In the Laboratory

Methylene Blue–Ascorbic Acid

An Undergraduate Experiment in Kinetics
T. Snehalatha, K. C. Rajanna, and P. K. Saiprakash
Department of Chemistry, Osmania University, Hyderabad – 500 007, India

Advanced concepts can be explained easily with Table 1. Effect of Variation of Ascorbic Acid Concentrationa
simple classroom experiments such as clock reactions (1– 102 [ascorbic acid] Reaction time, t v r = 103/t
8). A few clock reactions were earlier designed and re- Medium
(mol L{1) (s) (s{1)
ported by us for this purpose (6, 7). We have demon-
H2SO4b 1.0 75 13.3
strated the clock reaction involving methylene blue (MB,
a common dye) and L-ascorbic acid (vitamin C, a biologi- 2.0 38 26.3
cally important compound) to undergraduate students 3.0 26 38.5
who have basic knowledge in kinetics. The change in MB 4.0 20 50.0
color is quite visible and clear and is also very much ap-
preciated owing to its simplicity. Such clock reaction 5.0 18 55.6
studies have been undertaken with persulfate–iodide (8) HCl c 1.0 80 12.5
and bromination of phenols (5) and anilines (7), where 2.0 41 24.4
concepts such as effect of ionic strength, dielectric con-
stant, and substituent effects on reaction rate have been 3.0 30 33.3
successfully introduced and explained. The present clock 4.0 22 45.4
reaction kinetic study is entirely different from the fa- 5.0 19 52.6
mous “Blue Bottle” experiment reported by Campbell (9).
a
[H ] = 0.6 mol L ; [MB] = 2.0 × 10 mol L ; temperature = 301 K.
+ {1 {5 {1

b
Experimental Reagents and General Procedure Figure 1(A).
c
Figure 2(A).
All chemicals used were analytical grade B. D. H.
Emerck samples. Stock solutions of decimolar ascorbic
acid, micromolar MB, and sulfuric and hydrochloric acid
(each 2.0 mol L{1) were prepared afresh and standard-
ized according to published procedures. A high-precision
stopwatch was used to monitor the time.
Method of Following the Reaction
Two sets of solutions, A and B, were kept ready for
each experiment. Solution set A consisted of MB and sul-
furic acid. Water was added to make a total volume (af-
ter addition of B) of 50 mL. Solution B was ascorbic acid.
Both solutions were thermostated at the desired tem-
perature and clamped onto a white porcelain tile to make
the color change clearly visible. Solution B was added
to solution A and the stopwatch was started midway
during the addition. Uniformity was ensured by a brief
stirring. The reaction time for the blue color of methyl-
ene blue to change to “colorless” was recorded.

Individual Experiments
Determination of Order of Reaction
Into five 150-mL conical flasks were placed con-
stant volumes of MB (2.0 × 10{5 mol L{1) and sulfuric
acid (0.6 mol L{1) (solution A). The required amount
of water was added. Ascorbic acid ranging from 0.01
to 0.05 mol L{1 (solution B) was placed in a test tube.
The temperatures of both solutions were thermostati-
cally controlled at room temperature. Solution B was
added to solution A and the stopwatch was started.
When the solution became colorless the reaction time
(t) was noted. The results are compiled in Table 1. Plots
of log rate or log(v r ) or log(1/t) vs. log[ascorbic acid]
were drawn, which gave a straight line with a slope Figure 1. Methylene blue–ascorbic acid reaction in H2SO4. (A) Plot
equal to unity (Fig. 1, line A). This experiment was re- of 3 + log v r vs. 2 + log [ascorbic acid]. (B) Plot of 7 + log v r vs. 6 +
peated with hydrochloric acid, and in each case the log [MB]. (C) Plot of 3 + log vr vs. 1 + log [H+]. (D) Plot of 3 + log vr
reaction order with respect to ascorbic acid was found vs. 103 /D. (E) Plot of 3 + log vr vs. 102 nx. (F) Plot of 3 + log vr vs.
to be very close to 1 (Fig. 2, line A). (D – 1)/(2D + 1).

