Stationary Phases for Affinity Chromatography

Support materials for low-pressure affinity chromatography include agarose (cross-linked with epichlorohydrin), cellulose, dextran, silica, and polyacrylamide 62 in HPLC a rigid, highly porous, hydrophilic polymer is typically used. Large pore sizes are necessary, as either the analyte or the affinity ligand are macromolecules. To provide unhindered access of 

Chapter 2 Separations in High-Performance Liquid Chromatography 

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Stationary Phases for Affinity Chromatography and Enzyme Immobilization... 

Affinity chromatography is a very powerful technique which separates biomolecules according to differences in their biological function or chemical stracture. The stationary phase for affinity chromatography consists of a matrix to which ligands are covalently attached. 

Abou-Rebyeh H, Korber F, and Schubert-Rehberg K. Carrier membrane as a stationary phase for affinity chromatography and kinetic studies of membrane-bound enzymes. J. Chromatogr. B 1991 566 341-350. 

Recently, PHEMA microspheres have been more and more extensively used for IMAC. Separon-IDA-Fe(III) or Cu(II) was prepared. It was found that Cu(II) interacted preferentially with histidine and tryptophan residues, while Fe(III) preferred phosphate residues, as demonstrated by the separation of lysozyme, ribonuclease A, myoglobin, and transferrin on the Cu(II) column and ovalbumin on the Ee(III) column, respectively.The PHEMA-Congo Red-Cu(II) and PHEMA-Cibacron Blue E3GA-Zn(II) microspheres were applied for adsorption of BSA. Without incorporating the metal ions, the dyed sorbents were already good stationary phases for affinity chromatography. As shown in Fig. 2, the addi-... 

Agarose is a cross-linked polysaccharide and is stable over the whole pH range 1-14. It can be derivatized to give, for example, stationary phases for affinity chromatography. 

Stationary phases" for affinity chromatography can be prepared by the user, often starting from diol- or aminosilica. However, it is much simpler to buy an activated gel which allows the desired ligand to be bound according to well known methods. A possible example for activation and binding is shown in Figure 16.3. For separation problems that require common ligands, ready-to-use stationary phases... 

Based on their specific and reversible interactions with thrombin, PAOM resins have been used as stationary phases for affinity chromatography of the protease (12). Thus, in a simple one-step chromatographic procedure, human thrombin was isolated from activated prothrombin complex concentrate in high purity and yield Because of their excellent mechanical properties the... 

The elution of [60]- and [70]fullerenes was measured in water-methanol as a function of temperature on a poly(octadecylsiloxane) phase.67 The retention was shown to be dependent on the surface tension of the stationary phase through a simple geometrical model in which the solute formed a cavity in the stationary phase. In affinity chromatography, it was demonstrated that low ligand density may be a requirement for specificity of binding.68... 

Imprinted materials can be used as stationary phase in affinity chromatography, especially for the separation of enantiomers. Compared to standard chiral stationary phases, these materials present the advantage of being synthesised to order for a given enantiomerically pure molecule. These chromatographic materials have allowed the separation of numerous compounds such as naprox-ene (anti-inflammatory) [139], timolol (beta-blocker) [50] and nicotine [140]. 

A third area of development in carbohydrate l.c. analyses is in the combined techniques (see Section IV,3) and other methods that provide qualitative, as well as quantitative, information about sample constituents, such as high-performance liquid affinity chromatography. The use of specific lectin- and monoclonal antibody-based, stationary phases for analytical and preparative applications is now being considered. The basic concepts of these techniques have been reviewed - and their applications to carbohydrates have been discussed. 

Affinity Chromatography was initially defined as a method based on specific and reversible molecular interactions between biologically active substances. However, the method has extended to non-biological stationary phases, such as metal-chelate complexes. This technique is used for separation and purification of proteins and other biologic materials, such as viruses and cells.47,48 A survey of various stationary phases and affinity interactions is given in figure 8.2. 

Methodology Prior to experimentation the stationary phase constmct is prepared and poured into the column. Notably, the columns used for affinity chromatography tend to be shorter than those used for other column chromatography methods. Once the stationary phase construct has settled (packed) in the column, a loading buffer is passed through to equilibrate the system before addition of loading buffer... 

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