Chinese Journal of Chemistry, 2009, 27, 1232—1236 Full Paper

* E-mail: fahim_uddin01@yahoo.com Received July 21, 2008; revised November 20, 2008; accepted December 10, 2008.

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Reduction Kinetics of Thionine in Aerobic Condition with D-Galactose

AHMED, Khalida UDDIN, Fahim*,a AZMAT, Rafiab a Department of Chemistry, University of Karachi, Karachi 75270, Pakistan

b Department of Chemistry, Jinnah University for Women, 5C Nazimabad, 74600 Karachi, Pakistan

Reduction kinetics of thionine (Th) with D-galactose (RH) was observed on a UV/Visible 1601 Shimadzu spec-trophotometer at λmax 599 nm. The results showed that the initially slow reduction kinetics got enhanced and pro-ceeded to completion within a few minutes. A pseudo first order kinetics was observed when influence of different parameters like concentration of dye and reductant, ionic strength and temperature was investigated. A significant shift in wave length from 599 to 517 nm was observed at alkaline pH whereas addition of a small amount of acid caused a shift in equilibrium. This resulted in the generation of oxidized form of thionine which was pragmatic in the presence of atmospheric oxygen. Change in ionic strength at elevated temperature lied to decrease in the rate constant. Thermodynamics activation parameters like Ea reflects a high amount of energy required for reduction of Th with RH whereas entropy of activation (∆S�) and free energy of activation (∆G�) show the highly solvated states of transient complex which was less disorderly arranged than the oxidized form of dye. A mechanism consis-tent with above findings has been discussed in the relevant section of paper.

Keywords thionine, galactose, shift in wave length, entropy of activation, free energy of activation

Introduction The redox reaction of thiazine dyes often occurs on a

time scale of a few seconds to minutes. They may be followed usually for quantitative interpretation and spectrophotometrically for quantitative determination.1 These dyes commonly used as an indicator and the color change results from the reversible oxidation reduction reaction of the methylene blue (MB) indicator. In alka-line solutions, glucose is oxidized to D-gluconic acid by following reaction (Scheme 1).2,3

Scheme 1

The reaction was first order with respect to the con-centration of MB and the observed rate constant in-creased with glucose (GH) concentration in a saturated mode.4 Thionine and methylene blue exist as monova-

lent cations in the ordinary pH region and their redox reactions are reversible. Since leuco methylene blue (MBH) and leuco thionine (LTh) are very unstable and easily oxidized by coexisting oxygen in solution, few studies on the reaction of these compounds with elec-tron acceptors have been reported before.5 Photo bleaching of methylene blue with two aldohexoses i.e. galactose and D-mannose in alcoholic aqueous medium showed that the reduction was effected by change in concentration of reductant, temperature and acidity whereas dissolved oxygen role was significant when methylene green reacted with glucose in alkaline me-dium and regeneration of color was observed.6,7

Detailed literature survey revealed that reduction ki-netics of thionine with reducing sugars had not been studied yet. Therefore, this study has been focused on the kinetics of reduction of thionine with reducing sugar D-galactose as no associated work with kinetics of re-duction of thionine has been found yet. The redox reac-tion of thionine with galactose in alkaline medium has been monitored by recording the change in optical den-sity in the presence of atmospheric oxygen because the spectral changes were large and the reactions were able to be followed. Results were discussed in relation with the effects of concentrations of galactose, Th, NaOH, temperature and ionic strength of the medium. Activa-tion parameters have been computed and mechanism has been proposed based on above investigations.

Thionine Chin. J. Chem., 2009 Vol. 27 No. 7 1233

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Material and methods An aqueous methanolic solution of D-galactose, so-

dium hydroxide, thionine (E-Merck) was prepared by dissolving the known amounts of samples in doubly distilled conductivity water and methanol in molar ratio 1∶1. Potassium nitrate was used to maintain the ionic strength of the medium. Kinetics measurements were made by preparing three sets of reaction mixtures in which one species was varied while other two were kept constant at given concentrations. The three contents were mixed together and the progress of the (inlet) reac-tion was monitored by recording the change in optical density at λmax＝599 nm during the reaction on a UV-Visible spectrophotometer (Shimadzu 1601). The order of reaction at different operational parameters and activation parameters were evaluated by measuring the specific reaction rate at various temperatures and dif-ferent ionic strengths.7 Percentage decrease in absorb-ance was calculated by using % of decrease in absorp-tion＝[(Af－Ai)/Af]×100, where Ai and Af represent initial and final absorption, respectively. The UV/ Visi-ble spectrophotometer (Shimadzu 1601) was used throughout the experiments.

Results and discussion

Kinetic investigation was perused by following the change in optical density of thionine at λmax 599 nm and the results were reported in Table 1, showing that reduc-tion of thionine with D-galactose followed pseudo first order kinetics with respect to dye, reductant and alkali. The redox reaction of dye was investigated under aero-bic condition and it was found that initially the reduc-tion proceeded very slowly, then sharply decreased and finally proceeded slowly. It was noted that the absorp-

tion value of dye became less with irradiation time, thus indicating the decoloration of dye solution.

