Asymmetrical flow field-flow fractionation (AF4 or Asymmetrical FFF) is the most widely used field-flow fractionation technique. AF4 fractionates analytes by size and has the particular advantage of allowing the investigation of a very broad range of sizes (nanometer to micrometer range). The fractionation takes place in an open channel without the presence of a packed stationary phase. This imparts low shear forces and low operating pressures during analysis, making it possible to fractionate polydisperse, fragile and/or shear sensitive samples.

A variety of in-line detection systems can be connected to the AF4 channel, such as multiangle light scattering (MALS), refractive index (RI), UV absorbance, fluorescence, x-ray scattering etc. Because AF4 is a fractionation technique, sample fractions are easily collected for further complementary investigations by, for example, ICP-MS, MALDI-TOF-MS, SDS-PAGE, among others.

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.

(Image from Wahlund, K. G.; Nilsson, L. Field-flow fractionation in biopolymer analysis;
Williams, S. K. R., Caldwell, K. D., Eds.; Springer-Verlag: Wien, 2012, p 1.)

The AF4 separation channel consists of an ultrafiltration membrane (typical cut off 5–10 kDa) placed on top of a porous frit. A carrier liquid is pumped into the channel to create a longitudinal flow called channel flow (Fin and Fout), and a cross flow (Fc) which is a perpendicular flow originated by liquid exiting through the ultrafiltration membrane.

After the sample is injected, the cross flow transports the sample components towards the accumulation wall. The transport induced by the cross flow is counteracted by the Brownian motion of the sample components which – at steady state – establishes a concentration profile for each hydrodynamic size present in the sample.

This means that small sample components (i.e. higher diffusion coefficient) are distributed further away from the accumulation wall compared to larger components (i.e. lower diffusion coefficient). As the longitudinal flow along the channel (channel flow) is laminar with a parabolic flow profile, the flow velocity increases as we move away from the accumulation wall towards the center of the channel. Hence, small sample components are transported more rapidly out from the channel compared to larger components and, thus, separation is achieved.