Field Flow Fractionation FFF

Field flow fractionation (FFF) is an elution technique suitable for molecules with a molecular weight 1000 up to a particle size of some 100 pm. Separating, driving, external field forces are applied perpendicular to a liquid carrier flow, causing different species to be placed in different stream lines (Fig. 26). Useful fields are gravity, temperature, cross flow, electrical charge, and others [128-131]. 

The range of the (molecular) size of the analytes usually exceeds that which can be determined by classical laboratory analytical methods such as size exclusion chromatography, etc. [351]. Reports on investigated substances are widespread and cover applications such as the separation and characterization of proteins [450] and enzymes [240, 241], of viruses [132], the separation of human and animal cells [50, 51], the isolation of plasmid DNA [367], and the molecular weight and particle size distribution of polymers [216,217]. The approach is relatively new in biotechnology therefore, practical experiences are not yet abundant. Langwost et al. [229] have provided a comprehensive survey of various applications in bio-monitoring. 

The classical way to determine biomass concentration is typically an off-line method, namely to harvest a known aliquot of the culture suspension, separate cells by centrifugation, wash the cells and dry them to constant weight at a few degrees above the boiling point of the solvent (i.e. aqueous medium, usually 

105 °C). After gravimetric determination of the dry mass in the vial or on a filter, the mass concentration in the original sample volume can easily be calculated if there are neither particulate components in the fresh medium nor precipitates formed during cultivation. 

What does the cell dry weight concentration mean and teach us about the progress and behavior of a bioprocess The analytical value reflects only the cellular mass and does not allow a distinction between structural or storage materials and biocatalytically active components of the cells on the one hand, or between live and dead cellular material on the other. 

Field flow fractionation was developed about the same time as size-exclusion chromatography. However, major instrumentation problems were not solved as quickly for FFF as they were for SEC, and hence it has lagged behind in terms of its commercial exploitation. FFF is a chromatography-like separation method which consists of a family of techniques applicable to the separation and characterisation of macromolecules and colloidal and particulate matter, from a molecular weight of a few thousand up to 100 xm particle diameter. 

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Another area of rapid growth for particle separation has been that of Field-Flow Fractionation (FFF) originally developed by Giddings (12,13>1 1 ) (see also papers in this symposium series). Like HDC, the separation in field-flow fractionation (FFF) results from the combination of force field interactions and the convected motion of the particles, rather than a partitioning between phases. In FFF the force field is applied externally while in HDC it results from internal, interactions. 

There are many combinations of separations techniques and methods of coupling these techniques currently employed in MDLC systems. Giddings (1984) has discussed a number of the possible combinations of techniques that can be coupled to form two-dimensional systems in matrix form. This matrix includes column chromatography, field-flow fractionation (FFF), various types of electrophoresis experiments, and more. However, many of these matrix elements would be difficult if not impossible to reduce to practice. 

In conclusion one can say that SEC is a very powerful method for polymer characterization, especially in combination with other composition sensitive or absolute calibration methods. A big advantage is also that the sample amount is fairly small, typically 10 mg. For more complex polymers, such as polyelectrolytes, enthalpic effects often become dominant and also for rather high molecular weight polymers chromatographic methods such as field-flow fraction (FFF) techniques might be more suitable. For fast routine measurements linear columns are often used. 

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... 

One such consequence is their use in the physical characterization of colloidal dispersions and macromolecular solutions. Let us highlight one such application through one element of a class of analytical separation techniques known as field flow fractionation (FFF). 

In principle, all powerful element-specific methods that are able to monitor continuously the effluents of separation processes commonly in the range of a few mimin-1 and in element concentrations of some Klpg liter-1. A well-suited method is based on modern element-specific quadrupole mass spectrometry (MS) with an inductively coupled plasma (ICP) interface to the separation unit [e.g., liquid chromatography (LC) or field-flow fractionation (FFF)].Tlie ICP-MS detection can also be used for continuously characterizing the effluent of any kind of packed column (Metreveli and Frimmel, 2007). By this, the transport and elution properties of... 

