Natural colloids are defined as macromolecules and nanoparticles with sizes ranging between 1 nm and 1 µm. Colloidal pool is complex "ill-defined" mixture of humic substances, exopolymeric substances (EPS), inorganic oxides, clays, etc. Aquatic colloids have been recognized as key mediators in the removal of trace elements, facilitating contaminant transport, organic carbon cycling and micronutrient bioavailability. The residence times of the colloidal pool (and associate contaminants) can be order of magnitude different from those of the particulate fraction, but unlike the "truly" dissolved fraction, can be subjected to different coagulation and sedimentation processes. Colloids may also influence metal bioavailability by affecting free metal ion concentrations, constraining diffusion and affecting the dissociation kinetics of biological processes. The important environmental role of natural colloids is largely influenced by their surface area and closely related colloidal size. Nonetheless, it is currently unclear to what extend colloidal size can influence their behaviour and contaminant (e.g. toxic metals) binding properties. In our view this is partially related to the difficulties in determination of the colloidal size distributions and their impact in metal behaviour, mainly due to the inherent heterogeneity of the environmental colloids and their low concentrations in the environment. Furthermore, in addition to average parameters, there is a lack of appropriate sample collection and processing procedures, as well as analytical techniques to measure simultaneously the size (or related parameter) distributions of the colloid associated metal. The objective of the present work was therefore to develop sensitive analytical procedures for the fractionation of natural colloids and to apply them to improve the current understanding on the fate of the colloids and associated toxic metals in the freshwaters. To achieve these goals, the capabilities of asymmetric flow-field flow fractionation (aFlFFF) coupled to several detectors was explored. aFlFFF is a versatile technique separating colloids according to their diffusion coefficient. The system was hyphenated with multi-angle laser light scattering (MALS), differential refractive index (DRI), UV detector and, when necessary, high resolution inductively coupled plasma mass spectrometry (ICP-MS). Correct interpretation of the experimental results and extrapolation of meaningful molecular parameters by using an analytical tool with such a level of complexity requires improvement of the knowledge of colloids behaviour in the aFlFFF channel and careful optimization of the separation conditions. Given the very complex and dynamic nature of environmental colloids, different experimental procedures was first optimized and validate under lab conditions by using "standardized" colloids (e.g. alginate and EPS isolate from lab bacterial cultures), then applied to characterize colloidal organic matter (COM) isolated from natural water systems. The influence of critical operating parameters, such as crossflow rate, carrier composition and concentration, and sample load, on the alginate retention was carefully evaluated. Combined information obtained simultaneously by DRI and MALS detectors, allowed to set the appropriate combination of optimal parameters for the characterization of alginate, as a model COM. The metal distributions of metal-alginate complexes were probed by aFlFFF - ICP-MS: average values and continuous distributions of molar masses, radius of gyration and hydrodynamic radius, critical for understanding their role as carriers of metal pollutants, were evaluated in presence of lead or cadmium and compared to those in metal-free solutions of alginate. Furthermore, the molecular characteristic of exopolymeric substances (EPS) secreted by bacterium Sinorhizobium meliloti wild type and exoY strains, which is deficient in the production of the succinoglycan, were studied. EPS were collected in the extracellular environment or desorbed from the cell surface using different extraction protocols. EPS fractions with different chemical characteristic and molar mass were found, depending on the strain and extraction protocol. Molecular parameters as the distributions and average values of molar mass and gyration radius, and the polydispersity index have been measured and compared with data obtained by the chemical characterization. Size exclusion chromatography coupled to UV detection was also employed to provide additional information on the small class of size. Moreover, the effect of the cadmium, lead and calcium addition to exopolymeric substances excreted in the extracellular environment was investigated, with the aim to study how does EPS composition, size and mass distributions, can affect the trace metal binding properties of EPS and how the metal addition affects the EPS conformation. Obtained results revealed that cadmium and calcium bound preferentially on the low-size species population and the amount of the bound metal increased with the increasing of the concentration of added metal. In contrast, lead bound on preferentially larger classes of EPS size. The aFlFFF separation parameters were also optimized for the fractionation and characterization of humic substances, as other COM component, and applied to probe the temporal and spatial variability in the size and mass characteristics of COM samples collected in the Amazon River basin. COM characterization demonstrated the existence of a clear decrease of size of the colloidal material when passing from small to large watercourses (spatial variation). No significant changes were found in size distributions of samples collected in different period of the year, during the low and high flow, respectively (temporal variation). The analytical methodology, adapted to determine not only mean characteristics, but the population distributions, developed in the present work, allowed to obtain new information about the colloidal organic matter size distribution, which play utmost role in colloids behaviour and fate in the environment, in particular as toxic metal carriers.