HEPES's dipolar ionic (zwitterionic) structure means it does not form significant complexes with most metal ions, making it a preferred non-coordinating buffer for solutions containing biologically relevant metal cations --- an advantage over phosphate or citrate buffers in many applications. In cell culture specifically, HEPES outperforms bicarbonate-based buffering systems by maintaining stable physiological pH independent of CO2 fluctuations from cellular respiration, making it especially valuable for open-air culture systems, live-cell imaging, and applications outside standard CO2 incubators. A key handling consideration: prolonged exposure to fluorescent light or sunlight causes HEPES to react with riboflavin in culture media, generating cytotoxic hydrogen peroxide.
HEPES is a zwitterionic N-substituted aminosulfonic acid buffer, recognized as one of the "Good's buffers" and among the most widely used all-purpose buffering agents in cell biology, biochemistry, and molecular biology research. With an effective buffering range of pH 6.8--8.2 (pKa 7.48 at 25°C), it excels at maintaining stable physiological pH conditions.
| Chemical Name | HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) |
| CAS Number | 7365-45-9 |
| IUPAC Name | 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid |
| Molecular Formula | C8H18N2O4S |
| Molecular Weight | 238.30 g/mol g/mol |
Standard buffering supplement maintaining stable physiological pH in mammalian, insect, and other cell culture systems.
Widely used in PCR, protein purification, enzyme assays, immunoprecipitation, and cell lysis buffers.
Applied as a running buffer in gel electrophoresis and as an eluent in cation exchange chromatography.
Preferred buffer for applications requiring pH stability outside controlled CO2 environments.
IRL Press (reference text)
In Vitro Cell. Dev. Biol.
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