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.     

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.     

Dynamic light scattering measurements of particle size development in aqueous humic materials

Dynamic light scattering (DLS) has been used to monitor changes in aggregate sizes of aqueous humic materials as a function of solution properties. Humic and fulvic acids were dissolved at relatively low concentrations (15-30 mg L(-1)) in solutions of different temperature, cation and ethanol content, and pH. The results could be explained in terms of intramolecular contraction and intermolecular aggregation of humic polymers. The former were prevalent in soil humic acids, and less so in aquatic humic acids and fulvic acids. Increasing the temperature of humic solutions generally led to an increase in particle sizes, which was ascribed to an effect akin to surfactant clouding. The addition of cations led to either contraction or expansion, depending on the charge and concentration of the ion, and the nature of the humic material. Reducing the pH initially caused contraction, followed by growth and precipitation in more highly acidic media.     

Potential of capillary zone electrophoresis for estimation of humate acid-base properties

Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA(s)) and chernozem (HA(ch)) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA(s) fractions obtained by salting-out with ammonium sulfate at a saturation of 0-40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of "humic hump" was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5-7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA(s) fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA(s) with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the pK(a) of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 x 10(-5) cm(2) V(-1) s(-1) can be used for approximate estimation of HA isoelectric point. The data were discussed and agreement with the random coil structural model has been shown.     

Interaction of the cesium cation with mono-, di-, and tricarboxylic acids in the gas phase. A Cs+ affinity scale for cesium carboxylates ion pairs

Humic substances (HS), including humic and fulvic acids, play a significant role in the fate of metals in soils. The interaction of metal cations with HS occurs predominantly through the ionized (anionic) acidic functions. In the context of the effect of HS on transport of radioactive cesium isotopes in soils, a study of the interaction between the cesium cation and model carboxylic acids was undertaken. Structure and energetics of the adducts formed between Cs⁺ and cesium carboxylate salts [Cs⁺RCOO⁻] were studied by the kinetic method and density functional theory (DFT). Clusters generated by electrospray ionization mass spectrometry from mixtures of a cesium salt (nitrate, iodide, trifluoroacetate) and carboxylic acids were quantitatively studied by CID. By combining the results of the kinetic method and the energetic data from DFT calculations, a scale of cesium cation affinity, CsCA, was built for 33 cesium carboxylates representing the first scale of cation affinity of molecular salts. The structural effects on the CsCA values are discussed.     

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.     

A speciation methodology to study the contributions of humic-like and fulvic-like acids to the mobilization of metals from compost using size exclusion chromatography-ultraviolet absorption-inductively coupled plasma mass spectrometry and deconvolution analysis

High performance size-exclusion chromatography (HP-SEC) with UV absorption for organic matter detection and inductively coupled plasma mass spectrometry (ICP-MS) for elemental detection have been used to study the mobilization of metals from compost as a function of pH and the molecular mass of their complexes with dissolved organic matter (DOM). Due to its heterogeneous nature, organic matter mobilized from compost shows a continuous distribution of molecular masses in the range studied (up to 80 kDa). In order to differentiate between the contribution of humic and fulvic acids (FA) to the organic matter mobilized in the pH range 5-10, their UV absorption chromatographic profiles have been deconvoluted with respect to the adjusted gaussian profiles of the humic and fulvic acids isolated from compost. Results show a preponderant contribution of fulvic acids at low pH values and an increasing percentage of humic acids (HA) mobilized at basic pH (up to 49% of total DOM at pH 10). A similar deconvolution procedure has been applied to the ICP-MS chromatograms of selected metals (Co, Cu, Pb and Bi). In general, both fulvic and humic acids contribute to the mobilization of divalent transition metals, such as copper or cobalt, whereas bismuth or lead are preferably associated to humic acids. Non-humic substances (HS) also contribute to the mobilization of cations, especially at acidic pHs. These conclusions have been extended to different elements based on deconvolution analysis results at pH 7.     

