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Ozone Quantification

Technical Overview

In this procedure, we quantify aqueous ozone concentration by allowing it to react stoichiometrically with a brightly-colored dye (indigo trisulfonate) to yield a colorless product (isatin sulfonate).  The dye has an extremely strong absorbance (~20,000 M⁻¹cm⁻¹ @600nm), so this method has a higher sensitivity and is less prone to interference than direct spectrophotometric quantification of O₃ at 260nm (3290 M⁻¹cm⁻¹ [Hart 1983]).  As-written, the dye solution is completely exhausted at an O₃ concentration of 0.6mg/L; samples at or above this range should be diluted before assaying.

     Ozonolysis   (x2)
 potassium indigo trisulfonate                                                                                    isatin sulfonate

Primary References

"Determination of ozone in water by the indigo method"

"The concentration of aqueous ozone can best be determined by the decolorization of indigo trisulfonate (600 nm, pH below 4) whenever the ozone cannot be measured directly by its u.v. absorption. The method is stoichiometric and extremely fast. The change of absorbance vs ozone added is −2.0 ± 0.1 × 104 M−1 cm−1and is independent of the concentration of aqueous ozone in the range 0.005–30 mg 1−1."

"Development of an assay for ozone-specific antioxidant capacity."

"aliquots of sample or of a saline control were then added and sufficient time was allowed for ozonation to reach completion; and an aliquot of indigo trisulfonate (ITS) was added to react with excess O(3). Because each molecule of O(3) rapidly bleaches one molecule of the deeply colored ITS, an OZAC value in concentration units was computed from the difference in light absorbance between the sample and the saline control multiplied by the extinction coefficient of ITS."

"ITS is a water-soluble derivative of a common blue dye that has a molar extinction coefficient of 600 = 23,800 M–1cm–1 for 600-nm light (Bader & Hoigne, 1981). Ozone attacks a carbon–carbon double bond of ITS in a rapid reaction (i.e., rate constant of 1 × 107 M–1s–1) that yields colorless products."

"Light-Scattering and Molecular Spectrophotometery: Ozone: Aqueous Phase"

"Aqueous ozone concentrations in pure (e.g., distilled) water may be conveniently determined by direct spectrophotometric measurement.

               CO3 (mg/L as O3)  =   14.59*(Abs @260 nm)                   (17.42)
Equation 12 is based on a molar absorptivity of 3290 M-1cm-1 (Hart et al., 1983).  Unfortunately, most solutes will interfere at this wavelength, so with actual environmental samples another method must be used.  Since the iodometric method is too nonspecific for aqueous determinations, the indigo method of Bader and Hoigne (1981) is recommended."

"Modified Indigo Method For Gaseous And Aqueous Ozone Analyses"

"The indigo method developed by Bader and Hoigné for aqueous ozone analysis was modified to allow for both gaseous and aqueous ozone determination. Gas or water samples were extracted with a gas-tight syringe containing a known volume of indigo reagent. The modified procedure provided a more consistent basis for gaseous and aqueous ozone determination allowing for more accurate ozone mass balance calculations. Direct gaseous ozone UV absorbance with molar absorptivity of 3,000 M−1cm−1 at 258 nm was used as primary standard to determine the molar absorptivity of the indigo reagent. The molar absorptivity of indigo reagent, assuming a 1:1 stoichiometric ratio for the reaction between indigo and ozone, was determined to be 23,150 ± 80 M−1cm−1, or approximately 16 percent higher than that of 20,000 M−1cm−1 suggested by Bader and Hoigné."

This paper details a possible improvement of the protocol; a request for this paper has been placed with the library (2013.08.28).


Potassium Indigo Trisulfonate (ITS)
Specification; MSDS (nonhazardous)
Formula:                C16H7K3N2O11S3
Formula Weight:     616.72 g/mol

Phosphoric Acid
Specification; MSDS (hazardous: acid)
Formula:               H₃PO₄
Formula Weight:    98 g/mol

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


- Reagents include strong acids. Handle with care.


Indigo Stock Solution: (pre-made stock solution lives in the Clark lab cold room on common shelf.)
100 mL distilled water
100 ul 85% phosphoric acid (H3PO4)
77 mg potassium indigo trisulfonate

Indigo Reagent: (make fresh the day of the assay)
For 10ml:
200 ul indigo stock solution
100 mg sodium phosphate (NaH2PO4) [buffer]
70 ul phosphoric acid (H3PO4)
+ distilled water to 10 ml


1. Prepare indigo reagent according to the recipe above.

2. Immediately after sample is taken, add indigo reagent (1:1 vol/vol ratio) to sample in a small eppendorf tube and vortex. (The half-life of ozone in solution is very short, so reagent should be added ASAP for an accurate reading. The mixture with reagent should be stable for several hours.)

Note: The indigo reagent is blue. If the sample contains ozone, it will react with the indigo in the reagent and reduce the color. The more clear the sample+reagent mixture is, the more ozone it contains.

3. Measure the absorbance at 600nm of a blank – indigo reagent + untreated sample (or water) – using the UV-vis spectrometer in the Clark lab (light side). The cuvette for the UV-vis in Clark Lab is hidden in MP’s stuff. Use this – you only need to add about 100ul of sample to the cuvette. Make sure the window heading in the program says “cuvette”. Then click “Ref”.

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

5. Add 100ul of sample+reagent mixture to the cuvette. Click “Read” to measure absorbance at 600nm.

6. ZM/MP use a correlation coefficient of -2 /0.389 x the raw absorbance to calculate the O3 concentration, in mg/L.

Notes and Calculations

MW, indigo = 616.72
MW, ozone  = 48.00
molar mass ratio: 12.85

indigo = 0.77g/L = 1.25mM

indigo reagent:
50x dilution

mix 1:1 with sample:
2x dilution
indigo = 12.5µM

0.077g → 100mL (stock; 1.25mM)
0.20mL → 10mL  (50x dilution)
1mL    → 2mL   (2x dilution)

Final concentration of indigo: 12.5µM
Indigo and O₃ react 1:1this can react away 12.5µM (=600µg/L=0.6mg/L) ozone.
On MP's July 2012 GRC poster, O₃ is reaching 20mg/L in the liquid phase; this MUST involve dilution.

The indigo reagent is good to mix 50:50 with the undiluted sample up to 0.6mg/L O₃; above that, the sample must be diluted!
Brandon Curtis,
Aug 28, 2013, 2:48 PM