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Chemical structure of HEPES = N-(2-hydroxyethyl)
(2-ethanesulfonic acid);
aka 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CAS number: 7365-45-9

HEPES is a common buffering chemical similar to Tris in Tris-HCl buffers and phosphates in PBS. Buffers are used to keep the pH at a certain value and can "buffer" the addition of small amounts of acids/bases.

A Good buffer [Good'66 PMID 5942950, Good'74 PMID 4206745, Blanchard'84 PMID 6717292].

Property of HEPES

Stable pH vs. temperature, no primary amine groups, no metal chelation, near physiologic pH range. HEPES is often used to maintain protein solubility in biochemical experiments.

  • pKa at 25C of 7.55 (7.31 at 37C)
  • a second pKa at pH 3 is not of interest
  • usable buffering range of 6.8 to 8.2
  • molecular weight 238.3
  • ΔpKa/ΔT = -.014
  • HEPES contains tertiary amines, which are reactive under certain conditions.
  • Chemical formula: C8H18N2O4S

Buffers are typically 1 M, prepared by neutralizing HEPES with sodium hydroxide. HEPES is essentially insoluble until it is neutralized. and