ABSTRACT
Reversed-phase high-performance liquid chromatography (RPC) has grown to become a mature and well-established technique in the analysis, structure elucidation, and isolation of peptides and proteins. In gradient elution RPC, peptides and proteins are retained essentially according to their hydrophobic character. RPC of peptides and proteins is carried out on n-alkyl bonded silicas with an acidic low ionic strength mobile phase as a starting eluent and applying a gradient of organic solvent. Hydrophobic peptides are preferably chromatographed on cyanopropyl bonded silicas. The type of n-alkyl silica is also fundamentally important with respect to column life-time and recovery. The shorter the chain length, the more sensitive the anchoring siloxane bonds are to hydrolytic cleavage. Furthermore, n-alkyl silicas with a polymer type of layer offer a superior stability compared to those of monolayer type. Retention studies of peptides and proteins on n-alkyl bonded silicas with various pore sizes in the range between 6 and 100 nm.
