Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

Affinity chromatography has the advantage of specific binding interactions between the analyte of interest (normally dissolved in the mobile phase), and a binding partner or ligand (immobilized on the stationary phase). In a typical affinity chromatography experiment, the ligand is attached to a solid, insoluble matrix—usually a polymer such as agarose or polyacrylamide—chemically modified to introduce reactive functional groups with which the ligand can react, forming stable covalent bonds. The stationary phase is first loaded into a column to which the mobile phase is introduced. Molecules that bind to the ligand will remain associated with the stationary phase. A wash buffer is then applied to remove non-target biomolecules by disrupting their weaker interactions with the stationary phase, while the biomolecules of interest will remain bound. Target biomolecules may then be removed by applying a so-called elution buffer, which disrupts interactions between the bound target biomolecules and the ligand. The target molecule is thus recovered in the eluting solution.^{[page needed]}

Affinity chromatography does not require the molecular weight, charge, hydrophobicity, or other physical properties of the analyte of interest to be known, although knowledge of its binding properties is useful in the design of a separation protocol. Types of binding interactions commonly exploited in affinity chromatography procedures are summarized in the table below.

Binding to the solid phase may be achieved by column chromatography whereby the solid medium is packed onto a column, the initial mixture run through the column to allow settling, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure. Alternatively, binding may be achieved using a batch treatment, for example, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the elute.^{[citation needed]}

Sometimes a hybrid method is employed such that the binding is done by the batch method, but the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.^{[citation needed]}

The ligands used in affinity chromatography are obtained from both organic and inorganic sources. Examples of biological sources are serum proteins, lectins and antibodies. Inorganic sources are moronic acid, metal chelates and triazine dyes.

A third method, expanded bed absorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase.^{[citation needed]}

Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic strength directly or through a gradient to resolve the particles of interest.^{[citation needed]}