Field flow fractionation
Field-flow fractionation, abbreviated FFF,[1] is a separation technique invented by J. Calvin Giddings.
Separation is achieved by applying a field (hydraulic, centrifugal,[2] thermal,[3] electric,[4] magnetic,[5] gravitational, ...) or cross-flow, perpendicular to the direction of transport of the sample, which is pumped through a long and narrow laminar channel.
As an example, for the hydraulic, or cross-flow FFF method, the property driving separation is the translational diffusion coefficient or the hydrodynamic size.
The transition from normal to steric mode takes place when diffusion becomes negligible at sizes above a micron.
FFF is unique in its wide dynamic range of sizes covering both soluble macromolecules[9] and particles or colloids which can be separated in one analysis.
Coupling with Multi angle light scattering allows to calculate the size of eluting fractions and compare to values obtained via FFF theory.
FFF has a well worked-out theory, which can be used to find separation conditions to reach the optimal result, without a series of trial-and-error experiments.
For an effective separation, the sample has to be concentrated very close to the accumulation wall (a distance less than 10 μm), which requires the drift velocity caused by the force field to be two orders of magnitude higher compared to the diffusion coefficient.
The maximum field strength which can be generated in an FFF channel determines the lower size range of separation.
FFF behaves differently from column chromatography and can be counter-intuitive for HPLC or SEC users.
Giddings, credited for the invention of FFF, was professor of chemistry and specialist of chromatography and separation techniques at the University of Utah.
As mentioned above, in field-flow fractionation the field can be hydraulic (with a cross flow through a semi-permeable membrane as the accumulation wall), gravitational, centrifugal, thermal, electrical, or magnetic.
In all cases, the separation mechanism is produced by differences in particle mobility under the forces of the field, in a stationary equilibrium with the forces of diffusion: The field induces a downward drift velocity and concentration towards the accumulation wall, the diffusion works against this concentration gradient.
This is best visualized as a particle cloud, with all components in constant motion, but with an exponential decrease of the average concentration going away from the accumulation wall up into the channel.
Giddings and co-workers have developed a theory describing the general retention equation which is common to all FFF methods.
When these two transport processes reach equilibrium the particle concentration c approaches the exponential function of elevation x above the accumulation wall as illustrated in equation (1).
of each component can be related to the force applied on each individual particle or to the ratio of the diffusion coefficient D and the drift velocity U.
From this basic principle many forms of FFF have evolved varying by the nature of the separative force applied and the range in molecule size to which they are targeted.
In FFF the display of detector signals as a function of time is called fractogram, in contrast to the chromatogram of column chromatography techniques.
The fractogram can be converted to a distribution plot of one or several physical properties of the analyte using FFF theory and/or detector signals.
The varying velocities of a particular species of particles may be due to its size, its mass, and/or its distance from the walls of a channel with non-uniform flow-velocity.
Flow FFF separates particles based on size, independent of density and can measure macromolecules in the range of 1 nm to 1 μm.
Main applications are in pharmaceutical research and development for proteins, virus and virus-like particles, and liposomes.
Thermal FFF was developed as a technique for separating synthetic polymers in organic solvents.
[15] Split flow thin-cell fractionation (SPLITT)[16] is a special preparative FFF technique, using gravity[17] or electric,[18] or diffusion differences for separation of over μm-sized particles on a continuous basis.
The use of gravity alone as the separating force makes SPLITT the least sensitive FFF technique, limited to particles above 1 μm.
The flow and sample are pumped into the channel and centrifuged, allowing the operator to resolve the particles by mass (size and density).
The unique advantage presented by centrifugal FFF comes from the techniques capability for high resolution given sufficient buoyant density.
With the addition of the third parameter of density to centrifugal FFF, this produces a ratio more akin to mass:time to the power of three.
EAF4 overcomes the limitation of pure electrical FFF which has poor resolution and suffers from electrolysis products and bubbles contaminating the channel outflow and compromising the detector signals.
