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ChemicalBook >> CAS DataBase List >>Tetramethylbenzidine

Tetramethylbenzidine

CAS No.
54827-17-7
Chemical Name:
Tetramethylbenzidine
Synonyms
TMB;TMB;3,3',5,5'-Tetramethylbenzidine;Tmbz;TMB SUBSTRATE;Tetramethylbenzidine (TMB);3,3',5',5-TETRAMETHYLBENZIDINE;3,3,5,5-TETRAMETHYLBENZIDINE, STANDARD F OR GC;4-(4-Amino-3,5-dimethylphenyl)-2,6-dimethylaniline;TMB/M
CBNumber:
CB6682185
Molecular Formula:
C16H20N2
Molecular Weight:
240.34
MDL Number:
MFCD00007748
MOL File:
54827-17-7.mol
MSDS File:
SDS
TDS File:
TDS
Last updated:2026-05-28 02:33:23
Product description Number Pack Size Price
3,3′,5,5′-Tetramethylbenzidine tablet, 1 mg substrate per tablet T5525 50 tabs $246
3,3′,5,5′-Tetramethylbenzidine tablet, 1 mg substrate per tablet T5525 100 tabs $420
3, 3′,5 ,5′-Tetramethylbenzidine Liquid Substrate, Supersensitive, for ELISA ready to use solution T4444 100 mL $135
3, 3′,5 ,5′-Tetramethylbenzidine Liquid Substrate, Supersensitive, for ELISA ready to use solution T4444 4 x 100 mL $406
BM Blue POD Substrate, soluble 3,3′-5,5′-Tetramethylbenzidine (TMB) 11484281001 100mL $160
More product size

Tetramethylbenzidine Properties

Melting point 168-171 °C(lit.)
Boiling point 100 °C
Density 1
bulk density 430kg/m3
refractive index 1.5519 (estimate)
storage temp. 2-8°C
solubility Slightly soluble. <0.1 g/100 mL at 20°C.
pka 4.49±0.10(Predicted)
form tablet
color Cream
Water Solubility Slightly soluble. <0.1 g/100 mL at 20 ºC
Sensitive Light Sensitive
BRN 2808541
Henry's Law Constant 1.3×105 mol/(m3Pa) at 25℃, HSDB (2015)
Stability Stable, but moisture sensitive and may be light sensitive. Incompatible with water, strong oxidizing agents.
InChI 1S/C16H20N2/c1-9-5-13(6-10(2)15(9)17)14-7-11(3)16(18)12(4)8-14/h5-8H,17-18H2,1-4H3
InChIKey UAIUNKRWKOVEES-UHFFFAOYSA-N
SMILES Cc1cc(cc(C)c1N)-c2cc(C)c(N)c(C)c2
CAS DataBase Reference 54827-17-7(CAS DataBase Reference)
FDA UNII 3B3T5CB8EO
EPA Substance Registry System
UNSPSC Code 41116133
NACRES NA.83

SAFETY

Risk and Safety Statements

Symbol(GHS)  Exclamation Mark (GHS07)Health Hazard (GHS08)Flame (GHS02)
GHS07,GHS08,GHS02
Signal word  Warning
Hazard statements  H302-H315-H319-H335-H225-H312-H332-H370
Precautionary statements  P210-P280-P303+P361+P353-P261-P280a-P304+P340-P305+P351+P338-P405-P501a-P260-P280h-P308+P311a
PPE dust mask type N95 (US), Eyeshields, Faceshields, Gloves
Hazard Codes  Xi,Xn
Risk Statements  36/38-36/37/38-22-41
Safety Statements  26-36-36/37-39
RIDADR  UN 2796 8/PG 2
WGK Germany  3
RTECS  DV2300000
8-10
TSCA  TSCA listed
HazardClass  6.1
HS Code  29215990
Storage Class 11 - Combustible Solids
Hazard Classifications Acute Tox. 4 Oral
Aquatic Chronic 4
Carc. 2
Hazardous Substances Data 54827-17-7(Hazardous Substances Data)
NFPA 704
1
2 1

Tetramethylbenzidine price More Price(134)

Manufacturer Product number Product description CAS number Packaging Price Updated Buy
Sigma-Aldrich T5525 3,3′,5,5′-Tetramethylbenzidine tablet, 1 mg substrate per tablet 54827-17-7 50 tabs $246 2026-04-30 Buy
Sigma-Aldrich T5525 3,3′,5,5′-Tetramethylbenzidine tablet, 1 mg substrate per tablet 54827-17-7 100 tabs $420 2026-04-30 Buy
Sigma-Aldrich T4444 3, 3′,5 ,5′-Tetramethylbenzidine Liquid Substrate, Supersensitive, for ELISA ready to use solution 54827-17-7 100 mL $135 2026-04-30 Buy
Sigma-Aldrich T4444 3, 3′,5 ,5′-Tetramethylbenzidine Liquid Substrate, Supersensitive, for ELISA ready to use solution 54827-17-7 4 x 100 mL $406 2026-04-30 Buy
Sigma-Aldrich 11484281001 BM Blue POD Substrate, soluble 3,3′-5,5′-Tetramethylbenzidine (TMB) 54827-17-7 100mL $160 2026-04-30 Buy
Product number Packaging Price Buy
T5525 50 tabs $246 Buy
T5525 100 tabs $420 Buy
T4444 100 mL $135 Buy
T4444 4 x 100 mL $406 Buy
11484281001 100mL $160 Buy

