CHT Ceramic Hydroxyapatite Mechanism
Hydroxyapatite contains two types of binding sites, positively charged calcium and negatively charged phosphate groups. These sites are distributed regularly throughout the crystal structure of the matrix. Solute species dominantly interact through cation exchange via the phosphate groups and/or metal affinity via the calcium atoms.
Cation exchange occurs when protein amino groups interact ionically with the negatively charged phosphates. The amino groups are similarly repelled by the calcium sites. Binding depends upon the combined effects of these interactions. These ion exchange interactions can be disrupted by adding neutral salts such as sodium chloride or buffering species such as phosphate to the mobile phase. Cation exchange interactions also weaken with increasing pH. Hence, the addition of salt or phosphate, or an increase in pH, can be used to weaken the interaction. Studies with model proteins have demonstrated that anion exchange, which might be expected from interactions of negatively charged surface residues with calcium, does not make a significant contribution.
Calcium affinity occurs via interactions with carboxyl clusters and/or phosphoryl groups on proteins or other molecules (e.g., nucleic acids); these groups are simultaneously repelled by the negative charge of the CHT phosphate groups. The affinity interaction is between 15 and 60 times stronger than ionic interactions alone and, like classical metal-affinity interactions, is not affected by increasing ionic strength using typical elution ions (e.g., chloride). Species binding through calcium affinity may adsorb more strongly as the ionic strength increase due to ionic shielding of the charge repulsion from the CHT phosphate sites. Metal affinity interactions can be dissociated by phosphate in the mobile phase.
Most large proteins bind by a combination of mechanisms:
Dominantly acidic proteins, such as albumin, bind chiefly by metal affinity interactions. Sodium chloride at 1.0 M reduces retention time by approximately 10% in the presence of phosphate gradients, indicating a minor contribution by cation exchange. To elute acidic proteins, phosphate buffers are required.
Dominantly basic proteins, such as IgG, bind chiefly by cation exchange interactions. Sodium chloride reduces retention time in the presence of phosphate gradients, indication a minor contribution by metal-affinity. Basic proteins may be selectively eluted with either phosphate or salts.
