4


Monolithic Chromatography and its Modern Applications


chromatography and discuss how this method has recently been adapted for work with columns that contain monolithic support materials. Figure 1.1 shows a typical separation scheme used in affinity chromatography


(including work with monolith columns) for compound isolation and measurement. This particular scheme is sometimes called the ‘on/off’ or step-gradient elution mode of affinity chromatography. In this scheme, a sample containing a target solute that can bind to the affinity ligand is passed through the column under conditions in which the solute will have tight binding to the ligand [1–3]. This step is performed in the presence of an application buffer that mimics the natural pH and solvent conditions that promote strong binding between the target and affinity ligand. These conditions result in the target being bound and retained by the affinity column. However, the high selectivity of this interaction causes other sample components to pass through non- retained or to be only weakly retained as the application buffer washes these solutes from the column. The next step in the on/off mode of affinity chromatography is to pass an elution buffer through the column that causes the retained target to dissociate from the ligand and also elute from the column. The elution step may be performed by


Apply


Regenerate


Wash


Elute


Figure 1.1 A typical on/off elution scheme used in affinity chromatography.


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