N-15 NMR spectra of naturally abundant nitrogen in soil and aquatic natural organic matter samples of the International Humic Substances Society

The naturally abundant nitrogen in soil and aquatic NOM samples from the International Humic Substances Society has been characterized by solid state CP/MAS ¹⁵N NMR. Soil samples include humic and fulvic acids from the Elliot soil, Minnesota Waskish peat and Florida Pahokee peat, as well as the Summit Hill soil humic acid and the Leonardite humic acid. Aquatic samples include Suwannee River humic, fulvic and reverse osmosis isolates, Nordic humic and fulvic acids and Pony Lake fulvic acid. Additionally, Nordic and Suwannee River XAD-4 acids and Suwannee River hydrophobic neutral fractions were analyzed. Similar to literature reports, amide/aminoquinone nitrogens comprised the major peaks in the solid state spectra of the soil humic and fulvic acids, along with heterocyclic and amino sugar/terminal amino acid nitrogens. Spectra of aquatic samples, including the XAD-4 acids, contain resolved heterocyclic nitrogen peaks in addition to the amide nitrogens. The spectrum of the nitrogen enriched, microbially derived Pony Lake, Antarctica fulvic acid, appeared to contain resonances in the region of pyrazine, imine and/or pyridine nitrogens, which have not been observed previously in soil or aquatic humic substances by ¹⁵N NMR. Liquid state ¹⁵N NMR experiments were also recorded on the Elliot soil humic acid and Pony Lake fulvic acid, both to examine the feasibility of the techniques, and to determine whether improvements in resolution over the solid state could be realized. For both samples, polarization transfer (DEPT) and indirect detection (¹H–¹⁵N gHSQC) spectra revealed greater resolution among nitrogens directly bonded to protons. The amide/aminoquinone nitrogens could also be observed by direct detection experiments.     

Spectroscopic studies on soil organic fractions—II. IR and 1H-NMR spectra of methylated and unmethylated fulvic acids

The IR and ¹H-NMR spectra of fulvic acids (FA) are discussed. The FA, extracted by traditional methods, were fractionated on the basis of molecular weight (m.w.). Three fractions were obtained labelled FA I (m.w. > ～ 2000), FA II (m.w. > ～ 12000) and FA III (～2000 < m.w. < ～ 12000). Fraction FA II was methylated with CH₃I-Ag₂O. The ¹H-NMR spectra of unmethylated FA fractions show some signals common to all three fractions and some differences in the 3–5 ppm range due to the resonances of OCH₃ and O-CH₂ groups. The proton spectrum of the methylated fraction shows absorption areas for OCH₃ groups of phenols and carboxyls. The results confirm that NMR spectroscopy is a convenient technique which can contribute to defining the chemical structure of humic substances.     

[1H] and [13C]NMR studies on the importance of aromatic structures in fulvic and humic acids

To obtain information on the contribution of aromatic fragments to the chemical structure of humic substances, we carried out a study on the [¹H]NMR and [¹³C]NMR spectra of humic and fulvic acids and their oxidative degradation products extracted from an Andosol soil.[¹H]NMR spectra of all organic fractions present considerable adsorption between 7.4 and 8.8 ppm, due to the presence of aromatic protons.The percentages of aromatic protons in respect to the total amount of protons are as follows: FA 20%, HA 19%, degraded FA < 12%, degraded HA 14%. The values indicate that the contribution of aromatic structures to the humic substances is significant, also considering that they are highly substituted.The degraded fractions contain smaller amounts of aromatic protons, because degradation causes the opening of the aromatic rings. Thus results obtained from the degradation do not seem to be reliable for defining the importance of aromatic structures in humic substances.Also the [¹³C]NMR spectra show signals in the aromatic region which derive from unsubstituted carbon atoms, while signals originating from tertiary carbon atoms merge with the noise. We believe that, at present, [¹H]NMR spectroscopy is more suited for studying the role played by aromatic compounds in organic soil fractions.     

