Interdisciplinary studies on aquatic environments and cross-validation of laboratory vs. field results will likely increase the need for simultaneous use of large- and small-scale ultrafiltration systems. In this study, a comparison of two ultrafiltration systems differing in scale (PrepScale and PelliconXL, Millipore; membrane areas 0.54 m2 and 0.005 m2, respectively), was made for the cut-offs 3 and 300 kDa. Large systems are useful for their high permeate throughput, while small systems are necessary when the amount of sample is limited. The ability of PrepScale and PelliconXL systems to provide comparable results for organic carbon fractionation was studied for polysaccharide solutions and natural freshwaters. In the latter, the colloidal proportions of different trace metals (V, Cr, Mn, Co, Ni, Cu, Sb, and U) were also determined. Although the colloidal proportions obtained with PelliconXL 3 kDa were sometimes slightly higher than with PrepScale 3 kDa (principally for DOC and U in natural waters), Mann–Whitney statistical test showed no significant difference in the overall fractionation properties of the two systems. Our observations show that reaching high concentration factors lead to a strong modification of colloids size distribution in the range 50–2,000 nm and thus low concentration factors are preferable to preserve the colloid integrity.