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3,3′,5,5′-tetramethylbenzidine as multi-colorimetric indicator of chlorine in water in line with health guideline values

  • Palladino, Pasquale1
  • Torrini, Francesca1
  • Scarano, Simona1
  • Minunni, Maria1
  • 1 University of Florence,
Published Article
Analytical and Bioanalytical Chemistry
Publication Date
Sep 01, 2020
DOI: 10.1007/s00216-020-02918-9
PMID: 32870352
PMCID: PMC7461152
PubMed Central


Sanitizing solutions against bacterial and viral pathogens are of utmost importance in general and, in particular, in these times of pandemic due to Sars-Cov2. They frequently consist of chlorine-based solutions, or in the direct input of a certain amount of chlorine in water supply systems and swimming pools. Colorimetry is one of the techniques used to measure the crucial persistence of chlorine in water, including household chlorine test kits commonly based on colorimetric indicators. Here, we show a simple and cheap colorimetric method based on 3,3′,5,5′-tetramethylbenzidine (TMB), commonly used as chromogenic reagent for enzyme-linked immunosorbent assays. TMB is converted by chlorine to a colored molecule through a pH-dependent multi-step oxidation process where the chromaticity of TMB is directly proportional to chlorine content. This molecule offers several advantages over other commonly used reagents in terms of safety, sensitivity, and, peculiarly, hue modulation, giving rise to the detection of chlorine in water with a multi-color change of the indicator solution (transparent/blue/green/yellow). Moreover, through the appropriate setting of reaction conditions, such coloration is finely tunable to cover the range of chlorine concentration recommended by international health agencies for treatment of drinking water and swimming pools and to test homemade solutions prepared by dilution of household bleach during health emergency events such as during the current pandemic. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-020-02918-9) contains supplementary material, which is available to authorized users.

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