A new approach for reduction of methylene green withascorbic acid by de-oxygenation through carbondioxide

doi:10.4236/ns.2011.37079

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Vol.3, No.7, 566-572 (2011) Natural Science

http://dx.doi.org/10.4236/ns.2011.37079

A new approach for reduction of methylene green with

ascorbic acid by de-oxygenation through carbon

dioxide

Rafia Azmat1*, Noshab Qamar2, Raheela Naz2

1Department of Chemistry, University of Karachi, Karachi, Pakistan; *Corresponding Author: [email protected]

2Department of Chemistry, Jinnah University for Women, Nazimabad Karachi, Pakistan.

Received 11 March 2011; revised 18 April 2011; accepted 25 April 2011.

ABSTRACT

Reduction kinetics of the methylene green (MG)

with ascorbic acid (AA) in acidic medium at



max

660 nm was monitored through visible spectro-

photomtry in absence and presence of sodium

carbonate. CO2 release through reaction of so-

dium carbonate and oxalic acid, created de-

oxygenated atmosphere for reduction of dye

which greatly boosted the reaction rate. Initially

slow reaction in presence of atmospheric oxy-

gen proceeded rapidly when sodium carbonate

was added. The reaction followed fractional or-

der kinetics with AA and zero order kinetics with

MG. The rate of reaction shows no linear de-

pendence on [H+] concentration as an acidic

medium. The rate of reaction is directly related

with the elevated concentration of salt, which

suggests that the two same charged species are

involved in the rate determining step. Secondary

reactions at elevated temperature showed com-

plex kinetics.

Keyw ords: Ascorbic Acid; Methylene Green;

Sodium Carbonate; Charged Species; Secondary

Reactions; Elevated Temperature

1. INTRODUCTION

Ascorbic acid (AA),a form of Vitamin C and water

soluble which comes primarily from fruit and vegetables

is an important micronutrient and plays many physio-

logical roles. It occurs as l-ascorbic acid (AA) and its

oxidized form, dehydro-l-ascorbic acid (DHAA), both of

which are biologically active. The formula of which is

C6H8O6, behaves as a vinylogous carboxylic acid, where

in the double bond (“vinyl”) transmits, electron pairs

between the hydroxyl and the carbonyl [1-2].

Cooper et al. [3] reported the photo electrochemical

analysis of ascorbic acid in aqueous solution at a plati-

num channel electrode, using the dissolved phenothiaz-

ine dyes methylene blue and methylene green. This is

achieved by measuring the current produced by immedi-

ate electro-oxidation of the reduced form of the dyes

produced during the 1:1 photoreaction between the dyes

and ascorbic acid induced by visible light of a wave-

length of 620 - 625 nm. Strizhak [4] propose a kinetic

scheme for reduction of methylene blue by ascorbic acid,

characterized by the existence of two pathways: outer-

sphere reduction of methylene blue according to a sec-

ond-order reaction, and reduction through formation of a

1:1 complex with ascorbic acid. Strizhak [5] reported the

reduction of methylene blue by ascorbic acid in the

presence of copper (II) ions. The introduction of copper

(II) ions increases the reaction rate, owing to the in-

creased concentration of ascorbic acid radicals in the

solution. It is shown that the inhibition of the reaction

that is observed with low concentrations of copper (II) is

a result of redox reactions proceeding in the system, in-

volving oxygen dissolved in the water. Yusuf and Giirel

[6] used methylene blue (MB), incorporated into tita-

nium phosphate for determination of AA and showed

that reaction can be helpful in some pharmaceutical

products and are for the detection of AA as the procedure

involving the reaction between triiodide and AA. AA has

been determined by photo bleaching of methylene blue

using continuous and FI systems, both procedures de-

termination of AA based on its photooxidation sensitized

by Toluidine Blue [7,8]. Cooper et al., [9] discussed the

photoelectrochemical analysis of AA and decolorizing

efficiency of AA to different synthetic dyes using Co/

H2O2 system [10].

Detail literature search showed that few reports were

available on the reduction of methlyene green with or-

ganic reductants but no reports were available on reduc-

tion of MG and AA in presence and absence of sodium

carbonate in acidic medium. This procedure is shown to

be a good alternative to routine vitamin C analysis in

pharmaceutical preparations, fruit juices and soft drinks.

