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
The principle and experimental set-up for flow FFF are shown schematically in Figure 1. The separation in flow FFF is performed
with a carrier liquid pumped through a flat channel, formed by a spacer between two walls (although a hollow fiber can also
be used). In the first (so-called symmetric) system both walls were porous, and a second pump was used that drove a flow of
the same carrier liquid in the perpendicular direction, through both walls of the channel. Macromolecular sample components
are retained in the channel by an ultrafiltration (UF) membrane on top of one of the porous walls. Later, Wahlund and Giddings
(14) proposed a simplified instrumental system, with only one porous wall, for what was called asymmetrical flow FFF. In asymmetrical
flow FFF the in-going flow (F
in) is split, with the help of flow regulators or an extra pump, in two parts. One part, the channel flow (F
OUT), is flushed through the channel in the axial direction towards the detection side outlet. The other part of the in-going
flow, the cross flow (F
C), passes through the ultrafiltration membrane and the porous wall. The asymmetrical flow FFF set-up requires one pump or
flow regulator less than a symmetric system. Commercial instruments use the asymmetrical flow FFF principle.
Figure 1: Experimental set-up for (a) symmetrical and (b) asymmetrical flow FFF.
The procedure for a separation by asymmetrical flow FFF includes different steps (see Figure 2). First, a specific volume
of the sample solution is introduced in the channel. This can be done with the inlet flow or through an extra inlet port close
to the carrier solution inlet. During and after sample injection a part of the in-going flow enters the channel from the inlet
side, but most of the carrier liquid is pumped into the channel from the back end. In this way macromolecules or particles
(anything that can not pass the ultrafiltration membrane) introduced into the channel are concentrated in a thin layer on
top of the ultrafiltration membrane and focused in a thin band close to the inlet port.
Figure 2: Flow regimes in asymmetrical flow FFF during (a) sample injection and focusing and (b) elution.
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.