Hydrophilic interaction liquid chromatography method for measuring the composition of aquatic humic substances

A hydrophilic interaction liquid chromatography (HILIC) method was developed to measure the composition of humic substances from river, reservoir, and treated wastewater based on their physicochemical properties. The current method fractionates the humic substances into four well-defined groups based on parallel analyses with a neutral and a cationic HILIC column, using mobile phases of varied compositions and pH. The results indicate that: (i) the proportion of carboxylic acids in the humic substances from terrestrial origins is less than half of that from treated wastewater (Jeddah, KSA), (ii) a higher content of basic compounds was observed in the humic substances from treated wastewater and Ribou Reservoir (Cholet, France) than in the sample from Loire River (France), (iii) a higher percentage of hydrophobic macromolecules were found in the humic substances from Loire River than in the other samples, and (iv) humic substances of treated wastewater contained less ionic neutral compounds (i.e., pK_a 5–9) than the waters from terrestrial origins. The physicochemical property disparity amongst the compounds in each humic substances sample was also evaluated. The humic substances from the lightly humic Loire river displayed the highest disparity, whereas the highly humic Suwannee river (Georgia, USA) showed the most homogeneous humic substances.     

On-line fractionation and characterization of aquatic humic substances by means of sequential-stage ultrafiltration

A five-stage tangential-flow ultrafiltration (UF) device equipped with advanced membrane filters (molecular weight cut-off: 1, 5, 10, 50 and 100 kDalton) of the polyethersulfone type is described and applied for the analytical on-line fractionation of a series of aquatic humic substances (HS) originating from surface or groundwaters. Fractionation patterns of HS (6 fractions each) evaluated by this UF device exhibit their particular dependence on the HS concentration, the pH-value and the salt content of the sample (10 ml) to be analyzed. Fundamental parameters (e.g., washing volume) governing the molecular-size fractionation of HS by means of multistage UF are discussed, too. The fractionation of an aquatic reference HS (BOC 3/9.5) by means of the above UF procedure reveals considerable differences preferably characterized by the UV-VIS absorption ratio E₃₅₀/E₄₅₀ and metal complexing capacity (Cu(II)) of the produced fractions. Moreover, molecular spectroscopy investigations (FTIR, ¹H-NMR) of the fraction series of this HS indicate that carbohydrate substructures (preferably found in fractions >50 k Dalton) and aromatic ones (preferably in fractions <5 k Dalton) are unevenly distributed.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Structural and elemental investigations of isolated aquatic humic substances using X-ray photoelectron spectroscopy

Structural and elemental investigations of aquatic humic substances (HS) by means of X-ray photoelectron spectroscopy (XPS) are described. For that purpose small amounts (10-50 microg) of dissolved reference HS, which were characterized within the German research program DFG-ROSIG, were dried as thin films on small pieces of a high-purity silicon wafer. The photoelectrons from such HS layers exhibited characteristic signals of carbon C1s, nitrogen N1s, oxygen O1s and sulfur S2s, which could be fitted by Gaussian curves and used for the quantification of various moieties of HS: carbon (C-C, C-O, C=O, O=C-O), oxygen (C-O, C=O), nitrogen (C-N, C-N+) and sulfur. Moreover, by adding up the element signals of the HS samples their elemental composition of C, O, N and S was assessed. A comparison of the data based on solution state NMR and conventional elementary analysis revealed a satisfactory accuracy with those obtained by XPS.     

