Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. V. Performance of non-porous monodisperse 1.5-microns bonded silicas in the separation of proteins by hydrophobic-interaction chromatography.

Non-porous monodisperse 1.5-microns silicas were allowed to react with (A) and (B) N-acetylaminopropyltriethoxysilane to generate bonded phases useful in high-performance hydrophobic-interaction chromatography (HIC). Differences in the selectivity were observed between the amide and the ether phase. Peak capacities between 10 and 30 were achieved for several proteins with the amide and ether phase packed into columns of 36 X 8 mm I.D. and elution of the proteins under chromatographic conditions in which the gradient volume, VG, was held constant by varying the gradient time between 20 and 2.5 min and the flow-rate between 0.5 and 4.0 ml/min. The S values derived from the dependences of log k' on the volume fraction of the low ionic strength buffer, phi b, were of the same magnitude as reported for porous HIC silicas and showed a dependence on the molecular weight of the protein. Using these HIC stationary phases based on non-porous 1.5-microns supports, fast separations (less than 5 min) could be carried out with high biological recoveries.