FIG 1. CPMAS 13C NMR spectra of humic acids isolated at pH 7(1), 10.6 (2), 12.6 (3), and that precipitated from dilute solution at pH 2 (4). Spectra 5, 6, and 7 are for the fulvic acids, and 8, 9, and 10 are for the XAD-4 acids isolated in the same pH sequence. Spectrum 11 is for neutrals isolated in ethanol during soxhlet extraction of XAD-4. Note the definite compositional differences between the same fractions isolated at the different pH values. This shows the effectiveness of using charge density properties for the separation of components of SOM. FIG 2. CPMAS 13 C NMR spectra of humic acids isolated at pH 12.6 (0.1 M NaOH) after exhaustive extractions at the lower pH values[lower], and for the HAs isolated in 0.1 M NaOH + 6 M urea 9 (upper). Note the very close similarities in the spectra. These data suggest that the material isolated in the urea medium was held by hydrogen bonding and/or by entrapment within the humin matrix, and released by the urea. We follow the ‘Splitters’ approach for studies of Soil Humic Substances. In that we isolate and fractionate the components of Soil Organic Matter (SOM), as opposed to the ‘lumpers’ approach in which studies are carried out on the whole of the SOM. The classical methods for studies of SOM involves dissolving the OM in 0.1 or 0.5 M NaOH, precipitating the humic acids at pH 1, recovering these and the soluble (in acid) fulvic acids. We introduce here novel procedures, based on charge density and polarity differences for the isolation and fractionation of the components of SOM, and characterization is carried out by solid and liquid state NMR. Soils were sequentially exhaustively extracted at pH 7, then 10.6, then 12.6, then with 0.1 M NaoH + 6 M urea, then with DMSO + 6% concd. H 2 SO 4, and the residual material was recovered after destruction of the clays with HCl/HF. The aqueous extracts were processed by the XAD-8 and XAD-4 resin in tandem procedure (Hayes, 2006). The DMSO was removed and the products recovered using XAD-8 resin (Hayes, 2006). The samples were characterized and compared using solid and liquid state NMR. Introduction Experimental Procedures Results and Discussion FIG 4 1 H NMR spectra for A), the UREA FA Cultivated soil (C); B), the diffusion edited 1 H spectrum of the UREA FA C; C), Bovine Serum Albumin; D), the diffusion edited 1 H spectrum of the UREA HA C. “Simple” assignments, shown in A, indicate strong contributions from P, proteins/peptides; L, lignin; C, carbohydrate; WCL, waxes cuticles and lipids; PG, peptidoglycan; LP, lipoprotein. More specific assignments shown in B refer to 1, amide; 2, phenylalanine; 3, aromatics in lignin; 4, anomeric protons in carbohydrates; 5,  -protons in proteins and peptides; 6, methoxyl in lignin; 7, other carbohydrate protons; 8, P-OCO-CH2-R methylene unit adjacent to the carbonyl in lipoprotein; 9, acetate group in peptidoglycan; 10, methylene units in an aliphatic chain  to an acid or ester; 11, methylene (CH 2 )n in aliphatic chains; 12, CH 3 (†note when this peak is large relative to 11 it often indicates strong contributions from proteins as in these examples; see Figure 4C). In Figure 4C only the dominant protein signals are labeled. These when combined are the most distinctive indicators of protein in humic materials. In figure 4D, assignments are as above. Si indicates a natural silicate species and not TMS (a commonly used NMR reference standard). Note the highly significant aliphatic functionality (10-50 ppm), the –Oalkyl functionality (60-90 ppm; would include carbohydrate/peptide), the evidence for anomeric C (105 ppm), the evidence for the carbonyl of carboxyl (180 ppm), and the lack of significant aromatic functionality. FIG 3 CPMAS 13 C NMR spectra of the clay organic complex after exhaustive extraction with 0.1 M NaOH + 6 M urea. The spectrum is contributed by classically defined humin material. FIG 5. 1 H diffusion edited NMR of the A) UREA HA, uncultivated soil [UC]; B) DMSO HA UC; C) DMSO FA UC. Unless otherwise depicted, assignments are the same as shown in FIG 4. †Note this peak is too large to be attributed to aliphatic species alone, and indicates a substantial contribution from protein, *It is very important to note the Humin HA is soluble in DMSO only in the presence of a strong acid. D 2 SO 4 was added which, in addition to solubilizing the humin, also deuterium exchanged the N-H (to N- D) and so the amide resonance in the humin HA is strongly attenuated. FIG 1 shows that isolation of soil humic substances on the basis of charge density differences gives rise to fractions of different compositions. In FIG 2 we see that the material removed in the urea is similar to that isolated at pH 12.6 (after prior exhaustive extractions at pH 7 and 10.6). That indicates that materials held by ion