Another technique widely used for size separation of humic materials is field-flow fractionation (FFF) (e.g., Baalousha et al., 2006 Boehme and Wells, 2006 Geckeis et al., 2003 Hassil ov et al., 2007 Siripinyanond et al., 2005 Suteerapataranon et al., 2006 Zanardi-Lamardo et al., 2002). This technique was developed and introduced in 1966 by Giddings (1966) as a method for the separation and characterization of materials ranging in size from macromolecules to particulates. Similar to SEC, FFF... 

Figure 13.6. Separation principle of field-flow fractionation (FFF) is based on physical interactions of particles within an applied field and subsequent field-induced migration to the FFF channel wall ( accumulation wall ). Molecules, depending on their size and diffusion coefficient, are distributed over different velocity lines of axial flow, and they separate accordingly. Larger particles possess less diffusional motion and higher interaction with the applied field hence, they will be caught up in slower-moving streams near the channel wall and elute later than smaller particles.

Some of these fractionation problems can be ameliorated by the use of the relatively new technique of field-flow-fractionation (FFF). Its advantages include high-resolution separation and sizing of particulate, colloidal and macromolecu-lar materials covering 105-fold range from about 10 3 to 1()2/rm (see Chapter 8). 

This paper outlines the basic principles and theory of sedimentation field-flow fractionation (FFF) and shows how the method is used for various particle size measurements. For context, we compare sedimentation FFF with other fractionation methods using four criteria to judge effective particle characterization. The application of sedimentation FFF to monodisperse particle samples is then described, followed by a discussion of polydisperse populations and techniques for obtaining particle size distribution curves and particle densities. We then report on preliminary work with complex colloids which have particles of different chemical composition and density. It is shown, with the help of an example, that sedimentation FFF is sufficiently versatile to unscramble complex colloids, which should eventually provide not only particle size distributions, but simultaneous particle density distributions. 

Clearly, sedimentation FFF is a separation technique. It is an important member of the field-flow fractionation (FFF) family of techniques. Although other members of the FFF family (especially thermal FFF) are more effective for polymer analysis, sedimentation FFF is advantageous for the separation of a wide assortment of colloidal particles. Sedimentation FFF not only yields higher resolution than nearly all other particle separation techniques, but its simple theoretical basis allows a straightforward connection between observed particle migration rates and particle size. Thus size distribution curves are readily obtained on the basis of theoretical analysis without the need for (and uncertainties of) calibration. 

In this broad definition some techniques which are not usually considered as chromatography are included, for example Field flow fractionation (FFF) techniques and electrophoresis. However, isotachophoresis is not included. 

The 2D approach to separation offers not only a great increase in separation power over one-dimensional (ID) methods, but also greater versatility. We have noted that 2D separation requires the use of pairs of ID displacements. If N kinds of ID displacements can be employed, then N2 different pairwise combinations can be found for 2D use. For example, dozens of 2D methods can be envisioned that use a field-flow fractionation (FFF) mechanism these methods fall in four categories in which a given FFF mechanism can be combined with (1) another FFF subtechnique, (2) a form of chromatography, (3) an applied field (e.g., electrical), and (4) bulk flow displacement [20]. For separations generally, literally thousands of kinds of 2D separation systems are possible, although only a handful have been developed [8]. 

Field-flow fractionation (FFF) is a family of separation methods first conceived in the 1960s [7]. The various subtechniques of FFF are best suited for the separation of macromolecules, colloids, and particles, including biological components ranging in size from proteins to living cells, environmental colloids and particles, and industrial polymers, powders, latexes, and emulsions. 

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...

Field flow fractionation (FFF) comprises a family of techniques that have demonstrated, over more than two decades, the ability to characterise supra molecular species in a size range spanning many order of magnitude, from macromolecules to micron-sized particles. The discovery of FFF dates back to the late 1960s due to Giddings and Myers (1957-1993). 

History/The Family of Field-Flow-Fractionation (FFF) Techniques.. 72... 

Composition Liquid chromatography, e.g., high-performance liquid chromatography (HPLC), size exclusion chromatography (SLC)-HPLC Field flow fractionation (FFF) UV-visible spectrophotometry Refractive Index... 

Commercially available fractionation methods include hydrodynamic chromatography (HDC), field flow fractionation (FFF) and disc centrifugation (DSC). One advantage of fractionation methods over nonfractionation methods is that the particles are separated physically according to size, prior to detection, which allows much higher resolution in determining the size distribution [40]. 

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