R. Azmat et al. / Natural Science 3 (2011) 566-572

567

2. EXPERIMENTAL

2.1. Materials and Methods

All reagents of pure quality (E-Merk) were used

throughout the experiment. Dye stock solution of 3 ×

10–5 mol·dm–3 was prepared in 100 ml of deionized dis-

tilled water. Solution of dye was preserved in polyethylene

bottle. Stock solutions of ascorbic acid of 0.1 mol ·dm–3,

Oxalic acid of 0.5 mol·dm–3, Na2CO3 of 0.1 mol·dm–3,

KNO3 of 2 mol·dm–3, was prepared in distilled water

2.2. Kinetic Measurements

Five sets of mixture were prepared from stock solu-

tion. In each set one specie was varied while concentra-

tions of other four were kept fixed. Each component

were mixed together and portion was transferred to the

cuvette (1 cm) to record a change in optical density of

methylene green. Kinetic measurements were made with

visible spectrometer 180 A at λmax 660 nm of methylene

green at room temperature in thermostatic water bath

(FJPSO UK) [11,12].

2.3. Stoichiometry

The stoichiometry of the reaction mixture of MG and

AA were determined by mixing the reagents in molar

ratios of 2:1,1:1 and 1:2 with oxalic acid in excess.

Quick de-coloration was observed in the bottle contain

molar ratio in 1:2 as compared to other ratios showed

that MG react with AA in 1:2 ratios in aqueous medium

according to following equation [13].

N(C2H5)2

NO2

N(CH4)2

NNO2

N(CH3)2

(H3C)2N

+ 2AA+ DHAA

methlyene greenleucomethylene green

3. RESULTS AND DISCUSSION

In the present work reduction of the methylene green

with ascorbic acid were studied with variable concentra-

tions of dye, ascorbic acid, and oxalic acid keeping one

parameter varied and other constant in acidic medium in

absence and presence of sodium carbonate [11,12,14].

The values of rate constant were determined through a

plot of lnA vs time for each parameter concentration

from the slope of line and reported in the Tables 1, 3-7.

A plot of log [dye] and log k were evaluated for each

variable. It was observed that rate of reaction was very

slow and took long time for complete de-colorization of

the dye in absence of sodium carbonate according to

following Eqs.1-3.

AHAHH fast



 (1)



MG HMGH

slow in absence of sodium carbonate



 (2)

MGH eMGH





 (3)

The above reactions were significantly enhanced in

presence of sodium carbonate with reduced de-colo-

ration time. The total de-coloration time reduced to 5

min. while at the same pH value, but in the absence of

sodium carbonate the total de-coloration was achieved in

a time more than 60 min. Addition of sodium carbonate

in reaction mixture results in the brisk effervescence of

CO2 (confirmed by using lime water which turn milky)

with rapid de-coloration of dye indicating that CO2 at-

mosphere helps in fast bleaching of the MG. The change

in concentration of sodium carbonate proves that release

of CO2 in the reaction mixture affected the reduction

kinetics of MG (Table 1) and showed that CO2 displace

the oxygen due to which equilibrium shift in forward

direction with the formation of leuco dye [15]. The de-

cline in the time of de-coloration due to the innovation

of CO2 during reaction by the addition of sodium car-

bonate throughout course of reaction is also supported

by the early study of Gest and Stokes [16] who reported

that the time required for the reduction of methylene

blue was greatly increased by CO2 removal when no

substrates were added. Spectral changes for reduction of

dye were recorded and presented in Figure 1. clearly

showed that dye reduced without shift in wavelength in

absence and presence of sodium carbonate.

Chemical oxygen demand (COD) decreases sharply

with evolution of CO2 which greatly influences the re-

duction of dye by providing the oxygen free atmosphere

for reduction (Table 2). Reduced COD showed that CO2

atmosphere helps in the reduction of MG through the

movement of H+ ion in the reaction mixture [16]. The

R. Azmat et al. / Natural Science 3 (2011) 566-572

568

Table 1. Effect of Sodium Carbonate (Na2CO3) on Rate of

Reduction [MG +] = 1 × 10–5 mol·dm–3, [Ascorbic Acid] = 5 ×

10–3 mol·dm–3, [H2C2O4] = 0.18 mol·dm–3, [KNO3] = 0.2 mol·dm–3,

[Na2CO3] = (1 - 5) × 10–2 mol·dm–3, Temperature = 303 K.s.

