## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Solid State Investigations
For investigations performed on soft materials, the AFM non-contact mode (or AFM intercept mode) is preferred, in which an oscillating cantilever is used to maintain a constant distance between the tip of the AFM and the sample surface. Characterization of HS aggregates by AFM suggested that FA and HA could form aggregates and clusters at nanometer scale with globular shape according the pH of the aqueous solutions [96]. An image of HA particles (extracted from Leonardite) adsorbed on mica surface is shown in Figure 2A. AFM images in tapping mode of HA aqueous dispersion absorbed on mica, showed separated small globular particles with sub-micron size [97]. In the isolated particle cluster (Figure 2B) continuous HA layers, in ring format, were observed. These homogeneous ring structures (see Figures 3 and 4), formed in saturated particle dispersed in water at low pH values, indicate evidences of supramolecular structures of HA in the natural environment, which in turn are strongly dependent on the dispersion method and pH. The hydrophilic surface of the HA aggregates interacts with the negatively charged mica surface, and promotes a large, swollen shape. If the structure is composed of isolated molecules, the dispersion procedure makes either slowly removing of water on the HA from mica surface and keep the initial ring structure, with small changes. Figure 2: AFM images in tapping mode of Leonardite dispersed in water and then adsorbed and dried on mica surface: (**A**) Typical rounded particles; (**B**) Isolated particle cluster. Figures 3 and 4, illustrating high nanoscale resolution AFM-C images, show several spherical ring-like structures of HA bearing a shallow depression in the middle, with apparent colloidal diameters < 500 nm and vertical size of 3-4 nm. The image of a mica surface is very smooth and the adsorption of HA on the mica is quite clear. The size and  
Figure 4: AFM\(-\)C (contact mode): (**A**) microtopography with 3D images of HA dispersed in pure water at pH 5, adsorbed and dried on a mica surface with section analysis (**B**) and amplitude (**C**). Figure 3: AFM\(-\)NC (non-contact tapping mode): (**A,C**) microtopography amplitude and (**B,D**) 3D microtopography images of HA dispersed in pure water at pH 5, adsorbed and then dried on a mica surface. shape of the pseudo-spherical colloids are in agreement with those observed by [PERSON] et al. [97] and also reported by other research groups for similar HS [98; 99]. It should be noted that those ring-like structures were better observed at nanoscale with few drops of water suspension directly dried on the mica heated at 80-90 \({}^{\circ}\)C. The pseudo-spherical structure may be due to the rapid air-drying process occurring on the mica surface. It is generally accepted that the hydrophobic character of HA, due to the hydrophobic compounds (long alkyl-chain alkanes, alkenes, fatty acids, sterols, terpenoids, and phenyl-alkyl residues of lignin degradation), allows their self-assembly into supramolecular structures separated from the water milieu [100]. From the sectional analysis results, shown in Figure 4B, it is observed that the average dimensions of the ring-like particles ranged from about 350 to 550 nm in lateral dimension and 3-4 nm in height. In Figure 4B, the images illustrate a cut along the solid line shown in the vertical profile. Moreover, particle sizes and shapes appear quite uniform inside the field of view. These ring structures, formed at subacid pH values, again indicate evidences of supramolecular arrangements [101]. Consequently, it can be deduced that the analysis of the AFM images supports the HA aggregation model, based on the cooperative association of small molecules. It is important to highlight that aggregation process of HA depends strictly on the solvent used for the dispersions and the dehydration conditions.