228 Journal of Chemical Education • Vol. 74 No. 2 February 1997

 In the Laboratory

Similar experiments were performed keeping the Determination of Free Radicals

concentrations of ascorbic acid (0.03 mol L{1) and sul- Addition of acrylamide to the reaction mixture re-
furic acid (0.6 mole L{1) constant while varying the con- sulted in formation of polyacrylamide, thus showing the
centration of MB from 4.0 × 10 {6 to 2.0 × 10 {5 mol L{1. presence of free-radical intermediates.
The time (t) required for the blue MB to become decol-
orized was recorded for the two acids. The rate v = Effect of Acid
[MB]/(reaction time) was calculated and the plot of log(v) Keeping the concentration of ascorbic acid (0.025
vs. log[MB] was drawn. The slope of the plot (order) was mol L{1) and MB (2.5 × 10{5 mol L {1) constant, the ef-
found to be less than unity in both acid media (Table 2; fect of [acid] was studied by changing the concentra-
line B, Figs. 1 and 2). tion of acid from 0.15 to 1.0 mol L{1. It was observed
that reaction time decreased with increase in concen-
tration of acid. The slope of the plot of log(1/t) vs.
Table 2. Effect of Variation of Methylene Blue
log[H +] was found to be less than 1 for the two acids
Concentrationa
(Table 3; line C, Figs. 1 and 2).
106 [MB] Reaction time, t v r = 107 [MB]/t
Medium
(mol L{1) (s) (L mol{1 s{1) Effect of Salt
H2SO4 b 4.0 18 2.22 Keeping the concentrations of MB (2.5 × 10{5 mol
8.0 22 3.64 L{1), ascorbic acid (0.025 mol L {1), and sulfuric acid (0.5
mol L{1) constant, the effect of added salt was studied
12.0 25 4.80
by varying the concentration of potassium sulfate in the
16.0 28 5.71 range of 0.01 to 0.05 mol L{1. It was found that salt did
20.0 31 6.45 not affect the reaction time to any significant extent.
HCl c 4.0 21 1.90
Similar observations were made for potassium chloride
in hydrochloric acid.
8.0 26 3.08 Ionic strength (µ) variation generally brings about
12.0 31 3.87 two types of effects on chemical reaction rates, referred
16.0 35 4.57 to as primary and secondary salt effects (10).
20.0 41 4.88
Table 3. Effect of Variation of Acid Concentrationa
a
[H+] = 0.6 mol L{1; [ascorbic acid] = 3.0 × 10 {2 mol L{1; temperature = 301 K.
b
Figure 1(B). [Acid] Reaction time, t v r = 103/t
Medium
c
Figure 2(B). (mol L{1) (s) (s{1)
H2SO4 b 0.15 94 10.6
0.25 64 15.6
0.45 49 20.4
0.60 35 28.6
0.75 30 33.3
HCl c 0.15 105 9.5
0.25 87 11.5
0.45 56 17.8
0.60 47 21.3
a[MB] = 2.5 × 10 {5 mol L{1 ; [ascorbic acid] = 2.5 × 10{2 mol L{1; tem-
perature = 301 K.
bFigure 1(C).

c Figure 2(C).

Table 4. Effect of Solvent Acetone Concentrationa

Acetone Reaction time, t v r = 103/t
Medium
(vol %) (s) (s{1)
H2SO4b 5 50 20.0
10 81 12.3
15 120 8.3
20 155 6.45
25 210 4.76
HCl c 5 55 18.2
10 85 11.8
15 135 7.41
20 160 6.25
Figure 2. Methylene blue–ascorbic acid reaction in HCl. (A) Plot
of 3 + log v r vs. 2 + log [ascorbic acid]. (B) Plot of 7 + log v r vs. 6 + 25 220 4.54
log [MB]. (C) Plot of 3 + log v r vs. 1 + log [H+ ]. (D) Plot of 3 + log vr a
[MB] = 2.5 × 10{5 mol L{1; [ascorbic acid] = 0.025 mol L{1; [H+] = 0.5 mol L{1.
vs. 10 3/D. (E) Plot of 3 + log v r vs. 102 nx. (F) Plot of 3 + log vr vs. b
Figure 1(D,E,F).
c
(D – 1)/(2D + 1). Figure 2(D,E,F).

