Fundamental study of hydroxyapatite high-performance liquid chromatography : III. Direct experimental confirmation of the existence of two types of adsorbing surface on the hydroxyapatite crystal

Abstract

By using several types of hydroxyapatite (HA) with different external shapes and surface structures, adsorption and the high-performance liquid chromatographic experiments for several proteins were carried out in parallel. Direct confirmation was obtained that two types of adsorbing surface, the a (or b) and the c crystal surface, generally appear on the HA particle. During the chromatographic process on the column, acidic proteins with isoelectric points lower than about 7 (and also nucleic acids including other nucleotides) are mainly adsorbed on the a (or b) surface, basic proteins with isoelectric points higher than about 7 are mainly adsorbed onto the c surface, and these occur independently of the external crystal shape of HA that is used. It is highly probable that the surface of a domain on the HA crystal on which the molecular adsorption takes place is smooth, and that the size of the domain under consideration is larger than the size of the proteins that have been applied, i.e., larger than 100–200 Å, at least with respect to the HAs that have been examined. Multilayers of protein molecules may be formed on the HA surface when the molarity of the phosphate buffer in the mobile phase is much lower than the molarity that is needed for the migration of the molecules on the column.