Determination of the phenolic-group capacities of humic substances by non-aqueous titration technique

The phenolic-group capacities of five humic substances, such as, the Aldrich humic acid, the humic and fulvic acids extracted from a soil, the humic and fulvic acids extracted from a peat have been precisely determined by the non-aqueous potentiometric titration technique. The titration by KOH in the mixed solvent of DMSO:2-propanol:water = 80:19.3:0.7 at [K⁺] = 0.02 M enabled to measure the potential change in a wide range of pOH (=−log [OH⁻]), and thus to determine the capacities of phenolic groups which could not be precisely determined in the aqueous titration. The results of the titration revealed that the mean protonation constants of the phenolic groups were nearly the same for all humic substances and close to that of phenol in the same medium, indicating that each phenolic-group in the humic substances is rather isolated and is not electronically affected by other affecting groups in the humic macromolecule.     

Development of an automated system for isolation and purification of humic substances

Characterization of humic substances (HS) in environmental samples generally involves labor-intensive and time-consuming isolation and purification procedures. In this paper, the development of an automated system for HS isolation and purification is described. The novelty of the developed system lies in the way the multiple liquids and columns used in the isolation/purification procedure are handled in both forward and back-elution mode by solenoid valves. The automated procedure significantly reduces the total throughput time needed, from 6–7 days to 48 h, and the amount of labor to obtain purified HS for further characterization. Chemical characterization of purified HS showed that results were in good agreement with previously published values for HS from a variety of sources, including the IHSS standard HS collection. It was also shown that the general properties of HS were consistent among the different source materials (soil, waste, aquatic) used in this study. The developed system greatly facilitates isolation and characterization of HS and reduces the risk of potential (time-dependent) alteration of HS properties in the manual procedure. Figure Photograph of the rear (left) and front (right) of the automated system for the isolation and purification of humic substances Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction of atrazine with Laurentian humic acid

Ultrafiltration fractionation and liquid chromatography have been applied to study the binding and hydrolysis of polar herbicide atrazine on a stoichiometrically well characterized Laurentian humic acid. The main advantage of this method over gas chromatography is the simultaneous determination of both free and bound atrazine, hydroxyatrazine and copper(II) ion with satisfactory accuracy and precision. Atrazine binding requires extensive carboxylate site protonation but the binding sites represent only a very small fraction of total carboxylate of humic acid. The results show that binding of atrazine is not competitive with binding of the hydrolysis product hydroxyatrazine, the binding capacity is reduced at higher ionic strength or by cation competition for carboxylate and the atrazine binding constant and free energy of binding can be fitted by a single value at all pH values. The differences between atrazine binding by fulvic acid and humic acid can be ascribed to the structure difference, one being a flexible linear polymer and the other a three-dimensional colloidal gel particle.     

Effect of humic substances on the electrochemical reduction of p -nitrophenol

p-Nitrophenol (PNP) is the main hydrolysis product of methylparathion (MP), one of the most commonly used organophosphate insecticides in the world. Such a product is very toxic for human and animals. Humic substances (HS) are natural recalcitrant organic matter found in soil and waters that have an ability to interact, immobilize, and degrade pesticides. This article presents electroanalytical and UV-Visible studies, conducted to understand the effect of the HS on the PNP reduction process and therefore to understand how the HS can influence PNP degradation in the environment. Electroanalytical results showed that the HS benefit the reduction of the nitro-group of PNP by electrocatalysis. UV-Visible spectra showed that the catalytic effect of HS occurs due to the interection between the PNP and the HS followed by a proton donor mechanism.     

Potentiometric studies on the complexation of copper(II) by phenolic acids as discrete ligand models of humic substances

Studies on the complexation of copper(II) by phenolic acids, as ligand models of humic substances were done by potentiometry. The acids under study were: 3,4-dihydroxyhydrocinnamic acid or hydrocaffeic acid (1), 3,4-dihydroxyphenylacetic acid (2) and 3,4-dihydroxybenzoic acid or protocatechuic acid (3). Acidity constants of the ligands and the formation constants of metal-ligand complexes were evaluated by computer programs. The carboxylic group of the phenolic acids has different pK_a1 values, being the dissociation constants intrinsically related with the distance between the function and the aromatic nucleus. The results obtained allow concluding that acidity constants of the catechol moiety of the compounds are similar with pK_a2 and pK_a3 values between 9.47-9.41 and 11.55-11.70. The complexation properties of the three ligands towards copper(II) ion are quite similar, being the species found not different either in nature or stability. Although the model ligands have some structural differences no significant differences were found in their complexation properties towards copper(II). So, it can be postulated that complexation process is intrinsically related with the presence of a catechol group.     

