Single-Reagent Colorimetric Test Matches Gold Standard for Drinking Water Chlorine Monitoring

Free chlorine is the workhorse disinfectant of the modern water supply, and the small residual that remains in treated water as it travels through pipes is the main barrier standing between consumers and waterborne pathogens. Utilities are therefore required to monitor that residual continuously, both to confirm that disinfection is working and to detect failures before they become outbreaks. Yet the analytical chemistry behind this routine task has remained surprisingly cumbersome. A new study published in Environmental Monitoring and Assessment reports a simplified colorimetric approach that could make online chlorine monitoring substantially easier to build, operate, and maintain, while matching the performance of the established reference method across a range of drinking water–related samples.

The study, a short communication by Hao Zhang of Onestep Technologies Inc. in Vancouver, evaluates a stabilized single-reagent method based on tetramethylbenzidine, commonly abbreviated TMB. TMB is a chromogenic substrate that reacts with free chlorine to form a colored oxidation product, allowing the chlorine concentration to be quantified by measuring how strongly the solution absorbs light. In this work, absorbance was read at a wavelength of 650 nanometers using a controlled benchtop fixed-time readout taken 30 seconds after reaction. The choice of a single, pre-stabilized reagent is the central design idea: instead of requiring operators or automated analyzers to mix multiple reagents in precise sequence, the entire chemistry is packaged in one solution that can be stored sealed and deployed directly.

To appreciate why that matters, it helps to consider how free chlorine is measured today. The dominant routine technique relies on N,N-diethyl-p-phenylenediamine, or DPD, a dye that free chlorine oxidizes to produce a pink-colored species measured photometrically. DPD methods are codified in international standards, including ISO 7393-2 for routine control purposes and the U.S. Environmental Protection Agency’s Method 334.0 for online chlorine analyzers, and they are enshrined in Standard Methods for the Examination of Water and Wastewater. DPD works well, but the reagent system is notoriously fussy. DPD solutions degrade on exposure to air and light, reagent blanking is often needed, and multi-reagent fluidic handling adds pumps, valves, and failure points to online instruments. For continuous monitors installed across a distribution network, every additional reagent and fluidic step translates into maintenance burden, consumable cost, and opportunities for error.

TMB has long been recognized as an alternative. It was proposed as a colorimetric chlorine reagent as early as the 1990s, and subsequent studies demonstrated its use for hypochlorite detection in tap water and for spectrophotometric sequential injection analysis of free chlorine in waters. TMB has also been promoted as a multi-colorimetric indicator whose response can be aligned with health guideline values. What has limited its practical adoption in online monitoring is the same problem that afflicts many chromogenic reagents: instability in solution. The new work addresses this directly by using a stabilized single-reagent formulation, and it backs the reagent-handling rationale with sealed storage observations described in the study.

The analytical core of the paper is a head-to-head quantitative comparison between the TMB method and DPD. The dataset comprised 32 paired measurements. Twenty of these were real tap-water or finished-water samples, while the remaining twelve were controlled matrix–concentration conditions: three defined mineral-background matrices, each evaluated at four free-chlorine levels. This design is important because drinking water is not chemically uniform. Mineral content, hardness, and background ions vary widely between sources and across distribution systems, and a monitoring chemistry that performs well in one water can drift in another. By deliberately spanning multiple defined mineral backgrounds, the study probes whether the TMB chemistry is robust to realistic matrix variation rather than performing only in clean laboratory buffers.

The comparison protocol was deliberately tight. TMB and DPD measurements were performed within five minutes of each other and in triplicate, minimizing the chance that chlorine decay between measurements would masquerade as analytical disagreement. Agreement was assessed with Deming regression, a statistical technique appropriate when both methods carry measurement uncertainty, rather than ordinary least squares which assumes the reference method is error-free. The results were strikingly close: the Deming regression yielded a slope of 0.985 and an intercept of 0.007 milligrams per liter expressed as chlorine. In practical terms, a slope near unity means the two methods scale almost identically across the concentration range, and an intercept near zero means there is essentially no systematic offset between them.

The paired-difference statistics reinforce that picture. The mean paired difference between TMB and DPD readings was just +0.001 milligrams per liter as chlorine, meaning the methods were unbiased relative to one another on average. The mean absolute difference was 0.013 milligrams per liter, a figure comfortably below the discrimination levels typically relevant to regulatory and operational decisions about residual disinfectant. Precision, assessed from the triplicate TMB readings, produced a median relative standard deviation of 2.4 percent and a mean relative standard deviation of 3.6 percent. For a simple fixed-time absorbance readout at 30 seconds, that level of repeatability indicates the reaction chemistry and the readout timing are well controlled, since kinetic colorimetric methods are sensitive to reaction time if the color development is not sufficiently stable or reproducible.

The author frames the method as a measurement layer for simplified online monitoring, and the study’s logic points toward a compact instrument in which a single sealed reagent reservoir feeds a mixing point, a short reaction delay, and an optical detector reading at 650 nanometers. Eliminating multi-reagent fluidics would reduce the number of pumps and valves, shrink the mechanical footprint, and simplify the reagent supply chain for distributed sensors. This matters because continuous residual monitoring is increasingly seen as essential for managing biological stability in distribution systems, a theme emphasized in guidance from Health Canada on monitoring drinking water biological stability, and because chlorine decay depends on factors such as pipe service age, as documented in studies of transmission and distribution networks. Reviews of continuous chlorine detection have repeatedly identified reagent handling and fluidic complexity as barriers to wider deployment of online analyzers, which is precisely the problem a single-reagent format attacks.

Several caveats deserve attention. This is a short communication based on a controlled benchtop evaluation, not a full validation across the enormous chemical diversity of real distribution systems, and the author notes that the results provide a quantitative basis for further translation into a compact monitoring platform rather than a finished field-proven instrument. The dataset, while well designed, is modest in size, and the competing-interest declaration is explicit: the author is the founder of a company developing stabilized TMB reagents and prototype online chlorine analyzers, so readers should weigh the promising numbers with that affiliation in mind. Independent replication, longer-term reagent stability data, and interference testing across a broader set of water chemistries would be the natural next steps before utilities adopt the approach.

Even with those qualifications, the study offers a tidy demonstration of how rethinking reagent packaging, not just detection physics, can advance environmental monitoring. Free chlorine sits at the intersection of public health and water infrastructure management, and the tools used to measure it have changed little in decades. A chemistry that agrees with DPD to within a hundredth of a milligram per liter on average, achieves a few percent repeatability with a 30-second readout, and arrives in a single stabilized bottle is exactly the kind of unglamorous but consequential engineering that could bring denser, cheaper, and more reliable residual monitoring to the pipes that deliver drinking water. The work suggests that the next generation of online chlorine analyzers may be simpler than the last, built around one reagent, one wavelength, and one short, well-timed wait.

Subject of Research: Colorimetric free chlorine monitoring in drinking water using a stabilized single-reagent TMB method compared with DPD

Article Title: A stabilized single-reagent TMB method for free chlorine monitoring in drinking water–related matrices

Article References: Zhang, H. (2026). A stabilized single-reagent TMB method for free chlorine monitoring in drinking water–related matrices. Environmental Monitoring and Assessment, 198(11), Article 1142. https://doi.org/10.1007/s10661-026-15979-7

Image Credits: AI Generated

DOI: 10.1007/s10661-026-15979-7

Keywords: free chlorine, drinking water, TMB, DPD, colorimetric analysis, online monitoring, Deming regression, water treatment, residual disinfectant, repeatability, analytical agreement, environmental monitoring