Alkaline cupric oxide oxidation of a methylated fulvic acid

Fulvic acid, (FA), extracted from the Bh horizon of a Podzol soil, was methylated and then oxidized with alkaline cupric oxide. The oxidation products were extracted into organic solvents, remethylated and separated by column-, thin layer-, and preparative gas chromatography into relatively pure components, which were identified by matching their mass and i.r. spectra and gas Chromatographic retention times with those of authentic specimens.The degradation products isolated and identified accounted for approximately 18 per cent of the weight of the initial methylated FA. Major oxidation products were: (a) methylated phenolic aldehydes and esters (66.9 per cent): (b) benzenecarboxylic acid methyl esters (14.6 per cent): (c) aliphatic dicarboxylic acid methyl esters (2.7 per cent): and (d) adsorbed materials such as n-alkanes (0.3 per cent), n-fatty acid methyl esters (0.3 per cent) and dioetyl adipate (15.2 per cent). The oxidalive degradation of methylated FA indicates the presence of two types of basic structural units: (1) those yielding phenolic aldehydes and esters, and (2) those producing benzenecarboxylic acids. Alkaline cupric oxide oxidation of methylated FA is relatively selective for the isolation of the phenolic components and appears to be a promising technique for structural investigations on humic substances.     

Permanganate oxidation of methylated and non-methylated aquatic humus in Norway

The chemical composition of aquatic humus was investigated by permanganate oxidation. Both methylated and non-methylated samples were investigated and the results compared with those of different soil humic fractions investigated earlier.The total amount of oxidation products identified from the methylated sample was 2%, and from the non-methylated sample 0.9%. The composition of the oxidation products from methylated aquatic humus was 42% benzenecarboxylic acid methyl esters (8 different compounds), 43% methoxy-benzenecarboxylic acid methyl esters (12 compounds), 10% dimethoxy-benzenecarboxylic acid methyl esters (4 compounds), and 5% of 1, 2, 3-propanetricarboxylic acid trimethyl ester. The unmethylated aquatic humus yielded 84% benzenecarboxylic acid methyl ester (7 compounds), 7% methoxy-benzenecarboxylic acid methyl esters (2 compounds), and 9% of 1, 2, 3-propanetricarboxylic acid trimethyl ester. Three diazines isolated from methylated material were believed to be artefacts from diazomethane treatment. Two of the diazines have earlier been found by oxidation of methylated soil samples, the third, C₁₀H₁₂N₂O₆, is an oxidation product of methylated aquatic humus only.Oxidation of aquatic humus yielded more benzenecarboxylic acids and methoxy-benzenecarboxylic acids than soil humic fractions, and less dimethoxy-benzenecarboxylic acids. No aliphatic dicarboxylic acids were detected among the oxidation products of the aquatic humus.The compounds identified are mainly the same as those found by oxidation of different soil humic fractions, although their yields clearly demonstrated that the aquatic humus differed in composition from the soil fractions.     

Land use effects on the composition of organic matter in soil particle size separates. III. Analytical pyrolysis

Soil samples from the A horizon of an Eutrochrept under spruce forest and permanent grass were fractionated into clay-, silt- and sand-size separates. Humic acids extracted from each fraction were analysed by pyrolysis-gas chromatography-mass spectrometry. Protection of functional groups by simultaneous pyrolysis and methylation yielded pyrolysates in which methyl esters of fatty acids, aliphatic dicarboxylic acids, abietic acids, phenolic acids and benzenecarboxylic acids were represented. However, methylation was not complete, and unmethylated compounds were also present. Spectra showed differences in humic acid composition between size separates as well as across land use regimes. The abundance of lignin-derived pyrolysis products increased with decreasing particle size, and was greater in soil under spruce than in soil under grass. Also, the lipid components differed, with hexadecanoic and docosanoic acid methyl esters being the dominant compounds in humic acids from soil under spruce and hexadecanoic and octadecanoic acid methyl esters in the humic acids from grassland. A good correlation was found between previous ¹³CNMR and wet chemical data and pyrolysis data, indicating that pyrolysis-methylation can be used for fast detailed chemical characterization of humic acids extracted from size separates.     

