Here we show, for the first time, evidence of the primary molecular structures in humic substances (HS), the most abundant naturally occurring organic molecules on Earth, and their associations as mixtures in terrestrial systems. Multi-dimensional nuclear magnetic resonance (NMR) experiments show us that the major molecular structural components in the mixtures operationally defined as HS are aliphatic acids, ethers, esters and alcohols; aromatic lignin derived fragments; polysaccharides and polypeptides. By means of diffusion ordered spectroscopy, distinct diffusion coefficients consistent with relatively low molecular weight molecules were observed for all the components in the mixtures, and saccharides were the largest single class of component present. Liquid chromatography NMR confirmed that HS components can be easily separated and nuclear Overhauser effect (NOE) enhancements support the finding that the components are of relatively low molecular weight
High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) allows the application of solution-state NMR experiments to samples that are not fully soluble and contain solids. Only the species in contact with the solvent system employed become NMR observable. In this study utilizing D2O as the solvent system we show it is possible to examine the structures at the solid-aqueous interface of a whole soil. Combining one- and two-dimensional HR-MAS NMR allowed, for the first time, the identification of fatty acids, aliphatic esters, and ethers/ alcohols as prominent species at the solid-aqueous interface of the soil with signals from sugars and amino acids also apparent. Few, if any signals from aromatic protons were observed when the soil was swollen in aqueous media, although these signals are observed in extracts from the same soil and when the soil is swollen with a more penetrating solvent(DMSO-d6)which is known to disassociate hydrogen bonds. These findings indicate that the soil aromatic moieties are protected in hydrophobic regions which are not water accessible. Furthermore, when the soil was amended with a herbicide (trifluralin), direct observations of interactions between the protons on a xenobiotic and the surrounding soil matrix were possible for the first time. HR-MAS promises to be a method that can be widely applicable to a range of complex environmental samples without the need for extraction, pretreatment, or purification.
The recent melamine crisis in China has pointed out a serious deficiency in current food control systems, namely, they specifically focus on selected known compounds. This targeted approach allowed the presence of melamine in milk products to be overlooked for a considerable time. To avoid such crises in the future, we propose that nontargeted screening methods need to be developed and applied. To this end, NMR has an extraordinary potential that just started to be recognized and exploited. Our research shows that, from the very same set of spectra, 1H NMR at 400 MHz can distinguish between melamine-contaminated and melamine-free infant formulas and can provide quantitative information by integration of individual lines after identification. For contaminated Chinese infant formulas or candy, identical results were obtained when comparing NMR with SPE-LC/MS/MS. NMR was found to be suitable for routine nontargeted and targeted analyses of foods, and its use will significantly increase food safety.
Diffusion ordered spectroscopy (DOSY) was applied to two extracts of organic matter from the surface horizon of an oak forest soil. It was possible to identify and confirm the presence of numerous aliphatic, aromatic, sugar, and amino acid components that could be separated on the basis of diffusion in DMSO-d6 and D2O. On average, sugar components were identified as the largest molecules in solution, with molecular masses up to approximately 1500 Da followed by the aliphatic and aromatic components. Amino acids with a range of molecular weights were also identified in the mixture. The summation of the individual slices from the DOSY experiment closely resembles the conventional 1H spectra of the material, indicating that the components identified with DOSY represent all the components present in the mixture. The separation of components in the mixture in organic solvent supports new findings that fulvic and humic acids are not cross-linked, high molecular weight macromolecules but are instead aggregates composed of relatively simple molecules that take on colloidal properties in the presence of metal ions in aqueous solution. Using the knowledge that these organic mixtures are combinations of relatively simple entities with well-documented reactivities and behavior will improve our ability to predict and model their interactions and fate under natural conditions.
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