Flow FFF

Flow FFF is obtained by a crossflow of the mobile phase, usually through a semipermeable membrane. This can be performed with a symmetric channel with two semipermeable walls, with an asymmetric channel with only one permeable wall, or with a hollow fiber channel. FFFF is the most universal FFF technique. 

This equation, combined with Equation 8.1, tells us that small particles are less retained than larger particles in FFFF, as long as the particle diameters are much smaller than the channel diameter. 

Flow field-flow fractionation (FFFF) has been until now the most universally used subtechnique of FFF. With this subtechnique, the flow of the solvent, perpendicular to the flow of the basic medium in the channel, is an external field. The earliest works belonging to this class of FFFF were published by Lee and co-workers 

Giddings and co-workers [53,54] designed the FFFF channel in a classical manner, i.e., using two planparallel semi-permeable membranes, and a thin (0.38 mm) spacer into which the shape of the channel was cut. They developed theoretical bases of FFFF and fractionated successfully a series of monodisperse spherical PS latex and a number of proteins. They reached an excellent agreement between the theoretical assumptions of the retention and the experimental results. As with other FFF subtechniques, the agreement between the theoretical and the experimental characteristics of dispersion was poorer. The problem further requires a more extensive study. 

In FFFF the perpendicular flow having the velocity U acts on all of the solutes uniformly. For this reason the separation in FFFF is determined only by the differences in the values of the diffusion coefficient, D, or the friction coefficient, /. The retention parameter. A, is then determined by the relationship [9] 

The effect of relaxation on the retention and resolution in FFFF was studied in further detail in the subsequent work [55]. A substantial improvement in the resolution of the fractionation of f2 virus was proved as long as the stop-flow technique was being applied after the injection into the channel within the relaxation time sufficient for the establishment of the quasi-equilibrium. 

FFFF can be applied as a dialysation or ultrafiltration cell [56] to a continuous 

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FFF selectivities assume limiting values at high retention level for sedimentation FFF (SdFFF), 5j=3, whereas S nj= 1 for flow FFF (FIFFF), the limiting value of 5 is unitary. For ThFFF, the 5 of dissolved polymers depends on the polymer-solvent system with typical values in the 0.5-0.7 range. In ThFFF particle separation, a wide range of values have been reported [3],... 

The most common, and commercially available, FFF variants are flow FFF (FIFFF), sedimentation FFF (SdFFF), and thermal FFF (ThFFF). These techniques are described in greater detail... 

Detailed Instrumentation and Separation Operation Modes 12.3.2.1 Cross-Flow FFF... 

Cross-flow FFF or, as it was known in the past, FIFFF draws its name from the field type used to transport sample components across the channel thickness to the accumulation wall [3,8]. The... 

To generate this kind of field, that is, the cross-flow, the channel walls must be made semiperme-able, and a membrane must act as an accumulation wall so that carrier liquid is allowed to pass but not the analyte. Cross-flow FFF can be broken down into symmetric, asymmetric, and cylindrical configurations (see Figure 12.8). 

FIGURE 12.8 Cross-flow FFF variants. Side view of (a) symmetric, (b) asymmetric, and (c) HF channels. Top view of the channels (a) symmetric, (b) trapezoidal, and (c) capillary. 

When the channel has a trapezoidal shape [asymmetrical flow FFF (AsFlFFF)—see Figure 12.8], the cross-flow rate can be programmed to decrease linearly or exponentially. The commercially available power cross-flow program (Nova FFF software) has the following expression [16] ... 

Field flow fractionation (FFF), as a gentle size fractionation coupled to ICP-MS, offers the capability to determine trace metals bound to various size fractions of colloidial and particulate materials.112 On line coupling of FFF with ICP-MS was first proposed by Beckett in 1991 -113 Separation is achieved by the balance between the field force and macromolecular diffusion in the FFF channel. Depending on the field force used, FFF is classified into different techniques such as sedimentation, gravitational, electrical, thermal and flow FFF.112... 

By coupling flow field-flow fractionation (flow FFF) to ICP-MS it is possible to investigate trace metals bound to various size fractions of colloidal and particulate materials.55 This technique is employed for environmental applications,55-57 for example to study trace metals associated with sediments. FFF-ICP-MS is an ideal technique for obtaining information on particle size distribution and depth profiles in sediment cores in addition to the metal concentrations (e.g., of Cu, Fe, Mn, Pb, Sr, Ti and Zn with core depths ranging from 0-40 cm).55 Contaminated river sediments at various depths have been investigated by a combination of selective extraction and FFF-ICP-MS as described by Siripinyanond et al,55... 

Cross-flow FFF (F1FFF) utilizes a second fluid flow to transport sample components across the channel thickness to the accumulation wall, and the position of individual species in the laminar carrier profile corresponds to their ordinary (Fick s) diffusion coefficient. As the particle size increases, the diffusion coefficient (decreases until it becomes a relatively insignificant transport process. For micron-size particles, the extent of protrusion into the channel becomes the decisive factor in determining the order of elution. 

Among the FFF techniques available, cross-flow FFF is the most used to separate components of humic materials. Size characteristics were reported by several authors (Baalousha et al., 2006 Boehme and Wells, 2006 Geckeis et al., 2003 Siripinyanond et al., 2005 Suteerapataranon et al., 2006 Zanardi-Lamardo et al., 2002). Over the past decade, about 20 papers were published on F1FFF fractionation of humic... 