Comment on the paper: “[1H] and [13C]NMR studies on the importance of aromatic structures in fulvic and humic acids” (Ruggieroet al., 1979)

Some statements referring to our work on ¹³C NMR of humic and fulvic acids have been recently reported in this journal by Ruggiero and co-workers. They are discussed in order to avoid possible misinterpretations.     

Solid-state (H and C) nuclear magnetic resonance spectroscopy of insoluble organic residue in the Murchison meteorite: a self-consistent quantitative analysis

Complementary, double- and single-resonance solid-state (¹H and ¹³C) nuclear magnetic resonance (NMR) experiments were performed on a solvent extracted and demineralized sample of Murchison meteorite organic macromolecule. These NMR data provide a consistent picture of a complex organic solid composed of a wide range of organic (aromatic and aliphatic) functional groups, including numerous oxygen-containing functional groups. The fraction of aromatic carbon within the Murchison organic residue (constrained by three independent experiments) lies between 0.61 and 0.66. The close similarity in cross-polarized and single-pulse spectra suggests that both methods detect the same distribution of carbon. With the exception of interstellar diamond (readily detected in slow magic angle spinning single-pulse NMR experiments), there is no evidence in the solid-state NMR data for a significant abundance of large laterally condensed aromatic molecules in the Murchison organic insoluble residue. Given the most optimistic estimation, such carbon would not exceed 10% and more likely is a fraction of this maximum estimate. The fraction of aromatic carbon directly bonded to hydrogen is low (∼30%), indicating that the aromatic molecules in the Murchison organic residue are highly substituted. The bulk hydrogen content, H/C, derived from NMR data, ranges from a low of 0.53 ± 0.06 and a high of 0.63 ± 0.06. The hydrogen content (H/C) determined via elemental analysis is 0.53. The range of oxygen-containing organic functionality in the Murchison is substantial. Depending on whether various oxygen-containing organic functional groups exist as free acids and hydroxyls or are linked as esters and ethers results in a wide range in O/C (0.22 to 0.37). The lowest values are more consistent with elemental analyses, requiring that oxygen-containing functional groups in the Murchison macromolecule are highly linked. The combined ¹H and ¹³C NMR data reveal a high proportion of methine carbon, which requires that carbon chains within the Murchison organic macromolecule are highly branched.     

Comparative characterization of humic substances from the open ocean, estuarine water and fresh water

Humic substances were isolated from ocean, estuarine water and fresh water using a two column array of XAD-8 and XAD-4 resins in series. The extracted fulvic acids and XAD-4 fraction from different origins were characterized using UV–vis., molecular fluorescence, Fourier transform infrared (FTIR) spectroscopy and cross polarization magic angle spinning (CPMAS)-¹³C nuclear magnetic resonance (NMR) spectroscopy. The isolation procedure allowed us to obtain the necessary amount of sample for characterization, even in the case of open ocean water, which has a very low amount of dissolved organic carbon (DOC). Humic substances from the open ocean showed the lowest chromophore and fluorophore contents and showed relatively greater fluorescence at lower wavelengths than those from fresh water. FTIR and ¹³C NMR spectra highlighted the idea that humic substances from a marine environment have a more branched aliphatic structure and less aromatic structure than those highly influenced by terrestrial sources. The spectra also suggest that the open ocean humic substances have a higher content of olefinic carbons than aromatic- or alkyl-substituted carbons.     

Analysis of methylated humic acids from fossil fuels by size exclusion chromatography and NMR

Molecular Mass (MM) distributions in humic acids from peat and two lignites have been investigated by high performance size-exclusion chromatography (SEC) after methylation and found to be in the range approximately 300 to more than 5000, with number average NM(M_n) near 800. Values of M_n determined by SEC were in good agreement with those from vapour pressure osmotery. High field¹³C NMR spectra of the methylated humic acids are extremely well resolved, and indicate that the samples are significantly different, despite their similar elemental compositions and MM distributions.     