Concentration

of Na2CO3 ×

102

mol· dm–3

Velocity

υ × 105

Specific rate

constant

(kobs) × 103 s–1

1/(kobs) %

decoloration

1.0 –10 .00 1.0 1000 55.0

2.0 –3.00 1.2 833.33 60.48

3.0 –3.00 1.2 833.33 73.134

4.0 –3.00 3.2 312.5 81.223

5.0 –3.00 2.9 344.828 90.415

Table 2. Chemical Oxygen Demand in presence of sodium

carbonate. [MG+] = 1 – 5 × 10–5 M Mixture, [MG+] = 1 × 10–5 M,

[Ascorbic Acid] = 6 – 9 × 10–3 M, [H2C2O4] = 0.06 – 0.3 M,

[KNO3] = 0.2 M, [Na2CO3] = 1 × 10–2 M, Temperature = 303 K.

[MG]  105

mol·dm–3 COD Ascorbic Acid × 103

mol·dm–3 COD

1.0 –307.2 5.0 585.6

2.0 –208 6.0 572.8

3.0 –195.2 7.0 572.8

4.0 –188.8 8.0 566.4

5.0 –256 9.0 579.2

Figure 1. Spectral scan of MG with ascorbic acid in presence

and absence of sodium carbonate. Where a = 5 × 10–3 mol·dm–3,

b = 6 × 10–3 mol·dm–3, c = 7 × 10–3 mol·dm–3, d = 8 ×

10–3 mol·dm–3, e = 9 × 10–3 mol·dm–3.

effect of change in concentration of oxalic acid on rate

of reaction showed no a linear dependence on [H+]. This

may be attributed with that the varied concentration of

oxalic acid as a medium, showed no effect on rate of

reduction (Table 3). This indicated that oxalic acid is

involved in the release of CO2 with sodium carbonate as

varied concentration of sodium carbonate decrease the

reduction time according to following equations no. (4 -

7).

2242 322422

HCONaCONaCO COHO

(4)

2H/2e

MG AHMGHAH





 

fast (CO2 atmosphere) (5)

MG AHMGHA



 fast (6)

MGHeMGHA







 fast (7)

The dye and ascorbic acid concentration were varied

to analyze the effect of reduction of MG on rate of re-

duction. Results showed that the reaction followed frac-

tional order kinetics with ascorbic acid and zero order

kinetics with methylene green (Tables 4 and 5). COD

increases with the increase in concentration of dye (Ta-

ble 2). The values of percent de-coloration decreases

with the increase in concentration of dye in reaction so-

lution (Table 4) [17-19]. Unexpectedly it was observed

that the rate constant of MG reduction appears to be the

same in concentration range of 2 × 10–5 to 5 × 10–5 mol·dm–3

also, when concentration of ascorbic acid was varied in

solution containing a fixed amount of the dye, the de-

-coloration percentage increases with the increase in the

concentration of dye. This reflects that the reaction de-

pends upon the concentration of ascorbic acid (Figure 2).

However, beyond certain limits the value of rate constant

decreases (Table 5). The hydrogen ion abstraction and

electron donation by ascorbic acid take place through

following reaction which leads to the formation of leuco

dye with dehydroxy –1- ascorbic acid (DHAA) (Eq.8 )

[1-3,19]..

Table 3. Effect of Oxalic Acid (H2C2O4) on Rate of Reduction

of MG With Ascorbic Acid. [MG+] = 1 × 10–5 mol·dm–3, [As-

corbic Acid] = 6 × 10–3 mol·dm–3, [H2C2O4] = (0.06 – 0.3) mol·dm–3,

[KNO3] = 0.2 mol·dm–3, [Na2CO3] = 1 × 10–2 mol·dm–3, Tem-

perature = 303 K.

Concentration

of H2C2O4

mol·dm–3

Velocity

υ × 105

Specific rate

constant

(k obs) × 103 s–1

1/(k obs) % decoloration

0.06 –3.0 1.3 767.23 77.241

0.12 –3.0 1.3 767.23 77.241

0.18 –3.0 1.3 767.23 77.241

0.24 –2.0 1.2 500.00 71.312

0.3 –3.0 1.3 767.23 77.241

Table 4. Effect of Methylene Green [MG+] on Rate of Reduc-

tion. [MG+] = (1 – 5) × 10–5 mol·dm–3, [Ascorbic Acid] = 6 ×

10–3 mol·dm–3, [H2C2O4] = 0.18 mol·dm–3 , [KNO3] = 0.2

mol·dm–3, [Na2CO3] = 1 × 10–2 mol·dm–3, Temperature = 303 K.

