HEPES, CAPS, MES and MOPS are zwitterionic organic chemical buffering agents. These are very polar compounds which are not retained by traditional reverse phase chromatography. These compounds are zwitterions in nature and can be separated by mixed-mode hydrophilic interaction chromatography on Obelisc N column. Retention is achieved by combination of HILIC and ion-exchange mechanisms. These buffering agents do not have UV active groups, but can be analyzed with ESLD, LC/MS, and CAD detection.