HPLC of humic substances fractionated by Flow FFF

This communication reports a study of the effect of ionic strength and electrolyte composition on fractions, separately collected by flow FFF, of a mixture of humic substances. Reverse phase HPLC analysis of three early eluting fractions suggests that the components released by the column behave as organic acids. The baseline‐resolved peaks of the first two fractions, subject to higher retention in solutions of lower pH and/or higher polarity, substantiate this suggestion. The fraction with larger components, as measured by flow FFF, also appears to contain acidic species. Their retention level, however, may not be accurately modulated by varying the mobile phase properties as these species are either totally retained in acidic phases or released before the void peak at pH ≥ 4.2. Besides showing the effective separation achieved in the flow FFF channel, this study reveals the pronounced difference in the physicochemical properties of some components of a humic mixture even with very close particle size.     

Hyphenated techniques of thermal analysis for characterisation of soil humic substances

Our aim was to investigate the thermal behaviour of humic substances extracted from temperate and tropical soils by means of hyphenated techniques of thermal analysis (e. g. simultaneous thermal analysis DSC/TG coupled with mass spectrometry, MS, for the analysis of evolved gas, EGA) in order (i) to verify whether the chemical composition of isolated humic substances also reflected the differences in microbial parameters previously measured in related soil samples and (ii) to identify suitable indices of thermal stability. Our results show that the investigation of humic substances by thermal methods can provide information on soil organic matter dynamics. Differences in thermal behaviour between the two groups of soils were found. The indices of thermal stability here proposed, IR (index of thermal recalcitrance), and ID (index of thermal decomposability) clearly showed that in humic substances from tropical soils the thermally recalcitrant organic fraction dominated, whilst in temperate humic substances the opposite held. This agrees with previous results on the microbial dynamics and organic matter turnover of the respective soils and indicates that these indices of thermal stability could represent a useful tool in soil environmental quality investigations.     

Characterization of humic substances: Implications for trihalomethane formation

Humic substances are precursors of carcinogenic trihalomethanes (THMs) formed during disinfection by chlorination in water treatment processes. In an effort to understand the relationship between trihalomethane formation potential (THMFP) and physicochemical properties of humic substances, UV-visible absorbance, fluorescence in emission and synchronous scan modes, and NMR spectra were measured for several aquatic fulvic and humic acids. For comparison, a soil fulvic acid was also examined using these methods. The feasibility of the gradient modified spin-echo (GOSE) NMR experiment to selectively measure singlet resonances arising from isolated protons was examined. In addition, diffusion coefficients were measured for DMSO solutions of the fulvic acids using BPPLED and GOSE-edited pulse sequences. Although none of the methods tested produced results that correlated with THMFP, the GOSE intensities determined for different regions of the NMR spectra did reflect the relative abundance of different types of functional groups produced by lignin oxidation. In addition, the GOSE-edited diffusion results suggest that the isolated protons, those most reactive to chlorination, are more likely contained in the larger molecular weight fractions of fulvic acids.     

Differential pulse polarographic determination of europium in the presence of humic substances

Humic substances (HS) and other substance of similar nature (“building blocks” of HS, such as salicylates or phthalates) influence strongly a polarographic behaviour of europium at a mercury drop electrode. An addition of these substances into the supporting electrolyte causes an enhancement of the differential pulse polarographic (DPP) peak of Eu and an anodic shift of the maximum peak potential. It was deduced from the dependence of the peak height on the pulse time and from other experimental dependencies that an adsorption of the Eu-HS (salicylate, phthalate) complexes on the working electrode is the main mechanism responsible for the peak enhancement. Because no peak distortion or splitting were observed, the DPP determination of Eu can be realised readily in the presence of HS, e.g. in environmental samples. The sensitivity of the measurements increased in the presence of HS, whereas the limit of detection (LOD) decreased (LODs were 255 and 140 nmol/l in the absence and in the presence of HS, respectively). However, the calibration dependencies were non-linear in the presence of HS. It was found that the non-linear calibration dependencies may be approximated by the Langmuir-type equation. Special procedures, such as a method of (at least) two standard additions and numerical iterative calculations, are necessary for an evaluation of measurements.     

