3,3′,5,5′-Tetramethylbenzidine

English Name: 3,3′,5,5′-Tetramethylbenzidine
English Synonyms: TMB; 4,4′-Bi-2,6-xylidine; 4,4′-Diamino-3,3′,5,5′-tetramethylbiphenyl; TMB substrate; BM blue
CAS No.: 54827-17-7
EINECS No.: 259-364-6
Chemical Formula: C₁₆H₂₀N₂
Molecular Weight: 240.34
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
Density 1.071 g/cm³
Boiling point 368.62°C / 100°C (Literature differences)
Flash point 210.81°C
Water solubility Slightly soluble in water (<0.1 g/100 mL at 20°C), readily soluble in organic solvents such as acetone, ether, DMSO, and DMF
Vapor pressure Data not explicitly recorded
Refractive index 1.5519 (estimated value)
Storage conditions 2-8°C, protected from light, sealed, and stored in a dry place
Sensitivity Sensitive to light and moisture
Appearance White to pale yellow crystalline powder
Specific gravity 1.071
Color White or off-white to pale yellow

BRN 2808541
MDL No. MFCD00007748
Hazard Symbol Xi (Irritant)
Risk Terms R36/37/38 (Irritation to eyes, respiratory system and skin)
Safety Terms S26 (In case of accidental contact with eyes, rinse immediately with plenty of water and seek medical advice); S36 (Wear appropriate protective clothing)
Customs Code 2921590090 (Other aromatic polyamines and their derivatives and salts)

I. Properties
Physical Properties: White to pale yellow crystalline powder, odorless and tasteless
Melting Point: 168.5-171°C (Literature value 168-171°C)
Bulk Density: 430 kg/m³ Slightly soluble in water, readily soluble in acetone, ether, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethanol, and other organic solvents.
Stability: Stable, but sensitive to moisture and light; incompatible with strong oxidizing agents.
Chemical Properties:
Acidity coefficient (pKa): 4.49 ± 0.10 (predicted value)
In the presence of hydrogen peroxide and peroxidase (such as horseradish peroxidase HRP), TMB is oxidized to produce a blue product (maximum absorption wavelength 370 nm or 620-650 nm), turning yellow upon acidification (450 nm). (nm) Heating decomposes it, releasing toxic nitrogen oxide fumes.

II. Applications
TMB is a novel, safe chromogenic reagent. As a non-carcinogenic alternative to the traditional carcinogen benzidine (negative Ames test), it is mainly used in:
Clinical Diagnosis and Biochemical Testing:
Peroxidase substrate in Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme Immunoassay (EIA) for detecting viruses (such as hepatitis viruses, HIV), tumor markers (such as PSA, CEA), hormones, etc.
Preparation of urine test strips
Pregnancy tests
Rapid determination of glucose, hemoglobin, and albumin in blood and urine
Fecal occult blood test
Determination of granulocyte count in blood
Detection of steroids and sex hormones
Forensic and Environmental Testing:
Lacquer fingerprint detection (criminal investigation)
Forensic bloodstain examination
Environmental monitoring (water quality testing, pollutant detection)
Rapid determination of alcohol in saliva Other Applications: Analysis and detection of antigens, antibodies, and genetic material; Immunohistochemical (IHC) staining; Lateral chromatography detection (POCT, such as pregnancy tests, drug screening); Signal molecules in biosensors.
III. Preparation Methods: The preparation of TMB is relatively complex. Industrially, the following route is commonly used:
Main Synthetic Route: Using 2,6-dimethylaniline as a raw material, the process involves three steps: activation, oxidative coupling, and purification:
Activation: 2,6-dimethylaniline is activated under appropriate conditions.
Oxidative Coupling: A biphenyl skeleton is constructed through an oxidative coupling reaction to form a 3,3′,5,5′-tetramethylbenzidine structure.
Purification: High-purity product (≥99%) is obtained through recrystallization and other purification steps.
Overall Yield: Approximately 65%
Laboratory Preparation: It can also be prepared through benzene ring-opening and methylation reactions, but the conditions are more demanding and must be carried out under strictly controlled conditions.

IV. Safety Information

Hazard Overview: Hazard Category: Irritant (Xi)

GHS Hazard Statement: Causes skin irritation, causes serious eye irritation, may cause respiratory irritation

Safety Precautions:
Personal Protection: Wear protective eyewear, gloves, and clothing. Avoid direct contact with skin and eyes.

Ventilation Requirements: Handle in a well-ventilated environment. Avoid inhaling dust.

Storage Safety: Store in a cool, dry, dark place (2-8°C), away from sources of ignition, oxidizers, strong acids, and moisture.

Spill Handling: Collect spilled material in a sealed container and absorb with inert absorbent material.

First Aid Measures: Skin contact: Immediately flush with plenty of water.
Eye contact: Immediately flush with plenty of water for at least 15 minutes and seek medical attention.
Inhalation: Move to fresh air immediately, keep the airway open, and seek medical attention if necessary.
Ingestion: Seek immediate medical attention.
Special precautions:
Although TMB has been proven to be non-carcinogenic and non-mutagenic (negative Ames test), and its safety is greatly improved compared to traditional benzidine reagents, prolonged or repeated exposure should still be avoided.
It is sensitive to light; avoid direct sunlight during handling.
It is sensitive to moisture; maintain a dry environment during weighing and use.
It is incompatible with strong oxidizers and strong acids; avoid mixing them for storage.