Boundary between protein and peptide shown by their chromatographic behavior.

It has previously been pointed out that two different mechanisms exist in the reversed-phase (RP) HPLC of polypeptides, including proteins. We systematically investigated the separation of various peptides and proteins over a wide range of molecular weight using a nonporous octadecylsilyl (ODS) silica-gel column to provide a precise explanation for the separation mechanism of polypeptides, including proteins in RP-HPLC. As a result, we clarified that a critical point between a typical reversed-phase partition mode applicable to small peptides (molecular weight < 3000) and a characteristic elution mode applicable to proteins is in the vicinity of the molecular weight of 3500-4500. We also proposed a new concept, the "Transitional Desorption Mode", as a separation mechanism that can precisely explain the RP-LC separation of a wide range of polypeptides including proteins.