A typical use of asymmetric flow field-flow fractionation (AF4) at SOLVE is for size and molar mass determination. It is applicable to samples with size above ~3 nm / 10 kDa. AF4 is also applicable to aggregate analysis and to the analysis of large polydisperse compounds with tendency to aggregate or adsorb to surfaces. AF4 is as an orthogonal technique to size exclusion chromatography (SEC). One recent application of AF4 is the analysis of protein and antibodies in complex matrixes such as serum and plasma. At SOLVE we use AF4 instrumentation from Wyatt Technology, coupled to UV, RI, MALS, and FL detectors.
Principle:
Asymmetric flow field-flow fractionation (AF4), sometimes more simply shortened field-flow fractionation (FFF), is a separation methodology that separates macromolecules and particles in solution according to differences in size. The separation is based on the macromolecule’s differential location in a flow under the influence of a field (in the case of AF4, a secondary flow). AF4 is commonly used in connection with detectors such as UV to monitor the amount of the size separated components as they are eluted from the channel.
Applicability:
AF4 is a method applicable to a very wide range of compounds, such as proteins, nanoparticles, viruses, and polymers. AF4 is applicable to compounds with molecular weight of ~ 10 000 g/mol and upwards to compounds of approximately 1 µm in size. Typical uses are for stability testing of proteins, aggregation analysis, batch to batch control, size and molar mass distribution.
Pros
- Very wide size range
- High resolution
- Quantitative (with UV or RI)
- Low shear and surface area
- Fractions can be collected
Cons
- Less applicable for <10 000 g/mol components
- Requires some experience to operate
- Molar mass and Mw through the use of standards unless it is coupled to MALS detector.