Hydroxy apatite chromatography should be performed within a pH-range from 6 to 9 to obtain the zwitterionic state of a given protein. Even though the binding mechanism is not completely understood, ionic interactions as well as adsorption effects seem to contribute to protein binding. This means that the distribution of charges within the biomolecule affects binding significantly. Experiments have shown that acidic proteins mainly bind to calcium ions whereas basic proteins mainly bind to phosphate groups. Some proteins can also be bound in the presence of high concentration of sodium chloride.
Elution is performed by increasing the phosphate concentration either in a linear or step gradient.
Other buffers and salt gradients like Tris and NaCl can also be used for chromatography on hydroxy apatite.