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3,3'-Diaminobenzidine (DAB) by LC-MS
LC-MS method with excellent retention and peak shape
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printable Application Sheet
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Note: 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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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| 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 |
|
| Gradient |
| time (min.) |
%B |
time (min.) |
%B |
| 0 |
80 |
9 |
30 |
| 4 |
30 |
10 |
80 |
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| Post Time |
5 min |
| Injection Vol. |
1 microL |
| Flow rate |
0.4 mL/min. |
| Detection |
ESI – POS - Agilent 6210 MSD TOF mass spectrometer |
| Sample |
Stock Solution: 1 mg/mL in DI water diluent. The solution was filtered through a 0.45 µm nylon syringe filter (MicroSolv Tech
Corp). 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 |
Discussion
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.
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