Asymmetrical flow field-flow fractionation (FFF) is a versatile technique for the size-based separation of macromolecules, molecular aggregates, colloids, and solid particles. The first experimental study on flow FFF by J.C. Giddings and colleagues (1) was published over 40 years ago, but for a long period the development and application of asymmetrical flow FFF (or its symmetrical variant) was largely limited to a small number of research laboratories. This was at least partly related to a lack of suitable ready-to-use instruments. In recent years, however, several reliable asymmetrical flow FFF instruments have become available on the market and the use of them in routine applications has become more widespread. Several recent literature reviews can be found on the application of FFF in various application fields (2–6).
In many laboratories it has been shown that asymmetrical flow FFF can be used as an alternative for size-exclusion chromatography (SEC), with a strongly extended molecular-size range. It is typically used for ultrahigh molecular weight (UHMW) polymeric materials such as starches and other carbohydrates (7,8), for the study of protein aggregation (9,11), or for the fractionation and characterization of synthetic and natural colloids (12,13). However, the separation principles and characteristics of asymmetrical flow FFF and SEC are quite different and optimization strategies and rules-of-thumb for SEC are not always applicable in asymmetrical flow FFF. In this study, we have tried to derive some rules for the optimization of asymmetrical flow FFF separations, starting from the first principles of the technique, and we have validated the outcome with experimental data on the separation of a number of model compounds (proteins).
The Principles of Asymmetrical Flow FFF
After this focusing step the elution can start. A valve is switched to change the flow regime. The carrier liquid now enters the channel from the inlet side, with a regulated part leaving the channel through the porous wall and the remainder flowing to the detector. The channel flow is laminar (that is, its velocity is high in the center of the channel and zero at the walls). Sample components, concentrated by the cross flow in a thin layer on top of the ultrafiltration membrane, are all flushed with the channel flow towards the detector, but the velocity of a sample band depends on the thickness of the layer into which the particles or macromolecules are concentrated: the smaller the layer thickness of a band, the lower its velocity in the axial direction. The layer thickness for a specific analyte, in the steady state situation after focusing, depends on the cross-flow velocity on the one hand and the molecular diffusion of the analyte on the other. Because the cross-flow velocity is an instrumental parameter, elution velocity and retention time differences between analytes are purely based on differences in diffusion constant and, by that, in molecular size.