Effect of column degradation on the reversed-phase high-performance liquid chromatographic separation of peptides and proteins.

Many reversed-phase separations of proteins and peptides are currently performed in acidic mobile phases, e.g., 0.1% trifluoroacteic acid in water (pH 2) with organic modifiers. Such conditions are known to promote the cleavage of the silane from the silica in bonded-phase columns, especially for monomeric stationary phases. The stability of some columns commonly used for proteins and peptides has been examined, and it has been shown by both chromatographic and elemental analysis that degradation occurs very rapidly with fresh, "totally covered" column materials. Despite the loss of over half of the bonded phase in some cases, certain columns still exhibit adequate chromatographic performance, although reproducibility can be affected. The implications of these results with respect to both bonded-phase synthesis and mechanistic interpretation of chromatographic data is discussed.