Tetramethylbenzidine Chemical Properties,Uses,Production

Description

Tetramethylbenzidine(TMB) is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA). It is a white solid that forms a pale blue-green liquid in solution with ethyl acetate. TMB is degraded by sunlight and by fluorescent lights. It is a soluble chromogen substrate for horseradish peroxidase detection systems. Tetramethylbenzidine is recommended for ELISA procedures.

Chemical Properties

Tetramethylbenzidine is a white or light yellow solid, odorless, tasteless, insoluble in water, easily soluble in acetone, ether, dimethyl sulfoxide, dimethylformamide and other organic solvents. It forms a pale blue-green liquid in solution with ethyl acetate. TMB is degraded by sunlight and by fluorescent lights.

Uses

3,3,5,5-Tetramethyl benzidine is used as a reagent in a sensitive staining procedure for the detection of low levels of heme-associated peroxidase activity of cytochrome P-450 on SDS-polyacrylamide or agarose gel; non-carcinogenic substitute for benzidine as reagent for the detection of blood and determination of hemoglobin content.

Preparation

The synthesis of 3,3',5,5'-Tetramethylbenzidine involved using 2,6-dimethylaniline as the raw material. The process included activation, oxidative coupling, and purification, resulting in the production of pure material. The yield of 3,3,5,5-tetramethylbenzidine was 65%.

Reactions

TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase.
Oxidation of TMB
Shows the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to 3,3',5,5'-tetramethylbenzidine diimine
The resulting diimine causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at the wavelengths of 370 and 650 nm.

General Description

3,3',5,5'-tetramethylbenzidine appears as pale yellow crystals or off-white powder. (NTP, 1992)

Air & Water Reactions

Insoluble in water.

Reactivity Profile

Tetramethylbenzidine is sensitive to prolonged exposure to light . Neutralizes acids in exothermic reactions to form salts plus water. May be incompatible with isocyanates, halogenated organics, peroxides, phenols (acidic), epoxides, anhydrides, and acid halides. Flammable gaseous hydrogen may be generated in combination with strong reducing agents, such as hydrides.

Fire Hazard

Flash point data for Tetramethylbenzidine are not available, however, Tetramethylbenzidine is probably combustible.

Biochem/physiol Actions

3,3′,5,5′-Tetramethylbenzidine/TMB can be used as a chromogen to increase the progression of product obtained in peroxidase reaction. In food and environmental decontamination procedures, TMB can be used in insitu free available chlorine (FAC) monitoring of chlorite-based sanitizers.

Synthesis

4-Bromo-2,6-dimethylaniline

24596-19-8

Tetramethylbenzidine

54827-17-7

2,6-Dimethylaniline

87-62-7

4-Bromo-2,6-dimethylaniline (24.0 parts) was added with 3% palladium charcoal (50% paste; 2.0 parts), cetyltrimethylammonium bromide (4.0 parts), 32% sodium hydroxide solution (13.5 parts), sodium formate (6.8 parts), and water (60 parts), and boiled and stirred under reflux conditions for 3 hours. Subsequently, sodium formate (6.8 parts) was added again and boiling at reflux was continued for 3 hours. Next, the reaction mixture was kept boiling under reflux conditions for 23 hours. Upon completion of the reaction, the water was removed by distillation and 2.2 parts (18.2%) of 2,6-dimethylaniline was collected. The remaining reaction mixture was cooled to room temperature and filtered, and the residue was continuously extracted with methanol (60 parts) for 5 hours. After the extract was cooled, a colorless crystalline precipitate was filtered out and dried to give 7.7 parts (63.3%) of 3,3',5,5'-tetramethylbenzidine.

Carcinogenicity

The carcinogenic potential of Tetramethylbenzidine is uncertain due to conflicting evidence. It is not mutagenic by the Ames test, and did not induce formation of tumors in a single-arm study of 24 rats. On that evidence, it has been used as a replacement for carcinogenic compounds such as benzidine and o-phenylenediamine.
DOI: 10.1177/26.2.24068

Background

TMB is used as a substrate to generate detectable signal in ELISA. The reaction between the TMB substrate and a peroxidase, typically horseradish peroxidase, produces a measureable color change that correlates with analyte level.

References

[1] Patent: US4022795, 1977, A

1004761-68-5
24596-19-8
54827-17-7
Synthesis of Tetramethylbenzidine from 2,6-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline and 4-Bromo-2,6-dimethylaniline
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