CZE), depending on the pH of the electrolyte. This CZE work is extended here through investigations involving the titration of humic and fulvic acids with Fe(III) and Cu(II) cations. As increasing amounts of these cations were added to the humic substances (HUS), flocculation of metal-humic complexes occurred. This is believed to be caused by binding of the metal cations with negative carboxyl and phenolic sites on the HUS, resulting in a decrease of the repulsive forces that keep the HUS in a conformation more suitable for water solubility. The flocculated complexes were separated from the supernatant by centrifugation, and the supernatants were characterized as to total organic carbon (TOC) content, molecular weight (MW) using gel permeation chromatography, and average electrophoretic mobility (AEM) using CZE. The extent of flocculation correlated with both TOC and quantitative CZE measurements. The MW of the HUS remaining in solution actually decreased, presumably because of precipitation of larger molecules as they became insoluble because of reactions with the metals. Humic acids showed total precipitation of TOC with both metals at a concentration equivalent to their measured acidity. CZE demonstrated that certain fulvic acid fractions (low molecular weight phenolic acids) remained in solution even at high metal concentrations. In summary, changes in electrophoretic behavior of the soluble HUS could be related to changes in charge-to-mass ratios (charge densities) of both humic and fulvic acids with increasing metal cation concentration (neutralization). The copper treated HUS showed changes in their electrophoretic behavior even at low metal concentrations before flocculation, whereas the iron treated HUS flocculated uniformally over the range of added iron without significant changes in AEM. Thus these changes in CZE patterns illustrate different specific binding sites of the HUS for each metal.     

Complexation of Eu and Tb with fulvic acids as studied by time-resolved laser-induced fluorescence

Complexation of Eu and Tb with fulvic acids (FA) has been studied by time-resolved laser-induced fluorescence. The lanthanide species are excited by a pulsed Xe/Cl excimer laser and the short-lived luminescence of the fulvic acids is eliminated by time gating. The conditional binding constants (K') and the total metal-binding capacities of the fulvic acids have been determined from the equilibrium titration curves. No significant variations of the log K' values (about 6) have been found in the pH range investigated (2.7, 5.5, 6.5) at ionic strengths of 0.1 and 1M (NaClO(4)). An inverse relationship of K' to total metal concentration is observed. Substitution of Eu for Al has been found to depress the fluorescence signal of the Eu-FA complex. The binding constants of Al(3+) and Eu(3+), derived from the competition experiments, are of the same order of magnitude. The effect of competition on metal binding and trace-metal transport is discussed.     

Fluorescence quenching of three molecular weight fractions of a soil fulvic acid by UO(2)(II)

A soil fulvic acid isolated from a Korean forest was divided into three different molecular weight fractions (F1: less than 220 Da; F2: 220-1000 Da; and F3: 1000-4000 Da) by gel filtration chromatography and the fractions were studied by synchronous fluorescence (SyF) spectroscopy. Analysis of the SyF spectra for the fulvic acid fractions showed that the fractions with molecules of larger sizes have a higher content of condensed aromatic compounds. The information about their interaction with UO(2)(II) ions in an aqueous solution (100 mg l(-1) of fulvic acid, in 0.1 M NaClO(4) at pH 3.5) was obtained from the measurement of SyF spectra at increasing concentrations of metal ions. Self-modeling mixture analysis of the quenching spectra gives two distinct peak components having a maximum peak position of 386 (type I) and 498 nm (type II) for all the size-fractionated fulvic acids. From the analysis of the quenching profiles of the peaks, using a non-linear method, the concentration of binding sites (C(L)), and the corresponding stability constants (logK) were calculated. The stability constants of the UO(2)(II)-fulvate complexes ranged from 4.10 to 5.33, and increased with higher molecular weight fractions, which indicates a stronger affinity for UO(2)(II) in the fraction with molecules of larger size.     

Calorimetric investigation of complex formation of some humic compounds

In the present paper we studied the complexation capacity towards copper ion of fulvic acids extracted from Arno River and Lake Como sediments, as well as Antarctic Sea water at different pHs in order to evaluate the interaction of different complexing groups of fulvic acids with copper ions. The binding capacity studies were carried out by means of titration with a copper-selective electrode and calorimetric measurements. For the same purpose, the heat of reaction in the complexation of copper ions with small molecules containing functional groups similar to fulvic acid was also determined. Titration results indicated that the fraction of bound metal increases with increasing pH (from 5 to 7). This may be accounted for by the increase in the ionisation of the fulvic acid molecule. Results obtained from calorimetric measurements show that the quantity of total heat involved in the metal ion-fulvate interactions determines a decreasing exothermic response with increasing pH values. This revised version was published online in July 2006 with corrections to the Cover Date.     

