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HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic buffer used in cell culture and biochemistry. It has a molecular formula of C8H18N2O4S and a molecular weight of 238.31, and its useful pKa sits near 7.5, so it holds pH in the physiological window without relying on carbon dioxide. This page gives the properties, the typical concentrations in culture media, how it compares with Tris, and the light-exposure caveat that most product pages skip.
Short answer: what is HEPES?
HEPES is a “Good” buffer: a zwitterionic sulfonic-acid buffer whose pKa falls between 6.0 and 8.0, which is the range Thermo Fisher describes for this buffer family. In mammalian cell culture it is added to media, commonly at 10 to 25 mM, to keep pH near 7.2 to 7.6 when cultures are handled outside a CO2 incubator. It does not replace the bicarbonate system; it supplements it.
Definition and scope: HEPES full name, formula, molecular weight and structure
- Full name: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (IUPAC), also written 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
- Molecular formula and weight: C8H18N2O4S; molecular weight 238.31 (the “HEPES molar mass” or “HEPES MW” searchers ask about). Source: PubChem compound record, CID 23831, read 2026-11-07.
- Structure: a piperazine ring carrying a hydroxyethyl group on one nitrogen and an ethanesulfonic acid group on the other, as the IUPAC name shows.
- Form: a solid, white powder in its free-acid form (PubChem physical description).
HEPES pKa and buffer range
A buffer works best within about one pH unit of its pKa. A 2015 AIMS Biophysics paper on universal biochemical buffers (Brooke, Movahed and Bothner) reports pKa values of 7.55 for HEPES and 8.01 for Tris, and describes HEPES as diprotic with two relevant pKa values. Using the one-unit rule of thumb, the range around 7.55 is roughly pH 6.5 to 8.5; Thermo Fisher gives pH 7.2 to 7.6 as the range media buffering aims for.
Temperature matters. The same paper measured a pKa shift of -0.014 per °C for HEPES-based universal buffers at pH 7, and states that a Tris-HCl buffer changes by about 2 pH units over a protein thermal-denaturation run from 298 to 373 K. A buffer pH-adjusted at room temperature is therefore not at that pH at 37 °C or 4 °C, and the effect is larger for Tris than for HEPES.
How HEPES is used in practice: concentrations in cell culture media
Thermo Fisher’s HEPES reagent page (read 2026-11-07) states that media typically contain 10 to 25 mM HEPES, with 25 mM the most common, and gives these examples:
| Medium | HEPES concentration (Thermo Fisher) |
|---|---|
| DMEM (HEPES-containing formulation) | 25 mM |
| DMEM/F-12 (HEPES-containing formulation) | 15 mM |
| Typical range across media | 10 to 25 mM |
For how the base media differ, see DMEM vs RPMI and types of cell culture media.
HEPES buffer recipe: the arithmetic
The molecular weight gives the mass directly: a 1 M stock needs 238.31 g of HEPES free acid per litre of final volume, and a 25 mM working concentration corresponds to about 5.96 g per litre. The free acid is then titrated to the target pH with a strong base at the temperature of use and brought to final volume. Sterilisation, water grade and storage follow your SOP and the supplier’s label; the general method is in buffer and solution preparation.
HEPES vs Tris, and vs bicarbonate
| Property | HEPES | Tris | Source |
|---|---|---|---|
| Reported pKa | 7.55 | 8.01 | Brooke et al., AIMS Biophys 2015 |
| Temperature sensitivity | -0.014 pKa/°C (HEPES-based mixtures at pH 7) | Larger: the Tris-containing mixture measured -0.020 pKa/°C, and Tris-HCl alone shifts about 2 pH units from 298 to 373 K | Brooke et al., AIMS Biophys 2015 |
In short, HEPES sits closer to physiological pH and drifts less with temperature, which is why it is a common choice for live-cell work, while Tris is common in molecular-biology stocks. Bicarbonate is the physiological system most media already use; it depends on a CO2 atmosphere, and that is the gap HEPES fills during handling outside the incubator (Thermo Fisher). For the general principles, see what is a biological buffer.
Where it matters for labs: light exposure and hydrogen peroxide
The caveat most product pages omit is photochemical. Zigler et al. (In Vitro Cellular and Developmental Biology, 1985) found that adding HEPES to RPMI 1640 markedly increased cytotoxic products when the medium was exposed to visible light; the effect needed only HEPES and riboflavin, and hydrogen peroxide was a principal cytotoxic agent. A later study (Mahns et al., Free Radical Research, 2003) found hydrogen peroxide at least three-fold higher in HEPES-buffered media after UVA irradiation and concluded that riboflavin and HEPES or tryptophan should be avoided during irradiation experiments.
The practical reading, an editorial interpretation of those papers, is that HEPES-containing media are worth keeping out of prolonged light, and that HEPES is a variable to control in imaging or irradiation experiments.
Common misunderstandings
- “HEPES replaces CO2.” It adds buffering that does not depend on CO2; it is not a stand-alone substitute for a medium’s bicarbonate system in an incubator.
- “More is always safer.” Typical media use 10 to 25 mM; the light-exposure results above show HEPES is not an inert additive.
- “pH set at room temperature holds at 37 °C.” Buffer pKa moves with temperature, by an amount that differs between HEPES and Tris.
FAQ
What is HEPES?
A zwitterionic “Good” buffer (C8H18N2O4S, molecular weight 238.31) with a pKa near 7.5, used to stabilise pH in culture media and biochemical solutions.
What is the difference between HEPES and Tris?
HEPES has a lower pKa (7.55 against 8.01 in the paper cited above) and a smaller temperature shift. Tris buffers best at higher pH and changes pH markedly with temperature.
How long is HEPES buffer good for?
No universal figure applies. Shelf life depends on the supplier’s label, the formulation, sterility, container and light exposure; follow the product’s stated expiry and your lab’s SOP, and protect HEPES-containing media from light.
What is the main function of a buffer?
A buffer resists change in pH when small amounts of acid or base are added, keeping a solution within a working range around its pKa.
Can you provide a recipe for HEPES buffer?
See the arithmetic above: 238.31 g per litre gives 1 M. Titrate to the target pH at the temperature of use and follow your protocol for sterilisation and storage.
Sources
- PubChem, compound CID 23831 (HEPES): pubchem.ncbi.nlm.nih.gov/compound/23831, read 2026-11-07.
- Thermo Fisher Scientific, HEPES (mammalian cell culture reagents): thermofisher.com HEPES page, read 2026-11-07.
- Brooke D, Movahed N, Bothner B. Universal buffers for use in biochemistry and biophysical experiments. AIMS Biophysics 2015;2(3):336. doi:10.3934/biophy.2015.3.336, PubMed Central PMC8956001, read 2026-11-07.
- Zigler JS Jr, Lepe-Zuniga JL, Vistica B, Gery I. In Vitro Cell Dev Biol 1985;21(5):282-7. doi:10.1007/BF02620943, PubMed 4019356.
- Mahns A, Melchheier I, Suschek CV, Sies H, Klotz LO. Free Radic Res 2003;37(4):391-7. doi:10.1080/1071576031000064702, PubMed 12747733.