Analysis of polynuclear aromatic and aliphatic components in soil humic acids using ruthenium tetroxide oxidation

Although condensed aromatic components are considered to be one of the major structural units of soil humic acids (HAs) and to be responsible for the dark colour of HAs, their amount and composition remain largely unknown. In ruthenium tetroxide oxidation (RTO), condensed aromatic components are detectable as their degradation products, mainly benzenepolycarboxylic acids (BPCAs). We applied this technique to soil HAs with various degrees of humification (darkening). The yields of water‐ and dichloromethane‐soluble products from HAs upon RTO after methylation ranged from 210 to 430 mg g⁻¹ and 10–40 mg g⁻¹, respectively. Eight kinds of BPCAs with two to six carboxyl groups, and seven kinds of BPCAs with additional side chains (tentative assignment) were obtained as methylated counterparts. The yield of each BPCA and the sum of the yields of BPCAs (12–85 mg g⁻¹ HAs) increased with increasing degree of humification and aromatic C content. The compositions of BPCAs indicated that the degree of condensation was greater in the HAs with greater degrees of humification. The sum of the yields of aliphatic compounds ranged from 0.1 to 6.5 mg g⁻¹, and decreased with increasing degree of humification. The C₁₂ to C₃₀ monocarboxylic acid methyl esters accounted for > 56% of the aliphatic compounds assigned, which may be present mainly as end alkyl groups in the HA molecules. We also obtained the methylated counterparts of C₁₄ to C₂₄ dicarboxylic acids; these were possibly derived from polymethylene bridges between adjacent aromatic rings.     

The alkaline hydrolysis of humic substances

A humic and a fulvic acid were subjected to four successive hydrolyses with 2NNaOH solution at 170°C for 3 h. Following each hydrolysis, the degradation products were extracted into ethyl acetate, methylated, separated by chromatographic techniques and identified by mass spectrometry and micro-IR spectrophotometry.One g of humic acid yielded 113.5 mg of aliphatic compounds (mainly n-C₁₆ and n-C₁₅) fatty acids), 72.1 mg of phenolics (principally guaiacyl and syringyl derivative) 17.1 mg of benzenepolycarboxylic acid esters and 30.4 mg of N-containing compounds (mainly secondary aromatic amides). Repeated attacks by the alkali on 1.0 g of fulvic acid released 103.8 mg of aliphatics, 133.8 mg of phenolics, 27.0 mg of benzenecarboxylic but 181.8 mg of N-containing compounds. Most of the latter appeared to have been formed during the second, third and fourth hydrolysis which produced reactive compounds that could abstract N from diazomethane which was used as methylating reagent. While 25.0% of the initial humic acid resisted repeated attacks by the alkali, practically all of the fulvic acid was converted into ethyl acetate-soluble products. The alkali-resistant humic acid residue produced high yields of benzenepolycarboxylic acids on alkaline permanganate oxidation.Alkaline hydrolysis, which is known to cleave CO bonds, was found to be relatively specific, although not very efficient, for the degradation of structural phenolic humic and fulvic acid components and for the concomitant liberation of adsorbed aliphatics and N-containing compounds, but was relatively ineffective for degrading aromatic structures linked by CC bonds.     

CHEMICAL DEGRADATION OF HUMIC AND FULVIC ACIDS EXTRACTED FROM MEDITERRANEAN SOILS

Humic and fulvic acids were extracted from two Israeli and tour Italian soils and oxidized with alkaline permanganate solution after methylation. Following oxidation, the degradation products were separated by solvent extraction and chromatographic methods and identified by gas chromatography-mass spectrometry. Major oxidation products were aliphatic, phenolic and benzenecarboxylic acids. In toto, 33 oxidation products were identified. These were essentially the same compounds as those produced by the permanganate oxidation of methylated humic and fulvic acids extracted from soils formed under widely differing climatic and geologic conditions, except that yields of phenolic acids from Mediterranean humic and fulvic acids were lower than those produced under similar conditions from humic materials extracted from other soils. The information provided by chemical degradation suggests that humic and fulvic acids from widely differing soils have similar chemical structures.     

