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.     

Organically bound sulfur in refractory organic substances

The sulfur compounds of refractory organic substances (ROS) of different origin have been characterized. Total organic sulfur was determined by elemental analysis. Sulfur-containing amino acids methionine and cystine were analyzed chromatographically after hydrolysis with HCl or by proteolytic digestion using enzymes. The results obtained from elemental analysis show that the total amount of sulfur is strongly dependent on the origin of the samples, because of different environmental factors during the formation of ROS. For naturally occurring samples isolated from soil seepage water, bog lake water and ground water the carbon-to-sulfur atomic ratios (C/S) decrease with the stage of humification, because of preferential loss of carbon. In humic acids (HA) isolated from secondary effluent the high value of the nitrogen-to-sulfur ratio (N/S) was indicative of a large amount of protein-derived nitrogen and sulfur compounds. In the solutions from acid hydrolysis the total amount of amino acid carbon related to the dissolved organic carbon (DOC) was generally less than 5%. Percentages of cystine related to all the amino acids detected were in the range 4 to 16%; methionine was below the detection limit for most samples. The results show that cystine is very important among the amino acids released. Enzymatic release generally resulted in smaller amounts of amino acids, indicating that these molecules are not only present in bioavailable protein-like structures. The data were compared with those from other approaches reported in the literature for the speciation of sulfur forms in ROS, including potentiometric titration, differential reduction methods, and spectroscopic investigations.     

Quantitative comparison of the nitrogen-containing pyrolysis products and amino acid composition of soil humic acids

Pyrrole and acetonitrile, major pyrolysis products of soil humic substances, vary characteristically in abundance with the degree of humification of the organic matter in the soil. Their origin, however, is uncertain because of the large amount of unidentifiable soil nitrogen. A quantitative relationship between amino acid composition and pyrrole and acetonitrile abundances has been established by Curie-point pyrolysis-gas chromatography of a group of test proteins, using polystyrene as an internal standard. The pyrolysis yields of these products from three humic acids extracted from the surface horizons of Scottish brown forest soils (Cambisols or Ochrepts) were entirely accounted for by the known hydrolysable amino acid content and composition, and the products were related to individual amino acids. The peptide amino acid was also found to be the overall source of the aromatic pyrolysis products toluene, phenol and p-cresol.     

Fire impact on forest soils evaluated using near-infrared spectroscopy and multivariate calibration

The assessment of physico-chemical properties in forest soils affected by fires was evaluated using near infrared reflectance (NIR) spectroscopy coupled with chemometric methods. In order to describe the soil properties, measurements were taken of the total organic carbon on solid phase, the total nitrogen content, the organic carbon and the specific absorbences at 254 and 280 nm of humic substances, organic carbon in humic and fulvic acids, concentrations of NH₄⁺, Ca²⁺, Mg²⁺, K⁺ and phosphorus in addition to NIR spectra. Then, a fire recurrence index was defined and calculated according to the different fires extents affecting soils. This calculation includes the occurrence of fires as well as the time elapsed since the last fire. This study shows that NIR spectroscopy could be considered as a tool for soil monitoring, particularly for the quantitative prediction of the total organic carbon, total nitrogen content, organic carbon in humic substances, concentrations of phosphorus, Mg²⁺, Ca²⁺ and NH₄⁺ and humic substances UVSA₂₅₄. Further validation in this field is necessary however, to try and make successful predictions of K⁺, organic carbon in humic and fulvic acids and the humic substances UVSA₂₈₀. Moreover, NIR coupled with PLS can also be useful to predict the fire recurrence index in order to determine the spatial variability. Also this method can be used to map more or less burned areas and possibly to apply adequate rehabilitation techniques, like soil litter reconstitution with organic enrichments (industrial composts) or reforestation. Finally, the proposed recurrence index can be considered representative of the state of the soils.     