Structural and elemental investigations of isolated aquatic humic substances using X-ray photoelectron spectroscopy

Structural and elemental investigations of aquatic humic substances (HS) by means of X-ray photoelectron spectroscopy (XPS) are described. For that purpose small amounts (10–50 μg) of dissolved reference HS, which were characterized within the German research program DFG-ROSIG, were dried as thin films on small pieces of a high-purity silicon wafer. The photoelectrons from such HS layers exhibited characteristic signals of carbon C1s, nitrogen N1s, oxygen O1s and sulfur S2s, which could be fitted by Gaussian curves and used for the quantification of various moieties of HS: carbon (C-C, C-O, C=O, O=C-O), oxygen (C-O, C=O), nitrogen (C-N, C-N⁺) and sulfur. Moreover, by adding up the element signals of the HS samples their elemental composition of C, O, N and S was assessed. A comparison of the data based on solution state NMR and conventional elementary analysis revealed a satisfactory accuracy with those obtained by XPS.     

Acid-base properties of aquatic humic substances isolated from unpolluted tropical black water rivers

Summary The acid-base properties of tropical aquatic humic substances isolated from 2 Venezuelan black water rivers were studied. A model with discrete pK values was applied to potentiometric titration data, since direct determination of pK and concentration values from titration curves is not possible. In the computational procedure simulated curves have been compared with the titration curves to improve the calculations for pK and concentration values. Both rivers (Yuruaní and Aponwao) are located in a zone where the human influence is still negligible. The geomorphological characteristics of this remote area (Gran Sabana) are singular in relation to the rest of the world. The rivers have been monthly studied at sampling site, during the rainy season (May–September). Six discrete acidic groups represented well the acidic characteristics of aquatic humic substances under consideration. The average pK values were 2.74, 4.37, 5.67, 7.09, 8.73 and 10.30, and the average concentration values for each group were 2.50, 1.80, 0.94, 0.62, 0.66, and 1.31 mmol/g humic substance, respectively. Considering the factors month and river, significant quantitative, but not qualitative, differences were in general found. A comparison with data from temperate ecosystems showed that the three most acidic groups presented similar acid-base characteristics, wheras the three other groups are quite different for the tropical aquatic humic substances under study.     

Labile/inert metal species in aquatic humic substances: an ion-exchange study

Summary An ion-exchange procedure has been developed for the analytical fractionation of metals (e.g. Al, Co, Cu, Fe, Mn, Ni, Pb, Zn) forming labile/inert complexes with aquatic humic substances (HS) isolated (XAD 2, XAD 8, ultrafiltration) from bog, forest, ground and lake water. Using 1-(2-hydroxyphenylazo)-2-naphthol groups immobilized on cellulose (Cellulose HYPHAN) as chelating collector (batch and column procedure, resp.) for reactive metal fractions in dissolved HS, the kinetics and the degree of separation (referred to the total metal content) serve for the operational characterization of the metal lability. According to the separation kinetics (96 h), mostly the reactivity order Mn>Zn>Co>Pb>Ni>CuAl>Fe is observed for the above metals in HS, resulting in recoveries of >98% for Mn and Zn, but strongly varying for the other metals (e.g., 44–95% Cu, 18–84% Fe). By means of cellulose HYPHAN four metal fractions (e.g. Cu) can be distinguished kinetically: (a) about 50% of Cu freshly complexed with HS are directly exchanged (2nd order kinetics, k=0.275 1 · mol^–1 · s^–1) followed by (b) a less labile fraction (20–30%) of 1st to 2nd order exchange; (c) a hardly reactive fraction (5–10%) revealing uniform half times t_1/2 of 25 h closes the Cu exchange from HS. Moreover the Cu fraction (d), being exchange-inert in HS, amounts to 5–10% and increases by slow transformation processes of the formed HS/Cu species.     

Quantification of partial structures of aquatic humic substances by 1H-NMR under WATR conditions

Aquatic humic substances (HS) isolated from surface water, leakage water and ground water have been investigated by ¹H-NMR. The overlapping HOD signal was eliminated by adding ammonium chloride and applying the multiecho method CPMG (Carr, Purcell, Meiboom, Gill) under WATR conditions (water attenuation by transverse relaxation) permitting quantification of partial structures of HS. The proportion of carbohydrates/alcohols/ethers decreases and the proportion of alkyl moieties increases with increasing water or soil depth and thus microbial diagenesis. Also, increasing deoxygenation of aromatic substituents is observed with increasing water or soil depth. In some cases, elimination of the NMR signal of HOD is accompanied by the appearance of another HOD signal which is slightly shifted and much smaller in intensity; this signal probably results from water strongly bound by hydrogen bonding within the HS macromolecules.     