Quantification of long‐chain fatty acids in dissolved organic matter and soils

The objective was to develop and adapt a versatile analytical method for the quantification of solvent extractable, saturated long‐chain fatty acids in aquatic and terrestrial environments. Fulvic (FA) and humic (HA) acids, dissolved organic matter (DOM) in water, as well as organic matter in whole soils (SOM) of different horizons were investigated. The proposed methodology comprised extraction by dichloromethane/acetone and derivatization with tetramethylammonium hydroxide (TMAH) followed by gas chromatography/mass spectrometry (GC/MS) and library searches. The C_10:0 to C_34:0 methyl esters of n‐alkyl fatty acids were used as external standards for calibration. The total concentrations of C_14:0 to C_28:0 n‐alkyl fatty acids were determined in DOM obtained by reverse‐osmosis of Suwannee river water (309.3 μg g^—1), in freeze‐dried brown lake water (180.6 μg g^—1), its DOM concentrate (93.0 μg g^—1), humic acid (43.1 μg g^—1), and fulvic acid (42.5 μg g^—1). The concentrations of the methylated fatty acids (n‐C_16:0 to n‐C_28:0) were significantly (r² = 0.9999) correlated with the proportions of marker signals (% total ion intensity (TII), m/z 256 to m/z 508) in the corresponding pyrolysis‐field ionization (FI) mass spectra. The concentrations of terrestrial C_10:0 to C_34:0 n‐alkyl fatty acids from four soil samples ranged from 0.02 μg g^—1 to 11 μg g^—1. The total concentrations of the extractable fatty acids were quantified from a Podzol Bh horizon (26.2 μg g^—1), Phaeozem Ap unfertilized (48.1 μg g^—1), Phaeozem Ap fertilized (57.7 μg g^—1), and Gleysol Ap (66.7 μg g^—1). Our results demonstrate that the method is well suited to investigate the role of long‐chain fatty acids in humic fractions, whole soils and their particle‐size fractions and can be serve for the differentiation of plant growth and soil management.     

The chemistry of humic and fulvic acids extracted from argentine soils—II. Permanganate oxidation of methylated humic and fulvic acids

Five humic and three fulvic acids, extracted from Argentine soils, were methylated and oxidized with KMnO* solution. The oxidation products were extracted into ethyl acetate, remethylated, separated by preparative gas chromatography and identified by comparing their mass and micro-IR spectra with those of authentic specimens.The major oxidation products from the humic acids were benzenetetra, -penta-, and -tricarboxylic and hydroxybenepentacarboxylic acid. The major compounds isolated from the fulvic acid oxidation products were aside from benzenecarboxylic and phenolic acids, substantial amounts of ethyl-benzylsulfonate and N-methyl-benzylsulfonamide, one complex aromatic ester and two anhydrides. The origin of the S-containing compounds is uncertain; they could be impurities. Weight ratios of benzenecarboxylic to phenolic acids averaged 5·8 for humic acids but only 0·9 for fulvic acids, suggesting an enrichment in phenolic structures in the fulvic acids. Possible structural arrangements for humic and fulvic acids are discussed.     

Permanganate oxidation of methylated fulvic and humic acids in chloroform

Methylated fulvic and humic acids were oxidized by permanganate in chloroform in the presence of a cyclic polyether, 18-crown-6. The total amount of oxidation products identified was 6.5% for fulvic acid and 6.7% for humic acid. About 46% of the oxidation products found from both fractions were dicarboxylic acids (15 compounds), the rest were benzenecarboxylic acids and their methoxyl derivatives (15 compounds). Alkanes and fatty acids were also found.Nonandioic acid and 3,4-dimethoxy-benzenecarboxylic acid are found in almost equal amounts. Each accounts for 1.3% of the starting material, and together they represent 40% of the oxidation products. 3,4-Dimethoxy-benzoic acid and 4-methoxy-benzoic acid are believed to result from the oxidation of terminal groups in the humic polymer.Proton resonance spectra show that about 25% of the aliphatic protons are part of methylene chains. The most prominent chain lengths consist of 6–8 methylene groups as shown by the dicarboxylic acids formed by oxidation.     

