The capabilities of the asymmetrical flow field-flow fractionation (AsFlFFF) technique coupled with inductively coupled plasma mass spectrometry and UV–Vis spectrophotometry in the characterization of synthetic and natural colloidal samples are demonstrated in two different systems. The first system is a sol of hectorite which was co-precipitated in the presence of Lu. The results show that hectorite nanoparticles can be mobilized from a bulk sample and that they still contain the dopant Lu, which is homogeneously incorporated into the hectorite crystal structure. The second system is a natural groundwater from the Gorleben site on the northern German plain, which is being tentatively explored to assess its suitability as a nuclear waste repository. Colloidal matter heterogeneity is evident in this system. Alkaline-earth elements are mainly found as ionic species. Rare earth elements (REEs) and actinides are distributed in two main colloidal fractions: the heavier REEs and U are concentrated in the <4 nm fraction corresponding to the size range of organic colloidal particles, whereas lighter REEs and Th are concentrated in colloidal particles between 4 and 18 nm in size that are both organic and inorganic in nature. Similar results are reported for another sample from the same site, collected ~2 km from the first one, demonstrating the homogeneity of the aquifer system and/or a possible colloid migration pathway. The extent of the reversibility of colloid–radionuclide interactions remains to be evaluated.