The Effects of a Strong Disaggregating Agent on Sec-Page of Aquatic and Soil Humic Matter

Abstract

Size-exclusion chromatographic (SEC) fractionation and electrophoretic separation of aquatic humic matter samples from a Finnish lake using Sephadex G-75 with 7 M urea solution as eluent and 10% polyacrylamide gel (PAGE) with urea and sodium dodecyl sulphate solution (SDS), respectively, were performed and compared to similar analyses performed on a Russian chernozem soil humic acid sample and Nordic reference fulvic and humic acid samples. The integrated whole of aquatic humic solutes and soil humic acids were found to exhibit similar SEC-PAGE behaviours. Humic matter was not excessively disaggregated by the 7 M urea and hence SEC-PAGE can with confidence be applied as a coarse, initial fractionation procedure or for certain predeterminations of the structural composition.     

Separation and determination of traces of heavy metals complexed with humic substances in fresh waters by sorption on diethylaminoethyl-sephadex a-25

Copper(II), lead and cadmium complexed with humic and fulvic acids in filtered 1-l samples of fresh water are sorbed on a column containing 0.5 ml of the macroreticular weak-base anion exchanger, diethylaminoethyl-Sephadex A-25 at a flow rate of 20 ml min^?1. Simple metal cations are not sorbed at all. The sorbed trace metals are quantitatively desorbed with 4 M nitric acid batchwise and determined by graphite-furnace atomic absorption spectrometry. For synthetic aqueous solutions containing traces of heavy metals and humic acid, the results are in conformity with those obtained by cationexchange separation. About 80% of the sorbed humic substances are eluted with 0.5 M sodium hydroxide solution from the A-25 column and its quantity is estimated spectrophotometrically at 400 nm.     

Comparative study for differentiation of aquatic humic-type organic constituents by capillary zone electrophoresis using polyvinyl alcohol-coated capillary

Capillary zone electrophoresis (CZE) with UV detection (254 nm) was applied to characterize aquatic dissolved humic matter (DHM) from different environmental sources (lake, river and sea waters, in all 15 different samples). A series of separation examples of DHMs using a polyvinyl alcohol (PVA)-coated silica open tubular capillary were carried out in a phosphate buffer (40 mM) as a background electrolyte at neutral acidity (pH 6.8). The separative power of electropherograms was reasonable and the reproducibility was above the mark. Each electropherogram was characteristic of the corresponding humic sample. Special functional fulvic and humic acids or their overall mixtures separated with XAD, DAX and DEAE sorbing solids as well as the original dissolved organic matter (DOM) were nicely differentiated according to their environmental sources. The PVA coating of open tubular silica capillaries seems to be very potential in electrophoretic characterization and separation of different humic solutes at neutral acidities with low sample concentrations thus permitting a workable technique, in a growing series of CZE studies, for better compared results from different studies.     

Characterization of groundwater humic and fulvic acids of different origin by GPC with UV/Vis and fluorescence detection

Gel permeation chromatography (GPC) was applied for recognizing the origin of groundwater humic and fulvic acids. GPC was performed with Fraktogel TSK HW-50 in 0.1 M NaCl, pH 8.5 (0.05 M phosphate buffer), 1 mM EDTA, with 10% by volume methanol added. Humic substances from groundwaters and sediments of four different aquifer systems in Germany were isolated, purified and characterized. Both UV/Vis and fluorescence detection were applied. UV/Vis detection was found to be more powerful in identifying differences between the various humic and fulvic acids. The four aquifer systems investigated (“Gorleben”, “Fuhrberg”, “Franconian Albvorland” and “Munich”) differed from one another with respect to hydrological and geochemical conditions. The results showed that the GPC-elution behavior reflects the geochemical environment and origin (source material and generation process) of aquatic humic and fulvic acids.     