Quantum-chemical simulation of the synthesis of structural fragments of humic substances analogs

Semiempirical calculations by the PM3 method were used to optimize the structural fragments of the precursors of synthetic humic and fulvic acids. Changes in the thermodynamic parameters (enthalpy of formation, entropy, and Gibbs energy) were determined and a conclusion was drowm if the postulated processes are thermodynamically possible.     

Pyrolysis and soft ionization mass spectrometry of aquatic/terrestrial humic substances and soils

The rapid, reproducible, chemical characterization of complex environmental materials such as plants, humic substances and whole soil can be performed by controlled thermal degradation. Except for drying and milling no pre-treatment of the samples is required. Biomacromolecular cleavage during a short degradation step directly in the ion source of a mass spectrometer results in the production of high-mass chemical subunits. Short reaction times and small amounts of sample favour the generation of large, thermal fragments, i.e., chemical building blocks, which can be identified and correlated with the structure of the polymeric biomaterials investigated. The principal aim is to monitor the primary, thermal fragmentation by high molecular ion intensities of the pyrolyzates and to avoid consecutive, mass spectrometric fragmentation as far as possible.For the detection and identification of the pyrolysis (Py) products, a combination with time-/temperature-controlled mass spectrometry (MS) is used. Typical heating rates are 0.2–10°C/s and the temperature range is 50–800°C. Soft ionization techniques such as field ionization (FI), field desorption, chemical ionization (CI) and, to some extent, fast atom bombardment are employed in the positive and negative modes. The results of direct Py-MS are supported by high-resolution mass measurements using electric or photographic detection and Curie-point pyrolysis in combination with gas chromatography-electron ionization/FI/CIMS and library searches for the identification of the pyrolysis products.Fingerprinting and time-resolved Py-MS of aquatic and terrestrial humic substances are reported. The methodology for the investigations of dynamic processes during the volatilization and thermal decomposition of these complex biomaterials is illustrated. Weight loss curves and the temperature function of accurate molecular weight averages for aquatic fulvic and humic acid are derived from the Py-FIMS data. Initial results on the differentiation of soil horizons in a moder profile by Py-FIMS and pattern recognition are presented. In particular, the chemometric evaluation appears promising for future Py-MS studies of humic substances and whole soils, but also for fossil fuels, synthetic polymers and food. In an integrated approach, the linking of conventional chemical and spectroscopic data with the high-mass signals in pyrolysis-mass spectra will be the focus of forthcoming work. Preliminary results for combining wet-chemical data with those of ¹³C nuclear magnetic resonance, Fourier transform infrared and electron spin resonance spectroscopy are put forward in this survey. Finally, initial results of pilot studies to detect biocides such as atrazine directly in soils using Py-FIMS are demonstrated.     

Thermofractography of humic substances

Thermofractography (TF) has been applied to humic and fulvic acids from four different soil types. Among the thermal products, 3,5-dihydroxybenzoic acid, catechol, 5-hydroxymethylfurfural, vanillin, phenol, furfural, guaiacol and indole were identified. These are typical fragments from lignins, microbial polyphenols, polysaccharides and proteins.Thermofractography appears to be an effective method for study of humic molecule structures by thermal degradation. Furthermore, it seems promising to differentiate humic acids from distinct soil types and to distinguish between humic and fulvic fractions.

