HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) is a zwitterionic organic chemical buffering agent and is categorized as a "Good" buffer which derives from a set of buffers described by Dr. Norman Good and his colleagues in 1966 (Good et. al., Biochemistry 1966). 

"Good buffers" such as HEPES are attributed with the following characteristics:

  • pKa values between 6.0 and 8.0
  • high solubility
  • membrane impermeability
  • limited effect on biochemical reactions
  • very low visible and ultraviolet light absorbance
  • chemically and enzymatically stable
  • easy to prepare.

HEPES is widely used in many biochemical reactions and as a buffering agent in some cell culture media.

HEPES Use in Cell Culture

The most commonly used buffering system for media is bicarbonate.  This is due to its nutritional benefits despite the reduced buffering capacity at physiological pH.  Addition of HEPES provides supplemental buffering to cell culture medium at pH 7.2 through 7.6.

The level of HEPES in cell culture media may vary from 10mM to 25mM.  In GIBCO® DMEM it is at a concentration of 25mM.  In GIBCO® DMEM/F-12 it is at a 15mM.  The most commonly used concentration is 25mM. 

HEPES has no nutritional benefit to cells.  It is added to the media solely for extra buffering capacity when cell culture requires extended periods of manipulation outside of a CO2 incubator.