Preparation and characterization of humic and fulvic acid working standards—practical experience

Two fractions of both fulvic acids (FA) and humic acid (HA) were prepared by fractionation method of Pierce and Felbeck⁵ involving acid hydrolysis of soil rests. This step increases recovery of both FA and HA considerably what suggest us need for slight modification of IHSS method in some cases. The weight loss, change in organic carbon content and visible spectra are figures of merit discussed. After detailed characterization these humic substances (HS) will serve as the working standards for study of interactions between organomercurials and organic part of soil.     

Determinations of humic substances and other dissolved organic matter and their effects on the increase of COD in Lake Biwa.

Humic substances and other dissolved organic matter (DOM) in Lake Biwa and the surrounding rivers were investigated to elucidate their origins and behavior. An annual increase in chemical oxygen demand (COD) has been observed in the northern basin of Lake Biwa since 1985. The concentrations of dissolved organic carbon (DOC) in the northern and southern basins of Lake Biwa were 1.7-2.4 mgC/l and 1.9-2.6 mgC/l, respectively. The DOC concentrations tended to be high in summer and low in winter, and the seasonal changes in the concentrations of humic substances were small. The humic substances content of DOM was considered to be comparatively small because the ratio of the concentration of humic substances to DOC was in the range of 0.14-0.32. From the results of the fractionation of DOM in lake waters, it was estimated that hydrophobic acids, such as humic substances and hydrophilic acids, were about 25% and 45%, respectively. The main origin of hydrophobic acids in Lake Biwa may be humic substances from soils around the rivers that flow into Lake Biwa, while hydrophilic acids may be due to the inner production by phytoplankton. Therefore, the increase of COD in the northern basin of Lake Biwa may be attributed to the contributions of not only humic substances but also hydrophilic acids.     

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.     

Binding of Organic Pollutants to Soil Organic Matter

Abstract

Binding of organic pollutants to water-soluble, low-molecular weight humic substances increases their mobility in soil and their leaching, whereas binding to high-molecular weight humic substances results in unextractable residues. Water-soluble pollutants in leachate may be bound to low-molecular weight humic material, or may be free water-soluble conversion products that are slowly released from a soil-bound form, as shown for a conversion product of the insecticide aldrin. Unextractable soil-bound 4-chloroaniline was isolated by supercritical methanol extraction 16 years after application of the phenylurea herbicide buturon to soil. Biodegradation and bioavailability of unextractable soil-bound residues are greatly reduced as compared to the free compounds. For some chlorinated anilines and phenols, biomineralization and plant uptake was shown to be highest for residues bound in fulvic acids and lowest for those in humic acids. Model experiments demonstrate that chlorinated anilines form covalent bonds with humic acid precursors.     

NMR characterization of humic acid fractions from different Philippine soils and sediments

Humic acids from a peat soil, an agricultural soil and a lake sediment from the Philippines were fractioned by sorption chromatography on cross-linked dextran gels (Sephadex¹). The NMR spectra of these fractions have sharp, well resolved bands which can be attributed to lignin-like structures, carbohydrates and aliphatic groups. Similar bands have been observed in the NMR spectra of humic acid fractions from other environments; however, the chemical shifts of some of the lines vary from one humic acid to another. These lines appear to arise from well defined, partially decomposed plant components and probably are diagnostic of the types of plants that decomposed into these humic acids. The lines in the carbohydrate region are the best resolved and may prove to be the most useful in identifying the plant precursors of any particular humic acid fraction.     

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.     