Concentration of humic and fulvic acids in large aqueous samples by continuous-flow coprecipitation-flotation

Summary Humic and fulvic acids are quantitatively coprecipitated in a stream (0.5l/min) of sample solution with indium hydroxide at pH 8 and continuously floated with the aid of sodium dodecyl sulphate and numerous tiny nitrogen bubbles. The precipitate and foam on the surface of the solution are collected by suction and the latter is ruptured with ethanol. By these procedures the original sample volume is reduced to less than 1/100. After dissolving the precipitate in 2 mol/l hydrochloric acid, the solution (pH 0.5) is introduced onto the pulverized XAD-2 resin to collect humic and fulvic acids, leaving indium ions in the solution. The humic substances are desorbed with 0.1 mol/l sodium hydroxide solution. The recoveries of humic and fulvic acids are ca. 95% for coprecipitation-flotation, >90% for sorption and 80–90% for desorption.     

Comparison of physico-chemical pretreatment methods to seawater reverse osmosis: Detailed analyses of molecular weight distribution of organic matter in initial stage

In desalination, effective pretreatment is the key to reduce membrane fouling that occurs during the seawater reverse osmosis (SWRO) process. However, it is difficult to compare the flux decline after different pretreatments using a small-scale reverse osmosis filtration unit. In this study, we successfully evaluated the effect of pretreatment on SWRO in terms of molecular weight distribution (MWD) of seawater organic matter (SWOM) after 20 h of SWRO operation. Microfiltration (MF), ultrafiltration (UF), ferric chloride (FeCl₃) flocculation and powdered activated carbon (PAC) adsorption, were used as pretreatment. The effluents and the retentates after each pretreatment and 20 h of SWRO operation were characterized in terms of MWD.Although the normalized flux of SWRO showed similar flux decline (J/J₀ = 0.17) with/without pretreatment, SWOM concentration in the retentates after different pretreatments was different in quantity and it increased linearly with time. The slope of the SWOM increase was 0.110, 0.096, 0.077 and 0.059 after MF, FeCl₃ flocculation, UF and PAC adsorption pretreatments, respectively. MW peaks for the seawater used in this study consisted of 1200 Da (biopolymers), 950 Da (fulvic acids), 650 Da (hydrolysates of humic substances), 250 Da (low MW acids) and 90 Da (low MW neutrals and amphiphilics). FeCl₃ flocculation preferentially removed 1200 Da (biopolymers), while PAC adsorption mostly removed 950 Da (fulvic acids). UF and NF removed only a marginal amount of relatively large organics, while RO removed the majority of organics. The intensity of 1200, 950, 650 and 250 Da MW in the RO retentates increased with the RO operation time. The organics of MW around 1200 Da (biopolymers) had a relatively low rate of increase with time compared with those of lower MW. This suggests that the SWOM of 1200 Da MW was preferentially retained on the membrane surface.     

Separation and determination of traces of heavy metals complexed with humic substances in river waters by sorption on indium-treated amberlite xad-2 resin

A nonionic macroreticular styrene/divnylbenzene copolymer, Amberlite XAD-2 resin is pulverized to 1–10 μm and treated with indium ions to saturate traced of cation exchange sites for the quantitative separation of humic complexes from cations. A 100-ml filtered sample is passed through an indium-treated XAD-2 column (16 diameter, 5 mm tall) at pH 5 at a flow rate of 2 ml min^? to sorb heavy metals complexed with humic and fulvic acids. Inorganic cations and anions, EDTA complexes and colloidal hydrated iron(III) oxide are not retained on the column at all. The heavy metals sorbed on the column are then ultrasonically desorbed with 0.5 M nitric acid and determined by graphite-furnace atomic absorption spectrometry. The results fo two river water samples obtained are in good agreement with those obtained when the macroreticular weak-base anion-exchanger DEAE-Sephadex A-25 is used.     

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.     

