## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Liquid State Investigations
#### 3.3.1 Scattering Techniques
About 30 min after filtration, DLS revealed the presence of a polydisperse collection of assembled polymeric aggregates with sizes in the range 0.2-1 um. A nonlinear growth process was observed and the increase in particle size followed a sigmoidal trend recalling second-order kinetics. Both methods showed that microgels continued growing, reaching an average equilibrium size of about 5 um in 50-83 h.  
The aggregation process of FA and HA certified by the International Humic Substances Society (IHSS) [139] as a function of solution properties such as temperature, pH and metal ions of different charge, was explored through DLS technique by [PERSON] et al. [140]. The authors reported average particle sizes of 210-280 nm and showed that the evolution of particle sizes under different solution conditions progressed from unfolded anionic polymers at high pH, low cation concentrations, and low temperature, through micelle-like structures and finally to colloidal precipitates as these conditions were changed. The observation of a limited ionic strength effect on the \(\mathrm{R_{h}}\) derived from diffusion coefficients measured by Fluorescence Correlation Spectroscopy (FCS) was reported by [PERSON] et al. [141], who supported the hypothesis of HS as rigid spheres. The observed slight compression of the tested HS standards at high ionic strength was found in agreement with the viscosity measurements made by [PERSON] et al. [142] on several HA. Although viscometry was not sensitive to aggregation, the determinations of 35-65% molecular volume reduction for an increasing ionic strength (1-100 mM) followed the same trend of slightly decreasing \(\mathrm{R_{h}}\) obtained from FCS. DLS technique was also used to study the effect of pH on the aggregate size of a gray HA extracted from a commercial HS derived from brown coal [143]. The decrease of zeta potential, determined from laser Doppler electrophoresis (LDE) technique, from -27 mV (pH 1.5) to -63 mV (pH 8.8) was accompanied by a concomitant drop in particle size from 310 to 70 nm within the same pH interval. The authors justified that behavior on the basis of dissociation of acidic groups, which accordingly could lead to depletion of protons able to form H-bonds and hindering HA aggregation. [PERSON] et al. [144] determined the \(\mathrm{R_{h}}\) of Suwannee River HA (SRHA) in NaCl and CaCl\({}_{2}\) solutions at different concentrations in the range 10\({}^{-3}\)-0.5 M and pH 4.5-9.3. The small size of SRHA (<10 nm) measured at low electrolyte concentrationswas attributed to the presence of small base units, which in turn were able to interconnect and produce supramolecular structures at higher salt concentrations. DLS measurements, in combination with ultrafiltration (UF) and SEC techniques, were performed on alkaline aqueous dispersions of Aldrich humic acid (AHA) and HS derived from peat and compost [62]. The combined DLS-UF results indicated the presence of the following three main molecular fractions: R\({}_{\text{h}}\) = 0.5-2 nm (corresponding to the UF molecular fraction lower than 10 kDa), R\({}_{\text{h}}\) =2-5 nm (corresponding to the 10-100 kDa UF fraction) and R\({}_{\text{h}}\) =11-20 nm (corresponding to the 100 kDa and 0.45 um UF fraction). Those three main molecular fractions were also observed in the SEC study performed by the same authors. Using time-resolved DLS measurements of HS in seawater conducted over a time-scale of days, within the first 2 h following filtration [PERSON] et al. [145] identified free dissolved organic carbon (DOC) polymers with average sizes in the range 5-50 nm. However, 100-200 nm sized nanogels appeared after 5-10 h, which in turn began to restructure to form larger particles until reaching final dimensions of 3-6 um (microgels) at equilibrium after 60 h. Overall, the self-assembly process followed a nonlinear sigmoidal relationship, leading to the conclusion that low-energy physical interactions could be responsible for DOC self-assembly [146].