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     

Chromatographic determination of amino acids in foods

Amino acids in foods exist in a free form or bound in peptides, proteins, or nonpeptide bonded polymers. Naturally occurring L-amino acids are required for protein synthesis and are precursors for essential molecules, such as co-enzymes and nucleic acids. Nonprotein amino acids may also occur in animal tissues as metabolic intermediates or have other important functions. The development of bacterially derived food proteins, genetically modified foods, and new methods of food processing; the production of amino acids for food fortification; and the introduction of new plant food sources have meant that protein amino acids and amino acid enantiomers in foods can have both nutritional and safety implications for humans. There is, therefore, a need for the rapid and accurate determination of amino acids in foods. Determination of the total amino acid content of foods requires protein hydrolysis by various means that must take into account variations in stability of individual amino acids and resistance of different peptide bonds to the hydrolysis procedures. Modern methods for separation and quantitation of free amino acids either before or after protein hydrolysis include ion exchange chromatography, high performance liquid chromatography (LC), gas chromatography, and capillary electrophoresis. Chemical derivatization of amino acids may be required to change them into forms amenable to separation by the various chromatographic methods or to create derivatives with properties, such as fluorescence, that improve their detection. Official methods for hydrolysis and analysis of amino acids in foods for nutritional purposes have been established. LC is currently the most widely used analytical technique, although there is a need for collaborative testing of methods available. Newer developments in chromatographic methodology and detector technology have reduced sample and reagent requirements and improved identification, resolution, and sensitivity of amino acid analyses of food samples.     

Simultaneous analysis of small organic acids and humic acids using high performance size exclusion chromatography

An accurate and fast method for simultaneous determination of small organic acids and much larger humic acids was developed using high performance size exclusion chromatography. Two small organic acids, i.e. salicylic acid and 2,3‐dihydroxybenzoic acid, and one purified humic acid material were used in this study. Under the experimental conditions, the UV peaks of salicylic acid and 2,3‐dihydroxybenzoic acid were well separated from the peaks of humic acid in the chromatogram. Concentrations of the two small organic acids could be accurately determined from their peak areas. The concentration of humic acid in the mixture could then be derived from mass balance calculations. The measured results agreed well with the nominal concentrations. The detection limits are 0.05 mg/L and 0.01 mg/L for salicylic acid and 2,3‐dihydroxybenzoic acid, respectively. Applicability of the method to natural samples was tested using groundwater, glacier, and river water samples (both original and spiked with salicylic acid and 2,3‐dihydroxybenzoic acid) with a total organic carbon concentration ranging from 2.1 to 179.5 mg C/L. The results obtained are promising, especially for groundwater samples and river water samples with a total organic carbon concentration below 9 mg C/L.     

Immunological determination of triazine pesticides bound to soil humic acids (bound residues)

An immunological method for the determination of triazine herbicides covalently bound to soil humic acids has been developed. A sandwich-immunoassay has been performed, based on both polyclonal humic acid-antibodies and monoclonal triazineantibodies. A peroxidase-labelled third antibody has been used for the photometric detection. A triazine-humic acid conjugate served as calibration standard. The coupling density for this conjugate has been determined by measuring the difference of free amino groups both with ninhydrin and with the trinitrobenzene sulfonic acid method. In addition, the coupling density has been confirmed by scintillation counting using a (14)C-atrazine derivative. Due to nonspecific interactions between antibody proteins and humic acids, different blocking steps had to be performed. Finally, the assay has been applied to a triazine contaminated soil sample. Humic acids (including bound residues) have been extracted by diluted sodium carbonate solution. Concentrations of bound atrazine residues have been found in the range of 2 mg/kg soil on fields where triazine herbicides has been applied over a period of 21 years. These results are comparable to both the applied amount and the nonextractable fraction.     

Immunological determination of triazine pesticides bound to soil humic acids (bound residues)

An immunological method for the determination of triazine herbicides covalently bound to soil humic acids has been developed. A sandwich-immunoassay has been performed, based on both polyclonal humic acid-antibodies and monoclonal triazineantibodies. A peroxidase-labelled third antibody has been used for the photometric detection. A triazine-humic acid conjugate served as calibration standard. The coupling density for this conjugate has been determined by measuring the difference of free amino groups both with ninhydrin and with the trinitrobenzene sulfonic acid method. In addition, the coupling density has been confirmed by scintillation counting using a ¹⁴C-atrazine derivative. Due to nonspecific interactions between antibody proteins and humic acids, different blocking steps had to be performed. Finally, the assay has been applied to a triazine contaminated soil sample. Humic acids (including bound residues) have been extracted by diluted sodium carbonate solution. Concentrations of bound atrazine residues have been found in the range of 2 mg/kg soil on fields where triazine herbicides has been applied over a period of 21 years. These results are comparable to both the applied amount and the nonextractable fraction.     

