Synonym(s): 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), HEPES

Description

HEPES is one of the most widely used zwitterionic buffers in biology, valued for its stable buffering capacity across the physiological pH range of 6.8–8.2. Biofargo's HEPES Biochemical Grade is supplied as the free acid at ≥99.5% purity — a dependable, cost-effective choice for routine cell culture, protein work, and general laboratory buffer preparation.

A key practical advantage of HEPES is that it does not significantly bind metal ions, making it suitable as a non-coordinating buffer in systems containing metal cofactors — unlike many other buffers, it stays out of the way of metal-dependent reactions. It is also commonly used in cell culture media to hold pH steady, particularly during work outside a CO₂ incubator.

This Biochemical Grade is intended for general-purpose use. For high-sensitivity workflows — PCR, fluorescence detection, and other applications where UV-absorbing impurities matter — see our HEPES Ultrapure Grade, which is further refined for lower UV absorbance.

Specifications

Item	Value
Chemical Name	4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
Appearance	Pure White Powder
CAS No.	7365-45-9
Molecular Formula	C₈H₁₈N₂O₄S
Molecular Weight	238.31
Grade	Biochemical Grade
Purity	≥99.5%
Heavy Metals	≤5ppm
pKa at 20℃	7.4-7.7
Sulfates	≤0.05%
Color (1M Reagent Solution)	Passed the Test
Loss on Drying (105℃, 3 hours)	≤1%
UV Absorbance at 260nm (1mol/L, 1cm)	≤0.5
UV Absorbance at 280nm (1mol/L, 1cm)	≤0.5
UV Absorbance at 440nm (1mol/L, 1cm)	≤0.2
Storage / Handling	Store at Room Temperature.

Features

Biochemical grade, ≥99.5% purity — High-purity free acid form of HEPES, with minimal impurities, suitable for demanding biochemical and cell culture applications.
Optimal buffering at physiological pH — pKa ~7.5, effective range ~pH 6.8–8.2; stable pH control around physiological conditions (~7.2–7.6).
Zwitterionic, non-chelating buffer — Negligible binding to metal ions, minimizing interference in metal-dependent enzyme assays.
Low UV/visible absorbance — Suitable for absorbance, fluorescence, and protein assays without significant background. (For the lowest UV background, see Ultrapure Grade.)
Chemically and enzymatically stable, membrane-impermeable — Stable under typical lab conditions; does not readily cross cell membranes, maintaining extracellular buffering capacity in cell culture.
High solubility — Dissolves readily in water for straightforward, reproducible buffer preparation.

Applications

Cell culture media and cell-based assays — Maintains stable pH, especially during work outside CO₂ incubators. Typical working concentration 10–25 mM.
Protein purification, enzymatic reactions, biochemical assays — Non-chelating and minimally interfering; suitable for enzyme assays, affinity and ion-exchange chromatography, and protein extraction.
Molecular biology and electrophoresis — Used in DNA/RNA handling buffers, electrophoresis sample/running buffers, and isoelectric focusing.
Cell lysis, immunoprecipitation, immunoassays — Stable pH and minimal metal chelation for antibody-based workflows.
Compatible with the BCA protein assay.

Safety Disclaimer

This product is intended for laboratory research use only. Not for drug, human, or veterinary use. Review the Safety Data Sheet (SDS) provided by BioFargo for detailed handling, storage, hazard, and disposal information. Use appropriate personal protective equipment (PPE) including gloves, lab coat, and eye protection when handling. Dispose of waste and residual buffer in accordance with local, state, and federal regulations. Ensure proper ventilation if preparing large volumes or heating solutions.

FAQ

What is HEPES used for?

HEPES is a zwitterionic buffer used to maintain stable pH in biological systems, most commonly in cell culture media, protein purification, enzyme assays, and molecular biology buffers. It is effective across the physiological pH range (about 6.8–8.2), which is why it is so widely used in life science research.
What is the pKa and buffering range of HEPES?

HEPES has a pKa of approximately 7.5 at 20°C (this product specifies pKa 7.4–7.7). Its effective buffering range is roughly pH 6.8–8.2, which covers most physiological conditions.
Why is HEPES preferred over other buffers like phosphate or Tris?

HEPES does not significantly bind metal ions, so it does not interfere with metal-dependent enzymes the way phosphate buffers can. Unlike Tris, its pKa changes relatively little with temperature, giving more consistent pH control. HEPES also interferes less with DNA–restriction enzyme reactions than Tris.
Is HEPES toxic to cells?

At typical working concentrations (10–25 mM), HEPES is widely used in cell culture and is generally well tolerated. However, researchers should be aware that HEPES is not completely inert — it can affect some cellular processes, and it is known to generate radicals under light exposure, so it is not recommended for redox-sensitive reactions or experiments involving strong illumination. For sensitive cell-based work, optimal concentration should be confirmed for your specific system.
What concentration of HEPES should I use in cell culture?

Typical working concentrations in cell culture media are 10–25 mM. HEPES is especially useful for maintaining pH during procedures performed outside a CO₂ incubator.
What is the difference between HEPES Biochemical Grade and HEPES Ultrapure Grade?

Both grades are supplied at ≥99.5% assay purity. The Ultrapure Grade is further refined to deliver lower UV absorbance (lower A260/A280), indicating reduced organic impurities — which matters in applications sensitive to background signal, such as PCR, fluorescence detection, and optical assays. Biochemical Grade is the cost-effective choice for routine cell culture, protein work, and general buffer preparation. (See our HEPES Ultrapure Grade page.)
How should I store HEPES?

Store the dry powder at room temperature. Prepared buffer solutions should be stored as appropriate for your application; HEPES solutions are generally stable but should be protected from prolonged light exposure.
What package sizes are available?

HEPES Biochemical Grade is available from 25g up to 5kg. For bulk, OEM, or IVD raw material needs, use the Request Bulk Quote button or contact contact@biofargo.com.

Documents

INFO

COA

SDS

Publications

Recent research using HEPES buffer (source: PubMed)

Corazolla, E.M., et al. 2026. HEPES in Cell Culture Alters the Multi-Omics Profile Exhibited by Gaucher Disease Fibroblasts. J Cell Biochem 127(1), e70080. DOI — Study examining how HEPES in culture media influences cellular metabolism and diagnostic outcomes; relevant context for choosing buffer grade in sensitive cell work.
Merli, M., et al. 2022. Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics. Front Bioeng Biotechnol 10, 1032542. DOI — HEPES buffer used as a component in optimizing pH-controlled bioink formulations for 3D-bioprinted neural cell models.
Yeung, S.Y., et al. 2022. Reversible Self-Assembled Monolayers with Tunable Surface Dynamics for Controlling Cell Adhesion Behavior. ACS Appl Mater Interfaces 14(37), 41790–41799. DOI — HEPES buffer solution (pH 8) used as the medium for preparing reversible self-assembled monolayers for cell adhesion studies.

HEPES Biochemical Grade, ≥99.5%