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Zusammenfassung Die Thermofraktographie (TF) wurde zur Analyse von Humin- und Fulvinsäuren aus vier verschiedenen Bodenarten eingesetzt. Unter den Produkten der Thermolyse wurden die 3,5-Dihydroxy-benzoesäure, Brenzkatechin, 5-Hydroxymethyl-furfurol, Vanillin, Phenol, Furfurol, Guaiakol und Indol identifiziert. Diese sind typische Fragmente aus Lignin, mikrobiellen Polyphenolen, Polysacchariden und Proteinen.Die Thermofraktoraphie scheint eine wirksame Methode zum Studium der Struktur des Huminmoleküls durch thermische Zersetzung zu sein. Außerdem scheint sie auch zur Unterscheidung von Huminsäuren aus bestimmten Bodentypen und zur Unterscheidung von Humin- und Fulvinfraktionen geeignet.

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Résumé La thermofractographie (TF) a été appliquée à l'analyse des acides humiques et fulviques de quatre types différents de sols. L'acide 3,5-dihydroxybenzoïque, le pyrocatéchol, le 5-hydroxyméthyl-furfural, la vanilline, le phénol, le furfural, le guaacol et l'indole ont été identifiés parmi les produits de la thermolyse. Tous sont des fragments typiques des lignines, des polyphénols microbiens, des polysaccharides et des protéines.La thermofractographie paraît être une méthode efficace pour l'étude par dégradation thermique de la structure moléculaire des substances humiques. En outre, elle semble intéressante pour différencier les acides humiques dans les types de sols particuliers et pour distinguer les fractions humiques et fulviques.

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F. J. G. V. thanks DAAD (Deutsche Akademischer Austauschdienst) for providing a grant.     

Analytical fractionation of aquatic humic substances and their metal species by means of multistage ultrafiltration

The molecular-size fractionation of aquatic humic substances (HS) and their metal species by means of a novel sequential-stage ultrafiltration (UF) device equipped with five appropriate ultramembranes (1, 5, 10, 50 and 100 kD) is described. First of all, the concentration dynamics of macromolecules, particulary HS, during five-stage UF and its subsequent washing step has been modelled. Based on these results, the fractionation of aquatic HS (from ground and bog water) by means of multistage UF has been optimized for an analytical scale (10 ml sample, 1 mg/ml HS, 10 ml washing solution, pH 6.0). The molecular size-distribution of selected aquatic HS (BOC 1/2 from the DFG-Versuchsfeld Bocholt, VM 5 from Venner Moor, Germany) studied by five-stage UF exhibited strong systematic influences of the procedure used for their isolation. The molecular-size distribution of HS obtained by on-line UF and gel permeation chromatography (GPC) showed a satisfactory agreement in the range 1–50 kD. Moreover, when interrupting multistage UF for > 48 h a slow transformation in the HS samples has been found as gradually additional HS fractions of < 1 kD have been formed. Besides unloaded HS molecules, the molecular-size distribution of freshly formed metal species of HS (1.0 mg metal/g HS of Al(III), Cd(II), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), Zn(II), each) has been characterized by multistage UF as a function of pH-value, degree of loading and complexation time. Metal determinations as carried out by flame AAS, showed that considerable metal fractions in HS especially are present in molecules > 50 kD, which seemed to be rather acid-inert. With complexation times of < 2 days a transient shift of the molecular size distribution of both HS and their metal species (e.g., Al(III), Fe(III) to higher values (> 10 kD) has been found.     

Analytical fractionation of aquatic humic substances and their metal species by means of multistage ultrafiltration

