The main objective when utilizing field-flow fractionation is to separate a sample into different components. Asymmetrical (flow) field-flow fractionation, AF4, is the most widely used field-flow fractionation technique. AF4 fractionates analytes by size and a unique feature is the extremely broad range of sizes that can be investigated (approximately 2 nm–50 µm). In AF4, separation occurs in an open channel without the presence of a packed stationary phase. This imparts a tremendous capability to fractionate polydisperse, fragile and/or shear sensitive samples because of the relatively low operating pressures and shear forces which result in a mild treatment of the sample during separation.

A variety of in-line detection systems can be connected to the channel such as light scattering, refractive index, UV absorbance, fluorescence, ICP-MS, x-ray scattering etc. Also, because AF4 is an elution technique, sample fractions are easily collected for additional off-line analysis by MALDI-TOF-MS, SDS-PAGE, etc.

AF4 applications include synthetic, biological and natural macromolecules, proteins and protein aggregates, nanoparticles, latex and silica particles, clay particles, and more. Field-flow fractionation is listed by US FDA (Feb 2012) as a preferred technique for the characterization of protein aggregation in protein based therapeutics.

The field-flow fractionation channel with two sample components. In AF4, the field applied is a cross flow (Fc) passing through the accumulation wall counteracted by molecular diffusion (D). From Wahlund, K. G., & Nilsson, L. (2012). In S. K. R. Williams & K. Caldwell (Eds.), Field-flow fractionation in biopolymer analysis (pp. 1-21). Wien: Springer Verlag.