Field-flow fractionation: New and exciting perspectives in polymer analysis

Field-flow fractionation (FFF) is a family of flexible elution techniques capable of simultaneous separation and measurement. Its sample domain extends across a broad macromolecular-colloidal-particulate continuum from about 1 nanometer to more than 100 micrometers and incorporates both simple and complex macromaterials of biological, biomedical, industrial, and environmental relevance. Complex materials are separated into components to simplify measurement. Component properties measurable by FFF include mass, size, density, charge, diffusivity, and thickness of adsorbed layers. When characterization by these properties is inadequate, other measurement tools can be readily coupled to FFF, either off-line or on-line, by virtue of its flow-elution operation. This article describes the principles and major subtechniques of the FFF family along with application of its measurement and separative capabilities.

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.