## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Liquid State Investigations
#### 3.3.1 Scattering Techniques
The aggregation process of HS isolated from Rendzic Leptosol (RLHA) and IHSS HA standard from Elliot soil (EHA) was examined in the 2-11 pH range at constant ionic strength through DLS to monitor particle sizes and LDE technique for the determination of zeta potential [147]. At pH values > 6, particle sizes smaller than 10 nm were interpreted as small individual HA molecules or basic units. With the decrease in pH, the evidence of a sharp increase in diameters in the 6-4 pH range was attributed to the occurrence of the aggregation process of the HA polymers, indicating carboxylic hydrogen as responsible for the intermolecular H-bond and, consequently of the formation of aggregates. Aggregates formed at pH 2 were found of different sizes (from 600 nm to 1.5 um) and dependent on the HA origin and concentration in the aqueous dispersions. The zeta potential values were more negative with increasing pH in the range 2-6 as a result of the functional-group dissociation. Interestingly, the authors documented the existence of a zeta-potential minimum, reached in the 5-7 pH range, which was not yet reported in literature. The explanation of the increase in the surface charge of the particles observed at higher pH values has been articulated on the basis of the existence of two related mechanisms: dissociation of acidic functional groups and consequent dissociation of HA aggregates, caused by the breaking of H-bonds, with opposite effects on the zeta potential values. In the first part of the zeta-potential vs. pH curve (from pH 2 to pH where the zeta potential minimum was reached), dissociation of acidic groups could be considered as the prevailing process leading to a negative-charge increase. At higher pH values (from the zeta potential minimum upward), disaggregation process likely predominated over dissociation, resulting in the z-average particle size decrease at higher pH values. As a consequence of smaller particles formed, negative charge per HA particle decreased causing zeta potential to become less negative. Experimental evidences of dynamic processes related to the HS aggregation kinetics were also reported by [PERSON] et al. [148], although for shorter time scales, in aqueous dispersions of HA extracted from Leonardite. Indeed, time-resolved DLS measurements revealed a particle size growth scaling linearly with time for HA aqueous suspension at pH 2 and NaCl 0.015 M. The zeta-average D\({}_{\text{h}}\) of HA aggregates increased monotonically from \(\sim\)750 nm up to \(\sim\)1.3 um in 70 min for a sample left mechanically unperturbed in the measuring cell for the duration of the DLS experiment. However, the selected time window did not allow to observe a threshold limit value for the growth of particle sizes. After an hour of monitoring, a bimodal particle size distribution (PSD) on a numerical basis was found showing maxima centered, respectively, at 300 nm and 1.5 um. On the contrary, the time-dependent increase in the aggregates' size was not detected at pH > 2, where instead a monomodal PSD on a numerical basis centered at about 80 nm was obtained (see Figure 5 in ref. [148]). Both DLS measurements and zeta potential determined from LDE technique, have been proved valuable for increasing comprehension of the aggregation behavior of HSin simulated low electrolytic conductivity aqueous solutions (16-192 \(\mu\)S/cm) [74]. The humic materials selected by the authors were standard HS such as SRHA, SRFA and AHA sodium salt, and two types of actual environmental aqueous samples. The HS aggregate sizes measured within the range 0.01-1 \(\mu\)m were postulated to be composed of subunits as precursor molecules for the formation of the 5-\(\mu\)m-sized particles, which in turn were observed to occur on a time-scale of minutes not hours/days as observed, e.g., by [PERSON] et al. [145; 146], in both simulated and actual environmental water samples. Interestingly, the authors explored the influence of degree of turbulence on the extent of disaggregation and self-assembly process.