Field flow fractionation (FFF) is a robust separation technique used to characterize molecules by size. It is a chromatographic-like technique that relies on differences in a molecules laminar flow. The system is similar to an HPLC. However, instead of a column packed with a stationary phase, the FFF system is equipped with a thin flow channel that contains no stationary phase. Molecules nearest to the walls travel the slowest through the channel, while particles at the center of the channel move the fastest. A cross flow/secondary force is applied to the channel that creates a diffusion gradient, which moves larger particles toward the wall.

Asymmetric-flow field flow fractionation (AF4) employs two planar walls with different permeability properties that sandwich the mobile phase. The bottom wall of the separation channel is constructed using a frit with an overlaid membrane, while a liquid-impermeable glass is used for the top wall. The bottom wall allows liquid to pass through, leaving the analytes of interest in the flow channel. The resulting downward cross flow moves the larger particles to the bottom of the channel, where the laminar flow rate is much slower than it is above. A light-scattering detector, which is more commonly associated with size exclusion chromatography (SEC), can be used to measure the particles as they exit the channel. In contrast to SEC, where the larger particles elute first, it is the smaller particles that elute first in AF4.

AF4 is often used to separate and identify particles that are difficult to separate by other methods. LC, electrophoresis/capillary electrophoresis and centrifugation are more common separation techniques, but can be too harsh for certain sample types. AF4 is more gentle and offers a broader application range than competing techniques. Typical biotech and pharmaceutical applications include the separation of viruses, proteins and antibodies. AF4 is also used in environmental, materials science and polymer applications.

There are few commercial systems currently available. Postnova Analytics offers three different AF4 systems in its AF2000 Series product line. Each of the AF4 instruments is separated by the system’s operating temperature. The ambient and mid-temperature systems are used for aqueous or organic measurements, while the high-temperature system is designed for organic separations. Wyatt Technology offers the Eclipse AFFF, which incorporates its multiangle light-scattering instrument.

The total worldwide market for AF4 is less than $10 million and is expected to post solid growth fueled by innovative applications involving biopolymers and nanoparticle research for use in biotechnology and drug discovery. There also continues to be strong interest in other forms of FFF such as micro FFF (lab-on-a-chip) and electric field systems, although these technologies are still under development.

Asymetric-Flow FFF at a Glance:

Leading Suppliers

• Postnova Analytics

• Wyatt Technology

• ConSenxus GmbH

Largest Markets

• Academia

• Biopharmaceutical

• Polymers

Instrument Cost

• $20,000–$40,000