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     

Molecular features of organic matter in diagnostic horizons from andosols as seen by analytical pyrolysis

Andosols are usually formed from volcanic substrates, with thick A horizons high in organic carbon mainly in the form of stabilized humic fractions. The peculiar properties of these soils are, to large extent, affected by poorly crystalline materials like allophanes, imogolite and other Fe and Al oxyhydroxides that induce intense organo-mineral interactions which are considered to play a relevant role in OM protection against biodegradation as regards other soils developed under similar climatic conditions. Little is known about the molecular composition of this stabilized OM in a soil scenario often considered as an efficient C-sink in terms of C sequestration processes.

Whole soil samples in addition to isolated humic and fulvic acids from organic A horizons of three soils with andic properties and one non-andic soil (Sodic Cambisol) from the island of Tenerife (Canary Islands, Spain) were analysed by double shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) in order to get some insight on the molecular composition of different structural domains of progressive structural stability.

Clear differences were found between soils, both in thermal desorption and pyrolysis behaviour of humic substances. In general the results suggest large yields of aliphatic (both alkyl and carbohydrate-derived pyrolysis compounds) pointing to high-performance processes of incorporation of aliphatic humic constituents in andosols probably favoured by interactions with amorphous minerals, whereas in non-andic soils the latter are comparatively less stabilized and are removed in early pyrolysis stages as if they occurred as loosely joined, thermoevaporation-released products.

In fact, compared to Cambisol, humic and fulvic acids from andic soils led to comparatively richer pyrograms and compound assemblages. The lack of similar amounts of these loosely joined, mainly aliphatic structures after thermal desorption of the whole andic soils indicate an origin for humic substances based on rapid sequestration of C-forms of recent (litter or microbial) origin.     

Molecular features of organic matter in diagnostic horizons from andosols as seen by analytical pyrolysis

Andosols are usually formed from volcanic substrates, with thick A horizons high in organic carbon mainly in the form of stabilized humic fractions. The peculiar properties of these soils are, to large extent, affected by poorly crystalline materials like allophanes, imogolite and other Fe and Al oxyhydroxides that induce intense organo-mineral interactions which are considered to play a relevant role in OM protection against biodegradation as regards other soils developed under similar climatic conditions. Little is known about the molecular composition of this stabilized OM in a soil scenario often considered as an efficient C-sink in terms of C sequestration processes.Whole soil samples in addition to isolated humic and fulvic acids from organic A horizons of three soils with andic properties and one non-andic soil (Sodic Cambisol) from the island of Tenerife (Canary Islands, Spain) were analysed by double shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) in order to get some insight on the molecular composition of different structural domains of progressive structural stability.Clear differences were found between soils, both in thermal desorption and pyrolysis behaviour of humic substances. In general the results suggest large yields of aliphatic (both alkyl and carbohydrate-derived pyrolysis compounds) pointing to high-performance processes of incorporation of aliphatic humic constituents in andosols probably favoured by interactions with amorphous minerals, whereas in non-andic soils the latter are comparatively less stabilized and are removed in early pyrolysis stages as if they occurred as loosely joined, thermoevaporation-released products.In fact, compared to Cambisol, humic and fulvic acids from andic soils led to comparatively richer pyrograms and compound assemblages. The lack of similar amounts of these loosely joined, mainly aliphatic structures after thermal desorption of the whole andic soils indicate an origin for humic substances based on rapid sequestration of C-forms of recent (litter or microbial) origin.     

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.     

Analysis of cyclic pyrolysis products formed from amino acid monomer

Amino acid was mixed with silica and tetramethylammonium hydroxide (TMAH) to favor pyrolysis of amino acid monomer. The pyrolysis products formed from amino acid monomer were using GC/MS and GC. 20 amino acids of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were analyzed. The pyrolysis products were divided into cyclic and non-cyclic products. Among the 20 amino acids, arginine, asparagine, glutamic acid, glutamine, histidine, lysine, and phenylalanine generated cyclic pyrolysis products of the monomer. New cyclic pyrolysis products were formed by isolation of amino acid monomers. They commonly had polar side functional groups to 5-, 6-, or 7-membered ring structure. Arginine, asparagine, glutamic acid, glutamine, histidine, and phenylalanine generated only 5- or 6-membered ring products. However, lysine generated both 6- and 7-membered ring compounds. Variations of the relative intensities of the cyclic pyrolysis products with the pyrolysis temperature and amino acid concentration were also investigated.     

