## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Liquid State Investigations
#### 3.3.1 Scattering Techniques  
Studies of DOM and humic colloid structures in both natural aquatic and artificial aqueous environments have, until recently, largely relied upon scattering techniques [130]. Scattering techniques measure dimensional quantities on submicrometer length scales and on short time scales, for samples large enough to be considered statistically significant. For light scattering, an incident radiation having wavelengths much greater than the average size of the colloidal particles is selected. This implies that the light scattered by a particle is in phase. Consequently, the scattered intensities are greater and more efficient than X-ray scattering. Ultrasounds can be used to investigate even turbid or highly concentrated systems but use longer wavelengths than light. X-ray and light scattering methods can be applied to measure a similar range of colloidal properties such as the radius of gyration (R\({}_{\rm g}\)), the cross-sectional radius of gyration, colloid particle volume, the cross-sectional area of the particle, molecular weight per unit length, and particle shape. Dynamic light scattering (DLS) also known as quasi-elastic or photon correlation spectroscopy (PCS), can measure particle sizes down to a few nanometers, but only by making assumptions about the shape and diffusive behaviour of the object being studied. It is an inelastic scattering technique, not a type of diffraction measurement. It uses a laser beam to measure the fluctuation in the intensity of the scattered light originating from Brownian motion, defined by the translational diffusion coefficient, which in turn is converted into the hydrodynamic particle diameter D\({}_{\rm h}\) (or radius R\({}_{\rm h}\)) using the Stokes-Einstein relationship [131]. R\({}_{\rm h}\) corresponds to the apparent particle size, which includes also the hydration shell that surrounds it. In particular, the polydispersity index (PdI) and the z-average mean diameter of particles are determined from intensity-based DLS spectra, and represent a single average even if spectra are multimodal. DLS is a technique that measures the dynamics of fluctuations of scattered light to obtain the mobility of colloidal particles subject to Brownian motion [132]. Details on DLS data treatment such as, e.g., converting autocorrelation functions to particle size distributions, can be found elsewhere [133; 134]. Experimental data provided through DLS-based studies or in combination with other techniques, consolidated the hypothesis that HS consist of low molecular weight compounds able to aggregate step by step to give supramolecular structures. DLS measurements of a peat HA in water and also in NaCl solutions of varying concentrations yielded low values for \(\mathrm{D_{h}}\) = (2-9)\(\cdot\)10\({}^{-11}\) m\({}^{2}\cdot\)s\({}^{-1}\), leading to the conclusion that such particles can be interpreted as consisting of large macromolecules or aggregates of smaller molecular species [135]. The effect of pH and ionic strength on the aggregation of both peat and commercial HA was studied by [PERSON] et al. [136] through DLS and voltammetry, who reported large aggregates in the range 30-185 nm. [PERSON] et al. [137] studied the fractal dynamics of HA colloids and data were interpreted according to the dynamic scaling theory developed by [PERSON] and co-workers [138], that described power law-polydisperse colloids. For example, the assembly kinetics of DOM polymers in 0.22 um-filtered seawater was monitored by measuring the particle size as a function of time using DLS and flow cytometry [36]. It is worth noting that both of these methods are complementary: while flow cytometry provides accurate measurements of large particles (>1 um), DLS yields reliable measurements of particle size in the sub-micrometre range. DLS showed that fresh filtrate contained a polydisperse set of polymers with sizes ranging from 2 to 200 nm.