3,3',5,5'-Tetramethyl-[1,1'-biphenyl]-4,4'-diamine hydrochloride hydrate
[CAS# 207738-08-7]

Identification

• Classification: Biochemical >> Biochemical reagent >> Biological dye
• Name: 3,3',5,5'-Tetramethyl-[1,1'-biphenyl]-4,4'-diamine hydrochloride hydrate
• Molecular Formula: C₁₆H₂₀N₂.2(HCl).xH₂O
• CAS Registry Number: 207738-08-7
• EC Number: 688-511-0
• SMILES: CC1=CC(=CC(=C1N)C)C2=CC(=C(C(=C2)C)N)C.O.Cl.Cl

Properties

• Solubility: 20 mg/mL (acetic acid: water (1:2))

Safety Data

• Hazard Symbols: GHS07;GHS08 Warning
• Risk Statements: H302-H312-H332-H341
• Safety Statements: P203-P261-P264-P270-P271-P280-P301+P317-P302+P352-P304+P340-P317-P318-P321-P330-P362+P364-P405-P501

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Germ cell mutagenicity	Muta.	2	H341
Acute toxicity	Acute Tox.	4	H302
Acute toxicity	Acute Tox.	4	H312
Acute toxicity	Acute Tox.	4	H332

Discovery and Applications

3,3',5,5'-Tetramethyl-[1,1'-biphenyl]-4,4'-diamine hydrochloride hydrate, also known as TMB (tetramethylbenzidine), is an organic compound with the molecular formula (CH3)2C6H3NHC6H3(CH3)2•HCl•xH2O. The compound is commonly used as a chromogenic substrate in biochemical assays. It was developed to provide a safer alternative to benzidine substrates, which were found to be carcinogenic. The discovery of TMB involved extensive research to identify a compound that could react with peroxidase to produce a measurable color change, thus facilitating its use in diagnostic applications.

TMB has a biphenyl structure with two amine groups, each bound to a benzene ring substituted with two methyl groups. This structure gives TMB unique properties as a chromogenic substrate. The hydrochloride hydrate form enhances its stability and solubility in aqueous solutions. TMB is a white to pale yellow crystalline powder that is light sensitive and should be stored in a cool environment.

The color development reaction of TMB involves oxidation by HRP in the presence of hydrogen peroxide. The reaction produces a diimine compound that appears blue. When the reaction is stopped with acid, the diimine compound is converted to a yellow product, enhancing the versatility of TMB in different assay formats. The intensity of the color is proportional to the amount of HRP and, therefore, the target analyte in the sample.

TMB is widely used in ELISA (enzyme-linked immunosorbent assay) kits, where it serves as a substrate for horseradish peroxidase (HRP). After oxidation by HRP, TMB produces a blue color that is quantified by measuring absorbance at 370 nm or 650 nm after stopping the reaction with acid, which turns the color yellow.

TMB is used in a variety of clinical diagnostic tests, including those used to detect antibodies or antigens in blood samples. Its ability to produce a distinct color change upon interaction with an enzyme makes it very useful for visual confirmation of test results.

TMB is used in molecular biology to detect the presence of specific proteins or nucleic acids in a sample. Its high sensitivity and rapid color development are essential for accurate and timely analysis in a research setting.

While TMB is safer than its benzidine predecessor, it still requires careful handling. It should be used in a well-ventilated area with appropriate protective equipment to avoid inhalation or skin contact. Disposal of TMB should follow the institution's hazardous waste guidelines to prevent environmental contamination.

References

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