## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Liquid State Investigations
#### 3.3.2 Ultrasonic Velocimetry
In particular, below 1 g/L the slope changed from negative to positive (see Figure 8b), which was attributed to the change in prevailing role of different types of interactions influencing the  
Figure 8: Temperature dependence of ultrasonic velocity of sodium fulvates Suwannee River at concentrations (**a**) 1 and (**b**) 0.1 g/L (based on the results of ref. [69]). Figure 7: **(a)** Comparison of aggregation behavior of DOM and triton using increment of ultrasonic velocity (based on the results of ref. [180]). (**b**) Kinetic curve—ultrasonic velocity corresponding to the reconformation of DOM after addition of propionic acid as indicated by the change in \(U_{12}\), which is the difference between cell 1 with DOM sample and cell 2 filled with water (based on the results of ref. [47]). adiabatic compressibility. Specifically, below 1 g/L the most important factor was change in structural compressibility, which is caused by strengthening of van der Waals forces upon heating while hydration component of compressibility plays only minor role. In contrast, at higher HS concentration, only weakening of hydration shell plays an important role and the structural compressibility could be neglected since the structure is stabilized by weak interactions such as H-bonds, charge-charge interactions and possibly hydrophobic interactions. The observations about a change in the HS structure at 1 g/L were later confirmed by other authors [153,188]. Results obtained by FFC NMR relaxometry showed that also at extremely high concentration of DOM (30 g/L) in the structure occur changes, which are, however, not visible by velocimetry under applied conditions, i.e., around 6 MHz in the temperature range from 25 to 60 \({}^{\circ}\)C [71]. The heat-cool experiments were also used for testing of solutions of HS modified by either HCl or propionic acids. The results revealed a great difference in physical structure between resulted structures at pH 3.5. In case of HCl the structure resembled the original system with increasing strength of H-bonds, reflected by the increase in the transition temperatures. The addition of propionic acid induced a complete reconformation and strengthening of the HS structure [70]. The comparison of the heating and cooling cycles highlighted the structural hysteresis of HS, proving that the physical structure of HS also depends on the thermal history (Figure 9a) [69]. This problem was in detail investigated by [PERSON] et al. [189], who found that the increase of the heating/cooling amplitude caused an increase of the relaxation time needed for HS to reach the equilibrium. In addition, the authors confirmed that in the thermally-induced hysteresis were not involved aromatic structures whereas alkyl moieties, carboxylic and carbonyl carbons influenced the hysteresis positively. On the contrary, negative effect was observed for peptide and O-alkyl groups. The structural hysteresis was confirmed by FFC NMR. In addition, the method also unraveled that correlation time of water in DOM is lower after than before heating. Decreasing of correlation time indicates lower restriction of rotational and translational motions of water in DOM thereby indicating the delay in re-formation of DOM structure or potentially less rigid structure upon cooling. The ultrasonic velocimetry showed a fast reconfiguration dynamics of HS aggregates in water (up to 10 min) which was in contrast to processes taking part at the water/air interface (10 h) [190]. Ultrasonic velocimetry data allowed calculation of fractal dimensions of DOM aggregates [191] and calculation of changes in thermodynamical parameters caused by aggregation during progressive increase in concentration such as entropy of the DOM/water  
Figure 9: (**a**) Hysteresis in ultrasonic velocity of Elliot Soil sodium fulvate 0.01 g/L (based on the results of ref. [69]). (**b**) Fractal dimensions of Na\({}^{+}\) salts of Elliot soil HA and FA and H\({}^{+}\) form of Elliot soil FA (based on the results of ref. [70]). system [179].