Factors affecting measurement of channel thickness in asymmetrical flow field-flow fractionation

• Factors affecting channel thickness (w) measurement in AF4 was investigated.
• Applicability of retention equations for w measurement in AF4 was discussed.
• Effect of sample–membrane interaction on w measurement in AF4 was studied.

Asymmetrical flow field-flow fractionation (AF4) has been considered to be a useful tool for simultaneous separation and characterization of polydisperse macromolecules or colloidal nanoparticles. AF4 analysis requires the knowledge of the channel thickness (w), which is usually measured by injecting a standard with known diffusion coefficient (D) or hydrodynamic diameter (dh). An accurate w determination is a challenge due to its uncertainties arising from the membrane's compressibility, which may vary with experimental condition. In the present study, influence of factors including the size and type of the standard on the measurement of w was systematically investigated. The results revealed that steric effect and the particles–membrane interaction by van der Waals or electrostatic force may result in an error in w measurement.