Application

Note: 51867

Analysis of Methylene Blue Reduction

by Ascorbic Acid
Nicole Kreuziger Keppy, Michael W. Allen, Ph.D., Thermo Fisher Scientific, Madison, WI, USA

Introduction
Key Words
Ascorbic Acid
Kinetics
Methylene Blue
Reaction Rate
Reaction
Mechanism

Chemical kinetics is the study of properties of chemical

processes such as reaction rates, mechanisms and transition
states. The mechanism and rate of chemical reactions can
be determined using UV-Visible spectrophotometry. In this
application note, the reaction of methylene blue with
ascorbic acid is monitored with a Thermo Scientific
Evolution Array UV-Visible spectrophotometer. The rate
constant is also determined using Thermo Scientific
VISIONcollect software.

Methylene blue (MB+) is a water-soluble dye molecule.

Under acidic conditions, it is easily reduced to the
colorless hydrogenated molecule leucomethylene blue
(LB+) by ascorbic acid (H2A) as shown in Figure 1. The
stoichiometry of the overall reaction is 1:1 and is
represented in the equation:
MB+ + H2A LB+ + D

UV-Visible
Spectroscopy

Background

Figure 1: Reaction Mechanism of methylene blue with ascorbic acid

The mechanism of a chemical reaction can be identified by

measuring a change in the overall reaction rate caused by
changes in the concentration of individual reactants. In the
simple chemical reaction:
A+BP

the reaction rate (V) is proportional to the concentration

of reactants, such that:
V = k [A][B]

In this rate expression, k is the rate constant, is the

order of the reaction with respect to A, and is the order
of the reaction with respect to B. Thus, the overall order
of the reaction is:
n=+

A rate constant (kexp) is determined experimentally by

measuring the change in concentration of methylene blue
at 665 nm. As a first approximation, the rate of
disappearance of MB+ can be expressed as in Equation 1
below. By strategically varying the individual reactants, a
reaction mechanism can be determined by calculating
constants k0 and k1 in Equation 1 using the experimental
results.1
Equation 1:

d[MB+]
dt

= -{k0 + k1[HCl]}[H2A][MB+]

= -kexp[MB+]

Experiment and Results

Stock 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 prepared
from these stock solutions according to the conditions
defined in Table 1. A blank was prepared for each sample
by mixing together all of the reagents in the corresponding
sample except methylene blue. All measurements were
performed immediately following the addition of methylene
blue to the sample due to the fast nature of the reaction.
Time-based Kinetics mode was programmed using the
parameters shown in Figure 2.

Sample #

Ascorbic
Acid (M)

HCl (M)

Methylene
Blue (M)

Total Run
Time (s)

Set A: Varying Methylene Blue Concentration [MB+]

A1
A2
A3
A4
A5

0.3
0.3
0.3
0.3
0.3

0.025
0.025
0.025
0.025
0.025

3.5 x 10-5
2.5 x 10-5
1.5 x 10-5
9.0 x 10-6
4.0 x 10-6

200
120
120
120
120

Figure 2: Method parameters for methylene blue reduction analysis

Set B: Varying Ascorbic Acid Concentration [H2A]

B1
B2
B3
B4
B5

0.2
0.2
0.2
0.2
0.2

0.000
0.004
0.008
0.016
0.036

1.5 x 10-5
1.5 x 10-5
1.5 x 10-5
1.5 x 10-5
1.5 x 10-5

600
600
360
180
120

Set C: Varying Hydrochloric Acid Concentration [HCl]

C1
C2

0
0.1

0.045
0.045

1.5 x 10-5
1.5 x 10-5

C3
C4
C5

0.2
0.4
0.5

0.045
0.045
0.045

1.5 x 10-5
1.5 x 10-5
1.5 x 10-5

150
120
120
120
120

Table 1: Sample preparation and experimental conditions for the analysis of

methylene blue reduction
Figure 3: Absorbance decay of methylene blue under condition A1

Sample #

Start Time (s)

End Time (s)

Kexp

A1
A2
A3
A4
A5

0
0
0
0
0

B1
B2
B3
B4
B5
C1
C2
C3
C4
C5

0
0
0
0
0
0
0
0
0
0

80
80
80
80
80
Average
600
450
266
150
70
150
120
90
46
36

0.0616
0.0645
0.0607
0.0631
0.0619
0.0624
0.0000
0.0097
0.0172
0.0333
0.0693
0.0179
0.0454
0.0645
0.1076
0.1310

Table 2: Rate calculation range and experimental rate for each

experimental condition

The Relationship Between Rate Constant and Methylene

Blue 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 first
order fit of the absorbance between 0 and 80 seconds
was used to determine an experimental rate constant (kexp)
of 0.0616. The data collected for the remaining samples
(A2-C5) shows a similar pattern and their rate constants
are shown in Table 2.

