Separation of Pyrazines by Mixed-Mode HPLC

This application using the Zodiac HST B column illustrates the flexibility in tuning separation methods developed on mixed-mode columns. The pyrrole compounds, 5-formyl-1H-pyrrole-3-carboxylic acid and pyrrole-2-carbaldehyde, are efficiently separated, with the elution order easily reversed by switching the mobile phase modifier from trifluoroacetic acid (TFA) to acetic acid (HOAc). This switch impacts both the mobile phase pH and the counterion interaction with the column’s embedded functionality, resulting in changes to retention behavior. The method provides excellent peak shapes using a mass spectrometry-compatible mobile phase of water, acetonitrile (MeCN, ACN), and either TFA or HOAc as the acidic modifier.