The molecular-size fractionation of aquatic humic substances (HS) and their metal species by means of a novel sequential-stage ultrafiltration (UF) device equipped with five appropriate ultramembranes (1, 5, 10, 50 and 100 kD) is described. First of all, the concentration dynamics of macromolecules, particulary HS, during five-stage UF and its subsequent washing step has been modelled. Based on these results, the fractionation of aquatic HS (from ground and bog water) by means of multistage UF has been optimized for an analytical scale (10 ml sample, 1 mg/ml HS, 10 ml washing solution, pH 6.0). The molecular size-distribution of selected aquatic HS (BOC 1/2 from the "DFG-Versuchsfeld Bocholt", VM 5 from "Venner Moor", Germany) studied by five-stage UF exhibited strong systematic influences of the procedure used for their isolation. The molecular-size distribution of HS obtained by on-line UF and gel permeation chromatography (GPC) showed a satisfactory agreement in the range 1-50 kD. Moreover, when interrupting multistage UF for > 48 h a slow transformation in the HS samples has been found as gradually additional HS fractions of < 1 kD have been formed. Besides unloaded HS molecules, the molecular-size distribution of freshly formed metal species of HS (1.0 mg metal/g HS of Al(III), Cd(II), Cu(II), Fe(III), Mn(II), Ni(II), Pb(II), Zn(II), each) has been characterized by multistage UF as a function of pH-value, degree of loading and complexation time. Metal determinations as carried out by flame AAS, showed that considerable metal fractions in HS especially are present in molecules > 50 kD, which seemed to be rather acid-inert. With complexation times of < 2 days a transient shift of the molecular size distribution of both HS and their metal species (e.g., Al(III), Fe(III) to higher values (> 10 kD) has been found.     

Quantification of carbohydrate structures in size fractionated aquatic humic substances by two-dimensional nuclear magnetic resonance

Two-dimensional phase sensitive C,H correlation spectra were successfully applied to the quantification of carbohydrate substructures in aquatic humic substance (HS) fractions obtained by tangential flow multistage ultrafiltration (TFMSTUF) of a selected bog water HS (HO13, German Research Program DFG-ROSIG) as well as a river HS (Suwannee River Fulvic Acid Reference of the International Humic Substances Society, IHSS). It turns out that after size fractionation the HS samples give very well resolved C,H-correlation spectra which offer a great potential for substructure quantification. Details of the combined substructure quantification technique, novel in HS characterization, are presented. The results of the combined procedure point out that carbohydrate moieties predominantly occur in higher molecular mass fractions (> 10 kDa) of isolated HS.     

Quantification of carbohydrate structures in size fractionated aquatic humic substances by two-dimensional nuclear magnetic resonance

Two-dimensional phase sensitive C,H correlation spectra were successfully applied to the quantification of carbohydrate substructures in aquatic humic substance (HS) fractions obtained by tangential flow multistage ultrafiltration (TFMSTUF) of a selected bog water HS (HO13, German Research Program DFG-ROSIG) as well as a river HS (Suwannee River Fulvic Acid Reference of the International Humic Substances Society, IHSS). It turns out that after size fractionation the HS samples give very well resolved C,H-correlation spectra which offer a great potential for substructure quantification. Details of the combined substructure quantification technique, novel in HS characterization, are presented. The results of the combined procedure point out that carbohydrate moieties predominantly occur in higher molecular mass fractions (> 10 kDa) of isolated HS.     

Characterisation of structure and aggregation processes of aquatic humic substances using small-angle scattering and X-ray microscopy

Aquatic humic substances (HS), an important part of the dissolved organic carbon in freshwater systems, are polyfunctional natural compounds with polydisperse structure showing strong aggregation/coagulation behaviour at high HS concentrations and in the presence of metal ions. In this study, small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS) and X-ray microscopy (XRM) were applied to characterise the structure and aggregation processes of HS in solution. In SAXS and XRM the high brilliant synchrotron radiation was used as X-ray source. Applying small-angle scattering, information about the size distribution and shape of aquatic HS was obtained. Spherical HS units were found which were stable in a wide concentration range in a kind of "monomeric" state almost independent of pH and ionic strength. At higher concentrations they formed chain-like agglomerates or disordered HS structures. In studies on the coagulation behaviour of HS after addition of copper ions, a linear relationship between Cu(2+) concentration and the formation of large disordered HS-Cu(2+) agglomerates was obtained. By using X-ray microscopy, single "huge" particles were found in older solutions and in solutions with high HS concentrations. Over a threshold Cu(2+) concentration of approx. 300 mg/L, the formation of an extensive HS-Cu(2+) network structure was observed within a few minutes. The presented structures show the ability of the methods used to characterise processes between diluted phase and suspended matter, which play an important role particularly in the region of phase interfaces.     

