HEPES

Technical Specifications

Product Description & Scientific Applications

HEPES is a zwitterionic N-substituted aminosulfonic acid and one of the most widely used Good's buffers in biochemical, physiological, and cell-based research. Its conjugate-acid pKa is ≈ 7.48–7.50 at 25 °C, with an effective range of pH 6.8–8.2, covering the near-physiological window. It is highly water-soluble (≥ 400 mg/mL); the piperazine nitrogen is the titratable buffering group and the sulfonate confers zwitterionic character.

Buffer chemistry and temperature dependence. HEPES has a small temperature coefficient for a biological buffer (dpKa/dT ≈ −0.014 per °C; pKa ≈ 7.48 at 25 °C, ≈ 7.31 at 37 °C), less temperature-sensitive than Tris (≈ −0.028 per °C) and useful across cold-room, room-temperature, and physiological conditions. ITC studies show no detectable affinity for Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, or Ni²⁺, mild affinity for Cu²⁺ and Pb²⁺, and weak affinity for Zn²⁺ and Cd²⁺ — supporting Mg²⁺- or Mn²⁺-dependent enzyme assays, though HEPES is not fully non-coordinating: a CuL⁺ complex with log β ≈ 3.22 is documented, so copper-dependent assays and metal-speciation work need controls. HEPES media should be protected from prolonged visible light when riboflavin or other photosensitisers are present, since irradiation can generate hydrogen peroxide and alter readouts.

CO₂-independent buffering in cell culture and live-cell imaging. HEPES is widely used as a supplementary or primary buffer in mammalian cell culture when bicarbonate/CO₂ buffering cannot be maintained, stabilising extracellular pH in imaging media, balanced salt solutions, perfusion buffers, and short-term handling buffers under ambient atmosphere. This suits live-cell and confocal microscopy, benchtop manipulation between incubators, harvesting and washing, transport assays, and electrophysiology recording solutions. Typical media levels run from low-millimolar to tens of millimolar, depending on whether it supplements bicarbonate or serves as the main non-CO₂-dependent buffer.

Calcium phosphate transfection and molecular biology. HEPES-buffered saline (HBS) is the classical system for calcium phosphate transfection of eukaryotic cells: slow mixing of HBS containing sodium phosphate with a CaCl₂/DNA solution generates a fine calcium phosphate–DNA co-precipitate taken up by endocytosis. Formulations commonly use 2× HBS at pH 7.05, reflecting the narrow pH dependence of precipitate formation and transfection performance. HEPES also serves washing, short-term storage, transfection handling, and cell-suspension preparation for downstream analysis.

Protein biochemistry, enzyme assays, and analytical workflows. Its low interaction with Mg²⁺, Mn²⁺ and Ca²⁺ supports kinase, phosphatase, polymerase, and other metal-dependent enzyme assays. HEPES is also used in protein purification by ion-exchange, size-exclusion, and affinity chromatography; in capillary electrophoresis and isoelectric focusing; in native PAGE of protein complexes; and in protein biophysics including ITC, surface plasmon resonance, and fluorescence spectroscopy. Low absorbance at 260/280 nm supports UV/visible and fluorescence-compatible assays with matched blanks. Further uses include selected nucleic-acid hybridisation and in situ-hybridisation/FISH workflows, and protein-crystallography screening and optimisation buffers.

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