Figure 14.3. Representative chromatogram obtained with SEC. in .NET framework

Generating Code 128 Code Set A in .NET framework Figure 14.3. Representative chromatogram obtained with SEC.
Figure 14.3. Representative chromatogram obtained with SEC.
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CHROMATOGRAPHY OF BIOMOLECULES
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Figure 14.4. Selectivity curve from SEC of DNA (&) and protein (&) molecular weight standards on a 106 10 mm Superose 6 gel ltration column with 0.02 M Tris-HCl pH 7.6 containing 0.15 M NaCl as the eluent.4 [Reprinted, with permission, from H. Ellegren and T. Laas, Journal of Chromatography 467, 1989, 217 226. Size - Exclusion Chromatography of DNA Restriction Fragments. Fragment Length Determinations and a Comparison with the Behaviour of Proteins in Size-Exclusion Chromatography . # 1989 Elsevier Science Publishers B.V.]
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where a 1 for rods, a % 0:5 for exible coils, and a 0:3 for spheres. From this equation, it is apparent that the radius of gyration increases more rapidly with molecular weight for rods (DNA) than for spheres (proteins). Figure 14.4 shows the dependence of Kav on log(MW) for a series of protein and DNA standards.4 For proteins, a mathematical model of retention has been developed that works well for Sephadex gels.5 The solute is treated as a sphere of radius rs , while the gel is a network represented by in nitely long, straight rods of radius rx . The rods are randomly distributed, and have an average density of L units of rod length per unit volume of gel. The values of L and rx may be calculated from known dimensions of dextran chains, and then Kav may be found from Eq. 14.13: Kav expf pL rx rs 2 g 14:13
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Equation 14.13 has been used to evaluate the hydrated radii of proteins by determining Kav experimentally on three Sephadex gels, G-75 (fractionation range 3 80 kDa), G-100 (fractionation range 4 150 kDa) and G-200 (fractionation range 5 600 kDa). The results of these experiments are shown in Table 14.1. In addition to steric exclusion, other types of interactions between solute and gel are possible. In particular, electrostatic interactions may be very signi cant with some gels, especially if the gel has charged groups and can act as an ion exchanger. If the eluent has low ionic strength, these charged groups create a Donnan potential
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SIZE EXCLUSION (GEL FILTRATION) CHROMATOGRAPHY
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TABLE 14.1. Determination of rs for Proteins Using SEC on Sephadexa Kav on Sephadex G-75 G-100 G-200 0.43 0.04 0.00 0.59 0.19 0.05 0.72 0.41 0.21
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Substance Cytochrome c Human serum albumin Human immunoglobulin G
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rs (A) 16.4 36.1 55.5
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See Ref. 5.
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between the inside and outside of the gel particles. This can happen if, for example, a solute has a nonzero net charge but is too large to enter the pores, while its small counterions can enter the gel. This results in ion-exchange properties in localized areas of the gel. These types of electrostatic interactions may be suppressed by increasing the ionic strength of the eluent, which can be accomplished using volatile electrolytes that are readily removed from the separated components following the chromatographic run. Adsorption of the solute onto the gel matrix can also occur, but is not normally encountered in SEC. It may be counteracted by changing the eluent. The ideal eluent is a good solvent for both the solute and the substance forming the gel matrix. In addition, hydrogen-bond-breaking agents such as urea or guanidinium, or chaotropic ions, or surfactants may be added to eluents to improve SEC characteristics. To test for either adsorptive or electrostatic interactions, the SEC separation is performed at a variety of temperatures. If the separation occurs by size alone, the retention coef cient R Vo =Ve is independent of temperature; very small variations may be observed as a result of gel swelling or microstructural changes to the gel. The presence of a signi cant dependence of R on T indicates the presence of a mechanism other than size exclusion. While R should not vary with T, diffusion coef cients increase with T and so zone broadening occurs, leading to decreased resolution with increasing separation temperatures. Normally, an approximately linear relationship is observed between Kav and log(MW) over the fractionation range of the gel. The expanded relationship is, in fact, sigmoidal, as shown in Figure 14.5. The calibration curve can be linearized using the logit transformation, where logit Kav ln Kav = 1 Kav is plotted against log(MW). Resolution and separations in SEC are described by the van Deemter equation (Eq. 14.7). The main contributions to zone broadening, in order of importance, are as follows. The rst factor involves the kinetics of partitioning between the mobile phase and the gel; there is a limited rate at which equilibrium can be established, and this rate depends on the solute s diffusion coef cient. Second, differences in the lengths of different stream paths in the packed bed of irregularly shaped particles result in eddy diffusion ; with an ideally packed column, this is not a signi cant problem, but in practice, eddy diffusion may be signi cant. Finally, longitudinal
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