Application scientist Felix Deluweit, Wyatt Technology Europe, Dernbach, Germany

Characterization of Proteins, Large Biomolecules and Particles Using Multi Angle Light Scattering and Flow Field-Flow Fractionation

Originally invented by Calvin Giddings in 1966 Field-Flow Fractionation (FFF) has been massively developed over the last decades. The asymmetrical flow field-flow fractionation (AF4) – a part of the FFF family - is a widely accepted orthogonal method to size exclusion chromatography (SEC). It is a one-phase chromatography technique with a separation range from a couple of thousand Daltons to the colloidal range of hundreds of nanometers size. Thus, it is used for the separation of many types of molecules, such as proteins, synthetic polymers, biopolymers, but also for nanoparticles and sub-cellular structures. The main advantage of AF4, compared to SEC, is the possibility to control the separation force. Moreover, AF4 creates less shear forces and offers molecules and particles less surface. Therefore, the possibility of unwanted interactions is reduced. Following separation, molar mass, size and concentration over every peak of the chromatogram can be calculated using multi-angle light scattering (MALS). Online MALS measures the molar masses of the eluting molecules utilizing the amount of their scattered light. At least one concentration sensitive detector is required for the molar mass measurement using MALS. UV or differential refractive index (dRI) detection is typically used in combination with the MALS detector, often both to enable co-polymer or protein-conjugate analysis.