The interactions and structural formation of polyelectrolytes in solution and environmental stimuli-responsive water-soluble polymers are subjects of intense academic research, development, and application. An increasing number of novel materials are being created by synthesizing new molecules but also by assembly of existing molecules via intermolecular interactions. Such materials have found practical applications in cell and drug immobilization, environment protection, or sophisticated analytics and sensors. The reproducible synthesis, well-defined assembly, and full understanding of the molecular impact on the application behaviour require knowledge of structure-property relationships. These needs have motivated the selection of the research performed in this thesis. To establish such relationships and to gain more insight on the molecular level, powerful characterization methods must be employed. In this context, analytical ultracentrifugation was used. On the one hand, conventional analytical ultracentrifugation techniques were applied to complicated molecular characterization. On the other hand, analytical ultracentrifugation principles were extended to problems not studied before with this technique. Specifically, the following problems were addressed with polyelectrolytes as primary polymeric substances: Online study of hydrogel formation by interaction of oppositely charged polyelectrolytes using a modified synthetic boundary experiment Characterization of polydisperse branched high molar mass charged copolymers Molecular assembly of di-block copolymers containing one polyelectrolyte block Temperature-induced concentration dependent phase transition in aqueous solution. Overall, results were obtained from analytical ultracentrifugation experiments which were not accessible by other characterization techniques. Systematic online studies of hydrogel formation at the interface of aqueous solutions of oppositely charged polyelectrolytes revealed the impact of chemical structure, concentration, molar mass, and pH on the network quality of three combinations of polyanions and polycations: sodium alginate/oligochitosan, sodium alginate/poly(L-lysine), and sodium cellulose sulfate/poly(diallyldimethyl ammonium chloride). These combinations are of practical interest. The results are expected to contribute to the optimization of network properties for specific applications in medicine, biotechnology, and pharmacy. High molar mass polyelectrolytes, which were polydispersed with regard to molar mass, charge density, and chain architecture were characterized by hydrodynamic methods, particularly analytical ultracentrifugation. By combining synthetic boundary and sedimentation velocity experiments at various speeds, the degree of homogeneity of the various distributions of the samples were identified. The correlation of molecular characteristics and application properties corresponded to predictions from molecular simulations. Sedimentation velocity studies revealed concentration- and temperature-dependent aggregation of stimuli-responsive pre-associated polymers. The monotonic increase of the sedimentation coefficient with concentration and temperature confirmed pre-association prior to the phase separation. The aggregation process was proven to be completely reversible. Moreover, absorbance concentration profiles identified a temperature-dependent portion of polymer not participating in the aggregation process. Sedimentation equilibrium experiments allowed the estimation of the influence of concentration on the molar mass.