Background: Retention mechanism of proteins in hydroxyapatite chromatography (HAC)
was investigated by linear gradient elution experiments (LGE).
Materials and Methods: Several mobile phase (buffer) solution strategies and solutes were evaluated in
order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site
due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic
proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C)
and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion
due to P-site was eliminated. The number of the binding site B values determined from LGE also increased,
which also showed reduction of repulsion forces.
Conclusion: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different
from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by
using NaCl gradient.