Lability of heavy metal species in aquatic humic substances characterized by ion exchange with cellulose phosphate

Labile metal species in aquatic humic substances (HSs) were characterized by ion exchange on cellulose phosphate (CellPhos) by applying an optimized batch procedure. The HSs investigated were pre-extracted from humic-rich waters by ultrafiltration and a resin XAD 8 procedure. The HS-metal species studied were formed by complexation with Cd(II), Ni(II), Cu(II), Mn(II) and Pb(II) as a function of time and the ratio ions to HSs. The kinetics and reaction order of this exchange process were studied. At the beginning (<3 min), the labile metal fractions are separated relatively quickly. After 3 min, the separation of the metal ions proceeds with uniform half-lives of about 12-14 min, revealing rather slow first-order kinetics. The metal exchange between HSs and CellPhos exhibited the following order of metal lability with the studied HSs: Cu > Pb > Mn > Ni > Cd. The required metal determinations were carried out by atomic absorption spectrometry.     

二维核磁共振技术在结构解析中的应用

进入21世纪以来,核磁共振技术有了飞速的发展.在硬件方面:9000 MHz核磁共振仪(900MHz,场漂移＜1.5 Hz/hr)的投入使用,标志着超导核磁共振仪向大型化又迈出了重要的一步.与此同时,H/C/N超导核磁共振仪又为生物大分子的结构剖析提供了新的手段和方法.在软件方面:新的脉冲系列不断地被开发,针对某一领域的软件系统也在不断地更新.核磁共振技术发挥着重要的作用.     

Analytical fractionation of aquatic humic substances by metal affinity chromatography on iron(III)-coated cellulose

The chromatographic fractionation of aquatic humic substances (HS) onto iron(III)-coated cellulose (Cell-Fe(III)) as a metal-loaded adsorbent is described, analogously to the separation principle of the well-established metal affinity chromatography (MAC). For that purpose the sorption of HS from different aquatic origin on that collector was characterized by their kinetics and equilibrium distribution coefficients Kd. Based on Kd values of 10³ to 10⁴,mL/g, and fast sorption kinetics a preparative HPLC procedure, using stepwise increased pH-values (pH 8–12.5, borate buffer) as an eluent, was developed for the fractionation of dissolved HS (up to 7 fractions of different amount). The fractions obtained by this MAC procedure from selected aquatic HS samples were different in their Cu(II) complexation capacity, absorbance ratio E_{265 nm}/E_{365 nm} and Fourier transform infrared spectra. Received: 14 June 1999 / Revised: 6 August 1999 / Accepted: 10 August 1999     

Analytical fractionation of aquatic humic substances by metal affinity chromatography on iron(III)-coated cellulose

The chromatographic fractionation of aquatic humic substances (HS) onto iron(III)-coated cellulose (Cell-Fe(III)) as a metal-loaded adsorbent is described, analogously to the separation principle of the well-established metal affinity chromatography (MAC). For that purpose the sorption of HS from different aquatic origin on that collector was characterized by their kinetics and equilibrium distribution coefficients Kd. Based on Kd values of 10³ to 10⁴,mL/g, and fast sorption kinetics a preparative HPLC procedure, using stepwise increased pH-values (pH 8–12.5, borate buffer) as an eluent, was developed for the fractionation of dissolved HS (up to 7 fractions of different amount). The fractions obtained by this MAC procedure from selected aquatic HS samples were different in their Cu(II) complexation capacity, absorbance ratio E_{265 nm}/E_{365 nm} and Fourier transform infrared spectra. Received: 14 June 1999 / Revised: 6 August 1999 / Accepted: 10 August 1999