## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Solid State Investigations
#### 3.2.2 Nuclear Magnetic Resonance Techniques
Due to the low sensitivity, FFC NMR prevents any spectroscopic investigation, while it is applied to monitor molecular dynamics at frequency intervals lower than those span by the traditional NMR spectroscopy [119]. The Limits of the NMR Techniques in Humic Chemistry  
All the aforementioned NMR techniques are widely applied to unveil the chemical-physical characteristics of HS. However, some issues must be accounted for when dealing with NMR data. From the one hand, solid state NMR spectroscopy suffers for the limits due to the cross-polarization (CP). CP is based on the energy transfer from hydrogens to the closest carbon nuclei, thereby \"permitting\" the observation of the less abundant \({}^{13}\)C nuclei via the population of the most abundant \({}^{1}\)H systems. However, when the carbon backbone is made mainly by aromatic systems or quaternary, and tertiary alkyl C, lack of hydrogen nuclei is obtained, thereby enabling low CP efficiency. As a consequence, NMR sensitivity lowers, and signal disappearance occurs. In addition, if humic samples are not well packed in the zirconia containers used to perform solid state NMR investigations, a vortex effect occurs thus providing NMR artifacts [104]. On the other hand, due to the colloidal nature of HS, liquid state NMR spectroscopy suffers for the lack of information contained in the solid parts of the colloidal systems. In fact, liquid state NMR spectroscopy performed on HS permits analyses only of the surface groups in colloids [121]. The best solution to try to overcome the problems of solid and liquid state NMR spectroscopy is the application of the high-resolution magic angle spinning (HRMAS) technique. By means of HRMAS, the colloidal dispersions made by HS are placed in the zirconia rotors used in solid state NMR and oriented at the magic angle (54.7\({}^{\circ}\)). The efficiency of the solid-state NMR spectroscopy is coupled with that of the liquid state NMR one, thus permitting the analysis of both surface and inner solid part of the colloidal humic particles. Sensitivity and lack of mathematical models for data elaboration are the main problems affecting application of FFC NMR relaxometry on HS [119]. NMR Results of Humic Substances  
Over the years, modern NMR technology, such as the cryo-probe and hyphenated-NMR, along with the latest generation of NMR experiments, has been found very useful to explain the primary makeup and associations of HS. DOSY results, an NMR technique that directly correlates the diffusion coefficient of different molecular species to the proton chemical shift in a 2-D plot, allowed [PERSON] et al. [122] to identify a complex mixture of relatively simple compounds, such as fatty acids/esters, sugars, amino acids, and lignin-derived aromatic systems as the major components present in alkaline extractable soil HS. Those findings corroborated the concept of molecular aggregation of relatively low molecular weight components (<2000 Da), where metal ions play a crucial role in aggregate formation and stability [123]. Results similar to those revealed by [PERSON] et al. [122] have been obtained by [PERSON] et al. [124] by applying 2-D solid state NMR pulse sequences. In particular, those authors could distinguish different humic hydrophobic/hydrophilic domains via the evaluation of 2-D maps where the x-axis reports the \({}^{1}\)H-NMR spectrum, while the y-axis reports the \({}^{13}\)C NMR spectrum. The interpretation of CPMAS-NMR spectra made by [PERSON] et al. [125] allowed to speculate that humic aggregates can be considered as \"highly condensed or strongly associated rigid aromatic systems interacting with carboxyl groups, involved in hydrogen-bond formation, and mobile C-O/C-N systems that, in turn, should be mainly displaced, as well as the mobile alkyl moieties, on the surface of the solid state HAs\". Finally, recent applications of FFC \({}^{1}\)H-NMR relaxometry revealed that a \"hydrogel-like\" structure is recognizable in HS as concentration increases.