HEPES

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Chemical structure of HEPES = N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid)
Chemical structure of HEPES = N-(2-hydroxyethyl)
piperazine-N’-
(2-ethanesulfonic acid)

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.

Contents

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); (2nd pKa at pH 3 is not of interest)
  • usable buffering range of 6.8 to 8.2
  • molecular weight 238.3 g/mol
  • ΔpKa/ΔT = -.014
  • HEPES contains tertiary amines, which are reactive under certain conditions.
  • Chemical formula: C8H18N2O4S; N-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid); aka 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; CAS number: 7365-45-9

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

1M HEPES KOH buffer 7.5

  • 700 ml ultrapure water
  • 238.3 g HEPES
  • KOH (potassium hydroxide) pellets to adjust pH to 7.5
  • to 1L w ultrapure water; sterilise by filtration

1M HEPES-NaOH pH 7.5

  • 700 ml ultrapure water
  • 238.3 g HEPES
  • ~5.5 g NaOH (sodium hydroxide) pellets to adjust pH to 7.5
  • to 1L w ultrapure water; sterilise by filtration

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