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Reaction Monitoring with the QE Pro Spectrometer – Chemical Stoplight Reaction

Written by  Yvette Mattley, Ph.D.

The chemical stoplight reaction is a reversible oxidation reduction reaction featuring the redox indicator dye indigo carmine. During the reaction, the solution changes from green to red to yellow as the indicator dye is oxidized and then reduced when oxygen levels decrease. Vigorous mixing to reintroduce oxygen restarts the reaction. While the color change is the star of many classroom demonstrations and online videos, the change in absorbance during the reaction can be used to characterize the kinetics of the reaction. In this application note, we describe the use of the QE Pro spectrometer for measuring the change in absorbance during the chemical stoplight reaction. Enhanced spectrometer features including onboard buffering for data integrity are described.


The study of chemical kinetics provides important information on the rate and mechanism of the chemical reactions that occur all around us — from inside the cells of the human body to the ozone layer in the atmosphere. Characterizing the impact of parameters such as reactant concentration, temperature, pH and the presence of a catalyst are vital to optimizing reaction conditions and understanding the mechanism of the reaction. In an industrial or process setting, detailed knowledge of the chemical kinetics for a reaction enables the use of the optimum conditions and reactant concentrations to maximize product yield while minimizing reactant waste. In the human body, chemical kinetics measurements are made to characterize the impact of enzyme catalysts on metabolism and to understand the factors critical to the accurate dosing and release of a medication.

Reaction-Monitoring-Page-1-ImageIn this application note, the QE Pro is used to collect the absorbance data needed to characterize the chemical kinetics for a reversible oxidation reduction reaction featuring indigo carmine indicator dye.  The redox (reduction oxidation) indicator dye indigo carmine exists in oxidized, reduced and intermediate forms depending on its environment. Each form has a slightly different chemical structure resulting in the absorption of different wavelengths of light. When indigo carmine is mixed with a reducing agent (dextrose) in a basic solution (NaOH), it acts an indicator of the state of the redox process. When the solution is first mixed by shaking the solution to introduce oxygen, the indicator is in its green oxidized form (most exposed to oxygen in the air). The reaction mixture color changes to red and then to yellow as the indigo carmine goes from an oxidized to a reduced state.

Indigo carmine changes color as a result of changing levels of oxygen in the solution. The solution is yellow in color. When the solution is mixed, oxygen dissolves into the mixture oxidizing the indicator and changing the color to red. When the flask is shaken more vigorously, the levels of oxygen increase even more, oxidizing the indicator further and causing it to turn green. When the solution is left alone, the oxygen concentration drops due to a reaction with dextrose and the solution returns to its original yellow color. The formula for this chemical reaction is shown in Figure 1. Figure-1-oxidation-of-indigo-carmine-e1397062503329Figure 1: Oxidation of indigo carmine (from

The QE Pro spectrometer is ideally suited for kinetics measurements. With the highest dynamic range in its spectrometer class, the QE Pro is able to detect a wide range of light levels while pulling peaks out of the noise. When the goal of the measurement is monitoring reaction kinetics, the QE Pro’s onboard buffering of up to 15,000 spectra ensures data integrity. This means no data points are missed during critical stages of the reaction when the computer fails to request a spectrum because it is burdened with other tasks. This buffering is very important for kinetics measurements, where the assurance of data integrity means superior kinetics data.

Experiment Details

The chemical stoplight reaction mixture is prepared by adding 1.5 mL 2.5% dextrose solution, 1.5 mL 1 M NaOH solution and 2 drops of 1% indigo carmine solution to a CVD-VIS-1M disposable cuvette. Note that the NaOH solution used for this reaction is very caustic. Handle the NaOH solution and mixture with care and take the necessary safety precautions while working with these solutions. Place a CVD-COVER on the cuvette and shake the cuvette to introduce oxygen into the solution until the solution has a greenish color. Place the cuvette into the CUV-UV 1-cm pathlength cuvette holder and measure absorbance using OceanView spectroscopy software. Monitor the reaction using the OceanView strip chart feature set to measure absorbance at 553 nm and 759 nm.

