Analysis of Methylene Blue Reduction by Ascorbic Acid

ApplicationNote: 51867Analysis of Methylene Blue Reductionby Ascorbic AcidNicole Kreuziger Keppy, Michael W. Allen, Ph.D., Thermo Fisher Scientific, Madison, WI, USAKey Words• Ascorbic Acid• Kinetics• Methylene Blue• Reaction Rate• ReactionMechanism• UV-VisibleSpectroscopyIntroductionChemical kinetics is the study of properties of chemicalprocesses such as reaction rates, mechanisms and transitionstates. The mechanism and rate of chemical reactions canbe determined using UV-Visible spectrophotometry. In thisapplication note, the reaction of methylene blue withascorbic acid is monitored with a Thermo ScientificEvolution Array UV-Visible spectrophotometer. The rateconstant is also determined using Thermo ScientificVISIONcollect software.Methylene blue (MB + ) is a water-soluble dye molecule.Under acidic conditions, it is easily reduced to thecolorless hydrogenated molecule leucomethylene blue(LB + ) by ascorbic acid (H 2 A) as shown in Figure 1. Thestoichiometry of the overall reaction is 1:1 and isrepresented in the equation:MB + + H 2 A → LB + + DBackgroundThe mechanism of a chemical reaction can be identified bymeasuring a change in the overall reaction rate caused bychanges in the concentration of individual reactants. In thesimple chemical reaction:A + B → Pthe reaction rate (V) is proportional to the concentrationof reactants, such that:V = k[A] α [B] βIn this rate expression, k is the rate constant, α is theorder of the reaction with respect to A, and β is the orderof the reaction with respect to B. Thus, the overall orderof the reaction is:n = α + βFigure 1: Reaction Mechanism of methylene blue with ascorbic acidA rate constant (k exp ) is determined experimentally bymeasuring the change in concentration of methylene blueat 665 nm. As a first approximation, the rate ofdisappearance of MB + can be expressed as in Equation 1below. By strategically varying the individual reactants, areaction mechanism can be determined by calculatingconstants k 0 and k 1 in Equation 1 using the experimentalresults. 1d[MB + ]Equation 1: = -{k 0 + k 1 [HCl]}[H 2 A][MB + ]dt= -k exp [MB + ]

Experiment and ResultsStock solutions of methylene blue (4.0 x 10 -4 mol/L),ascorbic acid (0.5 mol/L) and hydrochloric acid (2.0 mol/L)were prepared. Fifteen sample solutions were preparedfrom these stock solutions according to the conditionsdefined in Table 1. A blank was prepared for each sampleby mixing together all of the reagents in the correspondingsample except methylene blue. All measurements wereperformed immediately following the addition of methyleneblue to the sample due to the fast nature of the reaction.Time-based Kinetics mode was programmed using theparameters shown in Figure 2.Ascorbic Methylene Total RunSample # HCl (M) Acid (M) Blue (M) Time (s)Set A: Varying Methylene Blue Concentration [MB + ]A1 0.3 0.025 3.5 x 10 -5 200A2 0.3 0.025 2.5 x 10 -5 120A3 0.3 0.025 1.5 x 10 -5 120A4 0.3 0.025 9.0 x 10 -6 120A5 0.3 0.025 4.0 x 10 -6 120Set B: Varying Ascorbic Acid Concentration [H 2 A]B1 0.2 0.000 1.5 x 10 -5 600B2 0.2 0.004 1.5 x 10 -5 600B3 0.2 0.008 1.5 x 10 -5 360B4 0.2 0.016 1.5 x 10 -5 180B5 0.2 0.036 1.5 x 10 -5 120Set C: Varying Hydrochloric Acid Concentration [HCl]C1 0 0.045 1.5 x 10 -5 150C2 0.1 0.045 1.5 x 10 -5 120C3 0.2 0.045 1.5 x 10 -5 120C4 0.4 0.045 1.5 x 10 -5 120C5 0.5 0.045 1.5 x 10 -5 120Table 1: Sample preparation and experimental conditions for the analysis ofmethylene blue reductionFigure 2: Method parameters for methylene blue reduction analysisFigure 3: Absorbance decay of methylene blue under condition A1Sample # Start Time (s) End Time (s) K expA1 0 80 0.0616A2 0 80 0.0645A3 0 80 0.0607A4 0 80 0.0631A5 0 80 0.0619Average 0.0624B1 0 600 0.0000B2 0 450 0.0097B3 0 266 0.0172B4 0 150 0.0333B5 0 70 0.0693C1 0 150 0.0179C2 0 120 0.0454C3 0 90 0.0645C4 0 46 0.1076C5 0 36 0.1310The Relationship Between Rate Constant and MethyleneBlue Concentration, [MB + ] (Set A):The change in absorbance of methylene blue at 665 nm,plotted as a function of time, is shown in Figure 3. A firstorder fit of the absorbance between 0 and 80 secondswas used to determine an experimental rate constant (k exp )of 0.0616. The data collected for the remaining samples(A2-C5) shows a similar pattern and their rate constantsare shown in Table 2.Table 2: Rate calculation range and experimental rate for eachexperimental condition

