ANALYSIS OF SYNTHETIC PEPTIDES BY CAPILLARY-ZONE-ELECTROPHORESIS IN ORGANIC AQUEOUS BUFFERS/

Whereas synthetic peptides have been routinely analyzed for purity by reverse phase high performance liquid chromatography (RPHPLC) for a nu mber of years, it is only in the last decade that the use of capillary zone electrophoresis (CZE) in aqueous buffers has been taken advantag e of as an orthogonal method for the detection of impurities. However, we have found that hydrophobic amino acids and peptides often migrate as very broad, tailing absorbances or even precipitate in the aqueous buffers during CZE analysis. As a result, alternative buffer systems containing organic modifiers were sought. Varying concentrations of ac etonitrile, methanol and isopropanol in sodium phosphate and triethyla mmonium phosphate buffers were used to study their effects on the elec trophoretic migration of several synthetic peptides [gonadotropin rele asing hormone (GnRH), corticotropin releasing factor (CRF) and analogs ] and an enantiomeric synthetic amino acid. The organic/aqueous buffer s used to obtain the best conditions for separation of porcine gonadot ropin-releasing hormone (GnRH) and chicken II GnRH were then used to o ptimize a separation of nine native forms of GnRH decapeptides. Intere stingly, several of these GnRHs have identical formal charges and yet could be separated. This suggests a mixed mechanism of separation that discriminates not only on the basis of peptide charge and structure b ut also of adsorptive properties (Van der Waals forces, dipole-dipole interactions and hydrogen bonding) of the capillaries. (C) Munksgaard 1998.