Support in affinity chromatography

Cellulose is another example of polysaccharides which is used as support in affinity chromatography. Cellulose has a historical significance. Phospo- and DNA-cellulose are used especially for DNA related separations [14]. Antibody and enz)nne purifications have also been carried out. However its fibrous and non-rmiform character limits its use since cellulose detains macromolecules [11]. 

The use of other coupling techniques with agarose is facilitated by the formation of agarose-spacer derivatives, thus increasing the versatility of the gel as a support in affinity chromatography. These derivatives are described in more detail in the Section on Spacer gels (p. 112). 

Whereas in packed bed chromatography, mass transfer rates and pressure drop may be limiting, in monoliths surface interactions determine the overall reaction rate . The feasibility of using ceramic monoliths as support in affinity chromatography has been clearly establishedt ... 

Table 2 Materials used as supports in affinity chromatography...

Compared with enzymes fewer reports are available on immobilization of antibody (Ab) in sol-gels and their applications in immunosensing. Immobilization of Abs on a solid support was first reported in 1967 [128] and the technology has widespread application in affinity chromatography and other areas. However, the major problem associated with covalent immobilization of antibody on solid surface is partial loss of biological activity due to the random orientation of the asymmetric macromolecules,... 

Table 13.3 summarizes various covalent immobilization methods that are used in affinity chromatography. Each of these methods involves at least two steps (1) an activation step, in which the support is converted to a form that can be chemically attached to the ligand and (2) a coupling step, in which the affinity ligand is attached to the activated support. With some techniques, a third step, in which remaining activated groups are removed, is also required. The methods listed in Table 13.3 can be performed either in-house or can be used in the form of preactivated supports available from commercial suppliers (see list in Table 13.2) [25,36]. 

For successful separation in affinity chromatography, the important parameter is that solute of interest should be bound firmly and specifically while leaving all other molecules. This requires that the support within the column contain an affinity ligand that is capable of forming a suitably strong complex with the solute of interest [8]. The other important property is that the, support material must be biologically and chemically inert to avoid... 

Polj/meric supports based on polyacrylamide are s)mthesized by copol3tmerization of acrylamide and a cross-linking reagent and can be used directly in affinity chromatography due to its more hydrophilic properties than polystyrene supports. Polyacrylamide gels are either soft... 

As discussed earlier, the proteins used as chiral selectors in affinity chromatography cannot be used under the high-pressure HPLC with a variety of mobile phases therefore, these proteins were immobilized with some solid support such as hydroxyethylmethacrylate, polystyrene-divinylbenzene, polyethylene fibers, and silica gel [14,15]. Avariety of techniques have been used for the immobiliza-... 

The basis for selectivity in affinity chromatography is the use of immobilized biochemicals, known as affinity ligands, that are covalently attached to a support matrix, as illustrated in Figure 2.17. The primary criteria that govern the suitability of a support matrix for affinity chromatography include (1) the mechanical and flow properties of the matrix, (2) the ease of covalent coupling of the ligand to the matrix, and (3) the stability of the... 

Dehydrogenases. One recent development in affinity chromatography is that of the use of group specific supports, already mentioned briefly. The use of group specific supports has found particular application in dehydrogenase purification with affinity columns composed of immobilised nucleotides. 

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