The preparation method for the surface zinc-modified hydroxyapatite particles that we developed as immobilized metal ion affinity chromatography medium, required decantation process to remove the excess zinc ion. Therefore, large-scale production was sometimes difficult because of this process. In this report, we describe our improved method to synthesize zinc-substituted hydroxyapatite (CaZnHAp) particles and their further chromatographic properties. This method is simple, easy to scale up, and does not require decantation process. CaZnHAp was synthesized from Ca(OH)(2), H3PO4, and ZnO by a wet method with spray-dryer. This method proved suitable for large-scale production and the quantity of substituted zinc ions was stable without decantation process, as indicated by X-ray photoelectron spectroscopic analysis. The analysis showed that Zn ions were located on the surface and further inside CaZnHAp, while located on only the surface of the conventional material, therefore, CaZnHAp showed stronger adsorbability and durability. Chromatographic separation of histidine oligomers (monomer, dimer, tetramer, and hexamer of histidine) and histidine-tagged protein from CaZnHAp was compared with the results obtained with other materials. These peptides and proteins were more strongly retained by CaZnHAp than other materials and could be separated in one step. This preparation method can serve to obtain hydroxyapatite with stronger affinity. (C) 2012 Elsevier B.V. All rights reserved.