Detection of glucosamine in the acid hydrolysis solution of humic substances

Several humic substances isolated from water and soil have been characterized applying acid hydrolysis to release defined components bound in these high molecular organic compounds. The hydrolysis solution have been analyzed by HPLC after precolumn derivatization with o-phthalaldehyde and 9-fluorenylmethylchloroformate. Under these conditions the nitrogen containing carbohydrate glucosamine could be detected and separated besides seventeen amino acids. The results show that 8.0 to 40.1% of the total nitrogen content of the investigated samples contributes to amino acids and up to 7.9% to the carbohydrate glucosamine. It could be deduced that defined amino acid carbohydrate building blocks were bound in humic substances, because the glucosamine nitrogen content correlates with the calculated amino acid nitrogen. Furthermore the total amino acid nitrogen amount of natural organic matter can be estimated from the results of the determination of glucosamine.     

Simultaneous presence of diverse molecular patterns in humic substances in solution

The chemical and structural nature of humic substances (HS) is the object of an intense debate in the literature involving two main theoretical positions: the classical view defending the macromolecular pattern, and the new, more recent, view proposing a supramolecular pattern. In this study, we observe that both molecular patterns are present in different whole humic systems in solution. We also identify these molecular patterns with a specific fraction of HS. Thus, the HS family formed by the gray humic acids studied presented a clear macromolecular pattern, whereas the HS family formed by the fulvic acids studied presented the coexistence of supramolecular assemblies and individual molecules. The third HS family studied, the brown humic acids, presented both the macromolecular pattern and the supramolecular pattern. We also find that molecular aggregation-disaggregation has a strong influence in the fluorescence pattern of HS, thus indicating that the current concepts of HS structure derived from fluorescence studies need revision.     

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.     

UVC-induced photodegradation of p-arsanilic acid assisted by humic substances

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Analysis of humic acids from various soils using acid hydrolysis

Structural changes in humic acids from various soils, produced by acid hydrolysis, are studied by elemental and thermal analyses and by IR and NMR spectroscopy.     

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.     

Separation methods in the chemistry of humic substances

Separation methods are widely used to isolate humic substances (HSs), to fractionate them before further investigation, and to obtain information about their structure and properties. Among the chromatographic methods, techniques based on a size-exclusion effect appear to be most useful, as they allow us to relate elution data to the molecular mass distribution of HSs. The limitations of this approach are discussed in this review. Gas chromatography with mass spectrometric detection is typically used to identify the products of pyrolysis or thermochemolysis of HSs; this technique is considered most important in the structural investigation of HSs. Electrophoretic methods (especially capillary zone electrophoresis) provide detailed characterization of HSs, but it is very difficult to relate the electrophoretic data to any specific subfraction, structure or properties of HSs. The electrophoretic patterns are often called "fingerprints" and can potentially be used for the identification and classification of HSs. This is limited, however, by the great diversity of the procedures employed and by the low degree of harmonization--no data on reproducibility and between-laboratory comparability are available. The same holds true, to a certain degree, for most methods utilized for the characterization of HSs. Separation methods play an important role in the examination of the interactions of HSs with heavy metals and other chemical pollutants. They allow us to determine binding constants and other data necessary to predict the mobility of chemical pollutants in the environment.     

Analytical chemistry of freshwater humic substances

Dissolved organic carbon (DOC) in aquatic environments represents one of the largest active organic carbon reservoirs in the biosphere. Current ideologies concerning the sources of DOC, how it is formed and utilized, and what determines the quality of DOC are examined. Humic substances can comprise a significant fraction of the DOC and developments in methods of analysis including the isolation and characterization of this fraction are reviewed.     

