## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Liquid State Investigations
#### 3.3.2 Ultrasonic Velocimetry
\[V^{0}=\frac{M}{\rho_{0}}-\frac{(\rho-\rho_{0})}{\rho_{0}C} \tag{5}\]  
Therefore, the partial molar adiabatic compressibility of a solute can be calculated from precise density and sound velocity in solutions. Measurement of sound velocity, however, requires a strict temperature control such as thermal stability of the order \(10^{-3}\)\({}^{\circ}\)C. Therefore, the measuring of a relative change in a physical characteristic per unit of solute concentration rather than its absolute value is often more important, more precise and easier. That is the reason why the modern devices use the differential arrangement: two identical resonator cells are placed in one termostated volume and the difference in ultrasonic velocities is measured [186]. In this way the increment of ultrasonic velocity is easily and precisely determined under the thermal stability of \(10^{-2}\)\({}^{\circ}\)C.  
The arrangement of modern devices, i.e., using the so-called resonator method [182] enables using of two modes such as ultrasonic velocimetry and ultrasonic spectroscopy. Spectroscopic approach utilizes measurements of the attenuation of ultrasound over a wide range of frequencies in order to determine data about the kinetics and thermodynamics of fast molecular equilibrium. The ultrasonic velocimetry is based on the accurate measurement of the ultrasonic velocity, optionally in combination with density measurement to determine the compressibility and volume of a molecule or aggregate in solution [187]. The results of investigation of physical structure of HS using ultrasonic velocimetry demonstrated the progressive aggregation of various humates from \(10^{-3}\) to \(10\)\(\mathrm{g}/\mathrm{L}\) under various conditions such as neutral and alkaline pH and at increased salinity of the solution [47]. It has also been demonstrated the difference in aggregation of HS and classical surfactants monomers such sodium dodecyl sulphate, (CMC = 2.3 \(\mathrm{g}/\mathrm{L}\)) [47] and polymers (Triton) [179] (Figure 7a). The increment of ultrasonic velocity (defined as a difference between ultrasonic velocity in HS (\(U_{1}\)) and water (\(U_{2}\)) divided by \(U_{1}\), (see Figure 7a) was used. The increment is constant when no interaction between sample molecules occurs (below CMC) and when CMC is reached, it progressively decreases. HS in solution showed a steep decrease already at concentration hundreds to thousand times lower that the CMC reported for classical surfactants (Figure 7a). This confirmed the view that HA form rather premicellar aggregates than classical micelle with a precisely defined CMC. Similar experiments conducted in 1M NaCl and in NaOH at pH 12 showed similar, but less intensive, aggregation of HS in solutions [47]. The rearrangement kinetics of the HS aggregates was tested by injection of various molecules including HCl, propionic acid and propanol into HS solution (Figure 7b). The results showed the specificity in interactions with molecules of various chemical properties. Whereas HCl and propanol showed a rearrangement within several minutes, the propionic acid treatment caused the long-term reconformation taking several hours. This supported the previously reported view [52] that organic acid can cause a reversible disruption of hydrophobic interactions stabilizing HS molecules at neutral pH and shift to HS structure to a new equilibrium at pH = 3.5 in which HS structure is stabilized via H-bonds. In order to test the nature and stability of HS, the salts of HA and FA from various sources in the concentration range from 0.001 to 10 g/L were repeatedly heated from 5 to 90 \({}^{\circ}\)C [69; 179]. The change in ultrasonic velocity in heating run indicated no transition above 4 g/L; below this concentration existence of transitions was demonstrated as breaks on the curve (Figure 8a). Decreasing concentration caused the weakening of the stabilizing interactions and several transitions could be observed (breaks in Figure 8a). The most important change could be seen in the slope of the record.