Comparison of reversed-phase and cation-exchange high-performance liquid chromatography for separating closely related peptides: separation of asp 76-human parathyroid hormone (1–84) from asn 76-human parathyroid hormone (1–84)

Abstract

Cation-exchange high-performance liquid chromatography (CE-HPLC) was compared with ordinary reversed-phase high-performance liquid chromatography (RP-HPLC) for separating closely related peptides. Some synthetic samples of bradykinin and angiotensins, which were homogeneous according to RP-HPLC, were found to be inhomogeneous when analyzed by CE-HPLC. On the other hand, diastereomeric peptides could be separated much more efficiently by RP-HPLC than by CE-HPLC. These results indicated that the purity of synthetic peptides chould be checked not only by RP-HPLC but also by ion-exchange HPLC. In the case of human parathyroid hormone (hPTH), baseline separation of Asp 76-hPTH from Asn 76-hPTH by RP-HPLC was not possible, but was by CE-HPLC. Using this method we confirmed that the Asn residue in hPTH at position 76 could not be converted into the Asp residue under the conditions used to isolate and purify it from human organs.