The present invention relates to a method of separating one or more immunoglobulin containing proteins from a liquid. The method includes first contacting the liquid with a separation matrix comprising ligands immobilized to a support; allowing the immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands; followed by an optional step of washing the matrix containing the immunoglobulin containing proteins adsorbed thereon; and recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins. The method improves upon previous separation methods in that each of the ligands comprises one or more of a protein A domain (E, D, A, B, C), or protein Z, or a functional variant thereof, with at least one of the monomers having a substitution of the Asparagine or Histidine at the position corresponding to H18 of B domain of Protein A or Protein Z, and wherein the ligand provides an increase in elution pH compared to non-substituted ligand.