Influence of amide linkages on acidity determinations of humic substances Testing with model-mixtures

A set of experiments, using model compounds, was carried out in order to evaluate the influence of amide linkages on acidity measurements of humic substances (HS). Three benzene-carboxylic acids (2,4-dihydroxybenzoic acid; 3,5-dihydroxybenzoic acid, and phthalic acid) and two peptides (dl-alanyl-dl-alanine, and glycil-l-leucine) were employed to simulate the major acidic functional groups present in humic material. The acidity of the samples was measured, using the Schnitzer and Gupta methods, for each compound separately, as well as for selected mixtures with different compound combinations. General results showed that, in the absence of peptides, phenolic groups with high pK_a values are not detected in the barium hydroxide reaction and the data so obtained do not represent the real concentrations of the acidic groups. For the mixtures containing peptides, the presence of amide linkages distorts the results relative to the total acidity (TA) mainly because under the extreme conditions of the reaction with Ba(OH)₂ hydrolysis of the peptide occurs, consuming extra hydroxyl groups and increasing artificially the phenolic content. Such a condition is minimized by the reduction of the reaction time.     

Distribution of humic substances dissolved and particulated in water column in Ross Sea, Antarctica

Dissolved (DFA) and particulate (PFA) fulvic acids were studied in some areas of the Ross Sea (Antarctica) characterised by different water masses. Our interest was focused on their distribution in the water column. Moreover, their correlation with fluorescence data and structural characterisation in relation to the water masses was also studied. We found that PFA concentration was mainly influenced by the primary production in surface layers and by stratification of water masses along the water column. Conversely, the DFA are less influenced by primary production and by the stratification of the water masses. An early stage of the humification process can be hypothesised for PFA related to DFA as shown by elemental analyses and spectroscopic data. PFA collected in the bottom depths of the water column showed a relationship with age of water mass (the highest concentration was found for the oldest circulating water mass (CDW)). This relationship was not observed for DFA. Moreover, any significant difference in structure was found in the DFA and PFA extracted from samples collected at bottom depths.     

Capillary electrophoretic separation of humic substances using hydroxyethyl cellulose as a buffer additive and its application to characterization of humic substances in a river water sample

We have developed a concise tool for the investigation of the transition of humic substances in environmental water. The separation of water-soluble humic substances was achieved rapidly and effectively by capillary electrophoresis using a polyacrylamide-coated capillary and a phosphate electrophoretic buffer solution (pH 7.0) containing hydroxyethyl cellulose. The separation mechanism was assessed using the ultrafiltration technique. The effect of the complexation of humic substances with metal ions was studied by using the proposed method. When Fe(III) ions or EDTA was added to the sample solution of fulvic acid, a distinct change in the electropherogram pattern based on the conformational change of fulvic acid was observed. The successful application of the proposed method to the characterization of humic substances in a river water sample was also demonstrated. Figure Addition of Fe(III) ions or EDTA to a solution containing fulvic acid (FA) results in a distinct change in the electropherogram pattern, which reflects the conformational change of FA: this forms the basis for the characterization of humic substances in river water samples     

A rapid quantitative method for humic substances determination in natural waters

A simple and rapid method was proposed for humic substances (HS) determination at microgram levels in natural waters. This assay method is based on the binding of a dye, Toluidine Blue (TB), to HS molecules to produce a dye-HS complex, which causes a decrease in absorbance at 630 nm. This method was calibrated with HS samples with up to a concentration of 40 mg L⁻¹, which covered the range of dissolved HS concentrations present in natural waters. The detection limit was 0.8 mg L⁻¹ of HS, and the relative standard deviation of 10 replicate measurements for a 20-mg L⁻¹ standard sample was 3.5%. From the Langmuir adsorption isotherm theory, the binding equilibrium constant and total number of binding sites at neutral pH were calculated to be (8.17 ± 0.42) × 10⁵ L mol⁻¹ and N of 1.45 ± 0.04 mmol g⁻¹ HS, respectively. The determination results with five water samples from lake, river and pond were consistent with those measured with the reference methods, demonstrating that this quantification method for HS determination was rapid, sensitive and feasible.     

