Optimization of asymmetrical flow field-flow fractionation (AF4)

Asymmetrical flow field-flow fractionation (AF4) can be used on a routine basis to separate high-molecular-weight compounds as an alternative to size-exclusion chromatography (SEC), but the principle of separation is very different and a different approach is required for optimization. This article illustrates how to choose the best experimental parameters. Two optimization parameters are proposed: the time required (TREQ) to separate two compounds that differ by a factor of two in molecular weight and the dilution factor (DILF), which describes the ratio of the actual analyte concentrations during the separation process and after elution from the channel. The TREQ parameter should be minimized to obtain fast separations and the DILF parameter should be minimized when detectability is an issue and the overloading of the separation system is a limiting factor. It is shown that in practice a compromise between these two optimization objectives has to be found.