This application note describes how to use microbore reversed-phase HPLC to separate peptide mixtures, to characterize proteins or to isolate peptides for further studies.
This document details the instrumental prerequisites for successful peptide mapping on 1-mm internal diameter (id) reversed-phase columns. It highlights the importance of a high-performance HPLC system with low delay volume, excellent mixing, and precise column thermostatting to achieve high-resolution separations. The study demonstrates how these factors influence the qualitative and quantitative aspects of chromatography for characterizing proteins or isolating peptides.
Column temperatures between 30 and 50 °C usually provide the best resolution.
The system delay volume should not exceed the column volume by more than a factor of 1-2.
The experiment utilized a flow rate of 0.12 ml/min.