## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Liquid State Investigations
#### 3.3.2 Ultrasonic Velocimetry  
Over past years, it became clear that the research of DOM is closely connected with its interaction with water [2] but the studies on solvation of DOM [69] and understanding of interaction of water with DOM and its structural stabilization role [179] are rare. In this respect the combination of old approaches and introduction of new methodologies is necessary to understand all the aspects of DOM environmental relevance [4; 14]. One of the methods that can analyze the properties of water in various materials is ultrasonic velocimetry. The sound wave is a wave of oscillating pressure and associated longitudinal deformation. When low-intensity ultrasonic wave propagates through the sample containing dispersed particles, it is scattered, diffracted and refracted. Change in ultrasonic velocity and wave attenuation are related to system composition, physical state, microstructure and molecular relaxation phenomena [180]. The amplitudes of deformations in the ultrasonic waves employed in analytical ultrasound are extremely small, making ultrasonic analysis a non-destructive technique. The resolution of modern devices, allowing precise determination of ultrasonic velocity, is down to \(\pm\)0.2 mm/s [181]. The speed of sound in liquids can be expressed by the Laplace equation  
\[U^{2}=\frac{1}{\rho\cdot\beta_{S}} \tag{2}\]  
where \(U\) stands for sound velocity, \(\rho\) for density and \(\beta_{S}\) for adiabatic compressibility. Virtually, all changes in physical and chemical state of solutions, suspensions or emulsions are associated with changes in density and compressibility, therefore, measurement of speed velocity in high resolution allows their monitoring. The adiabatic compressibility \(\beta_{S}\) is the fractional decrease of volume per unit increase in pressure, when no heat flows in or out,  
\[\beta_{S}=-\frac{1}{V}\bigg{(}\frac{\partial V}{\partial p}\bigg{)}_{S}=-\frac {1}{\rho}\bigg{(}\frac{\partial\rho}{\partial p}\bigg{)}_{S} \tag{3}\]  
where \(\partial V\) and \(\partial\rho\) are the changes in the volume \(V\) and density \(\rho\) at fixed entropy \(S\). Compressibility is a measurable parameter and it is sensitive to solute-solvent interactions, i.e., it can be utilized for characterization of hydration of a solute or for interaction of two solutes. In the study of hydration, apparent molar or specific compressibility of the solute is used. It is influenced by (i) interatomic interactions within the solute itself (intrinsic compressibility of a solute molecule), (ii) solute-solute interaction if any occur under experimental conditions and (iii) solute-solvent interactions (hydration in the case of aqueous solutions) [182]. The effect \"(ii)\" diminishes with decreasing concentration and at infinite dilution become equal to partial compressibility of the solute. In such case the partial compressibility of solute reflects only contribution of intrinsic compressibility and the hydration of solute [183]. Various functionalities and moieties differ in their hydration and thus their contribution to partial compressibility of the solvent/solute system. Since they are temperature dependent, their role differs with respect to the system temperature [184]. Differentiating Equation (2) with respect to molar concentration of solute \(C\), and performing simple arithmetical rearrangements, for infinitely diluted solutions Equation (4) can be derived:  
\[K_{S}^{0}=\beta_{S0}\bigg{(}2V^{0}-[2U]-\frac{M}{\rho_{0}}\bigg{)} \tag{4}\]  
where \(\beta_{S0}\) is the coefficient of adiabatic compressibility of the solvent, \(M\) is molecular weight of the solute, \(V^{0}=(\partial V/\partial N)_{T,P}\) is the partial molar volume of a solute; \([U]=(U-U_{0})/U_{0C}\) is the relative molar sound velocity increment of a solute and \(U\) and \(U_{0}\) are the sound velocities in the solution and solvent, respectively. The partial volume of a solute \(V^{0}\) can be calculated based on differential solution vs. solvent density measurement [185].