Molecular-exclusion partition

Gel filtration/permeation chromatography (also known as molecular exclusion chromatography) is a form of partition chromatography in which the solute molecules are partitioned between solvent and a stationary phase of defined porosity without an attractive interaction between the two phases. Gel filtration generally refers to aqueous systems while gel permeation is used in nonaqueous systems. The technique is normally used for the separation of biomacromolecules on the basis of size. Solutes are eluted in the order of decreasing molecular size. Gel filtration chromatography is not used as the first step in... 

I he liquid chromatography discussed in Chapter 22 separates solutes by adsorption or partition mechanisms. In this chapter, we consider separations by ion exchange, molecular exclusion, and affinity chromatography, which were illustrated in Figure 21-2. We also consider capillary electrophoresis, which separates species on the basis of their different rates of migration in an electric field. 

In newly formed plaque the matruc has a relatively loose and open structure but as the plaque ages the matrix forms a compact meshwork of entangled protein and polysaccharide with limited space for penetration by other molecules (Table 34.1). As the molecular weight of the polysaccharide components increases the permeability decreases and larger molecules are excluded. Molecular exclusion is responsible for an unequal partition of molecules in the mouth. The cells and large molecules already present are retained within the mesh-like structure of the plaque but the soluble salivary proteins are prevented fi-om entering. 

The elution volume, F/, and therefore the partition coefficient, is a function of the size of solute molecule, ie, hydrodynamic radius, and the porosity characteristics of the size-exclusion media. A protein of higher molecular weight is not necessarily larger than one of lower molecular weight. The hydrodynamic radii can be similar, as shown in Table 4 for ovalbumin and a-lactalbumin. The molecular weights of these proteins differ by 317% their radii differ by only 121% (53). 

Figures 6 and 7 illustrate the preposed mechanism in OC. Using the specific example of a separation of a styrene n-butyl methacrylate copolymer, the first SEC separates the copolymer according to molecular size in solution. At any desired retention time, the flow in the first instrument is stopped and an injection made into the second instrument of a single molecular size "slice" of the chrcoiatogram. The solvent running in the second instrument is a mixture of tetrahydrofuran (THF) and n-heptane. THF is a solvent for both styrene cuid n-butyl methacrylate portions of the polymer molecules. However, n-heptane is a nonsolvent for the styrene-rich portions. As a result, vrfien the injection is made into the second instrument, the styrene-rich molecules will shrink relative to the n-butyl methacrylate-rich molecules. An immediate size distribution will be present vrfiich will reflect the composition differences. The smaller styrene-rich molecules will enter more pores of the column packing than their n-butyl methacrylate-rich counterparts and so be fractionated. Furthermore, since the styrene-rich molecules "hate" the mobile phase, they should find the surface area of the packing more "sticky" than the n-butyl methacrylate-rich molecules. Thus, again the styrene-rich molecules should be retarded relative to the others. According to this picture, the mechanisms of size exclusion, adsorption and partition are thus able to act synergistic ally to accomplish a composition separation.

It is seen that an approximately linear relationship exists between the retention volume of each alkane and Its carbon number and that the smaller molecule exhibits the greatest retention. This is a direct result of the exclusion properties of the silica gel support. The fact that the data, taken at the two different temperatures, fall on the same straight line confirms that little or no partition is taking place and that the difference In retention between the Individual solutes is entirely related to their molecular size. 

Partitioning of the various modes of reorientation—even for the simplest member of this class, a disaccharide molecule—is not an easy task. For instance, separation of rotatory diffusion from internal oscillations around the glycosidic bonds is not feasible because no ring carbon atom in the disaccharide moiety relaxes exclusively via the overall molecular motion. This problem becomes more serious if the internal motion of exocyclic substituents, such as a hydroxymethyl group, is considered in the process of dynamic modeling. 

As the pore diameter increases in size (s decreases) relative to molecular or colloidal dimensions, less restrictions are imposed on the motions of contained species. Thus the exclusion effect gradually subsides as the pore size increases and consequently K-+1. For the separation of two molecules of different size, it is important to pick a pore diameter that will substantially exclude one species but not another. Pore size selection is thus of utmost importance in membrane science and in choosing a support for size exclusion chromatography (SEC). Aspects of pore size optimization in SEC based on the above partitioning theory have been developed [28]. 

The authors reported the investigation of random copolymers of styrene and -butyl methacrylate, containing the parent homopolymers PS and PnBMA. While in SEC 1 fractions of different molecular size were obtained, a separation with respect to chemical composition into fractions of PnBMA, P(St/nBMA) and PS could be achieved in SEC 2, the elution order being PnBMAsynergistic effect of different separation mechanisms including size exclusion, adsorption and partition. 

The equilibrium partition factor is defined as the ratio of the concentration of species A inside and outside the pores. This concept was first introduced by Ferry in terms of a geometric exclusion effect [25]. Since the center of mass of the molecule, assumed to be a hard sphere, cannot be closer to the pore wall than the distance of the molecular radius, Ferry obtained... 

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