Public Health Laboratory Service Board

The Public Health Laboratory Services Board

Christopher R Lowe

Anthony Atkinson

Klaus Mosbach

Roger F Sherwood

Peter Hughes

David A Small

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. Generally the contact solution is made up of the substance to be separated and a metal salt dissolved in a buffer solution, while the washing solution comprises the same metal salt dissolved in the same buffer. The eluting solution, may be a buffer solution, either alone or containing a chelating agent or it may be an alkali metal salt or a specific desorbing agent. Alternatively the eluting solution may be a mixture of two or more of these solutions or two or more of these solutions used consecutively.

Affinity chromatography using metal ions

A process for the high pressure liquid affinity chromatographic separation of at least one biological or related substance from a mixture in which the contact, washing and eluting phases are performed on a binding material made from a ligand, containing at least one of the groups anthraquinone, phthalocyanine or aromatic azo, coupled to a matrix through a spacer arm, the binding material being so constructed that at least one biological or related substance is retained on the binding material during the contact and washing phases. In one preferred embodiment the ligand is a reactive dye, especially a triazinyl dye, the matrix is agarose or silica and the spacer arm is a substituted aminohexyl group. The chromatographic procedure is preferably performed at 100-3500 psi, at a flow rate of 0.5-2.0 ml/min. The choice of washing and eluting solutions depends on the material to be separated. However buffer solution to wash the column and a desorbing agent to elute the material are preferred. When the biological substance is an enzyme the desorbing agent is preferably an enzyme substrate, cofactor, inhibitor or analogue thereof. A binding material for use in the above HPLAC procedure is also provided. This is made from a ligand containing at least one of the groups anthraquinone, phthalocyanine or aromatic azo, coupled to a matrix through a spacer arm.

High pressure liquid affinity chromatography

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Eastleigh, United Kingdom

Christopher R Lowe