Ultrafiltration of protein and humic substances: effect of solution chemistry on fouling and flux decline

The rate and extent of adsorption of a protein and a humic acid onto membranes was measured at varying conditions of pH and ionic strength. The resistance-in-series approach was used to calculate reversible and irreversible fouling resistances, which were then compared for static (no flow) and dynamic runs in order to determine the effect of convective flow and electrostatic interactions on fouling behavior. Although convective forces tended to increase the amount of material accumulated near the membrane surface, electrostatic interactions played a stronger role, as evident in the irreversible adsorption results for the static and dynamic cases. Electrostatic interactions affected reversible and irreversible resistances. Both resistances were higher at the isoelectric point (iep) of the protein and decreased at higher pH values. Humic acid adsorption decreased as pH was increased from 4.7 to 10. Humic acid filtration resulted in a higher resistance per unit mass than protein filtration.     

Pyrolysis of natural and synthetic humic substances

Thermogravimetric and Rock-Eval techniques were used for the characterization of natural (humic) and synthetic (melanoidins) substances and their hydrocarbon generation potential. A similarity between the thermal behaviour of humic substances and of melanoidins (prepared from an excess of sugar) and the unique thermal properties of melanoidins (prepared from basic amino acids) was observed. Rock-Eval analysis indicated that most synthetic melanoidins (also clay-complexed) generated more hydrocarbons and related compounds than terrestrial humic substances.     

Characterization of humic substances by capillary electrophoresis

Capillary electrophoresis was used for the separation of humic acid (HA) from peat, soil, and vermicompost. The electropherograms show the presence of at least three peaks eluted between 6 and 11 min for all HA. The best analysis resolution was obtained with the use of borate buffers at pH 8.9. The HA analyzed have structural and charge similarity, which increases the difficulty of separation. Therefore, the shape of the peaks is broad and the CE profiles of all HA are similar. It is reasonable to assume that the broad band in the three regions is due to the acidic groups that have a similar structure. By comparing the results obtained for HA extracted from soil, peat, vermicompost, and the commercial sample, HA from peat had the major carbon content.     

Enhanced adsorption of roxarsone onto humic acid modified goethite from aqueous solution

The adsorption of roxarsone (ROX) on the humic acid modified goethite (HA-α-FeOOH) was evaluated for several potential environmental factors. Results showed that 1) ROX had a higher adsorption capacity onto HA-α-FeOOH than unmodified α-FeOOH; 2) the adsorption of ROX increased with a decrease in pH; 3) the high ionic strength significantly inhibited the adsorption capacity of HA-α-FeOOH; and 4) a higher temperature yielded greater adsorption, since the process for ROX to be adsorbed by HA-α-FeOOH was a spontaneous endothermic reaction. The maximum adsorption capacity of ROX was found to be 80.71?mg?·?g^?1, when the temperature was 308?K. Meanwhile, the inhibitory effects of ionic strength and PO₄^3? on the adsorption of ROX onto HA-α-FeOOH were enhanced with an increase in concentration. In addition, the adsorption equilibrium data obeyed Langmuir isotherm model and the kinetic data were well described by the pseudo-second-order kinetic model. From the infrared spectra of HA-α-FeOOH, it could be deduced that the ROX adsorption onto HA-α-FeOOH was achieved via the ion exchange between the arsenic acid and the carboxyl group on adsorbent, as well as the formation of As-O-Fe bond between Fe-O and arsenic acid ions.     

Role of fractionation in studying the photochemical properties of humic substances

The purpose of the cooperative research between laboratories of Pushchino Biological Centre Russian Academy of Sciences and Laboratoire de Photochimie Moléculaire et Macromoléculaire, CNRS, France was the investigation of photochemical properties of humic substance fractions with different molecular sizes and electrophoretic mobilities. Humic acids from five different soils were used. The resulting data showed that a great part of photosensitizing fluorophores and chromophores are located in low-molecular fractions of humic acids, independently of their source.     

