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HEPES

Product NameHEPES
CAS7365-45-9
MFC8H18N2O4S
MW238.3
EINECS230-907-9
MOL File7365-45-9.mol

Chemical Properties

Melting point	234-238 °C
Boiling point	408℃[at 101 325 Pa]
bulk density	560kg/m3
Density	1.07 g/mL at 20 °C
vapor pressure	0Pa at 25℃
refractive index	n20/D 1.339
storage temp.	2-8°C
solubility	H₂O: 1 M at 20 °C, clear, colorless
form	powder
color	White
PH	5.0-6.5 (1Maqueous solution)
pka	7.5(at 25℃)
PH Range	7.0 - 7.6
biological source	synthetic
Water Solubility	Soluble
λmax	λ: 260 nm Amax: 0.06 λ: 280 nm Amax: 0.04
Merck	14,4654
BRN	883043
Stability	Stable. Combustible. Incompatible with strong oxidizing agents. Protect from moisture.
Major Application	diagnostic assay manufacturing
Cosmetics Ingredients Functions	BUFFERING
InChI	1S/C8H18N2O4S/c11-7-5-9-1-3-10(4-2-9)6-8-15(12,13)14/h11H,1-8H2,(H,12,13,14)
InChIKey	JKMHFZQWWAIEOD-UHFFFAOYSA-N
SMILES	OCCN1CCN(CC1)CCS(O)(=O)=O
LogP	-3.85 at 20℃
CAS DataBase Reference	7365-45-9(CAS DataBase Reference)
EPA Substance Registry System	1-Piperazineethanesulfonic acid, 4-(2-hydroxyethyl)- (7365-45-9)
Absorption	≤0.05 at 290 in H2O at 33%

Safety Information

Hazard Codes	Xi
Risk Statements	36/37/38
Safety Statements	24/25-22-36-26
WGK Germany	1
RTECS	TL6809000
TSCA	TSCA listed
HS Code	29335995
Storage Class	13 - Non Combustible Solids

MSDS

Provider	Language
2-[4-(2-Hydroxyethyl)-1-piperazine]ethanesulfonic acid	English
SigmaAldrich	English
ACROS	English
ALFA	English

Usage And Synthesis

Description

HEPES has been described as one of the best all-purpose buffers available for biological research. At biological pH, the molecule is zwitterionic, and is effective as a buffer at pH 6.8 to 8.2 (pKa 7.55). It is typically used in cell culture at concentration between 5mM to 30 mM. HEPES has been used in a wide variety of applications, including tissue culture. It is commonly used to buffer cell culture media in air. HEPES finds its usage in in vitro experiments on Mg.

Chemical Properties

White crystalline powder

Uses

Non-toxic to cell. It is used as a hydrogen ion buffer, which can control the constant pH range for long term. The concentration is 10-50mmol/L. Generally, 20mmol/LHEPES in nutrient solution can get buffer ability.

Uses

HEPES is common buffer for biological sciences, particularly used in cell culture to maintain physiological pH. It acts a buffering component, which is used in the preparation of buffers. It is described as one of the best all-purpose buffers available for use in biological research.
HEPES has been used as a component of:
Hank′s balanced salt solution, Dulbecco′s modified eagle′s medium and no-glucose DMEM, which are used for the preparation of tissue slices.
Buffers used in localized conductance measurements on the basolateral side of culture inserts to examine cation selectivity.

Uses

HEPES is a biological buffer. It may be used in isoelectric focusing applications and as a buffer in the quantitative and selective measurement of antigen-antibody reactions. HEPES is reportedly superior to NaHCO3 in controlling pH in tissue and organ culture. HEPES is not recommended for use with the Folin-Ciocalteu protein assay.

Definition

ChEBI: A HEPES that is ethanesulfonate substituted by a 4-(2-hydroxyethyl)piperazin-4-ium-1-yl group at position 2.

General Description

HEPES buffered saline is a tissue culture buffer, which is used to maintain the pH of cell culture. It is water soluble and atmosphere independent. HEPES destroys magnesium in sodium chloride solutions.

Biological Activity

Multi purpose buffer used in biological research.

Biological Activity

HEPES is a multi purpose HEPES buffer used in cell culture and other biological research. Working pH range in aqueous solution: 6.8 - 8.2. Does not form complexes with metal ions. Used in cell culture media.

Biotechnological Applications

HEPES serves as a buffering agent and transfection reagent that mediates cytoplasmic delivery of proteins into cells, and mixing it with proteins such as antibodies, recombinant proteins, and small peptides yields protein–HEPES mixtures suitable for transfection into cultured cells[1]. As a zwitterionic buffering agent in cell culture media, HEPES activates MiT/TFE-dependent lysosomal-autophagic gene networks and induces lysosome biogenesis, while its addition to culture media also triggers a lysosomal stress-related inflammatory phenotype and affects host-immune gene programs in RAW macrophages[2]. HEPES serves as a building block for molecular engineering of biomedical polymers, functioning as a pendant group in the zwitterionic polymer polyHEPES-pz intended for biomedical applications[3].

Synthesis

103-76-4

1562-00-1

7365-45-9

The general procedure for the synthesis of 4-hydroxyethylpiperazineethanesulfonic acid from N-hydroxyethylpiperazine and sodium 2-hydroxyethylsulfonate is as follows: 0.5 mole of N-hydroxyethylpiperazine, 0.565 mole of sodium 2-hydroxyethylsulfonate, and 500 ml of methanol were added to a 1000 ml four-necked flask fitted with a refluxing condenser tube, and the condensation reaction was carried out under sufficient stirring. The reaction was first carried out at 50 °C for 2.5 minutes, then gradually heated to reflux and continued for 3.0 hours. The reaction was cooled after completion of the reaction to give a reaction mixture containing 4-hydroxyethylpiperazine ethanesulfonate. The yield of 4-hydroxyethylpiperazine ethanesulfonate was 93.6% as analyzed by high performance liquid chromatography. A 20 liter solution of 4-hydroxyethylpiperazine ethanesulfonate at a mass concentration of 15 wt% was diluted to 5 wt% with deionized water under stirring, followed by removal of sodium chloride by dialysis. Then the purified HEPES product was obtained by evaporation concentration, cooling crystallization and filtration. Finally, the product was washed once with anhydrous methanol and dried under vacuum to obtain purified HEPES.A sample of purified HEPES was dissolved and tested for purity, which showed a purity of 99.9%, a yield of 85.6%, a pH of 6.46, and a concentration of chloride ions of 6.8 μg/g.

storage

Room temperature

Purification Methods

Crystallise the acid from hot EtOH and water. It is a useful buffer. [Beilstein 23 V 376.]

References

[1] Chen, S.-H., Chao, A., Tsai, C.-L., Sue, S.-C., Lin, C.-Y., Lee, Y.-Z., Hung, Y.-L., Chao, A.-S., Cheng, A.-J., Wang, H.-S., & Wang, T.-H. (2018). Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells. Molecular Therapy-Methods & Clinical Development, 13, 99–111. https://doi.org/10.1016/j.omtm.2018.12.005
[2] Tol, M. J., van der Lienden, M. J. C., Gabriel, T. L., Hagen, J. J., Scheij, S., Veenendaal, T., Klumperman, J., Donker-Koopman, W. E., Verhoeven, A. J., Overkleeft, H., Aerts, J. M., Argmann, C. A., & van Eijk, M. (2018). HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution. Autophagy, 14 3, 437–449. https://doi.org/10.1080/15548627.2017.1419118
[3] Tagad, H. D., Marin, A., Hlushko, R., & Andrianov, A. K. (2024). Hydrolytically Degradable Zwitterionic Polyphosphazene Containing HEPES Moieties as Side Groups. Biomacromolecules, 6791–6800. https://doi.org/10.1021/acs.biomac.4c01008

References

[1] Patent: CN106905262, 2017, A. Location in patent: Paragraph 0034; 0035; 0047

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