The complementary use of H NMR, C NMR, FTIR and size exclusion chromatography to investigate the principal structural changes associated with composting of organic materials with diverse origin

The aim of this work is to study the structural changes involved in humification processes. Total humic extracts (THE) obtained from five composted materials of diverse origin (solid wastes of wineries, solid mill olive wastes, domestic wastes, ovine manures plus straw, and a mixture of animal manures), and their corresponding initial raw fresh organic mixtures were studied using ¹³C nuclear magnetic resonance (NMR) using the cross-polarization magic angle spinning technique (CPMAS), ¹H NMR, Fourier transform infrared spectroscopy (FTIR) and high pressure size exclusion chromatography (HPSEC). One group of three humic acids extracted from soils, and a second group consisting of two reference humic acids and two reference fulvic acids (1S104H, 1R103H, 1R101F and 1R107F) obtained from the International Humic Substances Society were also characterized using these techniques, in order to compare the features of reference humic and fulvic acids with those of composted materials. Likewise, the results were compared with those obtained in previous studies, in which UV–Visible and fluorescence spectroscopies were employed to characterize the humification degree of the molecular systems.The results obtained by ¹³C CPMAS NMR, ¹H NMR and FTIR indicate that, in general, humification seems to be associated with an increase in the aromatic character of the systems, with the presence of phenol groups as principal substituents and a reduction in oxygen containing functional groups, principally carboxylic or carbonylic groups, as well as the development of molecular fractions with larger size. These results also support the suitability of UV–Visible and fluorescence spectroscopies in the assessment of the humification course of humic extracts in composting processes.     

Dissolution mechanisms of water in depolymerized silicate melts: Constraints from H and Si NMR spectroscopy and ab initio calculations

Dissolution of water in magmas significantly affects phase relations and physical properties. To shed new light on the this issue, we have applied ¹H and ²⁹Si nuclear magnetic resonance (NMR) spectroscopic techniques to hydrous silicate glasses (quenched melts) in the CaO-MgO-SiO₂ (CMS), Na₂O-SiO₂, Na₂O-CaO-SiO₂ and Li₂O-SiO₂ systems. We have also carried out ab initio molecular orbital calculations on representative clusters to gain insight into the experimental results.The most prominent result is the identification of a major peak at ∼1.1 to 1.7 ppm in the ¹H MAS NMR spectra for all the hydrous CMS glasses. On the basis of experimental NMR data for crystalline phases and ab initio calculation results, this peak can be unambiguously attributed to (Ca,Mg)OH groups. Such OH groups, like free oxygens, are only linked to metal cations, but not part of the silicate network, and are thus referred to as free hydroxyls in the paper. This represents the first direct evidence for a substantial proportion (∼13∼29%) of the dissolved water as free hydroxyl groups in quenched hydrous silicate melts. We have found that free hydroxyls are favored by (1) more depolymerized melts and (2) network-modifying cations of higher field strength (Z/R²: Z: charge, R: cation-oxygen bond length) in the order Mg > Ca > Na. Their formation is expected to cause an increase in the melt polymerization, contrary to the effect of SiOH formation. The ²⁹Si MAS NMR results are consistent with such an interpretation. This water dissolution mechanism could be particularly important for ultramafic and mafic magmas.The ¹H MAS NMR spectra for glasses of all the studied compositions contain peaks in the 4 to 17 ppm region, attributable to SiOH of a range of strength of hydrogen bonding and molecular H₂O. The relative population of SiOH with strong hydrogen bonding grows with decreasing field strength of the network-modifying cations. Ab initio calculations confirmed that this trend largely reflects hydrogen bonding with nonbridging oxygens.     

