Affinity column materials

Because of the complex nature of most biological samples, a single fractionation technique may not be adequate for the separation of the wide range of molecules present. Better resolution of some molecules is obtainal when properties other than differences in size are exploited. These include differences in ionic characteristics, affinity for other molecules and hydrophobicity. In separations that involve any one or more of these properties, the sample constituents interact with the column material and are then eluted with a suitable eluant. As a consequence of this interaction, and the use of eluants, whose properties may not closely resemble those of the medium found in vivo, the metal may dissociate from the ligand. In addition, as the complexity of the sample increases it is difficult to predict the behaviour of the various constituents. Undesirable effects leading to irreversible interaction between some molecules in the sample and the column packing material, degradation and decomposition of some constituents may result. Furthermore, it may be difficult to rid the column of certain trace metal contamination. 

Column procedures are useful in fractionating proteins with different affinity properties and sizes. By the use of different column materials in conjunction with specific eluting solutions, highly purified protein preparations can be obtained. 

Where in analytical chemistry can these features be advantageous Analytical chemists cannot always solve their problems with typical chromatographic or electrophoretic separations. In some of these cases they use affinity columns or affinity SPE. Affinity separations rely on reversible and very selective binding of the analyte to a biomolecule, e.g., antibody. Making the analyst s own preparation of affinity phases is not economical in most cases, so one has to rely on commercially available material. If this is not easily available the analyst may consider making an MIP, probably in the SPE format, because MIP preparations are fairly easy for any chemist. 

A) Samples are homogenized and centrifuged to remove insoluble material. (B) Samples are applied to and eluted from an affinity column. (C) The purified fractions are run by standard two-dimensional electrophoresis. (D) Target proteins can then be excised and identified. 

If the degree of purity achieved is still insufficient, further chromatographic steps have to be included. As the protein mixture at this point is already quite pure, so that only a few proteins tend to remain in the mixture, the next chromatography step has to be very specific. The method of choice is affinity chromatography, in which a specific group in the target protein is picked to interact with the column material. The principle is discussed with the two examples of his-tag and NAD+ affinity chromatography ... 

In certain cases, affinity columns can be used to fractionate within classes of bound materials for instance, protein A antibody columns have been used to separate the various subtypes of IgG. In this case, the packings are micro-porous, heavily cross-linked polymers and benefit from HPLC operating conditions. Eluting conditions are usually step gradients of buffers with different pHs. The last step of a protein G column is at a very acidic pH and the sample is eluted into a buffer solution that quickly raises the pH to prevent protein denaturation. 

C. Risks Associated with Leached Material from Affinity Columns... 

Useofavidin-biotin technology in immunoaffinity chromatography can lead to improved performance of the affinity column, which translates both into yields of the target material and stability of the column (1,2). The presence of free biotin-binding sites of the avidin-4 esin allows secondary interaction with biotinylated antibody or its complexes with avi-din that may have leaked from the affinity resin. 

The elimination of endotoxin from a recombinant or fermentation product is challenging because the host organism often contributes enormous amounts of endotoxin to the bulk material. The product is usually separated from endotoxin and feedstream impurities by affinity columns. 

If the starting material is reasonably clean a single step purification on a prepacked HiTrap affinity column may be sufficient to achieve required purity at the milligram scale, as shown in Figure 9. HiTrap affinity columns are available in a wide range of selectivities (see Table 6, page 34). 

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