Application Note | ABSOR.B™
UNDERSTANDING ABSORBANCE: MEASURING REACTION RATES IN CHEMISTRY
by Dimitria Camasão and CEP Noemie Deloire,
Application Scientists, Rheolution Inc.
Summary
- Chemical reactions in which colored reactants or products are involved can be analyzed through the changes in their absorbance capacity over time at specific wavelengths.
- ABSOR.B™ precisely measured the absorbance kinetics of a mixture composed of blue food colorant and bleach.
- The rate equation for the chemical reaction between the blue dye and bleach was determined using the absorbance kinetics.
INTRODUCTION TO ABSORBANCE MEASUREMENT IN CHEMICAL REACTION ANALYSIS
Introduction
Chemical reactions in which one of the reactants or products is colored can be monitored with absorbance measurements over time. For example, dye molecules can react with specific substances changing its chemical structure and therefore its ability to absorb/reflect light the same way. Similarly, the activity of enzymes can be monitored when the reaction between the enzyme and the substrate results in a colored product. In these two examples, the rate of the chemical reaction can be represented by the disappearance of reactants or the appearance of products, respectively.
These measurements (absorbance over time) give information about the reaction rate and the activity of the reactants. The reaction rate (or rate law) is a mathematical description of how the rate of a chemical reaction depends on the molar concentration of its reactants. Determining rate laws is fundamental for advancing the understanding of chemical reactions, optimizing reaction conditions, and ensuring the efficient and controlled production of desired products across various scientific and industrial fields.
In this light, the ABSOR.B™ was used herein to study the kinetics of the reaction between a food colorant (blue dye) with sodium hypochlorite (bleach). The hypochlorite ion (ClO-) is an oxidizing agent that removes one or more electrons from the dye molecules changing its chemical structure which in turn is no longer able to absorb light in the visible range. The decrease in the absorbance during the reaction was measured at two different concentrations of bleach. The obtained data were then used to determine the chemical equation for the reaction rate.
EXPERIMENTAL APPROACH TO MEASURING ABSORBANCE IN CHEMISTRY
Materials and Methods
Food colorant (blue color) was mixed with sodium hypochlorite (bleach, NaClO) in 10-mL vials at two concentrations (0.134 M and 0.269 M of ClO). Vias were immediately inserted in the ABSOR.B™ and tests were performed at room temperature and using the Emitt.635 cartridge, the emission optical cartridge that emits light at the wavelength of 635 nm (blue components have their absorbance peak at around this wavelength). Absorbance values over time were collected for each sample (n=3 for each condition). The rate law was calculated from the absorbance kinetics (1,2).
ANALYZING REACTION RATES THROUGH ABSORBANCE MEASUREMENTS
Results and Discussion
Experimental Monitoring of the Chemical Reaction
Figure 1 displays the results for each sodium hypochlorite concentration mixed with the blue dye in triplicate (n=3). As expected, the reaction happened quicker for the sample with the highest concentration of sodium hypochlorite as demonstrated by the faster decrease in absorbance. It is also possible to notice the great repeatability of the results among the replicates for each condition. inherent intra variability of animal tissues and the small number of samples (n=3), reinforcing the high repeatability and sensitivity of the results.
Figure 1: Absorbance (Abs) of food grade blue dye as a function of time when mixed with sodium hypochlorite at 0.269 M (orange) and 0.134 M (turquoise).
Theoretical Modeling of the Chemical Reaction
This chemical reaction with its stoichiometric coefficients (m and n) can be represented as follows:
The rate equation for this reaction as function of the molar concentration of its reactants can be written as:
![rate = k [blue dye]m[hypochlorite]n](data:image/svg+xml;base64,PHN2ZyB2aWV3Qm94PSIwIDAgNzAzIDM1IiB3aWR0aD0iNzAzIiBoZWlnaHQ9IjM1IiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==){kind=small}
Where k is the rate constant (specific for a particular reaction and temperature), and m and n are exponents that define the reaction order.
In this experiment, sodium hypochlorite was in great excess over the blue dye. Therefore, its concentration did not change significantly during this chemical reaction so it can be approximated as a constant.
To first determine the exponent m and given that the concentration of hypochlorite can be approximated as a constant in this experiment, the pseudo rate constant (k’) can then be determined as:
![k [hypochlorite]n](data:image/svg+xml;base64,PHN2ZyB2aWV3Qm94PSIwIDAgNzAzIDMyIiB3aWR0aD0iNzAzIiBoZWlnaHQ9IjMyIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==){kind=small}
Therefore the rate law can be re-written as:
By plotting Abs vs time, ln(Abs) vs time, and 1/Abs vs time, it is possible to determine the reaction order for the blue dye. If:
– Abs vs time is linear: zero-order reaction (m = 0)
– ln(Abs) vs time is linear: first-order reaction (m = 1)
– 1/Abs vs time is linear: second-order reaction (m = 2)
Figure 2 shows that the highest R² obtained was for the ln(Abs) vs time curves (R² > 0.99), therefore this is a first-order reaction in respect to the blue dye (m = 1). In addition, the pseudo constant (k’) is equal to minus the slope of the linear regression line. With this information, it is possible to calculate the rate law constant (k), as follows:
The rate constant (k) for this reaction is then 0.29 (M.s)⁻¹ and using the equation 2, n is also equal to 1. Therefore, the rate law for this chemical reaction can be written as:
Figure 2: Absorbance (Abs), ln Abs and 1/Abs average values as a function of time for food grade blue dye with both concentrations of sodium hypochlorite (n=3) with their respective linear regressions.
Conclusions and Perspectives
ABSOR.B™ was successfully used to measure the absorbance changes over time at 635 nm during the reaction between a food grade blue dye and bleach in solution. The rate law of this chemical reaction was calculated from the absorbance kinetics.
Overall, the ABSOR.B™ instrument facilitates the:
- Collection of continuous absorbance measurements into its Tablet App for the easy analysis of chemical reactions kinetics.
- Customization of the testing platform in terms of wavelength needed for the application (with the different optical cartridges of light emission), samples holders that better fit the experiment (among vials, cuvette, and micro-tubes), and testing capability by controlling multiple ABSOR.B™ units from a single wireless operating tablet.
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Absorbance is a crucial concept in the field of spectrophotometry, a technique used to measure the amount of light absorbed by a substance over a large range of wavelengths. It is a dimensionless quantity derived from the logarithm of the ratio of incident light to transmitted light through a sample. The absorbance spectrum, often represented as a graph, reveals the wavelengths at which a substance absorbs light most strongly (Figure 1). This information is useful in various scientific fields such as biology, chemistry, biochemistry, molecular biology for identifying and quantifying specific compounds...