HPLC separation of biomolecules using calcium phosphate supported on HPLC macroporous silica microparticles

The preparation of a chromatographic support showing similar selectivity and chemical inertness to hydroxyapatite (HA) and mechanical resistance to the pressures generally used in high-performance liquid chromatography is described. A mixed matrix was formed by covering macroporous silica microparticles with a thin layer of calcium phosphate (CaP-HA). The porous (500, 1000 and 4000 χ) silica microparticles (15 and 10 μm) had been previously inactivated with glycidoxypropyltrimethoxysilane in acidic medium, to convert the silanol groups into hydrophilic groups, producing a biocompatible support with excellent properties in terms of mechanical resistance to high pressure (5000 p.s.i.), selectivity, chemical inertness and efficiency. Columns packed with small particles (5 μm) were run at high pressure and high flow-rates for prolonged periods. Mixed silica WP-DIOL-/CaP-HA matrices were used to separate a standard protein mixture antimelanoma monoclonal antibody and some small molecules such as carnitine derivatives and sugars.