= Affinity chromatography
{wiki}
<Affinity chromatography> separates a target through its reversible, selective binding to a <ligand> immobilized on a <chromatographic stationary phase>. The <chromatographic mobile phase> carries a mixture through the material; components with little binding tend to pass through, while the target is retained. Washing removes unbound material, and changing suitable solution conditions or adding a competitor can release the target. Strong binding and recoverable binding are different design requirements.<Agarose Derivatives for Purification of Protein by Affinity Chromatography by Pedro Cuatrecasas>[Opening description: immobilized ligands and reversible macromolecular binding]{ref}
For a concrete experimental comparison, prepare two otherwise similar columns: one bearing the intended <ligand>, the other lacking it. Load the same <protein> mixture, collect the flow-through, wash, and then collect the released material. Target enrichment specifically on the ligand-bearing material supports the intended binding mechanism. Retention on both columns instead suggests an additional interaction with the support; disappearance from the flow-through alone does not prove successful purification.
An original numerical example separates recovery from purity. Start with $10$ milligrams of target in $100$ milligrams of total <protein>. If the collected fraction contains $8$ milligrams of target in $10$ milligrams total, its target purity rises from $10\%$ to $80\%$, while target recovery is $8/10=80\%$. An activity assay may answer a further question: whether the recovered target remains functional.
In his 1970 report, <Pedro Cuatrecasas> described agarose derivatives for presenting binding partners on an insoluble support. The arrangement makes molecular recognition usable as a preparative tool: retain a chosen partner in one place, pass a complex mixture over it, and recover material selected by that interaction.<Agarose Derivatives for Purification of Protein by Affinity Chromatography by Pedro Cuatrecasas>[Opening discussion of agarose support and ligand attachment]{ref}
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