Characterization of natural and synthetic humic substances (melanoidins) by stable carbon and nitrogen isotope measurements and elemental compositions

Stable carbon isotope ratios of the starting sugars (SG) and amino acids (AA) are mostly preserved in laboratory synthetic melanoidins. Stable nitrogen isotope ratios may not be “imprinted” in melanoidins by SG and AA precursors in the same way, indicating that nitrogen fractionation could occur during the rate-determining step in the Maillard reaction. Carbon isotope ratios support the stoichiometric ratios for combination of sugars with amino acids, which are based on the elemental composition data of melanoidins. These findings may provide clues to asses the role of the Maillard reaction in the formation of natural humic substances.     

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.     

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.     

Characterisation of dissolved combined amino acids in marine waters

Dissolved combined amino acids (DCAA) are important constituents of the dissolved organic nitrogen (DON) pool in marine environments, although little is known about their sources, dynamics and sinks. The DCAA pool consists of various compounds including proteins and peptides, proteins linked to sugars and amino acids adsorbed to humic and fulvic acids, clays and other materials. The proportions of each of these components and the extent to which they are used by microplankton living within the photic zone are not known. An investigation was carried out, using (15)N isotope dilution techniques, to determine the concentration and composition of dissolved amino acid pools in the marine environment. A near-shore seawater sample was collected and split into fractions to determine the concentrations of dissolved free amino acids (DFAA), DCAA and a <3 kDa dissolved peptide fraction (DPEP; obtained by ultrafiltration). DCAA and DPEP fractions were hydrolysed to yield free amino acids and all samples were analysed by gas chromatography/mass spectrometry (GC/MS) as isobutyloxycarbonyl/tert-butyldimethylsilyl derivatives. The DFAA was the smallest fraction representing approximately 1% of total dissolved amino acids. The majority of DCAA was contained in the low molecular weight DPEP fraction (90%) and was probably as a result of release from phytoplankton and degradation by heterotrophic bacteria.     

Hantzsch-type three-component approach to a new family of carbon-linked glycosyl amino acids. Synthesis of C-glycosylmethyl pyridylalanines

C-Glycosylmethyl pyridylalanines reported in this paper constitute a novel family of glycosyl amino acids that contain a pyridine ring linking the carbohydrate and amino acid residues. These amino acids may serve to prepare nonnatural glycopeptides displaying firmly bound carbohydrate fragments through a rigid and highly stable tether. A viable route to these new hybrid molecules has been opened via thermally induced Hantzsch-type cyclocondensation using an aldehyde-ketoester-enamino ester system. To one of these reagents was attached a C-glycosyl residue, while to another was bound an amino acid fragment. In a one-pot optimized methodology, the dihydropyridine was not isolated while its purification was carried out by removal of unreacted material and side products using polymer-supported scavengers. Then the dihydropyridine (mixture of diastereoisomers) was oxidized by a polymer-bound oxidant to give the target pyridine bearing the two bioactive residues. In this way a range of eight compounds (58-68% yield) was prepared in which the elements of diversity were (i) the gluco and galacto configurations of the pyranose ring, (ii) the alpha- and beta-configurations at the anomeric center, and (iii) the positions of the carbohydrate and amino acid sectors in the pyridine ring. The orthogonal functional group protection in these amino acids allowed their easy incorporation into oligopeptides via sequential amino and carboxylic group coupling.