Nitrogen-specific detection of peptides in liquid chromatography with a chemiluminescent nitrogen detector

Abstract

High-performance liquid chromatography with chemiluminescent nitrogen detection (HPLC-CLND) in the reversed-phase mode was used to quantitate peptides that were isolated from casein hydrolysate. When CLND is used simultaneously with a UV detector in peptide mapping, unique quantitative information about the nitrogen distribution of the sample is obtained. Nitrogenous compounds without UV chromophores are easily detected by CLND without pre- or post-column derivatization. Of further significance, the non-nitrogenous compounds in the sample matrix are transparent to the detector. This paper will focus primarily on the analysis of two peptides, identified as 1 and 2. The UV peptide map showed peptide 2 as the largest component. On the other hand, the CLND results indicated that peptide 1 was the major peak. RP-HPLC amino acid analysis of the peptides 1 and 2 confirmed the CLND results. This analysis shows that peptide 1 was the major component and did not contain aromatic amino acid residues. Peptide 2 however, contained aromatic groups with strong chromophores, thereby explaining the UV response.