When the absorbance of the peaks at 553 nm and 759 nm drops to the baseline, shake the cuvette thoroughly to reintroduce oxygen and restart the reaction. Shake the cuvette until the mixture is greenish in color and the indicator dye is oxidized. The reaction can be restarted several times by shaking the cuvette thoroughly to introduce oxygen and reoxidize the indigo carmine. If the reaction goes too fast or the peak intensities drop too low, the reaction mixture can be refreshed by adding a few additional drops of indigo carmine dye solution to the cuvette.


The visible absorbance spectra measured at different times during the chemical stoplight reaction are shown in Figure 2. After starting the reaction by thoroughly mixing the solution to introduce oxygen, the absorbance at 553 nm increases while the absorbance at 759 nm decreases. The absorbance for the peak at 553 nm increases until the oxygen in the mixture is depleted. As the oxygen levels decrease, the absorbance at 553 nm decreases to the starting absorbance level.

Reaction Monitoring-Figure-2Figure 2: Absorbance spectra measured during the chemical stoplight reaction

The absorbance trends measured during the chemical stoplight reaction for the peaks at 553 nm and 759 nm are shown in Figure 3. The absorbance trend over time corresponds to a change in the color of the solution as the indicator dye is oxidized and reduced. The solution starts out green and slowly turns red as the oxygenation state of the indigo carmine indicator dye changes. The solution then turns to yellow as the oxygen in the mixture reacts with dextrose, decreasing the oxygen available to oxidize the indicator dye. The absorbance trend repeats when oxygen is reintroduced into the reaction mixture via vigorous shaking.

Reaction Monitoring-Figure-3Figure 3: Absorbance trend measured during the chemical stoplight reaction for the peaks at 553 nm and 759 nm

The chemical kinetics for this reaction is characterized by varying the reactant concentrations and repeating these measurements at each reactant concentration. To ensure the most accurate kinetics measurements, it is important to collect as many data points as possible during the reaction. Data integrity can be a significant challenge with the multitasking nature of today’s computers. While the spectrometer continually acquires data at the acquisition time specified, the computer’s capacity to request spectra often falls behind, resulting in missed scans. Inaccurate reaction rates, rate law and rate constants could result if the computer lags behind during a critical point in the reaction.

The QE Pro overcomes data integrity issues related to computers that fall behind spectrometer acquisitions with the ability to buffer up to 15,000 spectra onboard. This means there will be no missed scans even if the computer falls behind and fails to ask for spectral data for up to 150 seconds (more than 2 minutes). The trend shown in Figure 4 for the number of spectra stored onboard the QE Pro over time was generated for approximately 50 minutes of continuous data acquisition.

For other spectrometers with minimal onboard buffering, each time the number of spectra stored onboard the spectrometer goes above 3 spectra (almost 20 times for the data shown), critical kinetics data would be lost as the computer falls behind the spectrometer. Fortunately, no data is lost during this 50-minute acquisition period when the QE Pro is used for these measurements due to its spacious onboard buffer that ensures complete data integrity and superior kinetics characterization.

Reaction Monitoring-Figure-4Figure 4: Trend for the number of spectra in the QE Pro onboard buffer during 50 minutes of continuous acquisition


The colorful chemical stoplight reaction is a staple in many undergraduate teaching laboratories. The changing color appeals to the visual senses as the solution goes from green to red to yellow in color. As demonstrated in the absorbance data acquired with the QE Pro spectrometer during this reaction, there is much more than just a color change going on in the solution. The changing oxidation state of the indicator dye results in a change in absorbance, which can be measured without losing a single data point during the entire reaction using the QE Pro spectrometer. For kinetics measurements like this one, where every data point is critical, the QE Pro spectrometer ensures that no data is lost during the reaction.

The QE Pro spectrometer used for these measurements is a high performance spectrometer with a cooled, back thinned detector, excellent signal to noise ratio and superior dynamic range. With the added feature of interchangeable slits, the QE Pro is an exceptional spectrometer with flexibility to enable a range of applications requiring different light levels and optical resolution needs. Combine the QE Pro with different sampling accessories and light sources to build your own modular toolkit with virtually endless measurement possibilities.