Characterization of groundwater humic and fulvic acids of different origin by GPC with UV/Vis and fluorescence detection

Gel permeation chromatography (GPC) was applied for recognizing the origin of groundwater humic and fulvic acids. GPC was performed with Fraktogel TSK HW-50 in 0.1 M NaCl, pH 8.5 (0.05 M phosphate buffer), 1 mM EDTA, with 10% by volume methanol added. Humic substances from groundwaters and sediments of four different aquifer systems in Germany were isolated, purified and characterized. Both UV/Vis and fluorescence detection were applied. UV/Vis detection was found to be more powerful in identifying differences between the various humic and fulvic acids. The four aquifer systems investigated (“Gorleben”, “Fuhrberg”, “Franconian Albvorland” and “Munich”) differed from one another with respect to hydrological and geochemical conditions. The results showed that the GPC-elution behavior reflects the geochemical environment and origin (source material and generation process) of aquatic humic and fulvic acids. Received: 17 November 1998 / Revised: 18 March 1999 / Accepted 23 March 1999     

Spurenanalyse von huminstoffen im trinkwasser: The d.c. polarographic determination of traces of humic substances in potable waters

(The d.c. polarographic determination of traces of humic substances in potable waters) The inhibiting effect of a tri-n-butylphosphate layer adsorbed at the mercury drop on the polarographic wave of copper(II) is reduced by humic substances. This effect can be utilized to determine humic substances in the range 0.05–1 mg l^-1. The standard substance used was isolated from peaty water. Humic and fulvic acids are not differentiated but amino acids, peptides and polyhydroxy compounds do not interfere.     

Application of resonance Raman spectrometry to the determination of vitamin B12.

A gel filtration technique is described for the study of the complexation of dissolved metals by humic and fulvic acids. Measurements can be made under realistic conditions of pH and free metal ion concentrations, both of which can be maintained by the use of TRIS as a buffer. The procedure permits the determination not only of the overall stability constant, but of the binding stoichiometries and the intrinsic stability constants associated with the various types of metal binding sites. The procedure has been applied to the investigation of the interaction of fresh-water fulvic acids with Cu, Zn and Ni, and has provided evidence for the existence of two different types of binding sites in the fulvic acid molecule.     

The uniqueness of humic substances in each of soil,stream and marine environments

Definitive compositional differences are shown to exist for both fulvic acids and humic acids from soil, stream and marine environments by five different methods (¹H and ¹³C NMR spectroscopy, ¹⁴C age and δ¹³C isotopic analyses, amino acid analyses and pyrolysis-mass spectrometry). Definitive differences are also found between fulvic acids and humic acids within each environment. These differences among humic substances from various sources are more readily discerned because the method employed for the isolation of humic substances from all environments excludes most of the non-humic components and results in more purified humic isolates from water and soils. The major compositional aspects of fulvic acids and humic acids which determine the observed characteristic differences in each environment are the amounts and composition of saccharide, phenolic, methoxyl, aromatic, hydrocarbon, amino acids and nitrogen moieties.     

Identification of diffuse and point sources of dissolved organic carbon (DOC) in a small stream (Alb, Southwest Germany), using gel filtration chromatography with high-sensitivity DOC-detection

Using gel filtration chromatography and high-sensitivity UV and organic carbon (OC) detection, the quality and quantity of organic matter in a stream (the Alb, Southern Germany) has been studied in the winter season. In the catchment area and upstream of two sewage plant effluents the organic load has been low (below 1 mg/l TOC) and has been dominated by humic substances (more than 80%). Downstream of the sewage plants, TOC increased up to 5 mg/l while humic constituents decreased down to 30%. A detailed analysis showed that in the catchment area the humic fraction has been composed of fulvic acids with relatively high amounts of aliphatic structures. Downstream of the sewage plants, the humic fraction has been composed of fulvic acid precursors with associated non-humic matter. It is concluded that the quality and quantity of organic constituents in the stream has been dominated by the secondary effluents.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday