Abstract: A process for the affinity chromatographic separation of at least one biological or related substance from a mixture wherein the at least one biological or related substance is bound to a binding material, having a ligand containing at least one of the groups anthraquinone, phthalocyanine or aromatic azo, in the presence of at least one metal ion selected from the group Ca.sup.2+, Sr.sup.2+, Ba.sup.2+, Al.sup.3+, Co.sup.2+, Ni.sup.2+, Cu.sup.2+ or Zn.sup.2+. The preferred metal ions are Co.sup.2+, Ni.sup.2+ and Zn.sup.2+, with Zn.sup.2+ being particularly preferred. The ligand may be linked directly to the matrix or via a spacer arm. The process may be performed at atmospheric pressure or under pressure, especially high pressure (100-3500 psi). The nature of the contact, washing and eluting solutions depends on the substance to be separated.