Table II.
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Reactions of activated supports
Elution methods Elution methods can be divided into biospecific and nonspecific methods. In biospecific elution, the mobile phase modifier (called the inhibitor) is a free ligand similar or identical to the immobilized affinity ligand or the solute. The inhibitor competes for sites on the ligand or solute and thus decreases the capacity factor (k') of the solute. Biospecific elution is most commonly used when a low-molecularweight inhibitor is available. For example, if immobilized glucosamine were used to purify a lectin, glucose or N-acetyl-D-glucosamine might be used as the inhibitor. In this case, the affinity ligand and inhibitor compete for the binding sites on the analyte. If an immobilized lectin were used to purify a glycoprotein, the inhibitor again might be glucose. In this case, the analyte and inhibitor compete for sites on the affinity ligand. This is sometimes called reversed-role affinity chromatography. Biospecific elution in normal and reversed-role affinity chromatography is illustrated in Figure 2. Nonspecific elution involves denaturation of either the ligand or analyte by means of pH, chaotropic agents (e.g., KSCN, urea), organic solvents, or ionic strength. Typically, these con-
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ditions are empirically determined, although workers such as van Oss have examined more fundamental and general ways to disrupt the hydrogen bonds, hydrophobic interactions, and coulombic attractions that exist in the binding site between ligand and analyte (11). The conditions chosen for nonspecific elution should be mild enough that the support and ligand are not irreversibly damaged. If the analyte is being purified for further use, it must not be irreversibly denatured. One subtle difference between biospecific and nonspecific elution is related to the kinetics of elution. The dissociation rate constant of many ligand-analyte complexes is in the range of 103 to 10 - 5 s _1 , leading to dissociation half-lives of 10 - 3 s to many hours. Clearly the longer half-lives (> 1 s) can lead to excessive band broadening. In the case of biospecific elution, it is generally thought that the competing inhibitor does not alter the dissociation rate constant of the analyte-ligand complex. Therefore, slow dissociation will lead directly to excessive band broadening. The worst case is isocratic elution, in which the analyte must adsorb and desorb many times as it passes down the column. Step elution helps to minimize this
