HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic sulfonic acid 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 aerobic 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.[1] Lepe-Zuniga et al. reported an unwanted photochemical process wherein HEPES when exposed to ambient light produces hydrogen peroxide,[2][3] 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 to prevent oxidation.

IUPAC name
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
Other names
3D model (JSmol)
ECHA InfoCard 100.028.098
EC Number
  • 230-907-9
RTECS number
  • TL6809000
Molar mass 238.3012 g/mol
Appearance white crystalline powder
Density Not applicable
Melting point >234-238°C (453-457K)
40 g/100 ml (20°C)
Acidity (pKa) 3 (pKa1),
7.5 (pKa2)
Main hazards Eye Irritant
Safety data sheet External MSDS
GHS pictograms
GHS Signal word Warning
H315, H319, H335
P261, P264, P270, P271, P280, P301+312, P302+352, P304+312, P304+340, P305+351+338, P312, P321, P322, P330, P332+313, P337+313, P362, P363, P403+233, P405, P501
NFPA 704 (fire diamond)
Flash point Non-flammable
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
N verify (what is YN ?)
Infobox references

HEPES has the following characteristics:

  • pKa1 (25 °C) = 3
  • pKa2 (25 °C) = 7.5
  • Useful pH range = 2.5 to 3.5 or 6.8 to 8.2

See also


  1. Baicu SC, Taylor MJ (2002). "Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency". Cryobiology. 45 (1): 33–48. doi:10.1016/S0011-2240(02)00104-9. PMID 12445548.
  2. Lepe-Zuniga JL, Zigler JS, Gery I (October 1987). "Toxicity of light-exposed Hepes media". J. Immunol. Methods. 103 (1): 145. doi:10.1016/0022-1759(87)90253-5. PMID 3655381.
  3. Zigler JS, Lepe-Zuniga JL, Vistica B, Gery I (May 1985). "Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium". In Vitro Cell. Dev. Biol. 21 (5): 282–7. doi:10.1007/BF02620943. PMID 4019356.
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