Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis

Reversed-phase high performance liquid chromatography continues to grow in importance for the analysis of peptides and proteins in biomolecular and pharmaceutical research. The mobile-phase conditions for separation of proteins and peptides are essentially fixed. Most separations of these solutes are conducted with shallow gradients using aqueous buffers modified with acetonitrile. Therefore, changing selectivity in peptide and protein separations is often best accomplished by a change in bonded-phase chemistry of the column (e.g. by using CN- or C3-bonded phases rather than C8 or C18). In general, however, short-chain bonded phases are more unstable and irreproducible than bonded phases having longer chains, due to increased susceptibility to hydrolysis as hydrophobicity of the bonded phase decreases. These problems are minimized using bonded phases that are protected through use of sterically bulky side-groups (StableBond Technology). This paper describes a series of comparisons between traditional polymeric bonded-phase silica columns and sterically protected, highly purified, silica stationary phases. These studies compare lot-to-lot reproducibility, stability of the bonded phase, selectivity effects between bonded phases, and operational advantages that can be obtained by high temperature operation using a series of stable bonded phases.