Asymmetric Flow-Field-Flow-Fractionation (AF4)
With Asymmetric Flow-FFF (AF4) the flow and sample are confined within a flat channel consisting of two plates that are separated by a spacer foil; the plates are then bolted together. The spacer foil has a typical thickness of 100 to 500 µm. The upper channel plate is impermeable. The bottom channel plate, on the other hand, is permeable, and made of a porous frit material. An ultra filtration membrane with a typical size barrier of 10kD, covers the bottom plate to prevent the sample from penetrating the channel.
Within the flow channel a parabolic flow profile is created because of the laminar flow of the liquid: the stream moves slower closer to the boundary edges than it does at the center of the channel flow. When the perpendicular force field is applied to the flowing, laminar stream, the analytes are driven towards the boundary layer the so-called "accumulation wall" of the channel.
Diffusion associated with Brownian motion, in turn, creates a counteracting motion. Smaller particles, which have higher diffusion rates, tend to reach an equilibrium position higher up in the channel, where the longitudinal flow is faster. Thus, the velocity gradient flowing inside the channel separates different sizes of particles.
The smaller particles are transported much more rapidly along the channel than the larger particles. This results in the smaller particles eluting before the larger ones; the opposite of a Size Exclusion/Gel Permeation Chromatography (SEC/GPC) separation in which the large molecules elute first.
With AF4/ FFF separation there is no column media to interact with the samples, so for very high molar mass polymers, there is no need to worry about shearing forces being applied. The entire separation is gentle, rapid, and non-destructive without a stationary phase that may interact, degrade, or alter the sample.
The separation process requires three steps: During the first two steps, injection and focusing, the main flow is split, enters the channel from both ends and is balanced to meet under the injection port. At this point the flow will move only down and permeate through the membrane. When the sample is injected it is focused in a thin band and concentrated towards the membrane. After complete transfer of the sample volume the injection flow is stopped and one typically allows for another minute of focusing before the flow pattern is switched to the elution mode. Now the flow enters only from the inlet port and exits at the outlet which is connected to the detectors. Sample constituents elute separated according to size and are monitored by the array of detectors.
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