Retention behavior of humic substances in reversed phase HPLC

Recovery as well as appearance and abundance (in percent) of different fractions of humic substances are found to depend on injected sample amounts in reversed phase HPLC. Sample amounts have been varied both by varying sample concentration and sample volume. In case of lowest amounts injected only two fractions were obtained for a commercial humic acid sodium salt, i.e. one for excluded molecules and one for hydrophobic components. The abundance of excluded molecules decreases upon increasing amounts injected. Another three fractions are obtained upon increasing amount injected: a hydrophilic fraction and two hydrophobic ones. This behavior is explained by auxiliary equilibria between excluded components and humic molecules previously adsorbed on the stationary phase.     

Capillary electrophoretic behaviour of humic substances in physical gels

We investigated the principles of the capillary electrophoretic behaviour of humic substances (HSs) in physical gels. Long chain (Mr 4000, 6000 and 20,000) polyethylene glycols (PEGs) at concentrations above their entanglement threshold caused the separation of HS fractions according to molecular size differences. Close linear relationships between effective mobilities and mean apparent molecular masses were observed at PEG concentrations between 2.5 and 15%. The efficiency of the separation does not increase in gels of increasing polymer concentrations. The possibility of interactions between HSs and gel-forming polymers was also investigated. Short chain (Mr 400) PEGs, added to the buffer at concentrations from 2.5 to 12.5%, increased the migration times of all HS fractions, but no separation was obtained even at large polymer concentrations, showing that gel formation was essential for the separation. In 2.5% polyvinyl alcohol (PVA) 49 000 all fractions show two unresolved, but well defined peaks. This separation is probably artefactual and depends on the relative concentration of HSs and PVA, as the relative abundance of the peaks changes with the sample concentration.     

Iodine emission in the presence of humic substances at the water's surface

Humic substances that preferentially adsorb at the air/water interfaces of water or aerosols consist of both fulvic and humic acid. To investigate the chemical reactivity for the heterogeneous reaction of gaseous ozone, O(3)(g), with aqueous iodide, I(-)(aq), in the presence of standard fulvic acid, humic acid, or alcohol, cavity ring-down spectroscopy was used to detect gaseous products, iodine, I(2)(g) and an iodine monoxide radical, IO(g). Fulvic acid enhanced the I(2)(g) production yield, but not the IO(g) yield. Humic acid, n-hexanol, n-heptanol, and n-octanol did not affect the yields of I(2)(g) or IO(g). We can infer that the carboxylic group contained in fulvic acid promotes the I(2)(g) emission by supplying the requisite interfacial protons more efficiently than water on its surface.     

Extraction and neutron activation analysis of humic substances

Methods are described for the isolation of humaic substances from a soil samples. The produres and based on extractions with sodium hydroxide or sodium pyrophosphate. Four of the humic samples were dried and then analysed by thermal neutron activation analysis. The humic samples along with three standard rocks were irradiated for 15 hours in a flux of 10¹² n·cm^–2·s^–1. Counting was carried out using both a large volume Ge(Li) detector and a high resolution LEP detertor. Quantitative analysis was based on the known element concentrations in the standard rocks. The elements determined were Sc, Cr, Fe, Co, As, Se, Br, Mo, Sb, La, Ce, Eu, Hf, Ta, Th, and U. concentrations ranged from 0.02 ppm (La) to 7900 ppm (Fe).     

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.     

Pyrolysis-GC-FTIR for structural elucidation of aquatic humic substances

Substructure components of various aquatic humic substances were investigated by a coupled pyrolysis — gas chromatography — Fourier-transform infrared spectroscopy (Py-GC-FTIR) procedure. The humic substances studied gave similar pyrolysis products, but in varying proportions. Many of the pyrolysis products (e.g. methanol, acetone, alkylbenzenes, cyclopentane, aliphatic and aromatic organic acids, acetamide, pyrrole and phenols) could be identified by their FTIR spectra using a digital library for automatic comparison. Some of the compounds are related to lignin fragments which form a large part of the humic substances investigated. Other products give hints to the involvement of tetrapyrroles, fatty acids, furanoses and amino compounds in the structure of humic macromolecules.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday