HEPES
[CAS# 7365-45-9]

Identification

• Classification: Analytical chemistry >> Analytical reagent >> Ion chromatography reagent
• Name: HEPES
• Synonyms: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid; N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 2-[4-(2-Hydroxyethyl)-1-piperazine]ethanesulfonic acid
• Molecular Formula: C₈H₁₈N₂O₄S
• Molecular Weight: 238.30
• CAS Registry Number: 7365-45-9
• EC Number: 230-907-9
• SMILES: C1CN(CCN1CCO)CCS(=O)(=O)O

Properties

• Solubility: 1 M (water 20 °C)
• Melting point: 234-238 °C
• Flash point: 124 °C

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H315-H319-H335
• Safety Statements: P261-P305+P351+P338

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Eye irritation	Eye Irrit.	2	H319
Skin irritation	Skin Irrit.	2	H315
Specific target organ toxicity - single exposure	STOT SE	3	H335
Acute toxicity	Acute Tox.	4	H302
Acute toxicity	Acute Tox.	4	H312
Acute toxicity	Acute Tox.	4	H332

Discovery and Applications

HEPES, chemically known as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, is a zwitterionic buffer developed by Norman Good and colleagues in the 1960s. It is part of a family of buffers designed to provide stable pH conditions for biological and biochemical research. These buffers, known as Good buffers, are designed to minimize interference with biochemical processes while maintaining stability over a wide range of temperatures and concentrations.

HEPES has the molecular formula C8H18N2O4S and is able to maintain a stable pH range of 6.8 to 8.2. Its pKa is 7.55 at 25°C, making it particularly useful for experiments requiring neutral to slightly alkaline pH. HEPES is highly soluble in water, providing a stable and reliable buffering capacity that is essential for a variety of laboratory applications.

HEPES is widely used in cell culture media to maintain a stable pH environment. It is particularly valuable in maintaining pH when cells are grown in environments with fluctuating CO2 levels, such as in open systems or when the culture medium is exposed to air.

In biochemical assays, especially those involving enzymatic reactions that are sensitive to pH changes, HEPES provides a stable pH, ensuring accurate and reproducible results. Its minimal interaction with metal ions makes it suitable for studies involving metalloproteins and metal-dependent enzymes.

HEPES is used in protein purification techniques, including chromatography, to maintain the pH of the buffer system. This stability helps to maintain the structural integrity and activity of the protein during the purification process.

HEPES is also used as a buffer in electrophoresis for the separation of proteins and nucleic acids. Its ability to maintain a stable pH ensures precise separation and resolution of biomolecules.

HEPES has several advantages, including low absorbance in the UV and visible spectra, which minimizes interference in spectrophotometric measurements. It is chemically stable and resistant to enzymatic degradation, ensuring its effectiveness over extended periods of time. In addition, HEPES does not form complexes with metal ions, making it ideal for studies involving metal-sensitive reactions.

References

2025. A Simple Selective Probe for Lysine Detection in Tablets, Food Samples and Cells. Journal of Fluorescence, 35(1).
DOI: 10.1007/s10895-023-03523-6

1970. Phenols, phenoloxidase, and photosynthetic activity of chloroplasts isolated from sugar cane and spinach. Planta, 92(1).
DOI: 10.1007/bf00387755