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3,3'-Diaminobenzidine (DAB) - LC-MS method with excellent retention and peak shape

Reference Number: AA-00818 Created: 05/18/2012 06:44 AM Last Updated: 09/04/2020 04:44 PM


Peak: 3,3'-Diaminobenzidine 215.1291 m/z (M+H)+

t0: 0.9 min

Method Conditions

Column: Cogent  Diamond  Hydride™, 4µm, 100Å

Catalog No.: 70000-15P-2

Dimensions: 2.1 x 150 mm

Solvents: A: 50% DI H2O/ 50% MeOH / 0.1% formic acid

B: Acetonitrile / 0.1% formic acid


        time (min.) %B

              0 80

              4 30

              9 30

            10 80

Post Time:  5 min

Injection vol.: 1µL
Flow rate:  0.4 mL/min
Detection: ESI – POS - Agilent 6210 MSD TOF mass spectrometer
Sample preparation: 
Working Solution: Stock solution was diluted 1:100 with

50/50 solvent A / solvent B mixture. Peak: 3,3'-Diaminobenzidine 215.1291 m/z (M+H)+ t0: 0.9 min
Stock Solution: 1mg/mL in DI H2O diluent. The solution was filtered through a 0.45µm nylon  syringe filter (MicroSolv Tech Corp.).


DAB (3,3'-Diaminobenzidine) is a very  challenging compound for analysis  by HPLC. It is highly polar  and hence  difficult to retain when RP-HPLC columns are used. Moreover, when  there are a significant number of silanol  groups present on the surface of the column packing material, the peak for DAB becomes very  broad (5 – 10 min peak width). As can be seen from the accompanying chromatograms, a Cogent Diamond Hydride column was an excellent choice for the analysis  of DAB. The peak shape is symmetrical with high  efficiency. The repeatability of the analysis  is also remarkable as can be seen in Figure B.


DAB reacts with hemoglobin (an oxidation reaction catalyzed by the heme groups) in the presence of hydrogen peroxide producing 
a dark brown color.  This reaction is used to stain cells that were prepared with hydrogen peroxidase enzyme. DAB tablets are used in immunohistology for the detection of peroxidase activity. Diaminobenzidine is a known mutagen (a compound that can induce changes in the genetic information of an organism).

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