Chloramine T Calibration Curve Protocol

To perform the calibration curve, we started with the Chloramine T solution at 100 μM (Tube 1). Tube 2 (Chloramine T 50 μM) was prepared with 400 μM of Chloramine T, and 100 μM is supplemented with 400 μM of distilled water. Tube 3 (Chloramine T 25 μM) was prepared by taking 400 μL from tube 2 and adding 400 μL of distilled water. Tube 4 (Chloramine T 12.5 μM) was prepared by taking 400 μL from tube 3 and adding 400 μL of distilled water. Tube 5 (Chloramine T 6.25 μM) was prepared by taking 400 μL from tube 4 and adding 400 μL of distilled water. Tube 6 (Chloramine T 3.125 μM) was prepared by taking 400 μL from tube 5 and adding 400 μL of distilled water. Tube 7 (Chloramine T 1.562 μM) was prepared by taking 400 μL from tube 6 and adding 400 μL of distilled water. Tube 8 (Chloramine T 0 μM) was prepared by taking 400 μL from tube 7 and adding 400 μL of distilled water, and 400 μL was mixed and withdrawn from the tube. To each of the previous solutions, 40 μL of Acetic Acid [Lot #01315KE Sigma-Aldrich Chemical Company, St. Louis, MO, USA] was added at 99%, and 20 μL of 1.16 M KI solution was added. This was stirred for a few seconds and immediately read on the spectrophotometer Synergy HT, Biotek^® (MA, USA) at a wavelength of 340 nm. The chloramine-T absorbance at 340 nm exhibited a linear relationship within the range of 0–100 µmol/L.