1H NMR spectra of humic and fulvic acids and their peracetic oxidation products

¹H NMR spectra of humic (HA) and fulvic (FA) acids and their oxidative degradation products are reported. The HA shows the presence of -(CH₂)_n - CH₃ (n > 6) chemical fragments belonging to n-alkanes and/or n-fatty acids physically adsorbed onto the macromolecule structure. These fragments are absent in the FA fraction. Both humic fractions reveal the presence of similar amounts of aromatic protons which partly undergo exchange phenomena. The importance of this experimental observation is discussed. Oxidative degradation seems to cause partial cleavage of aromatic rings, more pronounced in the FA than in the HA. The degraded FA shows a higher total acidity and a higher phenolic OH content than the degraded HA. Both degraded fractions display some sharp singlet signals at 1.9 and 3.9 ppm arising from protons belonging to repetitive chemical fragments probably formed during the oxidation reaction. Tentative assignments of these signals are given. A general analysis of the HA and FA degraded spectra seems to indicate that the chemical fragments which undergo peracetic oxidation are substantially similar. The extent of oxidation of the two humic fractions is different. The HA degradation products reveal the presence of oligomeric structures, whereas the degraded FA appears less resistant to the oxidizing agent.     

FTIR spectroscopic study of natural andalusite showing electronic Fe–Ti charge-transfer processes: zoning and thermal evolution of OH-vibration bands

We investigated a natural Brazilian Fe–Ti-containing andalusite and its thermal behavior by polarized infrared and optical spectroscopy. Polarized infrared spectra of the Brazilian andalusite and their evolution at thermal annealing in air clearly evidence that there are several types of OH-groups in the structure. Optical spectra and their evolution with temperature indicate that the incorporated iron (about 0.43 wt% calculated as FeO) is in the ferrous and ferric state. Incorporation of ferrous iron in the Al-sites of andalusite is discussed as a possible incorporation mechanism for hydrogen. The weakening and disappearance of the Fe²⁺/Ti⁴⁺ IVCT band in the andalusite spectra under annealing in air is caused by oxidization of Fe²⁺ to Fe³⁺ in Fe²⁺/Ti⁴⁺ IVCT pairs. The process of oxidation is accompanied by a rearrangement of the hydroxyl groups and dehydration of the sample, especially vivid at the final stage of the thermal annealing at 1,200 °C. During thermal annealing, structural hydroxyls of different types apparently transform into each other: the most distinct are the hydroxyls causing the doublet at 3,516 and 3,527 cm^?1 (i.e., H bonded to O1) which seem to transform into the hydroxyls causing the line at 3,461 cm^?1 (i.e., H bonded to O2). The infrared spectra scanned across differently colored zones of the crystal clearly show that some amount of hydroxyls is related to Fe²⁺/Ti⁴⁺ IVCT pairs which are the cause of the red-to-black coloration of the sample in E||c-polarized illumination: it is evident that in a part of the hydroxyl groups, OH-vector changes orientation aligning directly along crystallographic a-axis due to some kind of interaction with Fe²⁺/Ti⁴⁺ IVCT pairs.     

Investigation of marine and terrestrial humic substances by H and C, nuclear magnetic resonance and infrared spectroscopy

Humic acids were isolated from 5 sediments in which the origin nature of the organic matter are both typical and different. The humic acids were characterized on the basis of elemental compositions, infrared spectra and ¹H and ¹³C NMR. This last technique, especially ¹³C NMR, provides qualitative and semi-quantitative information regarding aromatic structure. Combined data from the three techniques permits differentiation of marine and terrestrial organic matter as well as identification of mixtures of humic acids from the two sources.     

Comparison of soil humic and fulvic acids of similar molecular weight

Soil fulvic acids were compared with humic acid of similar molecular weight. The elementary composition, total carbohydrate content and ¹³C-NMR spectral features differentiate these fractions and leads to consideration of fulvic acid as a separate chemical entity.     

