Asymmetric flow field flow fractionation (AF4) an analytical technique used for separating 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 method to separate larger molecules such as proteins, polysaccharides, aggregates such as 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. Hence AF4 can be used for separating various sizes in a mixture and obtaining their solution of physical properties.
The key features of the AF4 are:
- The ability to separate both soluble and colloidal components over a wide size range, There is little or no specific interaction that can affect the elution of the samples (as no column material is used).
- The AF4 system consists of multi-angle laser light scattering (MALLS from Wyatt, Dawn Heleos-II), differential refractive index cell (dRI from Wyatt, Optilab T-rex) and UV absorbance cell (DAD, Diode Array Detector from Dionex).
- The AF4 system can be used with both the flat plate and hollow fibre cylindrical channel type flow cells. The flow conditions and data collection is controlled by Wyatt Eclipse separation system- Dual Tec. Eclipse is coupled with Dionex ultiMate 3000 UHPLC, which contains pump, auto sampler, column compartments and DAD.
- AF4 results have been shown for the elution of single, monomeric proteins (e.g. bovine serum albumin, 66 kDa), larger proteins (Keyhole Limpet Hemocyanin 8.3 MDa), as well as silica particles (41-400 nm), illustrating the broad range of molecular weight / particle size that can (simultaneously) be analysed.