Study of the complexation of Cu(II) by fulvic acids extracted from a sewage sludge and its compost

 The complexation of Cu(II) by two samples of fulvic acids (FA) extracted from the raw sewage sludge (RsFA) of a waste water processing plant and from the composted sludge (CsFA) obtained upon aerobical digestion was studied at pH 6.0. Synchronous molecular fluorescence spectroscopy was used to monitor the association of FA with Cu(II) and a self-modelling mixture analysis method (SIMPLISMA) was used to preprocess the spectral data to calculate the number of components with different quenching profiles as function of the Cu(II) concentration and their spectra. The stability constants and a rough estimation of the binding site concentrations were obtained by a Stern-Volmer analysis, by a non-linear least-square method and by a linear procedure. The analysis of the SyF spectra allowed the identification of two binding sites for both samples. The logarithm of the conditional stability constant corresponding to the 1:1 complex formed between the stronger binding site and Cu(II) is about 4 for both samples. Received: 22 April 1996 / Revised: 5 August 1996 / Accepted: 7 August 1996     

Calcium binding by fulvic acids studied by an ion selective electrode an ultrafiltration method

The interaction between Ca(2+) and two well-characterized fulvic acids (Armadale and Laurentide FA) has been studied at 0.100 and 0.010M sodium nitrate using a fixed concentration of fulvic acid (100 ppm) and varying amounts of calcium (0.005-0.020 mmoles). Free calcium concentration was determined by in situ measurements employing a calcium electrode. For Armadale FA, free calcium was additionally determined via an ultrafiltration technique followed by atomic absorption measurements. For both fulvic acids, Ca(2+) binding was observed to be decreased by an increase in the ionic strength of the system. At the lower ionic strength the tendency for binding is dependent on the fulvic acid-to-metal ratio while at the higher ionic strength, the binding is insensitive to changes in the fulvic acid-to-metal ratio (an observation corroborating the contention that calcium binding to humic substances is primarily electrostatic). Comparison of the computed overall complex formation functions shows that values obtained from the ultrafiltration method were higher than those obtained using the calcium electrode. The binding of calcium was similar for the two fulvic acids.     

Conditional Stability Constant Determination of Metal-Fulvic Acid Complexes

Abstract

Conditional stability constants for metal complexes of a terrestrial fulvic acid were determined using an ion-exchange chromatography—atomic absorbance spectroscopy method. Employing the Scatchard model, conditional stability constants were determined for the metal (II) fulvic acid complexes of cadminum, copper, lead, nickel, manganese, and zinc. The order of metal binding by the fulvic acid was determined to be: Cu > Ni > Pb > Zn > Cd > Mn. Complexes of weakly bound metal ions were determined with an added metal ion concentration of 2 × 10^–5 M to 1 × 10^–4 M while complexes of strongly bound metal ions were determined with an added metal ion concentration of 1 × 10^–5 M to 8 × 10^–4 M. The fulvic acid concentration was kept constant at 4 × 10^–4 M. The effect of pH and ionic strength on the copper-fulvic acid complex also was investigated.     

Molecular and quantitative aspects of the chemistry of fulvic acid and its interactions with metal ions and organic chemicals : Part I. The electron spin resonance approach

A review is presented of the application of electron spin resonance (ESR) spectroscopy to the study of the chemistry of fulvic acid and its interactions with paramagnetic metal ions. After a short introduction on the nature and role of fulvic acids in the global environment, basic theory, instrumentation, procedures, methodology and limitations of ESR spectroscopy are summarized. This is followed by a discussion of the direct information that ESR data can provide on the status of organic free radical and paramagnetic metal species in fulvic acid. The effects of physical and chemical factors, including pH, ionic strength and state of aggregation, hydrolysis and alkylation, redox conditions and irradiation, on free radicals in fulvic acids are considered, also in relation to structural and reactive implications. Molecular and quantitative information furnished by ESR on both naturally occurring and laboratory-prepared paramagnetic metal ion-fulvic acid complexes are discussed. Emphasis is placed on the potential, sensitivity and advantages of this non-separative, non-destructive technique for the study of environmental samples such as fulvic acids.     

Determination of Dynamic Metal Complexes and their Diffusion Coefficients in the Presence of Different Humic Substances by Combining Two Analytical Techniques

The combined use of a competing ligand exchange (CLE) method and a diffusive gradient in thin films (DGT) technique in a quasi-labile system provides a better understanding of dynamic metal (Cu and Ni) complexes in the presence of humic substances of different origins. The CLE and DGT techniques provide total labile (dynamic) metal complexes (Cu and Ni) and their dissociation rate constants in environmental systems. DGT was found to estimate lower concentrations of labile metal complexes than CLE. These discrepancies were caused by diffusion controlled metal flux (towards the binding resin gel) in the diffusive gel of DGT. The interactions of Cu and Ni with humic acids are stronger than their interactions with fulvic acid and natural organic matter. Changes in the lability of Ni and Cu complexes (complexed with humic substances of different origins) with the changing analytical detection window indicate that the complexes of these metals were formed with different binding sites with diverse binding energies in the humic substances. The combination of these two techniques was found to be very useful in determining diffusion coefficients of labile metal-humate complexes in quasi-labile systems. The values of diffusion coefficients of labile Ni and Cu complexes determined in this study are in good agreement with limited results from the literature. This finding is novel and can be very useful in further improving our understanding of the metal-humate interactions in natural environments.     

