Field-flow fractionation (FFF) is a separation technique, but unlike chromatography methods, it does not have a stationary phase. Although the principle is similar to liquid chromatography due to it working on dilute solutions, separation is achieved by applying a field perpendicular to the movement of the sample being pumped through a narrow channel.
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As a result, the sample constituents are concentrated on one side of the channel wall due to the exerting force from the field, known as the accumulation wall. Separation of constituents occurs because of the differing mobilities from the exerting force of the field.
There are different types of forces that can be used to exert the force. These include electric, magnetic, gravitational, thermal, hydraulic and centrifugal.
FFF has a broad dynamic range of sample sizes that can be separated. It is often coupled to HPLC detectors and UV detectors. The primary applications are for separating large macromolecules, nanoparticles, viruses and other biological material.
Pros and Cons of Field-Flow Fractionation
There are several advantages of using FFF ahead of other separation techniques. One advantage is that FFF separates complex molecules that could not otherwise be separated by chromatography. As mentioned above, FFF does not have a stationary phase, so there is less interaction with surfaces.
The separation can be tuned by adjusting the intensity of the separation field. FFF is generally considered a soft technique for separating samples because it does not destroy them, which is especially important for delicate samples. Furthermore, the solution can be modified depending on the sample.
The theory of FFF is well characterized and understood, which means that ideal separation conditions for various samples can easily be achieved without needing method optimization through trial and error. In contrast, the main disadvantage of using FFF regards its application to the separation of small molecules: FFF cannot effectively separate them because of their fast diffusion.
Theory and Methodology
A channel composed of a top and bottom block separated by a spacer is where the separation occurs for FFF. A cavity is created via two methods: by a cut-out within the spacer to create the channel volume that is then sealed between the two blocks, or it is milled on the top block. This channel allows the force field to be applied to the sample.
Upon injection with the sample in dilute solution into the channel, a separation occurs as the sample migrates from inlet to outlet after being pumped through the channel. A detector is positioned after the outlet to analyze the fractions that have been eluted. The readout or graph produced by the separation is called a fractogram and is the detection of signal vs time, based on the sample’s flow velocities under certain fields. The varying degree of velocities of a given constituent may be due to its size or mass. Hence, the identification of the constituents in a sample can be detected by their different elution times.
Applications of Field-Flow Fractionation
Some of the applications of FFF include flow FFF, which separates particles based on size and can measure 1 nm to 1 µm macromolecules. In contrast, asymmetric flow FFF utilizes a semi-permeable membrane on the channel's bottom wall, producing a gentle separation and a broader separation range. Thermal FFF separates samples by applying a temperature gradient to the channel. The temperature differential causes the sample constituents to travel toward the cooler wall.
One of the primary functions of Thermal FFF is the separation of synthetic polymers in organic solvents. Still, it can also separate large molecules by molar mass and chemical composition, which allows for the separation of polymer fractions with the same molecular weight.
Additionally, a gravimetric separation of particles can be achieved by split flow thin-cell fractionation (SPLITT), which uses gravity as the force for separating particles and has the lowest sensitivity of the FFF techniques. Furthermore, centrifugal FFF involves the channel taking the shape of a ring and spinning at high speeds of approximately 5000 rpm.
The sample is pumped into the channel and centrifuged, which can then separate particles by size and density. Hence, two identically size particles can be separated by their differences in density. Finally, another form of FFF (electrical FFF) uses electrical current to create an electric field. This field is applied to the sample and, depending on the charge of the sample, can separate them by electrophoretic mobility and size.
References and Further Reading
Giddings, J. Calvin. (1966) A New Separation Concept Based on a Coupling of Concentration and Flow Nonuniformities. Separation Science. 1, pp. 123–125. https://www.tandfonline.com/doi/abs/10.1080/01496396608049439
Giddings, J.C., Yang F.J., and Myers M.N. (1976). Flow Field-Flow Fractionation: a versatile new separation method. Science, 193(4259), pp. 1244–1245. https://www.science.org/doi/10.1126/science.959835
Madou, Marc (2001). Fundamentals of Microfabrication. [Online] US: CRC. pp. 565–571. Available at: https://www.taylorfrancis.com/books/mono/10.1201/9781482274004/fundamentals-microfabrication-marc-madou
Lee H.L., Reis J.F.G., and Lightfoot E.N. (1974). Single-phase chromatography: Solute retardation by ultrafiltration and electrophoresis. AIChE Journal, 20, p. 776. https://aiche.onlinelibrary.wiley.com/doi/abs/10.1002/aic.690200420
W.J. Cao, P.S. Williams, M. N. Myers, and J.C. Giddings, (1999) Thermal Field-Flow Fractionation Universal Calibration: Extension for Consideration of Variation of Cold Wall Temperature. Analytical Chemistry, 71, pp. 1597– 609. https://pubs.acs.org/doi/10.1021/ac981094m