Pyrolysis—gas chromatography—mass spectrometry of a low organic matter calcareous soil

A modification to the general method of pyrolysis-gas chromatography of soils which improves the reproducibility of the technique is described. It has been applied to the study of a calcareous soil, low in organic matter and varations have been studied in programs from soil at different depths as well as from the corresponding humic acids, fulvic acids and extraction residues.Most of the major peaks have identified by gas chromatography-mass spectrometry: they correspond to those cited in earlier works. Data are presented on the characteristics of the pyrolysis products of this soil in the relation to their probobic origin. The evolution of organic matter through various pyrolysis relations is also discussed.     

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.     

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.     

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.     

Gas chromatography—mass spectrometry studies on the occurrence of acetamide,propionamide, and furfuryl alcohol in pyrolyzates of bacteria,bacterial fractions,and model compounds

Fused-silica capillary gas chromatography—mass spectrometry was employed to investigate the production of several bacterial pyrolysis products, including acetamide, propionamide, and furfuryl alcohol. A variety of model compounds, selected for their similarity with bacterial structures, were pyrolyzed and amounts of these pyrolysis products were quantitatively determined. Model compounds included several analogues of muramyl dipeptide differing in chain length at the lactyl—peptide bridge as well as several neutral and amino sugars, amino acids, and nucleic acids. Bacterial cell walls and whole microbial cells were also analyzed. Measurement of the amounts of a few chemically identified pyrolysis products provides some chemotaxonomic differentiation of microorganisms, as demonstrated by the pyrolysis of a group of Gram-positive and Gram-negative bacteria (including samples of Legionella pneumophila), and fungi.     

Determination of specificity of endopeptidases by combined high-performance liquid chromatography and amino acid analysis

The specificity of three neutral endopeptidases toward several biologically active peptides was determined by combined high-performance liquid chromatography and amino acid analysis of the degradation products. Incubation mixtures were chromatographed on a reversed-phase column equilibrated with a mixture of acetonitrile and potassium phosphate buffer (0.05 M; pH 2.0). Reaction products were eluted with a linear gradient of acetonitrile and the absorbance of the effluent monitored at 210 nm. Fractions corresponding to discrete peaks were subjected to quantitative amino acid analysis. The peptide bond undergoing cleavage is readily assigned from the knowledge of the primary structure of the peptide and the amino acid composition of the reaction products.     

Comparative characteristic studies on soil and commercial humic acids

Summary Two commercial and eight soil humic acids were characterized to evaluate the use of commercial humic materials in soil and environmental studies. Significant differences between commercial and soil humic acids were observed in ash content, major and micro-element constituents, E₄/E₆ ratio, total acidity and ¹³C-NMR spectroscopy; but data from stability constant and IR-spectroscopy were almost identical. The commercial materials appeared to be significantly less aromatic and more aliphatic in nature as compared with representative soil humic acids. The above discrepancies were interpreted as indicating an origin of the respective commercial product other than soil. The two commercial humic acids investigated may not originate from the same source material as evidenced by clear-cut differences in the intensity of the ¹³C-NMR spectra in almost all chemical shift regions. Due to the observed structural, compositional and characteristic dissimilarities, both between soil and commercial humic acids and between different commercial ones, the commercial humic products are not recommended to be used as analogues of real soil humic acids in soil and environmental studies.     

Simple off-line flash pyrolysis procedure with in situ silylation for the analysis of hydroxybenzenes in humic acids and coals

A simple device consisting of a glass pyrolysis chamber fitted for a commercial resistively heated pyrolysis probe and connected to a solvent desorption tube for air monitoring was applied to off-line pyrolysis under silylating conditions of humic acids (soil, lake) and coals. Samples were flash pyrolysed at 700 degrees C in the presence of excess hexamethyldisilazane, and evolved products were swept off by a nitrogen stream and trapped onto a charcoal filter from where they were desorbed with dichloromethane and analysed by gas chromatography (GC)-mass spectrometry. Humic acids afforded trimethylsilyl (TMS) ethers of phenols, 2-methoxyphenols (guaiacols), 2,6-dimethoxyphenols (syringols), and dihydroxy and trihydroxybenzenes as major products. TMSoxy benzenes were the principal products observed from pyrolysis/silylation of coals. In comparison with conventional pyrolysis, the in-situ derivatisation process enhances the levels of phenols with respect to hydrocarbons and improves the GC separation of isomers (e.g. meta- from para-cresol). With respect to tetramethylammonium hydroxide thermochemolysis, pyrolysis/silylation operates under milder conditions and permits discrimination between free and methylated hydroxy groups. The performance of the method for the quantitative determination of evolved product is described. Yields of evolved silylated mono and dihydroxybenzenes occur in the mg/g range with relative standard deviations generally between 16 and 30%.     

