Fast, low-pressure chromatographic separation of proteins using hydroxyapatite nanoparticles

Columns packed with ultrafine particles (e.g. sub-2 gm porous particles) are suitable for high-resolution, highspeed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm x 150 nm) and spherical ( < 200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 mM at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.