Asymmetric flow field flow fractionation (AF4) an analytical technique to separate larger molecules, aggregates and particles based on their size. It is an interesting alternative for size-exclusion chromatography (SEC).
The asymmetric flow field flow fractionation (AF4) is a technique to separate larger molecules (e.g. proteins, polysaccharides), aggregates (e.g. heat induced protein aggregates) and particles based on their size. This separation is obtained by the difference in mobility in the flow field induced by the liquid flow over the membrane and across the channel. AF4 can thus be used for separation of various sizes in a mixutre and obtaining their solution of physical properties. The set-up has a number of different detectors, together allowing the determination of:
- molar mass
- root mean squared radius (of gyration)
- second virial coefficient of the eluting compounds
Information obtained by AF4 analysis provides information on the diffusion coefficients, hydrodynamic radius and the shape of the aggregates.
Benefits of the AF4 over size-exclusion column chromatography are:
(1) the ability to separate both soluble and colloidal components over a wide size range, and (2) that in AF4 there is little or no specific interaction that can affect the elution of the samples (as no column material is used).