A study of the chromatographic determination of amino acids in the presence of large amounts of carbohydrate

Synthetic mixtures of 15 of the 18 most common amino acids (cystine, methionine and tryptophan excepted) were boiled with 6N HCl in the presence and absence of carbohydrate. The decomposition products from starch and glucose do not interfere with the chromatographic determination of the amino acids on ion exchange columns. In no instance was the observed recovery of an amino acid lowered by as much as 3 per cent by the addition of carbohydrate (2 g) to 25–50 mg of amino acids per 200 ml 6N HCl. The dilute conditions of hydrolysis correspond to those adopted for the determination of the amino acid composition of foods of high carbohydrate content.     

土壤胡敏酸的^13C核磁共振研究

胡敏酸(HA)是土壤有机质的重要组分,其结构性质与土壤形成和肥力特性有密切关系,长期以来一直是土壤化学研究的难点和重点之一。NMR是研究HA结构的有效手段。自Bar-ton和Schnitzer(1963)首次用~1H NMR研究土壤有机质以来,国外已先后应用~1H、~(13)C NMR和CP-MAS-~(13)C NMR等对土壤及其它来源的HA进行了许多研究,并取得引人注目的进展。但在国内,这方面研究还刚刚开始。本文应用~(13)C NMR方法对东北几种主要耕作土壤以及泥炭和猪粪的HA进行了结构表征,为土壤腐殖酸化学的基础研究提供了资料。     

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.     

Capillary zone electrophoresis of humic acids from the American continent

A multicomponent background electrolyte (BGE) was developed and its composition optimized using artificial neural networks (ANN). The optimal BGE composition was found to be 90 mM boric acid, 115 mM Tris, and 0.75 mM EDTA (pH 8.4). A separation voltage of 20 kV, 20 degrees C and detection at 210 nm were used. The method was applied to characterize several humic acids originating from various countries of the American continent: soil (Argentina), peat (Brazil), leonardite (Guatemala and Mexico) and coal (United States). Comparison with humic acids of International Humic Substances Society (IHSS) standard samples was also done. Well reproducible electropherograms showing a relatively high number of peaks were obtained. Characterization of the samples by elemental analysis and UV spectrophotometry was also done. In spite of the very different origins, the similarities between humic acids are high and by matrix assisted desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry it was shown that most of the m/z patterns are the same in all humic acids. This means that humic acids of different origin have the same structural units or that they contain the same components.     

Investigation of distribution of soil antimony using sequential extraction and antimony complexed to soil-derived humic acids molar mass fractions extracted from various depths in a shooting range soil

Trace metal contamination from bullet fragments in shooting ranges is a major environmental concern. In particular, trace metals such as lead, antimony, and copper are toxic and have the potential to enter groundwater supplies and to be absorbed by plants. Soil humic acids can play a critical role in mobilizing some of these released metals through complexation. The purpose of this study is to investigate the antimony complexed to soil-derived humic molar mass fractions extracted from various depths in a shooting range soil and to examine the distribution of antimony in various fractions of shooting range soils using sequential chemical extraction approach. The surface soil and soil core samples from a local shooting range were collected. Soil-derived humic acids were extracted from different depths of the top soil layer and characterized by various spectroscopic methods. Results of sequential chemical extraction demonstrated that Sb was found in shooting range in the upper 30 cm depth of the soil core. Highly elevated Sb is present in the exchangeable and ammonium acetate extracted fractions. Antimony is also present in the residual fraction in both surface and core soil samples, but is most likely present in a lithic phase which may not be readily bio-available. Leached antimony complexed to soil humic acid molar mass fractions was determined by size exclusion chromatography coupled to inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The results demonstrate that Sb is ‘tightly’ bound to humic acid mass molar mass fractions and confined in the top 10 cm of soil-derived humic acids.     

Comparability of composition of carbon functional groups in humic acids between inverse-gated decoupling and cross polarization/magic angle spinning 13C nuclear magnetic resonance techniques

To find a certain relation between the composition of carbon functional groups of humic acids derived from liquid state ¹³C nuclear magnetic resonance (NMR) spectra acquired with inverse-gated decoupling (IGD), known as a quantitative pulse sequence, and that by solid-state ¹³C NMR with cross polarization/magic angle spinning (CPMAS) techniques, fifteen soil humic acid samples with a wide range of aromaticity were analyzed. Relationships between the proportions of humic acid carbon as alkyl, O-alkyl, and aromatic carbon assessed by the two methods could be regressed to y = ax (r = 0.932-0.956; P < 0.005), respectively. The contents of alkyl, O-alkyl, and aromatic carbon assessed by CPMAS method were larger than those found by IGD method. However, the differences between the two methods were small and exclusive regression to y = x was also significant (r = 0.775-0.941; P < 0.005). Aromaticity calculated from ¹³C CPMAS NMR data also did not differ significantly from those computed from ¹³C NMR spectra with IGD. These observations indicated the comparability of the relative content of carbon functional groups in humic acids except for carboxyl and carbonyl carbon.     

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.