Vol. 74 No. 2 February 1997 • Journal of Chemical Education 229

In the Laboratory

The primary salt effect arises on the basis of the
different types of reactive species present in the slow
step. Reactions between two pairs of ions of like charge
are usually accelerated by increasing ionic strength
because of the favorable interactions of the activated
complex with the denser ionic environment. A reverse
trend is recorded in the case of reactions of oppositely
charged species. The rates of reactions between two
uncharged molecules or between an ion and a molecule
are usually only slightly affected by the addition of
salts. The secondary salt effect arises because of the
presence of acid–base equilibria prior to the rate-limit-
ing step.
When primary and secondary salt effects are opera-
tive in the same direction, either significant accelera-
tion or significant inhibition is observed. However, a neg-
ligible effect is recorded if primary and secondary salt
effects are operative in opposite directions.
Results of the present study appear to be in agree-
ment with a mechanism in which primary and second-
ary salt effects are operative in opposite directions. How-
ever, the participation of ion–dipole or dipole–dipole in
the slow step could not be ruled out.
Effect of Solvent
The effect of dielectric constant was studied by us-
ing a binary solvent mixture of water and acetone. So-
lution A consisted of MB (2.5 × 10 {5 mol L {1), sulfuric
acid or hydrochloric acid (0.5 mol L {1), acetone rang-
ing from 5 to 20%, and water added to make the total
volume of solution (including solution B) 40 mL. So-
lution B consisted of ascorbic acid (0.025 mol L{1). It
was found that as the concentration of acetone in-
creased the reaction rate decreased (Table 4; lines D–
F, Figs. 1 and 2) It is well known that an increase in
the organic component (acetone in this study) in a bi-
nary aquo-organic solvent mixture decreases the bulk
dielectric constant (D) of the medium. The kinetic re-
sults of this study indicate that the rates of the reac-
tion are faster in the solvent of low D. According to
Amis’ theory (11) of the effect of dielectric constant
(on reaction rates), such results lead to the formula-
tion of cation–dipole participation in the slow step.
Effect of Temperature
The reactions were studied at various temperatures
in the range of 300–325 K and the rate was found to in-
crease with temperature (Table 5).
Reactive Species and Mechanism
Ascorbic acid (H2A) in aqueous solution is known
to exist as protonated (H3A +) and dissociated forms
such as ascorbate monoanion (HA{ ) and ascorbate
dianion (A 2{) according to the following equilibria:
K1
H2 A HA{ + H+ (K1 = 5.01 × 10{5)

K2
HA{ A2{ + H + (K 2 = 1.51 × 10{12)

Since the acid concentration used in these studies is far

greater than the range of dissociation constants K1 and
K2 (or the experimental pH range is far less than ei-
ther pK1 or pK2), it is reasonable to consider that undis-
sociated H2A is the reactive species. In acid medium, MB
exists as I and II according to the following equilibrium
(12) (Fig. 5):
I II
First order dependence on [H2A] and fractional or-
der dependence on [MB] coupled with the results ob-
tained during studies of the effects of ionic strength, acid,
and solvents indicate that the most plausible mechanism
Figure 5. Structures of forms of methylene blue. In acid medium, involves a cation–dipole type of species (11) in the rate-
I and II exist in equilibrium. determining step (scheme I):
Scheme I
KD
Table 5. Effect of Temperaturea I II (1)
Temperature Reaction time, t v r = 103/t (D) (M)
Medium
(K) (s) (s{1) K1
H2SO4 303 47 21.3 H2 A + H + (H3A)+ (2)
308 30 33.3
313 22 45.4 (H 3A)+ + M →
k
III + HA ? + H+ (3)
slow
318 18 55.6
323 16 62.5 HA ? + M →
fast
III + A (4)
HCl 303 74 13.5
308 45 22.2 For the above mechanism the rate law could be derived
according to the following steps. From step 3:
313 33 30.3
318 22 45.4 – d MB +
323 18 55.6 = k M H 3A (5)
dt
[MB] = 2.0 × 10 mol L ; [ascorbic acid] = 0.02 mol L ; [H ] = 0.4 mol L .
a -5 {1 {1 + {1

230 Journal of Chemical Education • Vol. 74 No. 2 February 1997

 In the Laboratory

From equilibrium 1;

[M]
[M] = K D[D] or [D] =
KD

KD+ 1
[MB] T = [D] + [M] or [MB] T = [M] ⋅
KD

K D[MB] T (6)
[M] =
1 + KD

From equation 2 we can write

[H 2A] T = [H 2A] + [H 3A +]

[H 3A +]
and [H 2A] =
K 1[H +]

[H 3A +]
or [H 2A] T = [H 3A +] +
K 1[H +]

1 + K 1[H +]
or [H 2A] T = [H 3A +] ⋅
K 1[H +]