One benefit of the Evolution Array is its ability tocollect the entire spectrum during kinetics experiments.The full spectrum of methylene blue can be observed byclicking the full spectrum icon in Time Based Kinetics Modein the VISIONcollect software. The individual spectrafor sample A1 are shown in Figure 4. These spectra werealso used to construct the decay curve shown in Figure 3.Figure 5: Sample set B results show k exp as a function of ascorbic acidconcentrationFigure 4: Spectra of methylene blue reduction under condition A1As shown in Table 2, variations in k exp are small withrespect to methylene blue concentration. The averagevalue of k exp is approximately 0.0624. All of the measuredvalues fall within a small range of this average value anddo not exhibit a trend in variation as a result of a decreasein concentration. Thus, we can conclude that the initialconcentration of methylene blue is not a significant factorin the rate equation.The Relationship Between Rate Constant and AscorbicAcid Concentration, [H 2 A] (Set B):To determine the affect of ascorbic acid on the overallrate, the concentration of H 2 A was varied according tothe conditions defined in Set B, Table 1. A plot of k expagainst H 2 A concentration exhibits a linear relationship asshown in Figure 5. A linear fit of the data results in aslope of 1.9026 M -1 s -1 with a R 2 value of 0.9983. Theslope of this curve is the k exp and can be entered intoEquation 1:Equation 1: k 0 + k 1 [HCl] = 1.9026 M -1 s -1The concentration of HCl in this sample set is heldconstant at 0.2 M. Entering this value into the equationabove yields Equation 2:The Relationship Between Rate Constant and HClConcentration, [HCl] (Set C):In the final set of solutions, the concentration of HCl wasvaried according to Set C, Table 1. Plotting k exp againstHCl concentration illustrates the linear relationship of therate with respect to the concentration of HCl (Figure 6).The resulting linear fit has a slope of 0.2212 M -1 s -1 and aR 2 value of 0.9981. Taking the slope as the experimentalrate and substituting it into Equation 1 we find:k 1 [H 2 A] = 0.2212 M -1 s -1Entering the concentration of H 2 A (0.045 M), thevalue for k 1 is determined as follows:k 1 = 4.92 M -2 s -1Entering the value of k 1 into Equation 2, the value ofk 0 is determined as follows:k 0 + (4.92 M -2 s -1 )(0.2 M) = 1.9026 M -1 s -1k 0 = 0.92 M -1 s -1Figure 6: Sample set C results show k exp as a function of HCl concentrationExperimental Rate LawThe rate law is obtained by combining the experimentalresults from sets A-C. This rate law for the oxidation ofmethylene blue by ascorbic acid under acidic conditions isshown in Equation 3.Equation 2: k 0 + k 1 (0.2 M) = 1.9026 M -1 s -1 d[MB + ]Equation 3: = -{0.92 + 4.92[HCl]}[H 2 A][MB + ] = -k exp [MB + ]dt

ConclusionThe analysis of methylene blue reduction by ascorbic acidis performed quickly and easily with the Evolution Arrayand VISIONcollect software. In this experiment, rate datawas obtained for the analysis and rate constants weresimultaneously calculated using the kinetic analysis functionof the VISIONcollect software. The export function of theVISIONcollect software facilitates further data processingin EXCEL ® . From this analysis it was determined that therate constant of the reaction is dependent on theconcentration of both ascorbic acid and HCl and not onthe initial concentration of methylene blue.References1. Mowry, S. and Orgen, P.J. (1999) Kinetics of Methylene Blue Reductionby Ascorbic Acid, J Chem. Ed. 76(7), 970-974.In addition to theseoffices, Thermo FisherScientific maintainsa network of represen -tative organizationsthroughout the world.Africa-Other+27 11 570 1840Australia+61 2 8844 9500Austria+43 1 333 50 34 0Belgium+32 53 73 42 41Canada+1 800 530 8447China+86 10 8419 3588Denmark+45 70 23 62 60Europe-Other+43 1 333 50 34 0Finland/Norway/Sweden+46 8 556 468 00France+33 1 60 92 48 00Germany+49 6103 408 1014India+91 22 6742 9434Italy+39 02 950 591Japan+81 45 453 9100Latin America+1 608 276 5659Middle East+43 1 333 50 34 0Netherlands+31 76 579 55 55South Africa+27 11 570 1840Spain+34 914 845 965Switzerland+41 61 716 77 00UK+44 1442 233555USA+1 800 532 4752www.thermo.com©2010 Thermo Fisher Scientific Inc. All rights reserved. Excel is a registered trademark of Microsoft Corporation. All other trademarks are the property of Thermo Fisher Scientific Inc. and itssubsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.Thermo Electron ScientificInstruments LLC, Madison, WIUSA is ISO Certified.AN51867_E 01/10MPart of Thermo Fisher Scientific