PHENOLIC HYDROLYSIS PRODUCTS FROM GEL CHROMATOGRAPHIC FRACTIONS OF SOIL HUMIC ACIDS

Humic acids extracted from the Bh horizon of a kauri (Agathis australis) podzol and the A horizon of a yellow-brown earth under hard beech (Nothofagus truncata), both New Zealand soils, were fractionated by a ‘salt boundary’ technique using a dextran gel, and the fractions hydrolysed with 6 M hydrochloric acid. Lignin-derived phenolic acids were analysed by gas chromatography of the ether-soluble hydrolysis products of the humic acids. The lignin derivatives from the kauri podzol humic acid are probably modern products derived from the present-day scrub vegetation, whereas those from the yellow-brown earth humic acid are derived from the deciduous vegetation.     

Depolymerization and degradation of humic acids with sodium perborate

Two humic acids of different origin (peat and soil) were degraded with a 5% sodium perborate solution (140°C). This degradation process consists mainly of a stoichiometric production of hydrogen peroxide while the perborate is reacting with carboxyl groups of the oxidized polymers. A single perborate treatment degraded more than 40% of the humic acids to soluble products, but a 5-step oxidation was necessary for total degradation, the sample being transformed into soluble oligomers with properties similar to those of fulvic acids. The oligomeric fractions with lowest molecular weights, including individual molecules (soluble in ethyl acetate), were purified by adsorption chromatography and studied by GC-MS after methylation. The higher molecular weight fractions of oligomers were recovered over polyvinylpyrrolidone, eluted by alkali, and purified by ion-exchange chromatography (47% peat HA; 25% soil HA).Degradation products included alkanes, fatty acids and dicarboxylic acids. Aromatic compounds (mainly phenolic, benzenecarboxylic and cinnamic acids), amounted to 24–50% of the total volatile degradation products. There were striking differences between peat and soil humic acids, the former yielding typical lignin degradation products. Independently checked, the perborate degradation products were not the same as those obtained by mild treatment with hydrogen peroxide under alkaline conditions.     

The sodium-amalgam reduction of soil and fungal humic substances

Two soil humic acids and a “humic acid” synthesized in the laboratory by Stachybotrys chartarum were reduced with Na-amalgam. The reduction products were methylated, separated by preparative gas chromatography and identified by matching their mass and micro-infrared spectra with those of authentic specimsens.Yields of reduction products identified ranged from 2.7 to 4.2% of the initial weights of the humic materials. Major products identified were N-methyl-benzylsulfonamide, methylated phenolic acids, aromatic aldehydes and C₆C₂ - and C₆C₃ - compounds with 0 in the side chains. Since the Na-amalgam reduction of both soil and fungal humic materials produces the same or similar compounds, the method provides little information on the origin of these compounds, that is, whether they are lignin- or flavonoid-derived or synthesized by microorganisms. Compared with oxidative degradation methods, Na-amalgam reduction appears to be inefficient and tells little about the chemical structure of humic acid polymers.     

Structure of “unknown” soil nitrogen investigated by analytical pyrolysis

Curie-point pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and in-source pyrolysis-field ionization mass spectrometry (Py-FIMS) were applied for the first time to the structural characterization of organic nitrogen in hydrolyzates and hydrolysis residues resulting from the classical 6M HCl hydrolysis of mineral soils. Two well-described soils of widely different origin (i.e., a Gleysol Ah and a Podzol Bh) were investigated. Py-GC/MS was performed using a nitrogen-selective detector to detect and identify N-containing pyrolysis in the hydrolyzate (e.g., pyrazole and/or imidazole, N,N-dimethylmethanamine, benzenacetonitrile, propane- and propenenitriles) and the hydrolysis residue (e.g., pyrroles, pyridines, indoles, N-derivatives of benzene, benzothiazol, and long-chain aliphatic nitriles). Moreover, temperature-resolved Py-FIMS allowed us to record the thermal evolution of the N-containing compounds during pyrolysis. These were characterized by a particularly high thermostability compared to their thermal release from whole soils. The combination of pyrolysis with mass spectrometric methods permitted analyses of the identities and thermal stabilities of complex nitrogen compounds in hydrolysis residues of whole soils, which cannot be done by wet-chemical methods. Pyrolysis-methylation GC/MS with tetramethyl-ammonium hydroxide (TMAH) as methylating agent enabled the identification of N,N-dimethylbenzenamine and so confirmed the identification of benzeneamine by Py-GC/MS in nonmethylated hydrolysis residues. The described N-derivatives of benzene and long-chain nitriles are usually not detectable by pyrolysis-mass spectrometry of plants and microorganisms. These compounds are characteristic of soils, terrestrial humic substances and hydrolysis residues and seem to be specific, stable transformation products of soil nitrogen. Received: 15 April 1996     

