Separation Stokes radius

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

Although we often describe these as molecular weight columns, the separating parameter actually is their Stokes radius, the major axis of the molecule in its current configuration. The shape and folding of a protein molecule under differing solvent conditions affect their maximum radius and, therefore, their retention times. Only when extreme conditions are used to force all molecules into the same shape are we able to obtain a direct molecular weight relationship. 

The major components of beef glomerular basement membrane were solubilized with sodium dodecyl sulphate and resolved on 6% agarose [229]. 0.1% SDS has also been used in the separation of lipoprotein and apolipoprotein from human plasma [230] and 3% SDS used in gel chromatography of human erythrocyte membranes on Sepharose 6B [231]. Stokes radius determination of insulin-binding protein was performed on Sepharose 6B with 0.5% Triton X-100 [232]. 

The ability of gel electrophoresis to discriminate between conformational states that may differ in only minor degree in Stokes radius can be put to a number of good uses, for example in measuring the equilibria between the conformers of tRNA. Richards et al. (1973), working with 5 S RNA, have used gel electrophoresis to study the kinetics of interconversion between the native state (defined by its ability to recombine with the 50 S ribosome) and the denatured. Although these forms are practically identical in the extent of base-pairing, they separate readily in gel electrophoresis, and their relative proportions can be measured by densitometry of the stained... 

This method (FlFFF) is another member of the field-flow fractionation family, with measurement capabilities somewhat like those of SdFFF (18). The basic field-flow fractionation separation process is retained in this method. Particles separate because they are intercepted by different flow stream velocities near the accumulation wall. However, in this FFF method, particles equilibrate at distances from the wall strictly as a function of their size (Stokes radius). The nearness to the wall is a balance of the cross flow in the channel pushing particles toward the wall and normal diffusion tending to move them away. 

Trybus et al., 1982 Trybus and Lowey, 1984 Craig etal., 1983). Centrifugation separates the soluble folded monomer from filaments, whereas gel filtration can be used to separate folded from extended monomers because of their difference in Stokes radius (Trybus and Lowey, 1984, 1988). 

During SDS electrophoresis, the SDS-protein complex moves in the electric field toward the positive pole. The molecular sieve effect of a porous polyacrylamide matrix separates the SDS protein complexes according to their Stokes radius and thus according to their MW. 

Flow FFF is perhaps most promising in the area of water-soluble polymers. These polymers, which as a class are very difficult or impossible to separate by thermal FFF, can be fractionated according to diffusion coefficient or Stokes radius (which translate to molecular mass) in a flow FFF system using a water-compatible membrane such as cellulose acetate. Such a fractionation is shown in Figure 8.15, illustrating the programmed field separation of three sulphonated polystyrene components in a 510-//m-thick channel. The fact that the time of separation is somewhat longer than desired can be related to the excessive thickness of the channel, ten times thicker than the thinnest thermal FFF channel utilized. Recently we have been able to work successfully with a... 

The deviation of the nonionic micelle, OPDE, from the linear plot in Fig. 17 is not easily explained. However, since the separation parameter in exclusion chromatography is the Stokes radius (see next section), it may be argued that to the ellipsoidal shape of OPDE would give rise the large deviation, it is thus concluded that such molecular weight estimations. Fig. 17, can be only safe when... 

As shown in Fig. 1 gel filtration of d-bs gave two peaks suggesting that two species of d-bs, with different Stokes radius, exist and that these species can be separated by gel filtration (Calabro et al., 1976). 

A centrifuge is fitted with a conical disc stack with an included angle of 29, and there are n flow passages between the discs. A suspension enters at radius r and leaves at radius r2. Obtain an expression for the separating power E of the centrifuge. It may be assumed that the resistance force acting on the particles is given by Stokes law. 

Gel filtration is the last of the major chromatography techniques commonly applied in the resolving portion of a process. Of all the techniques discussed thus far in this chapter, gel filtration offers the lowest resolution. The separation is based solely on Stoke s radius of the protein molecule and is the most sensitive to flow rate and sample volume. To achieve significant resolution among sample components, the sample volume should be no greater that five percent of the column bed volume. 

Forced coalescence. Referring again to Stoke s equation. notice that the panicle radius occurs taken to (lie second power. II the particle can Ik- increased, the settling velocity increases by the square ol this change. Particle size then becomes the overriding parametei in the separation process. 

---