3,3′-Diaminobenzidine (DAB)

3,3’-Diaminobenzidine structural formula

CAS Number91-95-2
Molecular FormulaC12H14N4
Molecular Weight214.273
  • 3,3'-Diaminobenzidine
  • [1,1'-Biphenyl]-3,3',4,4'-tetramine
  • 91-95-2
  • 4-13-00-00530
  • BRN 1212988
  • 3,3'-Diaminobenzidene
  • EINECS 202-110-6
  • NSC 76152
  • (1,1'-biphenyl)-3,3',4,4'-tetramine
  • 3,3',4,4'-biphenyltetramine
  • 3,3',4,4'-diphenyltetramine
  • 3,3',4,4'-tetraaminobiphenyl
  • 3,3',4,4'-tetraaminodiphenyl
  • 3,3',4,4'-tetraminobiphenyl
  • 3,3'-diaminobenzidene
  • DAB
  • biphenyl-3,3',4,4'-tetrayltetraamine


HPLC Method for Analysis of 3,3′-Diaminobenzidine (DAB) on BIST B+ Column

July 6, 2023

HPLC Method for Analysis of 3,3′-Diaminobenzidine (DAB) on BIST B+ by SIELC Technologies

Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies

HPLC Method for Analysis of 3,3'-Diaminobenzidine (DAB) on BIST B+ Column
HPLC Method for Separation of 3,3′-Diaminobenzidine (DAB)

3,3′-Diaminobenzidine (DAB) is an organic compound that is often used as a staining reagent in various scientific applications. Here’s some basic information about DAB:

Chemical Structure: DAB is a derivative of benzidine, and its chemical structure consists of two benzene rings linked by a two-nitrogen “bridge”. Each benzene ring also has an amino group (-NH2) attached, hence the name “diamino”.

Uses: DAB is primarily used as a substrate for the enzyme horseradish peroxidase (HRP) in techniques such as immunohistochemistry (IHC) and immunoblotting. In the presence of HRP and hydrogen peroxide, DAB undergoes a reaction that results in the formation of a brown precipitate. This can be used to visualize the location of specific proteins or other molecules in biological samples.

Safety Concerns: It’s important to note that DAB is potentially carcinogenic, and should be handled with appropriate safety measures, including wearing gloves and protective eyewear, and using the chemical in a fume hood whenever possible.

Solubility: DAB is slightly soluble in water, but can be dissolved in organic solvents such as ethanol, or in weakly acidic solutions. For use in staining procedures, it is often dissolved in a buffer solution, sometimes with the addition of a small amount of detergent to improve solubility.

The 3,3′-Diaminobenzidine (DAB) can be retained and analyzed using a mixed-mode BIST B+, 4.6 x 100 mm, 5 µm, 100 A column. The mobile phase for this method consists of water, acetonitrile (MeCN), and Sulfuric acid, which serves as a buffer. This analytical method can be monitored using UV detection at 280 nm.

High Performance Liquid Chromatography (HPLC) Method for Analysis of 3,3′-Diaminobenzidine (DAB)


ColumnBIST B+, 4.6 x 100 mm, 5 µm, 100 A
Mobile PhaseGradient MeCN -80-30%, 10 min
BufferH2SO4 – 0.2%
Flow Rate1.0 ml/min
DetectionUV 280 nm


Class of CompoundsDiphenylamines
Analyzing Compounds3,3′-Diaminobenzidine (DAB)

Application Column


Column Diameter: 4.6 mm
Column Length: 100 mm
Particle Size: 5 µm
Pore Size: 100 A

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Application Analytes:
3,3′-Diaminobenzidine (DAB)

Application Detection:
UV Detection
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.

Separation of 3,3’-Diaminobenzidine on Newcrom R1 HPLC column

February 16, 2018
Separation of 3,3’-Diaminobenzidine on Newcrom C18 HPLC column

3,3’-Diaminobenzidine can be analyzed by this reverse phase (RP) HPLC method with simple conditions. The mobile phase contains an acetonitrile (MeCN), water, and phosphoric acid. For Mass-Spec (MS) compatible applications the phosphoric acid needs to be replaced with formic acid. Smaller 3 µm particles columns available for fast UPLC applications. This liquid chromatography method is scalable and can be used for isolation impurities in preparative separation. It also suitable for pharmacokinetics.

Application Column

Newcrom R1

The Newcrom columns are a family of reverse-phase-based columns. Newcrom A, AH, B, and BH are all mixed-mode columns with either positive or negative ion-pairing groups attached to either short (25 Å) or long (100 Å) ligand chains. Newcrom R1 is a special reverse-phase column with low silanol activity.

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Application Analytes:
3,3′-Diaminobenzidine (DAB)
The result was obtained by a proprietary SIELC algorithm. It may deviate from the actual experimental data. The experimental data are available upon request. Contact us by e-mail: support@sielc.com or by phone: 847-229-2629.