HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) is a zwitterionic organic chemical buffering agent; one of the twenty Good's buffers. HEPES is widely used in cell culture, largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration) when compared to bicarbonate buffers, which are also commonly used in cell culture. The dissociation of water decreases with falling temperature, but the dissociation constants ( pK) of many other buffers do not change much with temperature. HEPES is like water in that its dissociation decreases as the temperature decreases. This makes HEPES a more effective buffering agent for maintaining enzyme structure and function at low temperatures. Lepe-Zuniga et al. reported a phototoxicity of HEPES when exposed to ambient light by the production of hydrogen peroxide, which is not a problem in bicarbonate-based cell culture buffers. It is therefore strongly advised to keep HEPES-containing solutions in darkness as much as possible.
HEPES has the following characteristics:
- pK (25 °C) = 3
- pK (25 °C) = 7.5
- Useful pH range = 2.5 to 3.5 or 6.8 to 8.2