Field-flow-fractionation

Field-flow fractionation is a separation method which was introduced by Giddings18 around 1960. The polymer solution flows in a flat ribbon-shaped duct, (see Fig. 1.20). A field perpendicular to the plane of the ribbon interacts with the polymers this field may be a thermal gradient (or more simply the gravitation field). 

Let E be the thickness of the ribbon and h (0 h E) the coordinate of a point inside the ribbon along a direction perpendicular to this ribbon. The effect of the field is the creation of a concentration profile C(/i) of the form 

In all cases, the mean velocity of a polymer which flows inside the ribbon is given by 

Therefore, v depends on g and consequently on the mass of the polymer. This device can thus be used to separate polymers with different masses, and in this case, the light molecules come out first. 

(1969). Statistical Mechanics of Chain Molecules. Interscience-Wiley. 

This equation tells us that the retention is a function of the field strength, channel dimensions, and temperature. The magnitude of the field strength is related to the type of field and to the properties of the components. 

The force can be an electric field, a magnetic field, a thermal field, centrifugation or a crossflow of solvent. The latter is the more common mode. Instrumentation for both crossflow [flow FFF (FFFF)], sedimentation/centrifugation (SdFFF), and thermal (ThFFF) modes are commercially available. 

Chromatography Basic Principles, Sample Preparations and Related Methods, First Edition. 

Elsa Lundanes, Leon Reubsaet and Tyge Greibrokk. 

At the end of the channel, there is a detector A UV detector and a laser light scattering detector are the most common, the latter also for determining particle size. Off-line detection is also an alternative. 

In the last years, the separation method of field-flow fractionation (FFF) comes more into the focus of size determination and separation of polymer mixtures consisting of different components, for example, aggregates, gels, or defined assemblies. Here, the separation takes place in a long and narrow chaimel, where a carrier liquid transports the molecules. Perpendicular to the main flow direction, a force field will be applied, which influences the sample molecules in order to separate thena. Caused by the channel architecture, the 

Smaller components move into the faster flows farther away from the accumulation wall and therefore elute before large molecules. This normal elution mode is reverse to that observed in SEC. The separation can be performed with different force fields. In thermal FEE, the separation is based on thermal diffusion between temperature difference of top and bottom wall. In case of electric FEE, the separation is driven by charge differences and electrophoretic mobility of the molecules applying an electrical field. Another way to separate components depending on their density properties is sedimentation FEE, where a circular channel rotates and generates a gravitational force field. 

The group of flow FEE is the most improved separation technique within the FEE family. Particularly the asymmetrical flow field-flow fractionation (AF4) provides a broad range of application possibilities. In this case, the separation is caused by different diffusion coefficients (D) by inducing a flow field with a perpendicular liquid flow. A permeable wall (porous frit covered with an ultrafiltration membrane see Fig. 4.10b) allows the cross flow to act as force field. The retention time (t) is given by the following equation  

AF4 coupled with static and DLS detectors enables comprehensive information about structural and branching characteristics of biopolymers (e.g., starches), synthetic polymers, proteins, etc. [25, 26]. Especially in case of branched polymer stractures like dendronized glycopolymers, the separation and characterization with AF4-LS lead to comprehensive information and understanding in molecular structures and aggregation behavior [27]. Furthermore, studies of uptake studies of dendritic glycopolymers and dye molecules were performed for the first time by AF4-LS (see Fig. 4.12). Here, a good correlation was obtained between the increase of molar mass and the quantified amount of dye molecules, which were encapsulated by the glycopolymers [28]. 

Another technique suited to MW measurement of large molecules is field flow fractionation (FFF), in which separation occurs in a thin channel (10-250 pm wide, approximately 30 cm long). A field is applied perpendicularly to the channel flow. The field may be centrifugal, thermal, electrical, or solvent flow. For cereal proteins, flow FFF has been used. Solvent flows through the channel and a cross-flow of solvent is introduced. Because of the thinness of the charmel, a parabolic flow pattern is set up. The cross-flow causes the molecules (or particles) to be transported toward the channel wall. Smaller molecules with greater diffusivities remain near the center of the channel in the higher velocity flow. They therefore elute first—opposite to the case for SE-ITPLC. 

In studies of wheat proteins, Stevenson and Preston (1996) used symmetrical flow having upper and lower porous sintered glass channel walls. Wahlund et al. (1996) and Arfvidsson, Wahlund, and Eliasson (2004) used asymmetrical flow, which has only a lower porous sintered glass charmel wall. The lower channel in each case is overlaid with an ultrafiltration-type membrane that allows passage of solvent molecules only. 

The intrinsic diffusion coefficienf of a macromolecule depends on its effective hydrodynamic diameter. The latter is influenced by the shape 

Wahlund et al. (1996) estimated upper and lower limits for the molecular mass of glutenin fractions. The lower limit was defined as D = 0.0542M° for a flexible random coil polymer and D = 0.159Mi for a spherical shape. Values for the upper limit were in the range of 440,000 to 11 million. In order to obtain more precise measurements, the shape of the molecule needs to be determined. 

Arfvidsson, C., K. G. Wahlund, and A. C. Eliasson. 2004. Direct molecular weight determination in the evaluation of dissolution methods for unreduced glutenin. Journal of Cereal Science 39 1-8. 

Assume that the sample contains a mixture of charged particles. When these are in an electrical field in a narrow channel, they tend to stack vertically. A liquid flowing through the channel has a parabolic flow profile. The molecules near the center of the channel are pushed by a faster flow than those near the edge, and this results in a separation. Any field that stack molecules or particles can be used, and the particles need not be charged. This includes heat and centrifugal force. 

