Chemiluminescent nitrogen detection as a new technique for purity assessment of synthetic peptides separated by reversed-phase HPLC.

Abstract

Reversed phase high-performance liquid chromatography with chemiluminescent nitrogen detection (HPLC-CLND) was used for the quantitative analysis of peptides manufactured by solid-phase peptide synthesis. CLND provided quantitative information regarding the nitrogen distribution of peptide samples following HPLC separation. This technique permits the universal quantitation of the "peptide content" of synthetic peptides in an on-line mode without pre- or post-column derivatization and free of interference from non-nitrogen-containing UV chromophores. This paper will show the utility of this novel technique in measuring the peptide content of crude synthetic proinsulin chain C peptide. A mixture of five reference peptides was analyzed to show the homogeneity of the CLND response and used to determine peptide content. Application for the purified product is also discussed. The detection profiles were acquired in parallel with a UV detector.