Effect of Buffer on HPLC Separation of Buffers

HEPES, CAPS, MES, and MOPS are zwitterionic organic buffering agents commonly used in biological and biochemical applications. Due to their high polarity and zwitterionic nature, these compounds are not retained on traditional reversed-phase columns. However, effective separation can be achieved using the Zodiac HST N column operated in mixed-mode hydrophilic interaction chromatography (HILIC).

Retention is governed by a combination of HILIC and ion-exchange mechanisms, allowing for efficient resolution of these structurally similar buffer compounds. Since these buffers lack strong UV chromophores, detection is typically performed using ELSD, LC/MS, or CAD, making the method suitable for non-UV active analytes.

This approach offers a robust and selective platform for the analysis of polar, zwitterionic buffering agents in various complex matrices.