Effects of age on the chemical structure of paleosol humic acids and fulvic acids

Humic acids and fulvic acids were extracted from six paleosols in Southern Italy. Humic acids (HAs) constituted between 96.5 and 99.2% of the total extracts; the remaining materials consisted of fulvic acids (FAs). Radiocarbon ages of the HAs ranged from about 6,000 to close to 29,000 years B.P., δ¹³C values averaged $\text{?25.6 ± 0.3‰}$ The HAs were characterized by chemical (elemental and functional group analyses) and spectroscopic (IR, ESR, ¹³CNMR, E₄/E₆ ratios) methods. FAs were characterized by chemical methods, E₄/E₆ ratios and IR spectra.The chemical and spectroscopic analyses showed practically no differences in the chemical structure and composition of the six HAs and FAs, so that age appeared to have little effect on these parameters. The paleosols were found to be closed systems with low polysaccharide and protein contents, thus providing unfavorable substrates for microbial activity. The preservation of the humic materials in the paleosols may have been due to low biological activity and/or to retention by amorphous minerals. The HAs did not appear to be affected by temperatures higher than 170–200°C over the 23,000 year period which we observed.     

1H and 13C NMR of marine humic acids

¹³C and ¹H NMR spectra were obtained for humic acids isolated from marine sediments. NMR shows great promise in identifying structural components of humic acids as some new and interesting structural features are identified. Aliphatic structures were found to constitute a much larger fraction of humic acids than previously thought, and they appeared to be highly branched. Although the aromatic content of terrestrial humic acid was found to be lower than expected, the aromaticity appears to be a specific discriminator of terrestrial/aquatic source types. A humic acid isolated from an anoxic algal sapropel was found to be composed predominantly of polyuronic acids and different than other aquatic sedimentary humic substances.     

A study of the effect of igneous intrusions on the structure of an Australian high voltage bituminous coal

Samples of heat affected coal taken near two igneous intrusions (dykes) in the Victoria Tunnel Seam of the Newcastle Coalfield have been investigated by chemical analysis, petrography, Fourier transform infrared spectroscopy and solid state¹³C NMR spectroscopy. Visual inspection of the seam near the intrusion showed four distinct zones. These have been called, in order of increasing distance from the dyke, the massive cinder, the banded cinder, heat affected coal and apparently unaffected coal. The samples show an increase in structural change as the distance from the dyke decreases, with the samples taken from the massive cinder nearest the dyke being classified as semicokes.FTIR and¹³C NMR results, together with the results of dipolar dephasing NMR experiments, suggest that the predominant structural change in the heat affected zone is replacement of aromatic ethers and/or phenols with hydrogen, leading to an increase in proton aromaticity. Closer to the intrusion, bond rupture leads to a marked loss of aliphatic groups, while in the most affected region adjacent to the dyke aromatic crosslinking has also occurred to form coke-like material.     

Effects of oxygen and redox oscillation on degradation of cell-associated lipids in surficial marine sediments

Degradation patterns of sedimentary algal lipids were tracked with time under variable redox treatments designed to mimic conditions in organic-rich, bioturbated deposits. Uniformly ¹³C-labeled algae were mixed with Long Island Sound surface muddy sediments and exposed to different redox regimes, including continuously oxic and anoxic, and oscillated oxic: anoxic conditions. Concentrations of several ¹³C-labeled algal fatty acids (16:1, 16:0 and 18:1), phytol and an alkene were measured serially. Results showed a large difference (∼10×) in first-order degradation rate constants of cell-associated lipids between continuously oxic and anoxic conditions. Exposure to oxic conditions increased the degradation of cell-associated lipids, and degradation rate constants were positive functions (linear or nonlinear) of the fraction of time sediments were oxic. Production of two new ¹³C-labeled compounds (iso-15:0 fatty acid and hexadecanol) further indicated that redox conditions and oxic: anoxic oscillations strongly affect microbial degradation of algal lipids and net synthesis of bacterial biomass. Production of ¹³C-labeled iso-15:0 fatty acid (a bacterial biomarker) was inversely proportional to the fraction of time sediments were oxic, rapidly decreasing after 10 days of incubation under oxic and frequently oscillated conditions. Turnover of bacterial biomass was faster under continuously or occasionally oxic conditions than under continuously anoxic conditions. ¹³C-labeled hexadecanol, an intermediate degradation product, accumulated under anoxic conditions but not under oxic or periodically oxic conditions. The frequency of oxic: anoxic oscillation clearly alters both the rate and pathways of lipid degradation in surficial sediments. Terminal degradation efficiency and lipid products from degradation of algal material depend on specific patterns of redox fluctuations.     

