Field sedimentation FFF

Field-flow fractionation (FFF) technology is applicable to the characterization and separation of particulate species and macromolecules. Separations in FFF take place in an open flow channel over which a field is applied perpendicular to the flow. Among the various FFF subtechniques, depending on the kind of the applied external fields, sedimentation FFF (SdFFF) is the most versatile and accurate, as it is based on simple physical phenomena that can be accurately described mathematically. SdFFF, which uses a centrifugal grav-... 

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... 

One of the more advanced of the FFF techniques is sedimentation FFF (SdFFF), in which the applied field is a centrifugal force (see Fig. 2.1b). A typical separation achieved through SdFFF is also illustrated in Figure 2.1b. The SdFFF is suitable for species with molecular weights larger than about 106 and has proved useful for a large number of biocolloids (e.g., subcellular particles), polymers, emulsions, and natural and industrial colloids (Giddings 1991). 

Sedimentation FFF implies application of the centrifugal field, which is produced by placing the channel in a centrifuge basket. SdFFF instruments can be linked readily to analytical instruments to provide analysis in real time. For the first time, Beckett (1991) introduced FFF-ICP-mass spectroscopy (MS) as a powerful analytical tool for characterizing macromolecules and particles. Taylor et al. (1992) illustrated the characterization of some inorganic colloidal particles and river-borne suspended particulate matter of size range <1 pm using SdFFF and ICP-MS. 

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. 

We have noted that the combination of regular geometry, uniform field strength, and well-defined parabolic flow, make possible a rigorous theoretical treatment to relate particle properties to experimental observations. This theory serves as the basis for particle characterization in sedimentation FFF (2). Here, we will briefly review the main theoretical concepts and some of the equations needed for particle size calculations. 

Specifically, for sedimentation FFF, X relates to d (along with field strength (acceleration) G and the difference bp in density between the particle and the carrier) according to the expression... 

Figure 2. Fractionation of four samples of Dow polystyrene latex beads by sedimentation FFF. The nominal particle sizes are given in the figure. Flowrate = 12 ml/hr, channel thickness w = 0.0127 cm, void volume V° = 2.0 ml, and field strength G = 193.7 gravities. Reproduced with permission from Ref. 20. Copyright 1980 John Wiley.

The sedimentation coefficient s> often used to characterize biological macromolecules and particles, is equal to the ratio of the velocity U induced by a sedimentation field to the strength G of that field measured as acceleration. Show that s can be related to the retention parameter A in sedimentation FFF by s = D Gw. 

Of the many kinds of interactive fields or gradients possible, five principal types have proved the most practical sedimentation (Sd-FFF), electrical (El-FFF), thermal (Th-FFF), steric (St-FFF) and lateral cross flow (F-FFF). A commercial high-spin-rate sedimentation FFF instrument was developed by Kirkland and Yau, the SF3 technique (1982). 

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. 

When the diluted sample of solute is injected during rotation, it is concentrated at the beginning of the channel, due to the fact that the average volume flow rate of the retained solute is lower than the average flow rate of the injected solution. Hence diluted colloidal samples can be concentrated by sedimentation-FFF [189]. One can even operate such that the injection is run at a higher field force and, after the entire sample solution is injected, the field force is decreased to the required value. 

The techniques of field-flow fractionation appear to be well suited to colloid analysis. The special subtechnique of sedimentation FFF (SdFFF) is particularly effective in dealing with colloidal particles in the diameter range from 0.02 to 1 using the normal or Brownian mode of operation (up to 100 jU-m using the steric-hyperlayer mode). As a model sample for the observation of aggregate particles by SdFFF, of... 

The injection volume of the conventional stop-flow sample injection technique is limited typically to 2-20 /zL. This procedure is carried out using a sample loop and injecting the sample plug just onto the channel carried with the channel flow before stopping that flow and letting the sample attain its equilibrium distribution relative to the accumulation wall (relaxation). Sedimentation FFF (SdFFF) is the only technique where the field is not applied during the complete stop-flow procedure, but the centrifuge is started as soon as the sample is injected and the channel flow switched away for practical reasons. 

Further, it is possible to utilize different fields to yield the various FFF subtechniques. The two most common fields are centrifugal and fluid cross-flow, which give rise to the sedimentation and flow FFF subtechniques. Other fields currently in use include thermal, electrical, and magnetic fields. In the normal mode, it is possible to extract physical parameters from retention data. For example, sedimentation FFF using a centrifugal force gives information about the buoyant mass, and flow FFF gives information about the sample s diffusivity or hydrodynamic diameter. 

In their fundamental principle, field-flow fractionation (FFF) methods exploit the cell physical characteristics by means of their selective elution in a paraUelepipedic channel laminarUy flowed by a carrier phase under the effect of an external field apphed perpendicularly to the great surface of the channel and, by consequence, perpendicularly to the flow direction. In sedimentation FFF, the external field is gravitational (G-FFF) or multigravi-tational (Sd-FFF). 

Sedimentation FFF techniques encompass two different instrumentations one is very simple and uses the Earth s gravity (G-FFF), the second, much more complex, uses centrifugational field created by rotation (Sd-FFF). In any case, common features must be described. 

The particle sizing by field flow fractionation (FFF) is based on the different effect of a perpendicular applied field on particles in a laminar flow [63-66], The separation principle corresponds to the nature of the perpendicular field and may, for example, be based on different mass (sedimentation FFF), size (cross-flow FFF), or charge (electric-field FFF). Cross-flow FFF has been applied recently to investigate nanoemulsions, SLN, and nanostructured lipid carriers (NLC, particles composed of liquid and solid lipids) [58], Although all samples had comparable particle sizes in PCS, their retention in the FFF was very different. Compared to the spherical droplets of the nanoemulsion, SLN and NLC were pushed more efficiently to the bottom of the channel because of their anisotropic shape. Their very different shapes have been confirmed by electron microscopy. 

Particle-size and mass distribution curves, along with information on particle porosity, density, shape, and aggregation, can be obtained for submicrometer- and supramicrometer-size silica materials suspended in either aqueous or nonaqueous media by field-flow fractionation (FFF). Narrow fractions can readily be collected for confirmation or further characterization by microscopy and other means. Among the silicas examined were different types of colloidal microspheres, fumed silica, and various chromatographic supports. Size distribution curves for aqueous silica suspensions were obtained by both sedimentation FFF and flow FFF and for nonaqueous suspensions by thermal FFF. Populations of aggregates and oversized particles were isolated and identified in some samples. The capability of FFF to achieve the high-resolution fractionation of silica is confirmed by the collection of fractions and their examination by electron microscopy. 

Both Sa and tri are established by logarithmic plots of tr versus d of well-characterized standards such as polystyrene latex beads. The value of tri depends on the field strength and, for sedimentation FFF, also on the particle density. It will be shown later that this dependence makes possible the determination of particle density and porosity values. 

A battery of six different FFF systems was used to provide a comparison of results obtained not only from different systems of the same type but from systems of entirely different types (i.e., with different primary force fields). The characteristics of the six systems are summarized in Table I. Included in the collection are two sedimentation FFF systems, three flow FFF systems, and one thermal FFF system. The characteristics and operation of these different categories of instruments are described in more detail in this section. 

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