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Tetramethylbenzidine

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CAS:54827-17-7
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Related articles

  • What is Tetramethylbenzidine?
  • Tetramethylbenzidine(TMB) is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a vi....
  • Dec 31,2019
Tetramethylbenzidine Basic information
Background
Product Name:Tetramethylbenzidine
Synonyms:1’-biphenyl)-4,4’-diamine,3,3’,5,5’-tetramethyl-(;1’-biphenyl)-4,4-diamine,3,3’,5,5’-tetramethyl-(;1’-biphenyl]-4,4’-diamine,3,3’,5,5’-tetramethyl-[;TMB-E;TMB EASY;TMB/H;TMB-B;TMB BLUE ONE-STEP SUBSTRATE SYSTEM
CAS:54827-17-7
MF:C16H20N2
MW:240.34
EINECS:259-364-6
Product Categories:Biochemistry;Enzyme;Substrates;Enzyme substrates;Alphabetical ListingSubstrates, Buffers&Blockers;Detection Substrates;ELISA SubstratesELISA Substrates;Liquid Substrate SystemsEnzyme Substrates;Peroxidase;Peroxidase Substrates;Substrates by Enzyme;Substrates for Peroxidase;ELISA;Benzopyrans;Biphenyl & Diphenyl ether;Benzoquinones, etc. (Charge Transfer Complexes);Charge Transfer Complexes for Organic Metals;Functional Materials;54827-17-7
Mol File:54827-17-7.mol
Tetramethylbenzidine Structure
Tetramethylbenzidine Chemical Properties
Melting point 168-171 °C(lit.)
Boiling point 100 °C
bulk density430kg/m3
density 1
refractive index 1.5519 (estimate)
storage temp. 2-8°C
solubility Slightly soluble. <0.1 g/100 mL at 20°C.
pka4.49±0.10(Predicted)
form tablet
color Cream
Water Solubility Slightly soluble. <0.1 g/100 mL at 20 ºC
Sensitive Light Sensitive
BRN 2808541
Stability:Stable, but moisture sensitive and may be light sensitive. Incompatible with water, strong oxidizing agents.
InChI1S/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
InChIKeyUAIUNKRWKOVEES-UHFFFAOYSA-N
SMILESCc1cc(cc(C)c1N)-c2cc(C)c(N)c(C)c2
CAS DataBase Reference54827-17-7(CAS DataBase Reference)
EPA Substance Registry System3,3',5,5'-Tetramethylbenzidine (54827-17-7)
Safety Information
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 Class11 - Combustible Solids
Hazard ClassificationsAcute Tox. 4 Oral
Aquatic Chronic 4
Carc. 2
Hazardous Substances Data54827-17-7(Hazardous Substances Data)
MSDS Information
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3,3',5,5'-Tetramethylbenzidine English
ACROS English
SigmaAldrich English
ALFA English
Tetramethylbenzidine Usage And Synthesis
DescriptionTetramethylbenzidine(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 PropertiesTetramethylbenzidine 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.
Uses3,3,5,5-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.
PreparationThe 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%.
ReactionsTMB 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 Description3,3',5,5'-tetramethylbenzidine appears as pale yellow crystals or off-white powder. (NTP, 1992)
Air & Water ReactionsInsoluble in water.
Reactivity ProfileTetramethylbenzidine 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 HazardFlash point data for Tetramethylbenzidine are not available, however, Tetramethylbenzidine is probably combustible.
Biochem/physiol Actions3,3′,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 in?situ 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.

CarcinogenicityThe 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
BackgroundTMB 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
Tag:Tetramethylbenzidine(54827-17-7) Related Product Information
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