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

Description

HEPES is a buffering agent used in biochemistry and molecular biology that was selected and described by Good et al. It is a zwitterionic, piperazinic buffer that is useful for a pH range of 6.8 – 8.2. HEPES lacks the ability to form a significant complex with most metal ions and is recommended for use as a non-coordinating buffer in solutions with metal ions. It is commonly used in cell culture media and as an ampholytic separator to create a pH gradient in isoelectric focusing. HEPES interferes with reactions between DNA and restriction enzymes to a lesser extent than similar buffers with less substituted amine groups, such as Tris. This lack of interference occurs because steric hindrance of the protonated tertiary amine in HEPES shields the negative charge of DNA to a lesser extent than less substituted buffers. This buffer is capable of forming radicals and is therefore not suitable for redox reactions. It is suitable for use in the bicinchoninic acid (BCA) assay.

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

High-quality biochemical grade, ≥ 99.5% purity

The product is a high-purity free acid form of HEPES (CAS 7365-45-9), ensuring minimal impurities and contaminants, suitable for demanding biochemical and cell culture applications.
Optimal buffering capacity at physiological pH

With a pKa around 7.5 and effective buffering range ~ pH 6.8–8.2, HEPES provides stable pH control around physiological pH (≈ 7.2–7.6), ideal for cell culture and biochemical reactions.
Zwitterionic, non-chelating buffer — inert toward metal ions

HEPES exhibits negligible binding to metal ions, minimizing interference in metal-dependent enzyme assays or metal-ion containing systems.
Low UV/visible absorbance and minimal interference with biochemical assays

The low absorbance at UV/visible wavelengths makes HEPES suitable for sensitive detection methods (e.g., absorbance, fluorescence, protein assays) without significant background.
Chemical and enzymatic stability, membrane-impermeable

HEPES is stable under typical lab conditions (room temperature, aqueous solutions), and does not readily cross cell membranes — a key property for maintaining extracellular buffer capacity in cell culture.
High solubility, easy to prepare buffer solutions

As a highly soluble buffer, HEPES dissolves readily in water, making buffer preparation straightforward and reproducible.

Applications

Cell culture media and cell-based assays

HEPES is widely used as a buffering component in cell culture media to maintain stable pH — especially useful during manipulations outside CO₂ incubators, or for cell types sensitive to pH fluctuations. Typical working concentrations in cell culture range from 10–25 mM.
Protein purification, enzymatic reactions and biochemical assays

Because HEPES is non-chelating and has minimal impact on enzyme activity, it is ideal as a buffer in enzyme assays, affinity or ion-exchange chromatography, protein extraction, purification, and other biochemical workflows.
Molecular biology, nucleic acid handling, and electrophoresis

HEPES can be used in buffer systems for DNA/RNA extraction, in electrophoresis (e.g., as part of sample or running buffers), in isoelectric focusing, and other molecular biology protocols requiring stable pH and minimal interference.
Cell lysis, immunoprecipitation, immunoassays

Suitable for buffer formulation in cell lysis, immunoprecipitation, antibody-antigen binding reactions, and other antibody-based assays, where stable pH and minimal metal chelation are important.
Live-cell imaging, tissue culture, and sensitive detection (fluorescence / absorbance)

Because HEPES has low optical absorbance and does not interfere significantly with biological processes, it is compatible with fluorescent microscopy, spectrophotometric assays, and live-cell imaging where stable pH and minimal background are critical.

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.

Documents

INFO

COA

SDS

HEPES 99.5% Biochemical Grade