exchange or by steric constraints are released in the urea solution. FIG 3 that exhaustive extractions in base + 6M urea effectively removes the humic components derived from lignin. However, there is clearcut evidence for significant contributions from aliphatic components (which are likely to contain long chain hydrocarbons, fatty acids, waxes, etc.) and for carbohydrate and peptide functionalities in association with the soil clays. When the humin components were further isolated in DMSO + 6% H2SO4, and fractionated into materials that fall into the classical definitions of humic acids (but are not, of course such) we see in the spectra in FIGS 4, 5, and 6 that the humin samples studied contain varying contributions from five main categories of structures, namely protein, aliphatic species (including contributions from lipoproteins), carbohydrates, peptidoglycan (the main structural components in bacterial cell walls) and still some lignin-derived materials. Lignin is clearly identifiable as a plant input in humin materials while peptidoglycan is indicative of microbial inputs. It is not possible in the present studies to positively identify the source of the protein, aliphatic or carbohydrate species. In the case of the lipids, a fraction would appear to be present as lipoproteins (which may be from lysed microbial cells). Similarly, some of the carbohydrates can certainly be ascribed to microbial sources (attributable to the carbohydrate backbone in peptidoglycan); however, from the size of N-acetyl peak in peptidoglycan it is clear this peptidoglycan alone cannot account for all the carbohydrates present. The source of the other carbohydrates cannot be determined in this study and could include structural carbohydrate from plants (cellulose, hemicellulose etc.) as well as other microbial carbohydrates. The solubilised humin materials have components similar to those in the more traditional humic and fulvic acid fractions, with the exception that peptidoglycan is present at significant levels and these humin fractions have a macromolecular instead of supramolecular character, such as for traditional HAs an FAs. That indicates that the humin fraction contains, in addition to plant derived biomass, a significant amount of microbial biomass. Because the humin fraction is strongly associated with clays, and because bacteria are known to associate strongly with clay minerals, it is feasible that the clay component provides protection for microbial species that ultimately contribute, in terms of biomass, to the operationally defined humin fraction. In addition, it is feasible that some of the more labile components, especially proteins may be preserved through associations with the clays. However further work will be needed to determine if that is the case, or if the protein is present due to the release of cellular proteins during lyses from DMSO and UREA. Summary Reference M.H.B. Hayes Solvent systems for the isolation of organic components from soils. Soil Sci. Soc. Am. J. 70, FIG 6. Heteronuclear Multiple Quantum Coherence Spectra (HMQC) of the DMSO FA UC. A) depicts the complete spectrum. Assignments in Figure 6A are as follows: 1, protons in p-hydroxybenzoates (lignin); 2, phenylalanine (in protein); 3, mainly protons adjacent to an Ar-OR functionality in lignin; 4, units in syringyl units (lignin); 5, anomeric protons in carbohydrates; 6, other CH groups in carbohydrates; 7, CH 2 in carbohydrates; 8,  -protons in proteins and peptides; 9, methoxyl in lignin; 10, aliphatic linkages including numerous signals from various lipids, and side chain protons in proteins/peptides. B) shows an expansion of the aliphatic region. Assignments in Figure B are as follows; 1, R-OCO-CH 2 -R methylene unit adjacent to the carbonyl in lipids (including lipoproteins and cutins); 2, methylene units in an aliphatic chains  to an acid or ester; 3, methylene (CH 2 )n in aliphatic chains, 4, methylene units in an aliphatic chains  to an acid or ester; 5, CH 3 (a small contribution in this region will be from terminal CH 3 from lipids). However, the majority of signals are from proteins. In proteins a classic elongated CH 3 results as many of the side chain experience varying 13 C chemical shifts due to local environments with the macromolecular structure. Michael H.B. Hayes 1, Andre J. Simpson 2, Guixue Song 1, C. Edward Clapp 3, and J. Burdon 4 1 Chemical and Environmental Sciences, Univerrsity of Limerick, Ireland; 2 Departmen of Physical and Environmental Sciences, University of Toronto at Scarborough, Canada; 3 USDA ARS, Department of Soil, Water and Climate, University of Minnesot, St. Paul, USA; 4 Department of Chemistry, University of Birmingham, England COMPOSITIONS, ASPECTS OF STRUCTURES, and CLAY ASSOCIATIONS of SOIL HUMIC COMPONENTS