Resolving Heterogeneity in Bioconjugates, Biohybrid Assemblies, and Functional Nanocarriers by Flow Field Flow Fractionation and Multidetection

Abstract Bioconjugates and biohybrid assemblies are intrinsically heterogeneous, with distributions in size, molar mass, conjugation degree, composition, architecture, cargo loading, aggregation state, and functional accessibility governing their stability, performance, and biological activity. Flow field-flow fractionation (flow FFF), particularly asymmetric flow FFF (AF4), and hollow-fiber flow FFF (HF5) provide a mild separation platform for resolving such heterogeneous populations. Combined with orthogonal online detection and fraction-based analyses, flow FFF can relate hydrodynamic separation to molar mass, composition, cargo association, surface functionalization, internal organization, and biological function. This review evaluates multidetector and multidimensional flow FFF workflows across macromolecular conjugates, affinity-driven assemblies, biofunctionalized nanoparticles, nucleic acid complexes, nanocarriers, vesicles, and viral assemblies. We discuss key controls for reliable interpretation, including recovery, membrane and focusing effects, coelution, detector selectivity, and band broadening, and highlight emerging integrated FFF−LC−MS, structural, automated data analysis, and multi-attribute quality assessment workflows.