Asymmetric flow field flow fractionation is a type of field flow fractionation (FFF) separation technique. FFF has been coexisting with size exclusion chromatography (SEC) for several decades. However, unlike SEC, where the development of instrumentation has been driven by other liquid chromatography (LC) techniques, commercially available and reliable FFF instruments were, until recently, unavailable. Although there are many publications dealing with FFF theory (1–4), many fractograms published so far suffer from poor signal-to-noise ratio and the experimental results usually do not prove FFF to be a mature technique for routine applications. For that reason, SEC in either conventional mode using column calibration, or combined with molar mass-sensitive detectors, became a dominating method for the characterization of molar mass distribution of synthetic and natural polymers.

However, recent developments in asymmetric flow FFF provide several advantages for the separation of various polymers, proteins, and particles over traditionally employed SEC. The possible advantages of asymmetric flow FFF over SEC include a broad separation range accessible by a single channel, a significantly reduced risk of shearing degradation, elimination of polymer interactions with column packing, the possibility of injecting large sample volumes (needed for samples that can be prepared only in low concentrations), the rapid change of mobile phase, the control of resolution by cross flow and other separation conditions, and efficient separation of branched polymers. It is also worth mentioning that in the case of SEC, the expensive columns often are damaged by deposition of samples in the columns. This does not happen with FFF, in which the separation takes place in an empty channel. If the channel membrane is contaminated, its replacement is very easy.

The goal of this article is to present asymmetric FFF as a mature analytical separation technique, capable of providing separation efficiency comparable to SEC, and in addition, solving several traditional SEC drawbacks. The focus of the article is not on aqueous applications, in which asymmetric flow FFF–multiple angle light scattering (MALS) has been used successfully (5–7), but mainly on the characterization of synthetic polymers in tetrahydrofuran, an area in which the applications have so far been sporadic (8).

Asymmetric flow FFF is a separation analytical technique similar to LC. Unlike LC, the asymmetric flow FFF method has no stationary phase and the separation is achieved solely by a flow in an empty channel, where a perpendicular flow force is applied. The channel consists of two plates bolted together that are separated by a spacer foil with a typical thickness of 350 μm. The upper plate is impermeable, while the bottom plate is permeable, made of porous frit covered by a semipermeable membrane with a typical cutoff of 5–10 kDa. The membrane is permeable to the molecules of mobile phase but not permeable to the polymer molecules and colloidal particles, and therefore, it keeps the sample in the channel and directs the sample by flow to the channel outlet. The laminar flow of the mobile phase creates a parabolic flow profile within the channel; that is, the stream moves slower close to the channel walls than it does in the channel center.

The analyzed molecules and particles are driven by the cross flow toward the bottom wall of the channel. Diffusion creates a counteracting motion, causing smaller particles, which have a higher diffusion coefficient, to move closer to the channel center, where the longitudinal flow is faster. The velocity gradient inside the channel separates the molecules and particles according to their hydrodynamic size, so that smaller molecules are eluted before the larger ones. This means that the asymmetric flow FFF separation is opposite to SEC separation, in which the large molecules are eluted first. Note: Steric separation, which can occur for large molecules and particles and where the elution order is reversed, is not discussed here and the information can be found elsewhere (9).