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Peroxide (H2O2) Quantification

Technical Overview

In this procedure, we quantify the aqueous H2O2 concentration by allowing it to react with a titanium sulfonate reagent to produce pertitanic acid, which is yellow in color. The reaction is effectively instantaneous and the yellow-colored complexes are stable for at least 6 hours [Machala 2013].

Ti4+ + H2O2 + 2H2O = H2TiO4 (pertitanic acid) + 4H+

This method is based on the procedure reported by Eisenberg in 1943.

Primary References

"Colorimetric Determination of Hydrogen Peroxide" [DOI: 10.1021/i560117a011]

"It is evident that 1 volume of titanium sulfate reagent per 10 volumes of peroxide solution is required for maximum color development. As far as could be ascertained, the color is developed instantly and remains stable for at least 6 hours."

"Beer’s law is valid for measurements at 380 to 430 millimicrons." [Our lab typically measures absorbance at 407nm.]

"Formation of ROS and RNS in Water Electro-Sprayed through Transient Spark Discharge in Air and their Bactericidal Effects"

"Measurements of hydrogen peroxide formed in plasma treated water were performed by titanium sulfate colorimetric method... The intense yellow color is formed when an acidic solution of titanyl ions are mixed with H2O2. The method is specific to H2O2 and there are no interferences from other compounds present in water, while working under strong acidic conditions following the modified procedure of Satterfield and Bonnel [Anal. Chem. 1955]. The Lambert-Beer’s law is followed and the yellow color intensity is proportional to the H2O2 concentration giving a linear relationship. The molar extinction coefficient determined in our laboratory from the slope of the calibration plot at the
wavelength [of] 407nm was e=6.89 x 10^2 L/mol-cm. The method is independent of the pH since the measurement of H2O2 is carried out in strong acidic solution of sulfuric acid (dilute 1:1). In the presence of nitrites, a solution of sodium azide was added to the samples with H2O2 prior to mixing with titanium reagent to eliminate decomposition of H2O2 by nitrites under acidic conditions. With sodium azide, nitrites are reduced into molecular nitrogen under acidic conditions."

"Interferences in the Titanium Sulfate Method for Hydrogen Peroxide"
Satterfield, C.N., A. H. Bonnell 1955, DOI: 10.1021/ac60103a042

The modified procedure  described in this reference was used to prepare the titanium sulfate reagent:

"Prepare a reagent solution by adding 10 grams of titanium sulfate and 20 grams of concentrated sulfuric acid to 50 ml of distilled water. Allow the mixture to stand for 24 hours, then decant and filter on analytical filter paper to remove suspended solids. Dilute the test solution with sulfuric acid of such concentration that the diluted solution is about 3N and allow to stand for 2 hours. Add 1 ml of the reagent solution to 10 ml of the diluted test solution and determine the absorbance of [the yellow color] developed in comparison with a blank containing 10 ml. of distilled water and 1 ml. of the titanium sulfate solution. Compare this with a calibration curve determined from known concentrations of hydrogen peroxide."

Materials (needs updating)

Titanium Oxysulfate

Phosphoric Acid
SpecificationMSDS (hazardous: acid)
Formula:               H₃PO₄
Formula Weight:    98 g/mol

Sodium Phosphate, Monobasic
SpecificationMSDS (nonhazardous)
Sigma-Aldrich: ($0.10-0.25 per gram)
Formula:                NaH₂PO₄
Formula Weight:    119.98 g/mol


- Reagent include strong acids. Make sure that you have read and signed the lab standard operating procedures (SOPs) for "Strong Acids" and "Oxidizing Reagents" before using concentrated phosphoric acid or hydrogen peroxide. Always wear appropriate personal protective equipment (safety glasses, chemical-resistant gloves, lab coat) when working with these chemicals. 


Titanium sulfate reagent: (pre-made solution in the Graves Lab refrigerator, brown glass bottle.)
Dissolve 25g TiOSO4 (titanium oxysulfate) with constant stirring in 1L of a 2 M sulfuric acid solution. Cool reagent and store in a cool, dark place. [Stable for up to 4 months.]

"The titanium sulfate test reagent was prepared by digesting 1 gram of anhydrous titanium dioxide (Schering-Kahlbaum) with 100 ml. of sulfuric acid (specific gravity 1.84) for 15 to 16 hours on a sand bath at a temperature of 150 degC." [Eisenberg 1943]

Sodium azide solution (60mM NaN3): (pre-made solution in the Graves Lab refrigerator, brown glass bottle.)
Add NaN3 to distilled water to a concentration of 60 mM. 1-10 mM NaN3 (concentration in final solution) is sufficient to scavenge ROS that could otherwise react with H2O2


0. Absorbance is measured on the UV/vis spectrophotometer in the Clark Lab using either a 96-well plate (preferred for running many samples) or quartz cuvette. If using a cuvette, combine sample, sodium azide (if necessary) and titanium sulfate reagent in an eppendorf tube prior to measurement. Otherwise sample and reagents can be added directly to well(s) in 96-well plate. 

1. If nitrites are expected to be present, add 10ul sodium azide solution per 100ul of sample immediately after sample is taken! Vortex or pipette repeatedly to mix. 

2. Add 50ul of titanium sulfate reagent per 100ul of sample. The mixture with reagent should be stable for several hours. 

**If the sample contains peroxide, it will react with the titanium ions in solution to form pertitanic acid, giving the solution a yellow color. 

3. Measure the absorbance at 407nm using the UV-vis spectrometer in the Clark lab (light side). If using a plate, include a blank(s).  If using a cuvette, set an appropriate blank as a reference prior to measuring the absorbance of the sample. To do this, make sure the window heading in the program says “cuvette”. Then click “Ref”.  There is a cuvette for the UV-vis in the small bench drawer in Graves lab. Use this – you only need to add about 100ul of sample to the cuvette. 

4. Empty and rinse the cuvette with water (~3x) and dry (blow air or use vacuum…) in between blanks and samples. 

5. Add 100ul of sample+reagent mixture to the cuvette. Click “Read” to measure absorbance at 407nm. 

6. CEA measured a correlation coefficient of 5.3996 to convert from units of Absorbance to mM H2O2 (using 96-well plate, 160ul total well volume).  The data fit to obtain this coefficient are attached. 

Notes and Calculations

None at this time. 
Carly Anderson,
Nov 4, 2013, 2:51 PM