One benefit of the Evolution Array is its ability to

collect the entire spectrum during kinetics experiments.
The full spectrum of methylene blue can be observed by
clicking the full spectrum icon in Time Based Kinetics Mode
in the VISIONcollect software. The individual spectra
for sample A1 are shown in Figure 4. These spectra were
also used to construct the decay curve shown in Figure 3.

Figure 5: Sample set B results show kexp as a function of ascorbic acid

concentration

The Relationship Between Rate Constant and HCl

Concentration, [HCl] (Set C):

Figure 4: Spectra of methylene blue reduction under condition A1

In the final set of solutions, the concentration of HCl was

varied according to Set C, Table 1. Plotting kexp against
HCl concentration illustrates the linear relationship of the
rate with respect to the concentration of HCl (Figure 6).
The resulting linear fit has a slope of 0.2212 M-1s-1 and a
R2 value of 0.9981. Taking the slope as the experimental
rate and substituting it into Equation 1 we find:
k1[H2A] = 0.2212 M-1s-1

As shown in Table 2, variations in kexp are small with

respect to methylene blue concentration. The average
value of kexp is approximately 0.0624. All of the measured
values fall within a small range of this average value and
do not exhibit a trend in variation as a result of a decrease
in concentration. Thus, we can conclude that the initial
concentration of methylene blue is not a significant factor
in the rate equation.

Entering the concentration of H2A (0.045 M), the

value for k1 is determined as follows:
k1 = 4.92 M-2s-1

Entering the value of k1 into Equation 2, the value of

k0 is determined as follows:
k0 + (4.92 M-2s-1)(0.2 M) = 1.9026 M-1s-1
k0 = 0.92 M-1s-1

The Relationship Between Rate Constant and Ascorbic

Acid Concentration, [H2A] (Set B):
To determine the affect of ascorbic acid on the overall
rate, the concentration of H2A was varied according to
the conditions defined in Set B, Table 1. A plot of kexp
against H2A concentration exhibits a linear relationship as
shown in Figure 5. A linear fit of the data results in a
slope of 1.9026 M-1s-1 with a R2 value of 0.9983. The
slope of this curve is the kexp and can be entered into
Equation 1:
Equation 1:

k0 + k1[HCl] = 1.9026 M-1s-1

The concentration of HCl in this sample set is held

constant at 0.2 M. Entering this value into the equation
above yields Equation 2:
Equation 2:

k0 + k1(0.2 M) = 1.9026 M-1s-1

Figure 6: Sample set C results show kexp as a function of HCl concentration

Experimental Rate Law

The rate law is obtained by combining the experimental
results from sets A-C. This rate law for the oxidation of
methylene blue by ascorbic acid under acidic conditions is
shown in Equation 3.
Equation 3:

d[MB+]
dt

= -{0.92 + 4.92[HCl]}[H2A][MB+] = -kexp[MB+]

Conclusion

References

In addition to these

The analysis of methylene blue reduction by ascorbic acid

is performed quickly and easily with the Evolution Array
and VISIONcollect software. In this experiment, rate data
was obtained for the analysis and rate constants were
simultaneously calculated using the kinetic analysis function
of the VISIONcollect software. The export function of the
VISIONcollect software facilitates further data processing
in EXCEL. From this analysis it was determined that the
rate constant of the reaction is dependent on the
concentration of both ascorbic acid and HCl and not on
the initial concentration of methylene blue.

1. Mowry, S. and Orgen, P.J. (1999) Kinetics of Methylene Blue Reduction

by Ascorbic Acid, J Chem. Ed. 76(7), 970-974.

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