Applications of Focusing FFF

Focusing FFF methods can be classified according to various combinations of the driving field forces and gradients. The gradients proposed and exploited are:

• effective property gradient of the carrier liquid

• cross-flow velocity gradient

• gradient of the nonhomogeneous field action

Focusing can appear due to the effective property gradient of the carrier liquid in the direction across the channel combined with the primary or secondary transversal field. The density gradient in sedimentation-flotation focusing field-flow fractionation (SFFFFF) or the pH gradient in isoelectric focusing field-flow fractionation (IEFFFF) has already been implemented for separation of polystyrene latex particles and of biological samples. Separation by SFFFFF is carried out according to the density difference of the latex particles. An electric field can be applied to generate the density gradient in a suspension of charged silica particles. The separation by IEFFFF is carried out according to the isoelectric point differences by using the electric field to generate the pH gradient and to focus the sample components. A simple design of a channel for SFFFFF is shown in Figure 8 and an example of the separation of two latex particles according to small density difference is demonstrated in Figure 9. The separation is very rapid and much less expensive when compared to isopycnic centrifugation.

The effective property gradient of the carrier liquid, e.g., the density gradient, can be preformed at the beginning of the channel and combined with the primary or secondary field forces. A step density gradient is formed in such cases but the preforming is not limited to a density gradient.

Figure 8 Schematic representation of the channel for focusing FFF in coupled electric and gravitational fields: (1) flow in; (2) flow out; (3) channel walls forming electrodes; (4) spacer.

Figure 9 Fractogram of two samples of polystyrene latex particles showing a good resolution obtained by focusing FFF while no detectable resolution was achieved under static conditions: (1) injection; (2) stop-flow period; peaks corresponding to particle diameters of 9.87 |im (3) and 40.1 |im (4).

Figure 8 Schematic representation of the channel for focusing FFF in coupled electric and gravitational fields: (1) flow in; (2) flow out; (3) channel walls forming electrodes; (4) spacer.

Figure 9 Fractogram of two samples of polystyrene latex particles showing a good resolution obtained by focusing FFF while no detectable resolution was achieved under static conditions: (1) injection; (2) stop-flow period; peaks corresponding to particle diameters of 9.87 |im (3) and 40.1 |im (4).

The focusing appears in the gradient of transverse flow velocity of the carrier liquid which opposes the action of the field. The longitudinal flow of the liquid is imposed simultaneously. This elutriation focusing field-flow fractionation (EFFFF) method has been investigated experimentally by using a trapezoidal cross-section channel to fractionate micrometre-size polystyrene latex particles but the use of the rectangular cross-section channel is possible.

The hydrodynamic lift forces that appear at high flow rates of the carrier liquid combined with the primary field are able to concentrate the suspended particles into the focused layers. The retention of the particles under the simultaneous effect of the primary field and lift forces generated by the high longitudinal flow rate can vary with the nature of the various applied primary field forces.

The high shear gradient in a carrier liquid can lead to the deformation of the soft particles. The established entropy gradient generates the driving forces that displace the particles into a low shear zone. At a position where all the driving forces are balanced, the focusing of the sample components can appear. Although this method was originally proposed by applying a temperature gradient acting as a primary field and generating the thermal diffusion flux of the macromolecules which opposes the flux due to the entropy changes generated motion, it should be applicable to soft particles as well.

A nonhomogeneous high-gradient magnetic field can be used to separate various paramagnetic and diamagnetic particles of biological origin by a mechanism of focusing FFF. A concentration of paramagnetic particles near the centre of a cylindrical capillary and the focusing of diamagnetic particles in a free volume of the capillary should occur. No experimental results have yet been published.

Other gradients and a variety of the fields can be combined to produce the focusing and to apply these phenomena for PSD analysis. This review of the mechanisms used in focusing FFF should give an idea of their potential.

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