Ultrafiltration and determination of Zn- and Cu-humic substances complexes stability constants

This study exhibits that size fractionation of humic substances (HS) and their metal complexes by ultrafiltration is an efficient procedure for simultaneous determination of stability constants. Using sequential-stage ultrafiltration and a radiotracer technique the HS–Cu and HS–Zn complexes studied can gently be size-fractionated and their free metal fractions simply be discriminated. The conditional stability constants Ki obtained for size fractions of these HS metal complexes exhibit a clear molecular size dependence. Accordingly, the highest Ki values (6.6 for Zn and 6.4 for Cu) are found in the HS fractions of >105 kDa. Moreover, the overall stability constants K found for Cu (log K=5.5) and Zn complexes (log K=4.5) of the aquatic HS complexes studied are quite comparable to those reported in the literature.     

Fourier transform infrared study of mercury interaction with carboxyl groups in humic acids

An interaction between humic acid, an organic part of soil and mercury was studied by Fourier transform infrared spectroscopy (FTIR) and by ICP-AES analysis under given pH and concentration conditions. First the spectroscopic model was validated on the interaction of simple molecules representing the structural components of humic acid such as benzoic acid, catechol and salicylic acid with mercury. The interaction of carboxylic parts of humic acid with mercury is very interesting and easily characterised by infrared spectroscopy, an ideal mean for molecular study. Under the salt form (commercial humic acid Fluka TM: FHA), humic acid reacts with mercury in a different way from its acid form (FHA purified noted PFHA) and the Leonardite (LHA). Because of the straightforward exchange between Na⁺, Ca²⁺ and Hg²⁺, fixation of the latter is much more important with the salt form (FHA). However, this reaction is reduced under the acid form (PFHA, LHA) because the exchange with protons is difficult. The effect of this exchange was studied by FTIR showing the intensity decrease of ν_CO (COOH), the carboxylic functional group band of the acid, and the shifting of ν_as (COO⁻), the carboxylate functional group band under given pH and mercury conditions. For the FHA salt form, the characteristic band ν_CO (COOH) represented by a shoulder did not evolute, whereas the corresponding band to ν_as (COO⁻) strongly shifted (40 cm⁻¹) for a maximum Hg²⁺ concentration (1 g l⁻¹). On the other hand, for the acid form (PFHA, LHA), the intense band of ν_CO (COOH) disappeared proportionally to the increase of Hg²⁺concentration and the ν_as (COO⁻) band moved for about 20 cm⁻¹. The same results were reached with pH variations. Our results were confirmed by ICP-AES mercury analysis. This study shows that humic acids react differently according to their chemical and structural state.     

Characterisation of humic substances by acid catalysed transesterification

The chemical composition of low molecular weight moieties linked to the core structures of humic substances (HS) are of substantial importance for the understanding of the chemical structures and mode of interactions of HS with other substances in the environment. In this study a novel approach to characterise certain low molecular weight compounds bound to HS is suggested. The method includes transesterification (TE) of ester and amide bound structures, and esterification (E) of free carboxylic groups using acid catalysed methanolysis followed by gas chromatography (GC)-mass spectrometry (MS) and GC-FID analysis. Methanolysis of five HS of different origin, demonstrated the presence of multifunctional hydroxy-substituted benzoic acids, hexoses, and long chain fatty acids. Based on GC-FID and addition of the internal standard before methanolysis, the total amounts of low molecular weight material could be estimated. In case of HS from the aqueous sources the yields were below 1%, whereas in case of the HS derived from lignite the yield was significantly higher. The hydrophobic long chain fatty acids constituted about one-third of this material. Principal component analysis (PCA), which was used for extended data evaluation, differentiated between the samples in terms of chemical composition.     

