Affinity chromatography is based on the interaction strength differences between biomolecules in the mobile and stationary phases.
In the first step, the proteins are first loaded on the column under conditions that impact the interaction between the target protein and its ligand. The bound protein is washed under conditions that leave the specific interactions intact while disrupting any non-specific interactions between contaminating proteins and the stationary phase.
In the second step, the bound protein is eluted with a buffer containing a competing molecule or under conditions that interfere with all protein-protein interactions. The target protein gets displaced when competing molecules bind to the ligand.
In the third step, the target protein is separated from the competing molecule either through dialysis or another chromatographic procedure.