Concentration

of [MG+] × 105

mol·dm-3

Velocity

υ × 105

Specific rate

constant

(kobs) × 103 s-1

1/(kobs) % decoloration

1.0 –4.0 1.8 555.56 71.111

2.0 –4.0 1.0 1000 81.361

3.0 –5.0 1.0 1000 89.194

4.0 –5.0 1.0 1000 93.657

5.0 –7.0 1.0 1000 82.282

R. Azmat et al. / Natural Science 3 (2011) 566-572

569

Table 5. Effect of Ascorbic Acid on Rate of Reduction of MG.

[MG+] = 1 × 10–5 mol·dm–3, [Ascorbic Acid] = (6 – 9) ×

10–3 mol·dm–3, [H2C2O4] = 0.18 mol·dm–3, [KNO3] = 0.2

mol·dm–3, [Na2CO3] = 1 × 10–2 mol·dm–3, Temperature = 303

Concentration of

Ascorbic acid × 103

mol·dm–3

Velocity

υ× 105

Specific rate

constant

(k obs) × 103 s–1

1/(kobs) %

decoloration

5.0 –10.00 2.7 370.37 81.223

6.0 –4.00 1.8 555.56 71.111

7.0 –10.00 5.5 181.82 57.667

8.0 –20.00 14.3 69.93 67.025

9.0 –9.00 121.0 8.265 49.738

Figure 2. A plot of 0

ln t

AA A





 vs Time for reduction

of MG with variable concentration of AA. [MG+] = 1 × 10–5 M,

[Ascorbic Acid] = 5 – 9 × 10–3 M, [H2C2O4] = 0.18 M, [KNO3]

= 0.2 M, [Na2CO3] = 1 × 10–2 M, Temperature = 303 K.

HO OH

HO OO

HO OH

-H +

OOH

N(C2H5)2

NO2

N(CH4)2

NNO2

N(CH3)2

(H3C)2N

CO2

Atmosphere ___(8)

(MG) LMG DHAA

The general effect of an added electrolyte on the ob-

served rate constant of a reaction in solution is deter-

mined only by the ionic strength which showed that the

rate of reaction is directly related with the elevated con-

centration of salt, suggested that the two same charged

species are involved in the rate determining step [11,14].

The kinetic salt effect of added salt electrolyte was de-

termined through Debye-Huckel limiting law which

gives relationship between ionic strength and rate con-

stant. The equation may be given as

loglog1.02 AB

kk ZZ



 (9)

for the determination of reactive specie in the rate deter-

mining step. This equation shows that a plot of log k vs



should be linear, with the slope and intercept equal

to 1.02 ZAZB and log k0 respectively. The slope repre-

sents the product of charges on the species involved in

the rate timing stop. If the rate limiting step is between

the species of like charges, a positive slope is expected.

When the reaction is in between opposite charges, it re-

sults in a negative slope. In present work effect of salt

was studied at constant concentration of dye, reductant

and acid by varying concentration of salt which showed

that the rate constant increases with increase in an ionic

strength of the reaction (Table 6) indicating that the re-

action is in between ions of like charges in rate deter-

mining step. The plot of log k vs



is linear with

1.5012 slope [20]. The positive sign of the slope

R. Azmat et al. / Natural Science 3 (2011) 566-572

570

Table 6. Effect of Ionic Strength on Rate of Reduction of MG. [MG+] = 1 × 10–5 mol·dm–3, [Ascorbic Acid] = 6 × 10–3 mol·dm–3,

[H2C2O4] = 0.18 mol·dm–3, [KNO3] = (0.2 – 1) mol·dm–3, [Na2CO3] = 1 × 10–2 mol·dm–3, Temperature = 303 K.