Excluded from this list is sieving, to which the concept of selectivity is not applicable. For completeness, we have subdivided the FFF family into sedimentation FFF, thermal FFF, flow FFF, and steric FFF to show how the selectivity of each of these subtechniques compares to that of the other fractionation methods. The values reported here differ from S values reported elsewhere (12), which refer to mass rather than size selectivity. 

In the fourth subtechnique, flow FFF (F/FFF), an external field, as such, is not used. Its place is taken by a slow transverse flow of the carrier liquid. In the usual case carrier permeates into the channel through the top wall (a layer of porous frit), moves slowly across the thin channel space, and seeps out of a membrane-frit bilayer constituting the bottom (accumulation) wall. This slow transverse flow is superimposed on the much faster down-channel flow. We emphasized in Section 7.4 that flow provides a transport mechanism much like that of an external field hence the substitution of transverse flow for a transverse (perpendicular) field is feasible. However this transverse flow—crossflow as we call it—is not by itself selective (see Section 7.4) different particle types are all transported toward the accumulation wall at the same rate. Nonetheless the thickness of the steady-state layer of particles formed at the accumulation wall is variable, determined by a combination of the crossflow transport which forms the layer and by diffusion which breaks it down. Since diffusion coefficients vary from species to species, exponential distributions of different thicknesses are formed, leading to normal FFF separation. 

Flow FFF has been applied to a variety of species including proteins, water-soluble synthetic polymers, organic-soluble synthetic polymers, and various colloidal particles. The fractionation of proteins is illustrated in Figure 9.10. 

Flow FFF is the most versatile FFF subtechnique (and one of the most versatile of all separation techniques) because it requires nothing more than the interaction of the transverse stream with a suspended or dissolved component. This interaction is made up of the drag forces which induce... 

Figure 9.10. Rapid separation of three proteins by flow FFF at a channel flowrate of 8.0 mL/min and a crossflow rate of 6.8 mL/min. (Courtesy of Min-Kuang Liu, University of Utah.)...

Fig. 26. Schematic design of field flow fractionation (FFF) analysis. A sample is transported along the flow channels by a carrier stream after injection and focusing into the injector zone. Depending on the type and strength of the perpendicular field, a separation of molecules or particles takes place the field drives the sample components towards the so-called accumulation wall. Diffusive forces counteract this field resulting in discrete layers of analyte components while the parabolic flow profile in the flow channels elutes the various analyte components according to their mean distance from the accumulation wall. This is called normal mode . Particles larger than approximately 1 pm elute in inverse order hydrodynamic lift forces induce steric effects the larger particles cannot get sufficiently close to the accumulation wall and, therefore, elute quicker than smaller ones this is called steric mode . In asymmetrical-flow FFF, the accumulation wall is a mechanically supported frit or filter which lets the solvent pass the carrier stream separates asymmetrically into the eluting flow and the permeate flow which creates the (asymmetrical) flow field...

This review will introduce the basic principles, theory, and experimental arrangements of the various FFF techniques focusing on the most relevant for praxis Sedimentation-FFF (S-FFF),Thermal-FFF (Th-FFF) and Flow-FFF (Fl-FFF). In a second part,selected applications of these techniques both to synthetic and biological samples will illustrate applications under a variety of conditions, where problems and potential pitfalls as well as recent developments are also considered. 

FFF techniques were pioneered by Giddings in 1966 [1]. Starting from this point, a remarkable development has taken place resulting in a diversity of different FFF methods. Figure 1 gives an overview of the different techniques with their time of invention. The number of different methods is directly related to the variety of force fields which can be applied for the separation of the samples. Practically, only three of those FFF methods are commonly used and commercially available at the present time namely sedimentation-FFF (S-FFF), flow-FFF (Fl-FFF) and thermal-FFF (Th-FFF). The range of possible techniques was established in the early years whereas the main development of the last years is seen in a continuous optimization of the methodology and the instrumentation. This becomes most evident for the case of flow-FFF, where an asymmetrical channel with better separation characteristics has been developed. 

For samples with a broad size distribution in the micron range, it is important to avoid the transition region between the normal and the steric mode during the measurement. This can be achieved by proper adjustment of the channel thickness, channel flow and the strength of the applied field [69]. The transition region in Fig. 6 can be experimentally determined by plotting the retention ratio vs. the particle size, as illustrated in Fig. 7 for the example of flow-FFF. 

If a continuous viscosity detector is coupled to an FFF channel, viscosity distributions and intrinsic viscosities can be measured without calibrating the channel [76]. The coupling of one FFF instrument to another opens the possibility of obtaining two-dimensional property distributions of complex materials the combination of sedimentation- and flow-FFF provides the size-density distribution of complex colloids, whereas a combination of thermal- and flow-FFF yields the composition-molecular weight distribution of copolymers. 

Flow-FFF. The measurement of retention in flow-FFF directly yields the diffusion coefficient D and the related hydrodynamic diameter dH which is related to... 

Thermal-FFF. The retention rate directly yields the Soret coefficient DT/D. If D is known (for example from flow-FFF), the thermal diffusion coefficient DT can be obtained which can give information about the chemical sample composition. Unfortunately, no context is known which analytically relates DT with the sample composition [84]. On the other hand, for known DT values (material constant), the diffusion coefficient distribution is directly obtained. 

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