I'm attempting to follow the method outlined in the following publication for determining the concentration of hydrogen peroxide in aqueous solution:

Mathew, Sunitha B., Ajai K. Pillai, and Vinay K. Gupta. "Spectrophotometric determination of hydrogen peroxide using leucocrystal violet in micellar medium." Journal of Dispersion Science and Technology 30, no. 5 (2009): 609-612.

Their published procedure for generating the standard curve was as follows (the reagent preparation section outlines how to prepare each of these solutions):

• "To an aliquot containing 0.05 to 0.45 mg of hydrogen peroxide in a series of 25 ml graduated tubes, 2 ml phosphate buffer, 1 ml potassium iodide, and 0.2 ml ammonium molybdate were added and gently shaken for few seconds. Then 1 ml of leucocrystal violet solution was added followed by drop wise addition of 0.1 M sodium hydroxide till a violet colored dye was obtained. The reaction mixture was gently shaken with 1 ml of cetyl pyridinium chloride, which increased the intensity of dye and was kept in a thermostat maintained at 45oC for 2 minutes. The contents were diluted up to the mark with water and kept for 25 minutes at room temperature for complete color development; the absorbance was measured at 593 nm against a reagent blank, prepared in a similar manner, with out hydrogen peroxide."

In the conclusions, the authors claim that "The present method is nonenzymatic simple, highly sensitive, selective and rapid. The method is reproducible and had been successfully used to determine hydrogen peroxide in food products and rainwater."

I've attempted this protocol many times, and I have not been able to reproduce their results. I think I am overlooking some critical detail that the authors neglected to include in the paper, so I would like to get some feedback on what I am doing improperly.

I followed the instructions to the letter all the way up to the part where it says to dropwise add NaOH until a violet color is obtained. No matter how much or how little NaOH I add, I never get color development. According to the paper, there is no color development below pH ~ 3.5, and you get turbidity above pH ~ 5.0. I have gotten a turbid solution with no color development, but I have also carefully controlled the pH until it was ~ 4.3 (the authors say the optimal pH for the reaction is 4.5 +/- 0.5), and still had absolutely no color development.

I keep my pre-prepared stock solutions in the refrigerator before use, but I did experiment with warming up my samples before doing anything, and I still wasn't successful.

One other thing to note is that for the preparation of the phosphate buffer, the authors simply say to use "potassium hydrogen phosphate" buffered with phosphoric acid, so I wasn't sure whether to use monobasic or dibasic potassium phosphate.

Does anyone have any ideas to help me reproduce this assay?