Studying the interaction between triazines and humic substances--a new approach using open tubular capillary electrochromatography

The strength of the interaction between a pesticide and the soil organic matter is a key parameter to assess the risk of it reaching to groundwater with potentially harmful effects to human health. In this work, a new approach that allows measuring such interactions in a few minutes using a purified fraction of the soil organic matter (humic substances) is detailed. The strength of sorption is assessed via the normalised difference of elution (retention factor, k′) between the chemical of interest and a neutral marker transported via electroosmotic flow through an open tubular column supporting the immobilised humic substances (open tubular capillary electrochromatography). The immobilisation was achieved by incubating a capillary, pre-coated with a monolayer of humic acid, with an acidic solution of humic substances. This induces the formation of a supramolecular structure of humic substances as it occurs in soils. This aggregate can easily be removed using alkaline solutions, and a new structure assembled using other humic substances (HS) or different incubations conditions. The whole procedure takes 2 h. This approach has been tested using five triazines and three types of humic substances. The order of the strength of sorption of the triazines as expected from relevant literature and the relative standard deviation of k′ was between 1 and 6%. Good repeatability was also observed after long period of wash, between re-coating and repeating of the full coating with a new capillary.     

Spectral and temporal luminescent properties of Eu(III) in humic substance solutions from different origins

Although a high heterogeneity of composition is awaited for humic substances, their complexation properties do not seem to greatly depend on their origins. The information on the difference in the structure of these complexes is scarce. To participate in the filling of this lack, a study of the spectral and temporal evolution of the Eu(III) luminescence implied in humic substance (HS) complexes is presented. Seven different extracts, namely Suwannee River fulvic acid (SRFA) and humic acid (SRHA), and Leonardite HA (LHA) from the International Humic Substances Society (USA), humic acid from Gorleben (GohyHA), and from the Kleiner Kranichsee bog (KFA, KHA) from Germany, and purified commercial Aldrich HA (PAHA), were made to contact with Eu(III). Eu(III)-HS time-resolved luminescence properties were compared with aqueous Eu³⁺ at pH 5. Using an excitation wavelength of 394 nm, the typical bi-exponential luminescence decay for Eu(III)-HS complexes is common to all the samples. The components τ₁ and τ₂ are in the same order of magnitude for all the samples, i.e., 40 ≤ τ₁ (μs) ≤ 60, and 145 ≤ τ₂ (μs) ≤ 190, but significantly different. It is shown that different spectra are obtained from the different groups of samples. Terrestrial extract on the one hand, i.e. LHA/GohyHA, plus PAHA, and purely aquatic extracts on the other hand, i.e., SRFA/SRHA/KFA/KHA, induce inner coherent luminescent properties of Eu(III) within each group. The ⁵D₀ → ⁷F₂ transition exhibits the most striking differences. A slight blue shift is observed compared to aqueous Eu³⁺ (λ_max = 615.4 nm), and the humic samples share almost the same λ_max ≈ 614.5 nm. The main differences between the samples reside in a shoulder around λ ≈ 612.5 nm, modelled by a mixed Gaussian–Lorentzian band around λ ≈ 612 nm. SRFA shows the most intense shoulder with an intensity ratio of I_612.5/I_614.7 = 1.1, KFA/KHA/SRHA share almost the same ratio I_612.5/I_614.7 = 1.2–1.3, whilst the LHA/GohyHA/PAHA group has a I_612.5/I_614.5 = 1.5–1.6. This shows that for the two groups of complexes, despite comparable complexing properties, slightly different symmetries are awaited.     

Trace enrichment of polar pollutants from water and the influence of humic substances

Summary A practical liquid chromatographic early-warning system for polar pollutants in water ought to have detection limits at a level below a microgram per litre. Using ultraviolet absorbance detection this normally necessitates trace enrichment of the samples. In this study ten different sorbents have been tested with respect to their enrichment capacity. The hydrophobic polymeric PLRP-S material, in combination with a C-18 modified analytical column, proved to be the best choice. The influence of humic substances, present in surface water, was also studied. Lowering of the sample pH, resulted in a significant increase in the interferences due to the humic substances.