Isolation and fractionation of soil humin using alkaline urea and dimethylsulphoxide plus sulphuric acid

Humin, the most recalcitrant and abundant organic fraction of soils and of sediments, is a significant contributor to the stable carbon pool in soils and is important for the global carbon budget. It has significant resistance to transformations by microorganisms. Based on the classical operational definition, humin can include any humic-type substance that is not soluble in water at any pH. We demonstrate in this study how sequential exhaustive extractions with 0.1 M sodium hydroxide (NaOH) + 6 M urea, followed by dimethylsulphoxide (DMSO) + 6% (v/v) sulphuric acid (H₂SO₄) solvent systems, can extract 70–80% of the residual materials remaining after prior exhaustive extractions in neutral and aqueous basic media. Solid-state ¹³C NMR spectra have shown that the components isolated in the base + urea system were compositionally similar to the humic and fulvic acid fractions isolated at pH 12.6 in the aqueous media. The NMR spectra indicated that the major components isolated in the DMSO + H₂SO₄ medium had aliphatic hydrocarbon associated with carboxyl functionalities and with lesser amounts of carbohydrate and peptide and minor amounts of lignin-derived components. The major components will have significant contributions from long-chain fatty acids, waxes, to cuticular materials. The isolates in the DMSO + H₂SO₄ medium were compositionally similar to the organic components that resisted solvation and remained associated with the soil clays. It is concluded that the base + urea system released humic and fulvic acids held by hydrogen bonding or by entrapment within the humin matrix. The recalcitrant humin materials extracted in DMSO + H₂SO₄ are largely biological molecules (from plants and the soil microbial population) that are likely to be protected from degradation by their hydrophobic moieties and by sorption on the soil clays. Thus, the major components of humin do not satisfy the classical definitions for humic substances which emphasise that these arise from microbial or chemical transformations in soils of the components of organic debris.