Molecular and quantitative aspects of the chemistry of fulvic acid and its interactions with metal ions and organic chemicals : Part II. The fluorescence spectroscopy approach

Information in the literature on the use of fluorescence spectroscopy for the study of the chemistry of fulvic acid and its interactions with metal ions and organic chemicals is discussed. Basic principles, instrumentation, procedures, methodology and limitations of the fluorescence technique and the fluorescence polarization method are briefly summarized. This is followed by an extended discussion on the direct information that fluorescence spectra can furnish on the properties and nature of fluorescing structures in fulvic acids. The effects of molecular parameters and environmental factors (molecular weight, concentration, pH, ionic strength, temperature and redox potential) on the fluorescence behaviour of fulvic acids are discussed. Particular attention is devoted to the indirect information that fluorescence quenching and fluorescence polarization studies provide on molecular and quantitative aspects of the chemistry of fulvic acid in solution, especially in relation to molecular conformation and binding with metal ions and organic chemicals. The potential advantage of this non-separative, non-destructive technique for the study of environmental samples such as fulvic acids is emphasized.     

Ultraviolet absorbance titration for determining stability constants of humic substances with Cu(II) and Hg(II)

We describe an ultraviolet (UV) absorbance titration method that can be used to determine complexing capacities (C_L) and conditional stability constants (log K) of humic substances (HSs) with metal ions such as Cu(II) and Hg(II). Two fulvic acids (FA) and one humic acid (HA) were used for this study. UV absorbance of HSs gradually increased with the addition of Cu(II) or Hg(II) after blank correction, and these increases followed the theoretical 1:1 (ligand:metal ion) binding model. The results from the absorbance titration calculation for HSs with Cu(II) and Hg(II) compared well with those from fluorescence quenching titration. The titration of the model compound l-tyrosine with Cu(II) proved the validity of this method, and the K and C_L were within 2.3% and 7.4% of the fluorescence quenching titration. The results suggest that the UV absorbance titration can be used to study the binding capacities of HSs and/or dissolved organic matter (DOM) with trace metals. The advantages and disadvantages of the absorbance titration method were also discussed.     

Determination of stability constants for rare earth elements and fulvic acids extracted from different soils

Fulvic acids (FAs) were extracted by alkali extraction from different soil samples in China, then purified using resins and characterized by Fourier transform infrared spectroscopy. The complexing ability of FAs was investigated by measuring the stability constants of rare earth elements (REEs) (La³⁺, Ce³⁺, Sm³⁺, Gd³⁺, Y³⁺) with FAs by the ion exchange technique. The results indicated that maximum binding ability forY³⁺ (4.414.44) was higher than other REEs (La³⁺, Ce³⁺, Sm³⁺, Gd³⁺) (0.721.03). There were two types of binding sites in the functional groups of fulvic acids. The complexing reaction followed two steps. The stability constants (K₁ and K₂) of REEs with FAs were calculated from experimental data by division of Scatchard plots into two straight-line segments. Y³⁺ (log K₁=5.72±0.05, log K₂=4.83±0.01) also has higher stability constants than the other four REEs (log K₁=4.37±0.16, log K₂=3.62±0.28).     

Fractionation analysis of trace metals in humic substances of soils irrigated with wastewater in Central Mexico by particle induced X-ray emission

The Mezquital Valley in Central Mexico has received wastewater from Mexico City for nearly 100 years. Wastewater brings in organic matter and nutrients but also trace metals. Humic substances, the main components of organic soil matter, are responsible for retaining and regulating the mobility of trace metals in soils. In this study, humic substances were extracted from the soil and separated into distinct fractions (humic acids, fulvic acids and humins). The particle induced X-ray emission (PIXE) technique was applied to determine the metal content in bulk soil as well as in humic acids and fulvic acids not soluble in H₃PO₄. In order to assess whether the long-term input of organic matter and metals modifies the metal association with these humic substances, parcels irrigated for three time periods (5, 47 and 89 years) were selected for this study. It was observed that metals such as Zn and Cu are mainly associated with the humic acids. Fulvic acids retain mainly Cr while Pb is distributed among humic and fulvic acids. It was also observed that in general, metal retention by humic substances increases with irrigation time. Depth also affects metal association with the humic substances.     

