Affinity chromatography capacity

Patwardhan, A.V. and Ataai, M.M., Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on Chelating Superose, /. Chromatogr. A, 767, 11, 1997. 

In the downstream processing of bioprocesses, fixed-bed adsorbers are used extensively both for the recovery of a target and for the removal of contaminants. Moreover, their performance can be estimated from the breakthrough curve, as stated in Chapter 11. The break time tg is given by Equation 11.13, and the extent of the adsorption capacity of the fixed bed utilized at the break point and loss of adsorbate can be calculated from the break time and the adsorption equilibrium. Affinity chromatography, as weii as some ion-exchange chromatography, are operated as specific adsorption and desorption steps, and the overall performance is affected by the column capacity available at the break point and the total operation time. 

Adsorption techniques such as ion exchange, HIC, chromatofocusing, reversed-phase chromatography, and affinity chromatography can have high capacity factors because experimental conditions can be manipulated so that the elution volume for a peak can exceed the total bed volume (VM as is the case with peaks 2 and 3 in Fig. B4.2.4.) However, in gel filtration, which is a nonad-sorptive technique, all peaks must elute within the volume Vy- V(h as is the case with peak 1 in Fig. B4.2.4. 

Affinity chromatography combines the analytical and chemical capacities of chemically bonded stationary phases and immobilized enzymes. Technology and methodology of both techniques are joined in the development of affinity stationary phases. Since steric requirements are even more determining than in simple immobilized enzyme systems, spacer molecules have great importance in these modifications. Commonly used spacer arms are summarized in figure 8.3. 

Affinity chromatography (AC) Ligand specificity High capacity High resolution + + + + + + + +... 

In practice, affinity chromatography is usually designed to be the final (if not the sole) chromatographic step in a purification procedure, so as to protect the valuable affinity material from unnecessary contamination. It is normal to use short columns whose capacity is largely fully exploited. Elution can be by a batch process or gradi-... 

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