A comparison of four different raw humus types in Norway using chemical degradations and CPMAS 13C NMR spectroscopy

Crust, felty, greasy and granular raw humus were analysed by wet chemical methods and by ¹³C NMR. The amounts of amino acids, monosaccharides and aliphatic dicarboxylic acids were determined and the yields compared with the ¹³C NMR spectra. Protein carbon constitutes 9–13%, polysaccharide carbon 8–19% dicarboxylic acids 1–2% and free carboxylic acid groups 2–4% of the total sample carbon. Degradation of greasy raw humus yields half the amount of monosaccharides and twice the amount of aliphatic dicarboxylic acids found in the other raw humus types. This result is confirmed by ¹³C NMR. Forty to fifty percent of the soil carbon is unaccounted for among the degradation products identified. Based on estimates of ¹³C NMR data, the unknown part consists of aliphatic carbon, where the C:O ratio ranges between 1 1.1:1 and 1.8:1. All data indicate great similarity between crust and felty raw humus, whereas greasy raw humus differs clearly from those two. Granular raw humus gives approximately the same amount of degradation products as crust and felty raw humus but differs in its ¹³C NMR spectrum. The relative proportions of all compounds identified, including aliphatic dicarboxylic acids, are approximately constant, indicating a difference in degree rather than kind of the four raw humus types.     

Persulfate oxidation of humic acids extracted from three different soils

Three soil humic acids were degraded by a new mild chemical oxidation method using potassium persulfate at pH 2.0. The method degraded 30–40% of the starting material. The type and relative quantities of the products or fragments released as determined by GC/MS varied with the source of the polymers. Humic acid extracted from an Inceptisol yielded fatty, benzenecarboxylic and phenolic acids; humic acid from a Typic Chromoxerert produced largely dialkyl phthalates, and humic acid from an Humic Haplorthod gave only branched and straight-chain fatty acids. It is suggested that the method may be used to advantage as a first oxidant in a sequential degradation.     

Organic matter composition in sewage farm soils: Investigations by 13C-NMR and pyrolysis-field ionization mass spectrometry

The composition of soil organic matter (SOM) on sewage farms south of Berlin was investigated by solid-state CP/MAS ¹³C-NMR and pyrolysis-field ionization mass spectrometry (Py-FIMS) of freeze-dried sewage solids and soil samples of differing contamination. These were an untreated soil (USOIL), a former sewage farm used as arable land since 1990 (SF90A), and a recent sewage farm (SF1994). The CP/MAS ¹³C-NMR spectra showed enrichments of the sewage-treated soils with aliphatic C and C in OCH₃-groups and amino acids. In the Py-FI mass spectra the major markers of sewage and SOM in sewage farm soils were (i) N-containing compounds, in particular peptides, (ii) dimethylphthalate (m/z 194), (iii) sterols, and (iv) signals in the mass range m/z 502 to 554 of mono- and diaryl esters which were substituted by long aliphatic chains. The latter signals were intense in the sewage solids, increased in intensity from sample SF90A to SF1994; but they were not present in the USOIL, thus clearly indicating anthropogenic origin. Temperature-resolved Py-FIMS showed that the SOM compounds in the sewage farm soils were generally incorporated into bonds with widely different stabilities which could be relevant for SOM turnover and environmental effects. This is demonstrated for the trapping of dimethylphthalate in a modelled humic substance.     

Comparison of methylation procedures for conjugated linoleic acid and artifact formation by commercial (trimethylsilyl) diazomethane

Four different methods for methylating conjugated linoleic acid (CLA) were compared. The HCl/MeOH and BF(3)/MeOH methods were tested under different time and temperature combinations. Increasing temperature and/or incubation time for either method decreased the cis-9,trans-11 and trans-10,cis-12 isomers, but trans-9,trans-11/trans-10,trans-12 isomers and artifacts (allylic methoxide) were increased. In addition, the triacylglyceride form of CLA was tested using the above methods and NaOMe at various temperatures for 20 min. The NaOMe did not generate methoxy artifacts. However, there were impurities in GC after methylation with NaOMe as well as with BF(3)/MeOH. The (trimethylsilyl)diazomethane method, which is a mild and easy alternative, was tested. Free forms of fatty acids were easily, but not completely, methylated by this method. Also, the method generated artifacts (trimethylsilyl CLA esters) and impurities (trimethylsilyl) that would interfere with short-chain fatty-acid analysis by GC.     

