"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.
The level of HEPES in cell culture media may vary from 10mM to 25mM. In Invitrogen's GIBCO® DMEM it is at a concentration of 25mM. In Invitrogen's 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.