Metal distribution and nature of some Cu,Mn and V complexes in humic and fulvic acid fractions of soil organic matter

Organic matter from an arable soil derived from base rich parent material was extracted by alkali and fractionated on the basis of solubility in 0.1 N HCl, hot water and hot 6 N HCl and by selective adsorption on charcoal. The distribution of associated metals was determined and Cu had the largest proportion, 15%, associated with the organic matter. Moderate proportions of the total Al, Co, Ni, and V (3–8%) but only small amounts (?1%) of the Mn, Fe, Ti, Cr, Ba and Sr were extracted from the soil by alkali. The Fe and Ti were concentrated mainly in the humic fraction whereas Mn and V were both found largely in the fulvic acid.Electron paramagnetic resonance spectra of the various fractions were examined and attempts made to relate the spectra to the forms of some of the metals present. In the humic acid fraction Cu was present partly as a copper porphyrin-type complex but in the fulvic acid it was in some other complexed form. VO²⁺ occurred in complexed forms in the fulvic acid which were more covalent than VO²⁺ humic acid complexes, whereas the Mn²⁺ components of the humic and fulvic acids all had a high degree of ionicity.     

Proton-binding study of standard and reference fulvic acids, humic acids, and natural organic matter

The acid-base properties of 14 standard and reference materials from the International Humic Substances Society (IHSS) were investigated by potentiometric titration. Titrations were conducted in 0.1 M NaCl under a nitrogen atmosphere, averaging 30 min from start to finish. Concentrations of carboxyl groups and phenolic groups were estimated directly from titration curves. Titration data were also fit to a modified Henderson-Hasselbalch model for two classes of proton-binding sites to obtain “best fit” parameters that describe proton-binding curves for the samples. The model was chosen for its simplicity, its ease of implementation in computer spreadsheets, and its excellent ability to describe the shapes of the titration curves. The carboxyl contents of the IHSS samples are in the general order: terrestrial fulvic acids > aquatic fulvic acids > Suwannee River natural organic matter (NOM) > aquatic humic acids > terrestrial humic acids. Overall, fulvic acids and humic acids have similar phenolic contents; however, all of the aquatically derived samples have higher phenolic contents than the terrestrially derived samples. The acid-base properties of reference Suwannee River NOM are surprisingly similar to those of standard Suwannee River humic acid. Results from titrations in this study were compared with other published results from both direct and indirect titrations. Typically, carboxyl contents for the IHSS samples were in agreement with the results from both methods of titration. Phenolic contents for the IHSS samples were comparable to those determined by direct titrations, but were significantly less than estimates of phenolic content that were based on indirect titrations with Ba(OH)₂ and Ca(OAc)₂. The average phenolic-to-carboxylic ratio of the IHSS samples is approximately 1:4. Models that assume a 1:2 ratio of phenolic-to-carboxylic groups may overestimate the relative contribution of phenolic groups to the acid-base chemistry of humic substances.     

The reaction of ozone with isolated aquatic fulvic acid

Aquatic fulvic acid, isolated from a North Carolina bay lake, was reacted with ozone, an alternative oxidant in drinking water treatment. Ozonated samples were acidified, extracted with ether, dried, concentrated, and methylated prior to GC-MS analysis. Identified reaation products include mono-, di- and tribasic aliphatic acids and benzene tricarboxylic acids. The products with the highest relative concentrations were succinic and malonic acid, although all products were at relatively low concentration levels. Many of the products identified in this study have also been seen among the reaction products of fulvic acid and other oxidants, such as Cl₂, CIO₂ and KMnO₄.