HPLC Determination of HEPES on Newcrom AH Column

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HEPES is a zwitterionic organic chemical buffering agent.  It is a very polar compound which is not retained by traditional reverse phase chromatography.  HEPES can be separated by mixed-mode hydrophilic interaction chromatography on the Newcrom AH mixed-mode column, which has both hydrophobic and cationic exchange properties.  The mobile phase consists of H2O and Methanesulfonic acid.  It can be detected using a conductivity detector.


Column Newcrom AH, 4.6×150 mm, 5 µm, 100A
Mobile Phase H2O- 100%
Buffer Methanesulfonic acid  – 10 mM
Flow Rate 1.0 ml/min
Detection Conductivity



Class of Compounds
Analyzing Compounds HEPES


Application Column

Newcrom AH

The Newcrom columns are a family of reverse-phase-based columns. Newcrom A, AH, B, and BH are all mixed-mode columns with either positive or negative ion-pairing groups attached to either short (25 Å) or long (100 Å) ligand chains. Newcrom R1 is a special reverse-phase column with low silanol activity.

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Application Analytes:
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic Acid)
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.