Acid properties of fulvic and humic acids isolated from two acid forest soils under different vegetation cover and soil depth

We studied the acid‐base properties of 16 fulvic acids and 16 humic acids isolated from the surface (3–15 cm) and subsurface (> 45 cm) horizons of two types of acid forest soils, derived respectively from amphibolite and granite rocks, under five different types of vegetation. The observed differences between the contents of humic substances in the two types of soils were related to the degree of Al‐saturation of the soil organic matter, as indicated by the molar ratio between pyrophosphate extractable Al and C. Humic fractions were characterized in terms of elemental composition, and CPMAS ¹³C NMR spectrometry. The contents of carboxylic and phenolic groups were estimated by potentiometric titrations conducted in 0.1 m KNO₃ in a nitrogen atmosphere. The fulvic acids contained more carboxylic groups but less phenolic groups than the humic acids: the ratio of phenolic to carboxylic groups in the humic acids was 0.48 ± 0.10 and in the fulvic acids 0.23 ± 0.05. The mean values of the protonation constants of each of the humic substance fractions can be used as generic parameters for describing the proton binding properties. The fulvic acids isolated from the subsurface horizon of the soil contained between 2.6 and 23% more carboxylic groups, and the humic acids between 8 and 43% more carboxylic groups than those isolated from the surface horizon of the same soil.     

3C-NMR structural features of soil humic acids and their methylated,hydrolyzed and extracted derivatives

¹³C-NMR spectra of aqueous solutions of three humic acids (1 Typic Chromoxerert (HA-V), 1 Humic Haplorthod (HA-P), and 1 Inceptisol (HA-D)) are presented. The influence of chemical modifications (hydrolysis with 6N HCl, diazomethane methylation, and organic solvent extraction) of the humic substances and their spectra is studied. The spectra of the various humic substances show significant differences.Methylation of the compounds introduces changes in the aromatic and aliphatic (sp³) region of the spectra. Acid hydrolysis removes residual carbohydrates and amino acid signals from the spectra, whereas solvent extraction has the most pronounced influence upon the aliphatic high field region of the spectra, and shows also, that a significant amount of the paraffinic carbons is bound covalently to the aromatic nuclei of the compounds.     

Dissolved organic matter and its parent organic matter in grass upland soil horizons studied by analytical pyrolysis techniques

We have sought to understand the molecular mechanisms by which dissolved organic matter (DOM) forms and soil organic matter (SOM) degrades in upland peaty gley soil under grass. Pyrolysis mass spectrometry (Py-MS) and pyrolysis gas chromatography mass spectrometry (Py-GC/MS) were applied to characterize the DOM collected from lysimeters and its parent SOM. The macromolecular organic matter in the litter and fermentation (Lf) horizon of the soil consists primarily of little decomposed lignocellulose from grass, whereas the humus (Oh) horizon is characterized by an accumulation of selectively decomposed lignocellulose material, microbial metabolites and bound fatty acids. The mineral horizon produced a relative enrichment of furan structures derived from microbial reworking of plant polysaccharides but virtually no lignin signals. A series of exceptional long chain C₄₃ to C₅₃ fatty acids with odd over even predominance, probably derived from mycobacteria, were also identified in the Oh horizon. Side-chain oxidation and shortening, increase of carboxyl functionality and selective removal of syringyl (S) > guaiacyl (G) > p-hydroxyphenyl (P) lignin units were the main reactions when lignin degraded. Compared with SOM, the DOM shows a large accumulation of more oxidized lignin and aromatic structures, especially those containing carboxylic and dicarboxylic acid functionalities and with shorter side-chain length. The polysaccharide-type compounds in the DOM were more modified (greater abundance of furan structures in pyrolysis products), and had significantly lower molecular weight and more diverse polymeric structures than did those in soils. Increased temperature and rainfall appeared to result in greater relative abundance of lignin degradation products and aromatic compounds in DOM.