K 1[H +][H 2A] T

[H 3A +] = (7)
1 + K 1[H +]
+
Substitution of [H3A ] and [M] in equation 5 yields Figure 3. Rate constants and protonation constants of ascorbic
acid in H2 SO4 at various temperatures. (E) Plot of log k1 vs. 103/ T.
+ (F) Plot of K1 versus 10 3/T.
– d[MB] = v = k K DK 1[MB] T[H 2A] T[H ]
(8)
dt 1 + K 1[H +] 1 +K D

This equation explains the observed kinetics, thus keep-

ing consistent with the proposed mechanism. However,
the fractional order dependence on [MB] may be ex-
plained as due to the dimerization equilibrium of MB
species. The above rate equation may be simplified as

k K DK 1[H +] k 1K 1[H +]
k′′ = =
1 + K 1[H +] 1 + K D 1 + K 1[H +]

– d[MB]
where k′′ = dt
[H 2A] T[MB] T

KD
and k1 = k ⋅
1 +K D
Taking reciprocals, the above equation could be simpli-
fied as

1 = 1 + 1 (9)
k′′ k 1K 1[H +] k 1
The plots of 1/k99 vs. 1/[H+ ] should be linear with
positive slopes and intercepts for verification of the rate
equation. Such plots have been realized in this study,
showing the validity of the proposed mechanisms. From
the slope and intercepts, the rate constant (k1) and pro-
tonation constant of H2A (K 1) were determined at vari- Figure 4. Rate constants and protonation constants of H2A in HCl
ous temperatures. For H2SO4 (Fig. 3, lines A–D) and HCl at various temperatures. (D) Plot of log k 1 vs. 103/ T. (E) Plot of K1
(Fig. 4, lines A–C), corresponding thermodynamic param- vs. 103/ T.

Vol. 74 No. 2 February 1997 • Journal of Chemical Education 231

 In the Laboratory

Table 6. Thermodynamic and Activation Parameters For the above mechanism the rate law could be de-
Involving Rate Constant (k 1) and Protonation Constant (K 1) rived as
Temperature Medium
Parameter – d[MB] = v = k MH + H A
(K) 2 2 (13)
H2SO4 HCl dt
10–4 k 1 303 5.56a 5.13b From the equilibria of scheme 2
308 7.14c — +
[MB]T = [D] + [M] + [MH ] (14)
313 10.0d 9.5e
323 16.7f 15.4g Substituting for [D] = [M]/K D
∆H # (kJ mol{1) 43.4h 45.4i [M] +
[MB] T = + [M] + [MH ] (15)
∆G # (kJ mol{1) 46.7h 46.6i KD
{∆S # (J K{1 mol{1) 10.9h 3.99i
Rearranging the above equation in terms of [MH+]
10 K 1 303 3.89a 3.2b
4.64c [MH +] [MH +]
308 — [MB]T = [MH +] + + + (16)
313 5.0d 4.05e K 3[H ] K DK 3[H +]
323 6.18f 5.00g
∆H # (kJ mol{1) 18.4j 19.2k 1 + K D + K 3K D[H +]
[MB]T = [MH +] ⋅ (17)
∆G # (kJ mol{1) 8.18j 8.67k K DK 3[H +]
{∆S # (J K{1 mol{1) 33.7j 34.6k or
aFigure 3(A); b Figure 4(A); cFigure 3(B); dFigure 3(C); eFigure 4(B);
K DK 3[H +] [MB]T
f Figure 3(D); g Figure 4(C).
[MH +] = (18)
hFigure 3(E); iFigure 4(D). 1 + K D + K 3K D[H +]
jFigure 3(F); k Figure 4(E).

