The comprehensive characterization of a wide range of analytes and samples using Asymmetrical Flow Field-Flow Fractionation (AF4) coupled with various detection techniques has been demonstrated in numerous studies. In the present work, we describe the in-line coupling of AF4 with Small-Angle X-ray Scattering (SAXS) to enable detailed characterization of complex sample compositions. SAXS is a versatile technique for the characterization of pharmaceutically relevant macromolecules, providing information on their absolute concentration, size, shape, and internal structure [1,2]. A prerequisite for reliable SAXS analysis is the availability of highly pure and monodisperse analytes. This limitation can be addressed by coupling SAXS with AF4, which separates sample constituents according to their hydrodynamic size prior to SAXS analysis, thereby enabling size-resolved structural characterization. Here, we present the in-line coupling of multi- detector AF4 with SAXS detection, allowing quantitative, multi-attribute analysis of mRNA nanoparticles within a single measurement.
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