Comparative study of copper(II) interactions with monomeric ligands and synthetic or natural organic materials from potentiometric data

Potentiometric measurements were used to characterize the complexing properties of (a) a mixture of five monomeric ligands, (b) a synthetic humic-like substance and (c) fulvic acids extracted from soils, with copper (II). In order to compare the binding strengths involved, the same mathematical treatments of the data were used for all measurements. Calculations of the total ligand concentration with the Gran function give underestimated values for the multiligand mixture because of the known presence in the mixture of functional groups with pK^H ; 11 which > cannot be titrated and are revealed only by complexation phenomena. The acid-base properties of the humic substances are better described with a continuous model than with the descrete Henderson-Hasselbach model, because of the inability to distinguish properly between equivalent and independent types of functional groups. In the presence of copper (II), the formation functions show that a mixture of different kinds of complexes with different stability constants is probably formed. Extra protons released during the titration of the various copper (II) systems with alkali are attributed to untitrated functional groups with extremely low protonation constants in the case of the multiligand mixture, but ambiguity remains with the humic substances because their chemical structure is not known. Comparison of binding strengths can be made in terms of global conditional stability constants, by taking into account the three parameters, pH, concentrations of metal and of ligand. It is shown that the mechanisms of complexation are different for monomers and for polymers.     

COMPLEXES OF Al(III) WITH HYDROXYAROMATIC LIGANDS

Abstract

In order to assess the aluminium binding ability of humic and fulvic acids, important organic soil constituents, a pH-potentiometric study was made of the proton and aluminium(III) complexes of various bi-, tri- and tetradentate catechol and salicylic acid derivatives at 25°C and at an ionic strength of 0.20moldm^?3 (KC1). The stability data revealed that at low pH the salicylate function, and at high pH the catecholate function, is preferentially bound to the aluminium ion. In the intermediate pH range, mixed hydroxo complexes and other di/oligomeric species are also formed. With an increase of the number of available coordinating sites in the molecule, the tendency to oligomeric complex formation increases, while the tendency to metal ion hydrolysis decreases.     

Accurate analysis of Mg2+ binding to RNA: From classical methods to a novel iterative calculation procedure

Mg²⁺ acts as a catalytic cofactor in many ribozymes and specifically bound divalent metal ions have been implicated in the stabilization of structural motifs that are essential for RNA folding. The accurate calculation of intrinsic affinity constants of M²⁺ to specific binding sites in nucleic acids is therefore of high importance. Methods classically applied to determine the affinity constants of metal ions to RNAs are summarized in the first part of this review, e.g. hydrolytic cleavage experiments, equilibrium dialysis, and spectroscopic techniques like EPR and NMR. However, the fact that several binding sites of similar affinities are often present in a single RNA molecule is mostly neglected. The most immediate consequence of several binding sites is that less than the total amount of M²⁺ is available to bind to a particular binding site at a given total concentration. We have recently introduced a new iterative procedure that tackles this problem and have developed a rapid calculation tool (ISTAR) that is available from the authors. Here, we explain this procedure in detail under different assumptions and illustrate how the intrinsic affinity constants for Mg²⁺ to a short RNA hairpin, a minimal domain 6 from the group II intron Sc.ai5γ, change. We use ISTAR to calculate intrinsic affinities and to validate a particular binding stoichiometry by judging the quality of the fit to the experimental data for a given model. This is important since weak coordination sites exhibiting similar binding affinities, and being thus in direct competition to each other, are a characteristic feature of nucleic acids. With ISTAR these binding affinities can be calculated more accurately within minutes and we can gain a better understanding of these crucial metal ion–nucleic acid interactions.     

Application of sampled-d.c. anodic stripping voltammetry to metal/fulvic acid equilibria

The use of anodic stripping voltammetry (ASV) to determine the labile metal fraction in metal/ fulvic acid equilibrium systems is discussed. A method is described for distinguishing between the contributions of processes in the reduction and oxidation steps to the observed anodic (stripping) current. This method, which facilitates separate examination of the two processes, is based on timed addition of fulvic acids during the deposition step, on pH control, and on measurement of sampled-d.c. ASV peak areas (Faradaic charge) for metal/fulvic acid solutions. Results are presented for copper(Il) and lead(Il) complexes with six colloid-free soil-derived fulvic acids. In contrast to differential-pulse ASV, the stripping current measured by sampled-d.c. ASV showed no measurable contribution from ligand adsorption on the mercury drop. For heterogeneous ligand systems, such as fulvic acid, use of stripping peak heights over-estimates the fraction of non-labile metal complex because peak broadening results from the range of complexes formed in the anodic step.