eters were computed and compiled in Table 6 (Figs. 3 Substituting for [MH+ ] in equation 13
line E and 4 line D). Similarly, thermodynamic param- +
eters involving the protonation constant K1 were com- – d[MB] k 2 K D K 3[MB] T [H 2 A][H ]
= + (19)
puted by utilizing Vant Hoff ’s relationships and the dt 1 + K D + K 3 K D[H ]
Gibbs–Helmholtz equation. The data are compiled in
Table 6 and in Figures 3 line F and 4 line E for H2SO4 This rate law is also in accordance with the observed
and HCl media. kinetic features—namely, first order dependence on
By and large, similar values of observed ∆G# in HCl [H2A] and fractional order in [H+]. However, the frac-
and H2SO4 support the contention that the same mecha- tional order kinetics in [MB]T could be explained as due
nism is operative in both media. This could be seen from to the presence of dimeric species of MB in solution.
the similar values of k and K in the two acids. Although Equation 19 could be written as
the difference in k values was small, it seemed to be sig-
nificant. The entropy of activation ({∆S#) in H2SO4 ap- k 2K DK 3[H +]
k′′ = (20)
peared to be greater than in HCl, suggesting a loosely 1 + K D + K 3K D[H +]
bound transition state in H2SO4 medium.
where
Alternative Mechanism
– d[MB]T
Another possible mechanistic route could be the re- k′′ = dt
action between the protonated form of methylene blue [H 2A] [MB]T
(MH+) and the undissociated form of ascorbic acid (H2A). Reciprocals of equation 20 give
Since [H+ ] >> [H2A], dissociation of H 2A into HA{ and
A2- could be suppressed and at the same time protona-
tion of MB could also be more likely. The mechanism 1 = 1 ⋅ 1 + 1 + 1 (21)
could be written as shown in scheme II. k′′ [H +] k 2K DK 3 k 2K 3 k2
Scheme II +
The plot of 1/k99 vs. 1/[H ] was found to be linear
KD with a positive slope, and thus in keeping with the pro-
I II (1) posed mechanism. From the intercept the specific rate
(D) (M) constant (k2) could be evaluated as in the case of scheme
K3
I. However, owing to the complexity of the terms present
M + H+ MH + (10) in the slope term of equation 2, K3 and K D could not be
evaluated.
k2
MH+ + H2A III + HA? + H + (11) Conclusions
slow

HA ? + MH+ → III + A + H+
With a simple clock reaction technique, the mecha-
(12) nism of a complex reaction can be explained. If stock so-
fast
lutions are supplied to students each type of experiment
takes no more than half an hour and the entire investi-

232 Journal of Chemical Education • Vol. 74 No. 2 February 1997

 In the Laboratory

gation can be completed in three practical sessions of
three hours each. Hence, this would be a good under-
graduate and postgraduate laboratory exercise to illus-
trate the basic concepts of chemical kinetics. The advan-
tage of clock reactions over other methods is that they
can be used as simple demonstration experiments in the
classroom, consuming only 3–4 min, by means of which
the concepts in kinetics are easily understood.
Safety Measures
The micromolar concentrations of methylene blue
and decimolar L-ascorbic acid are found to be very safe
when allowed to react in acid media below 353–363 K.
Mineral acids of the required concentration should be
supplied to students, and it is advisable to use gloves,
safety glasses, and propipettes.
Acknowledgments
One of the authors (T. S. L.) is thankful to U. G. C.,
New Delhi, for granting an F. I. P. fellowship, and to
Sarojini Naidu Vanitha Maha Vidyalaya for providing
facilities. The authors are thankful to T. Naveneeth Rao
(former Vice Chancellor, Osmania University) for keen
interest and constant encouragement.
Literature Cited
1. Barrett, R. L. J. Chem. Educ. 1955, 32, 78.
2. Russel, R. A.; Switzer, R. W. J. Chem. Educ. 1987, 64, 445.
3. Alyea, H. N. J. Chem. Educ. 1977, 54, 167.
4. Lambert, J. L; Fina, G. T. J. Chem. Educ. 1984, 61, 1037.
5. Clarke, J. R. J. Chem. Educ. 1970, 47, 775.
6. Snehalatha, T. Clock Reactions as Chemical Demonstrations for
Understanding the Concepts in Reaction Kinetics; Ph.D. Thesis,
Osmania University, Hyderabad, India, 1994.
7. Snehalatha, T.; Rajanna, K. C.; Sai Prakash, P. K. Chem. Educ.
New Delhi, 1995, 11, 51.
8. Levitt, B. P. Findlay’s Practical Physical Chemistry; Longman:
London, 1973; p 351.
9. Campbell, J. A. J. Chem. Educ. 1963, 11, 578.
10. Laidler, K. J. Chemical Kinetics, 3rd ed.; Harper & Row: New York,
1987.
11. Amis, E. A. Solvent Effects on Reaction Rates and Mechanisms;
Academy: London, 1966; p 43.
12. Blaedel, W. J.; Meloche, V. W. Elementary Quantitative Analysis,
Theory and Practice, 2nd ed.; Harper & Row: New York, 1963; pp
447, 833.

Vol. 74 No. 2 February 1997 • Journal of Chemical Education 233