mol·dm–3

Concentration of

KNO3

mol·dm–3

Velocity

υ × 105

Specific rate constant

(kobs) × 103 s-1 1/(kobs) % decoloration

0.8809 0.2 –4.0 1.8 555.56 71.111

0.9879 0.4 –3.0 1.1 909.091 77.931

1.0844 0.6 –3.0 1.3 767.23 83.448

1.1730 0.8 –10.0 3.4 294.118 87.805

1.2554 1.0 –20.0 5.5 181.818 81.034

indicate that the species involve in the rate determining

step has like charges. If ZA and ZB have the same sign,

then ZA Z

B is positive and the rate constant increases

with ionic strength as observed in the present investiga-

tion. The value of ZA and ZB was obtained from the slope

of the plot of logk vs



which is 1.50. The pseudo

first-order rate constant (k') with respect to dye concen-

tration was found to non significantly correlated (R2 =

0.5717) as a function of ionic strength. From the data in

Table [4] it can be observed that higher rate constants are

attained at higher ionic strengths. This can be related to

alterations of ionic atmosphere and changes in the

charge densities around anions involved in the rate-de-

termining step. The formation of a single highly charged

ionic complex from two less highly charged ions is fa-

vored by a high ionic strength because the new ion has a

denser ionic atmosphere [20].Using rate constant data at

different ionic strengths, a graph was obtained employ-

ing the Brønsted–Bjerrum equation, based on the limit-

ing law of Debye–Hückel (Figure 3).The rate constant

at infinite dilution (k0) is 4.2 M [the product of charges

ZA ZB is 1.5. Considering the extended law of De-

bye–Hückel (Figure 3), k0 is 4.2 mol·dm–3 ZAZB is 1.50.

This value is close to the expected value of 2 considering

the proposed mechanism, which involves a collision

between a monovalent and a biivalent cation in the

rate-determining step [20].

The effect of temperature on rate of reaction indicated

that elevation in temperature results in the complex ki-

netics which may be due to the some secondary reac-

tions operating in the reaction mixture at a given tem-

perature [19, 21-29]. It was observed from the Table 7

that there is no linear relation in between the temperature

elevation and rate constant which showed that tempera-

ture elevation cause secondary reactions in which dye

molecule or reductant may be break down due to which

black particles were appear in the reaction mixture. The

values of activation parameters like entropy of activation,

enthalpy of activation and energy of activation support

secondary reaction predominantly degradation of dye

molecule instead of reduction [14,23].

4. REACTION MECHANISM

Reaction mechanism of the reduction of MG proposed

in which role of CO2 was considered to displace the

oxygen and provide deoxygenated atmosphere to in-

crease the rate of reduction. Ascorbic acid existed in

reaction mixture as follows (equation) and reduction

take place by abstraction of H from AA (Eqs.10 and 11).

AHAH H

k







 (10)

MG HMGH







 (11)

MGH +2AHMGH+A



Rate law for abstraction of H to convert dye into col-

orless form is as follows



d[MG] MG AH







(12)







dMGMG H







(13)

Rate of formation of active species of reductant ac-

cording to Eq. 2.

Figure 3. A plot of logk vs



. [MG+] = 1 × 10–5 M, [Ascor-

bic Acid] = 6 × 10–3 M, [H2C2O4] = 0.18 M, [KNO3] = (0.2 – 1)

M, [Na2CO3] = 1 × 10–2 M, Temperature = 303 K.

R. Azmat et al. / Natural Science 3 (2011) 566-572

571

Table 7. Effect of Temperature on Rate of Reduction. [MG+] =

1 × 10–5 M, [Ascorbic Acid] = 5 × 10–3 M, [H2C2O4] = 0.18 M,

[KNO3] = 0.2 M, [Na2CO3] = 1 × 10–2 M, Temperature = 303 –

323 K.

Temperature

(K)

Velocity

υ × 10–5

Specific rate

constant (kobs)

× 103 s–1

1/(kobs) %

decoloration

303 –3.00 1.6 625 65.87

308 –3.00 1.5 666.667 66.25

313 –8.00 3.3 303.03 88.22

318 –4.00 3.2 312.5

323 –5.00 2.5 400



mol· dm–3 ∆E≠

kj·mol–1

∆H≠

kj·mol–1

∆S≠

kj·mol–1

∆G≠

kj·mol–1

0.8809 28.337 258.765 380.392 143.506







dAHAH

 (14)

At equilibrium rate of formation of H = rate of reduc-

tion of dye

Hence









122

AHMG Hkk





(15)







MG H





 (16)

Taking k1/k2 = K







MG H





 (17)







 (18)

According to Eqs.1 and 2 reaction is in depended

upon H ion concentration







dMG











dMG

MG H













(19)







dMG



 (20)

d[M G]

[AH ]



 (21)

The above Eq.21 corresponds to our results that re-

duction is in depended upon concentration of dye and

fractional order with respect to ascorbic acid Tables 4

and 5.

5. CONCLUSIONS

It was concluded from above investigation that dehy-

drogenation of ascorbic acid with methylene green in the

presence of sodium carbonate decolorizes the dye more

rapidly in acidic medium which could be more eco-

nomical technique in future and has a significant advan-

tage over other studies reported in the chemical literature

for the dehydrogenation of ascorbic acid or in other

words it may be used for the detection of AA in different

fruit samples in very diminutive quantities.

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