Dissolved ozone concentration measures how much active ozone (O₃) remains stabilized within the glycerin matrix at the time of testing, expressed in milligrams per liter (mg/L). This is the primary indicator of product potency. Unlike indirect proxies such as ORP or pH, this is a direct, ozone-specific measurement — the gold standard for quantifying what’s actually in the product. Sources: Gordon et al. (1995); Rakness et al. (2010)
We use the indigo trisulfonate colorimetric method (Standard Methods 4500-O₃ B / Bader-Hoigné), adapted and validated for the glycerin matrix. Ozone selectively bleaches the blue indigo dye in proportion to its concentration; the absorbance change is read at ~600 nm with a Hanna Instruments HI83399 multiparameter photometer. Because glycerin is viscous and chemically reactive toward ozone (reported rate constants ~10⁻¹–10⁻² L·mol⁻¹·s⁻¹), we perform matrix-specific validation including spike-recovery studies (target 80–120% mean recovery) and repeatability testing (%RSD ≤10%) to ensure accuracy in our specific glycerin. Samples are collected immediately after the ozonation cycle in tared vessels with minimal headspace to prevent off-gassing losses, and reacted with indigo reagent within seconds. Sources: Rakness et al. (2010); Canterino et al. (2009)
Ozone is the active compound in ozonated glycerin. A higher dissolved ozone concentration means more potent antimicrobial, anti-inflammatory, and wound-healing properties. Critically, glycerin is not water — it reacts with ozone through direct molecular attack and radical pathways, so methods validated for water cannot be assumed accurate in glycerin without specific validation. This is why most competitors cannot make credible concentration claims: they haven’t validated their measurement method for the glycerin matrix.
SimplyO3 Glyzine must contain not less than (NLT) 1,500 mg/L of dissolved ozone by validated indigo assay. Our batches consistently achieve 2,000–3,000+ mg/L — significantly exceeding the minimum specification.
Of 10+ brands researched, only SimplyO3 publishes batch-level dissolved ozone data measured by a validated, ozone-specific method. Most competitors do not disclose concentration at all, or rely on non-validated methods. This lack of traceable, matrix-validated measurement makes independent comparison impossible — which is exactly why we publish every batch.
Stated accuracy: ±6% relative uncertainty. Here is how we arrive at that number and what the science says:
The Method — Standard Methods 4500-O₃ B: The foundational reference for ozone colorimetry states: “The proportionality constant at 600 nm is 0.42 ± 0.01/cm/(mg/L)” and “for the spectrophotometric volumetric procedure in the absence of interferences, the relative error is less than 5%. In laboratory testing this may be reduced to 1%.” Duplicate determinations gave “an average deviation of 1 to 1.5%.”
Reagent Variability — Rakness et al. (2010): “The molar absorptivity for seven sources of indigo ranged from 15,800 to 20,355 M⁻¹cm⁻¹.” This means “indigo purity (and stability) is potentially a significant source of measurement error when unvalidated indigo stock solutions are used.” Rakness further showed that “ITS solution stored on a shelf for several days can cause an appreciable under-estimation of ozone residual.” We mitigate this by using freshly prepared reagent from a single verified supplier. doi:10.1080/01919510903467864
The Instrument — Hanna HI83399: This multiparameter photometer has published absorbance accuracy of “±0.003 Abs @ 1.000 Abs” with spectral bandwidth 8 nm, silicon photodetector, and reference detector for source stability. We use it in absorbance mode at 610 nm with the indigo reagent (not the manufacturer’s DPD method), so the governing accuracy is the absorbance specification, not the DPD-specific ozone range.
Matrix Validation — ICH Q2(R1): The international standard for analytical method validation requires: “Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range.” We perform matrix-specific spike-recovery studies (target 80–120% mean recovery, %RSD ≤10%) following these guidelines.
Glycerin Matrix Complexity — Canterino et al. (2009): Glycerol reacts directly with ozone through multiple pathways; Canterino’s kinetic study found that “direct ozonation of glycerol results into a mixture of its oxygenated derivatives” with rate constants consistent with alcoholic C-H bond reactivity. This competing reaction is why aqueous-validated methods cannot simply be assumed to work in glycerin — and why we invest in matrix-specific validation. doi:10.1080/01919510903244610
Our ±6% Budget: Standard Methods base error (<5%) + instrument absorbance uncertainty (±0.003 Abs, which at a typical indigo delta-absorbance reading of ~0.1–0.2 Abs corresponds to ~1.5–3% relative) + glycerin matrix factor, combined in quadrature, yields approximately ±6% total relative uncertainty. This is conservative — controlled lab conditions routinely achieve <3%.
What ±6% Means For Your Batch: If your batch reports 2,500 mg/L dissolved ozone, the true value is between approximately 2,350 and 2,650 mg/L. Since our acceptance minimum is 1,500 mg/L, even the lower bound of a typical batch exceeds the specification by more than 50%.