A complete mathematical treatment of the theory for retention was developed by Giddings in 1968 (J. Chem. Phys., 49, 81). A simplified FFF as a random walk theory by Giddings is presented below. 

Vwiou nthet poly m e r s Crude oils ortd aspholtenes 

A molecule in the low velocity state falls behind by the same amount. Distance g may be regarded as the approximate length of step in a random walk process -the distance moved forward or backward with respect to the zone position before some random event (diffiision) reshuffles altitudes and thus velocity states. 

Column plate height, as in chromatography, is defined as H = (f/L, and is, therefore, equal to 

The value of X depends on V, which in turn depends on the type of the applied field. The following are some common field-flow fractionation methods  

Lower block cooled by water FIGURE 13.19 Thermal field-flow fractionation. 

Sedimentation Field-Flow Fractionation This method uses the centrifugal field to separate molecules. The value of A, is calculated by using 

Flow Field-Flow Fractionation This method is similar to dialysis or ultrafiltration, with the solvent acting uniformly on all the solutes. The field is generated by the flow of the solvent. The separation is mainly determined by the diffusion coefficient or frictional coefficient. The value of X is calculated using 

FIGURE 13.21 Separation of polystyrene sample in terms of X. [Adapted from Giddings (1974).] 

As in HDC, a liquid carrier flowing in the channel will have a parabolic flow profile due to the channel geometry the liquid near the walls of the channel will have near-zero velocities and its velocity reaches a maximum at the center of the channel. After the sample is introduced into the channel, the flow is stopped momentarily and the external field is applied. This field can take a variety of forms. The common ones are centrifugal, thermal, electric, magnetic, flow, gravitational, or the opposed flow sample concentration technique. In addition, there can be different operating modes for each field, such as in steric and hyperlayer sedimentation FFF. The purpose of the applied field is to partition particles into different velocity streamlines in the liquid flow according to their size. Completion of this process is based on the response of the particles to the applied field, due to one of particle s properties. For example, under a thermal 

Although the FFF technique has been demonstrated in sizing of a variety of particles across a size range of over five orders of magnitude (ftom 1 mn to 500 pm), and though research and development stdl continues, its commercialization has not been successful. During the past decade, a few commercial instruments have been manufactured and mailceted. They are not popular and their main applications are limited to academia. 

As a result of its versatility, FFF in one form or another has been applied to the characterization of particles or molecules whose sizes range over five orders of magnitude from particles as small as 0.005 pm to as large as 500 pm [80]. The total mass range covered is over 15 decades. Most of the reported data are for aqueous suspensions non-aqueous suspensions have also been used [81]. 

Materials analyzed by FFF range from high-density metals and low-density latex microspheres to deformable particles such as emulsions and biological cells. The particles need not be spherical since separation is based on effective particle mass. 

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Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Field-Flow Fractionation. Field-flow fractionation is a general name for a class of separation techniques that fractionate a particle population into groups according to size. The work in this area has been reviewed (59). 

Fig. 9. Principles of field-flow fractionation (a) sample equilibrium position before flow is initiated, (b) fractionated sample after flow initiation, and (c) a...

Fig. 10. Centrifugal sedimentation field-flow fractionation equipment deposits particles along the circumference of the disk by size. The fluid enters and...

Among the techniques employed to estimate the average molecular weight distribution of polymers are end-group analysis, dilute solution viscosity, reduction in vapor pressure, ebuUiometry, cryoscopy, vapor pressure osmometry, fractionation, hplc, phase distribution chromatography, field flow fractionation, and gel-permeation chromatography (gpc). For routine analysis of SBR polymers, gpc is widely accepted. Table 1 lists a number of physical properties of SBR (random) compared to natural mbber, solution polybutadiene, and SB block copolymer. 

Currently, there are several molecular weight separation techniques, such as OTHdC, PCHdC, SEC, thermal field flow fractionation (ThFFF), and sedimentation field flow fractionation (SdFFF). The molecular weight separation range... 

SEC, size exclusion chromatography OTHdC, open tubular hydrodynamic chromatography PCHdC, packed column hydrodynamic chromatography ThFFF, thermal field flow fractionation. 

Techniques which seem less suitable for routine size analysis are (1) analytical ultracentrifugation combined with a Schlieren optical system (Mason and Huang, 1978 Weder and Zumbuehl, 1984) (2) the sedimentation field flow fractionation (SFFF) technique to separate heterogeneous dispersions (e.g., Kirkland et al., 1982). 

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. 

Field-Flow Fractionation Analysis of Macromolecules and Particles,... 

Cdlfen, H. and Antonietti, M.t Field-Flow Fractionation Techniques for Polymer and Colloid Analysis. VoL 150, pp. 67-187. 

FFF Field-flow fractionation GD-(MS) Glow-discharge (mass spectrometry)... 

Lee, H. Williams, S. K. R. Wahl, K. L. Valentine, N. B. Analysis of whole bacterial cells by flow field-flow fractionation and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. Anal. Chem. 2003, 75,2746-2752. 

Reschiglian, P. Zattoni, A. Cinque, L. Roda, B. Dal Piaz, F. Roda, A. Moon, M. H. Min, B. R. Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 2004, 76,2103-2111. 

Schure, M.R. (1999). Limit of detection, dilution factors, and technique compatibility in multidimensional chromatography, capillary electrophoresis, and field-flow fractionation. Anal. Chem. 71, 1645-1657. 

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. 

Venema, E., deLeeuw, P., Rraak, J.C., Poppe, H., Tijssen, R. (1997). Polymer characterization using online coupling of thermal field flow fractionation and hydrodynamic chromatography. J. Chromatogr. A 765(2), 135-144. 

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. 

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