HEPES

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.

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.

• pK_a at 25C of 7.55 (7.31 at 37C); (2nd pK_a at pH 3 is not of interest)
• usable buffering range of 6.8 to 8.2
• molecular weight 238.3 g/mol
• ΔpK_a/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

Links

• original publications: Good'66 PMID 5942950, Good'74 PMID 4206745, Blanchard'84 PMID 6717292
• Wikipedia: buffer solution
• CP: common buffers
• List of common buffers from Smith College