HEPES buffer

🏾 Content :

🏾 Introduction

HEPES is a general purpose buffering agent widely used in biochemistry and molecular biology to maintain physiological pH (pH range 6.8-8.2).

It is a zwitterionic organic compound, it tends to capture or release H+ ions depending on the pH value of the aqueous solution where they are found. The zwitterion is in equilibrium with the cation and the corresponding anion.

HEPES Buffer is categorized as "Good buffers", which is derived from a set of buffers described by Dr. Norman Good et al. (Good et al., Biochemistry 1966).


Hepes buffer

Hepes free acid

CAS number 7365-45-9
Molecular mass 238.30 g/Mol
Formula C8H18N2O4S
Synonyms N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic) acid

HEPE sodium saltS

CAS number 75277-39-3
Molecular mass 260.29 g/Mol
Formula C8H17N2O4SNa
Synonyms sodium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate

🏾 HEPES Buffer Applications and Uses

A buffer is a solution that contains an acid and a base or a salt. Its main purpose is to maintain the concentration of hydrogen ions at an ideal level for specific purposes, such as a bacterial culture. Just like other types of chemical buffers, HEPES buffer maintains the ion concentration of a solution at an ideal level (= ideal pH level) for a certain use. Specifically, HEPES buffer is used to maintain ideal conditions for cell culture

  • HEPES is used in many environments because it has a higher buffering capacity than sodium bicarbonate at physiological pH (7.2 - 7.4) at 37°C. Sodium bicarbonate is nutritionally necessary for most cells, so HEPES should be added in addition to, not instead of, sodium bicarbonate. It is usually added at concentrations of 10-25 mM (higher levels may cause cytotoxicity).
  • HEPES does not have the ability to form a significant complex with most metal ions and is recommended for use as a non-coordinating buffer in solutions with metal ions.
  • HEPES is advantageously used in tissue culture. Fears that HEPES could serve as a nutrient source for aerobic bacteria have proven to be unfounded.
  • HEPES is recommended for the protection of frozen solutions of enzymes against freezing-induced pH changes. It is also useful in oxidative phosphorylation, protein synthesis with cell-free bacterial systems, photophosphorylation, CO2 fixation
  • In TEM (transmission electron microscopy) studies, HEPES does not affect metal substrates. It is a recommended buffer for the glutamate binding assay, which prevents binding to non-receptor materials.

🏾 Properties

HEPES is a general purpose buffer. It is water soluble, does not interfere with biological processes or biological membranes (penetration, solubilization, surface adsorption, etc.), has a known tendency to form complexes with metal ions, is non-toxic and has very low U.V. absorption at wavelength > 260 nm.

HEPES powder is high temperature resistant with its melting point reaching 200℃, so it will not degrade by autoclaving, or you can filter the HEPES buffer solutions using a syringe filter from 0.2 micrometer.

The aqueous solution of HEPES should be stored away from light. exposure to ambient light for three hours would produce cytotoxic hydrogen peroxide


  1. J Jeffrey Morris - Continuous hydrogen peroxide production by organic buffers in phytoplankton culture media
  2. hopaxfc - HEPES handling and storage tips that you must know
  3. goldbio - HEPES, Free Acid
  4. goldbio - HEPES, Sodium Salt
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  6. W Lang - Physiological HEPES buffer proposed as a calibrator for pH measurement in human blood
  7. fisherscientific - Acide libre HEPES
  8. W Lang - Physiological HEPES buffer proposed as a calibrator for pH measurement in human blood
  9. Reagent -
  10. Biowest - HEPES Buffer Solution
  11. Uptima - HEPES Buffer
  12. Capricorn Scientific - HEPES Buffer Solution