Retention and Separation is easy with the Cogent Diamond Hydride Column with the Method below. Normally this compound is very hard to get good peak shapes in Reversed Phase.

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

Method Conditions:
Column: Cogent Diamond Hydride™, 4µm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150 mm
50% DI Water/ 50% MeOH / 0.1% Formic Acid
B: Acetonitrile / 0.1% Formic Acid

time/(min) %B
0 80
4 30
9 30
10 80

t0: 0.9 min
Post Time:
5 minutes
Injection vol.: 1µL
Flow rate: 0.4 mL/minute
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.).

Discussion : 3,3′-Diaminobenzidine (DAB) 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.

Notes: 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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