Effect of dye, reductant and pH of the medium

Kinetics of reduction of thionine was followed by the change in dye concentration to check the effect on decoloration of the dye solution. It was observed that rate of reduction decreased with the increase in concen-tration of thionine (Table 1) while keeping other pa-rameters constant at fixed concentrations in these stud-ies. An inverse relation between the dye concentration and rate constant (k) (Table 1) showed that a less num-ber of protons available for the absorption to dye molecule resulted in the inhibition of rate of reduction (Table 1).8-11 A plot of optical density vs. time showed change in the absorption values, monitored at regular intervals of times and the representative plots were shown in Figure 1. Table 1 showed the maximum per- centage decrease in absorption at the maximum concen-tration of reductant, whereas very less absorption of radiation at high concentration of the dye was observed.

To optimize the decoloration kinetics, a systematic study was carried out by varying the concentration of D-galactose (Figure 2 ). It can be seen that the apparent rate of dye decoloration was directly proportional to galactose concentration; however, at high concentration of reductant the increase in dye decoloration was not linear (Table 1).12-15 This may be due to that at high con-centration the solution undergoes self quenching of OH－ radicals through added alkali and mixed organic solvent system

OH－

＋2OH－ → HO2＋H2O (1)

OH－

＋dye → P (2)

Table 1 First order kinetics of thionine at different operational parametersa

[Th＋]/(mol•dm－3) [NaOH]/(mol•dm－3) [Galactose]/(mol•dm－3) [dx/dt]/(mol•dm－3•s－1) k/s－1 Absorption/%

5.53×10－5 1.27 1.71×10－2 －14×10－4 1.42×10－2 62

5.53×10－5 1.27 1.57×10－2 －12×10－4 1.24×10－2 58

5.53×10－5 1.27 1.49×10－2 －10×10－4 1.22×10－2 50

5.53×10－5 1.27 1.35×10－2 －9×10－4 1.05×10－2 49

5.53×10－5 1.27 1.28×10－2 －7×10－4 0.68×10－2 42

5.53×10－5 1.46 1.42×10－2 －9×10－4 1.4×10－2 76

5.53×10－5 1.39 1.42×10－2 －7×10－4 0.97×10－2 72

5.53×10－5 1.33 1.42×10－2 －6×10－4 0.91×10－2 68

5.53×10－5 1.20 1.42×10－2 －5×10－4 0.87×10－2 61

5.53×10－5 1.14 1.42×10－2 －3×10－4 0.46×10－2 56

7.91×10－5 1.27 1.42×10－2 －4×10－4 0.62×10－2 35

7.12×10－5 1.27 1.42×10－2 －6×10－4 0.41×10－2 40

6.33×10－5 1.27 1.42×10－2 －8×10－4 0.36×10－2 43

5.53×10－5 1.27 1.42×10－2 －9×10－4 0.41×10－2 45

4.74×10－5 1.27 1.42×10－2 －5×10－4 0.23×10－2 48 a Solvent: 50% aqueous methanol, temperature: 30

℃.

1234 Chin. J. Chem., 2009, Vol. 27, No. 7 AHMED, UDDIN & AZMAT

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1 A plot of optical density vs. time of photoreduction of thionine with D-galactose.

Figure 2 First order kinetics of thionine decoloration with D-galactose in alkaline medium.

The effect of pH values by adding small amounts of acid and base showed that the reduction was more prominent in a higher pH range as shown in Figure 3 where it can be seen that the reduction proceeds more rapidly whereas addition of a small amount of acid shifts the equilibrium towards left hand side and regen-eration of oxidized form of thionine was observed ac-cording to the following mechanism (Scheme 2).16-18

Scheme 2

Spectral change for reduction of thionine with ga-lactose shown in Figure 3 showed a shift in wavelength from 599 to 517 nm by the addition of alkali in the reac-tion mixture whereas the sharp peak showed the oxi-dized form of thionine and the broader peak resulted in the fast reduction of thionine in alkaline medium. It was also shown that the photolytic dye decoloration ap-peared larger at alkaline pH and less at acidic pH. This

Figure 3 Spectral change for photodecoloration of Th with D-galactose in low and high pH ranges.

enhancement in dye decoloration in alkaline condition is most likely due to the fact that at alkaline pH, peroxide anions (HO2) are produced in solution by UV/Visible radiation, which in turn can generate more OH－ radicals according to following mechanism.19-21

HO2＋hv→OH－

＋O－ (3)

Effect of change in ionic strength

Ionic strength of the medium was changed by the addition of KNO3 to check whether a charged species is involved in rate determining step or not, because the rate of reaction between two uncharged molecules or between an ion and a molecule is usually only slightly affected by the addition of salt as shown in Table 2 which indicates that primary and secondary salt effects are operative in opposite directions.

Table 2 Effect of ionic strength on dye reductiona

µ /(mol•dm－3) k/s－1 ln(k/s－1) (1/k)/s

5.00×10－2 6.5×10－3 －5.04 153.85

8.75×10－2 5.8×10－3 －5.15 172.41

12.50×10－2 7.4×10－3 －4.91 135.13

16.25×10－2 6.9×10－3 －4.98 144.93

20.00×10－2 6.4×10－3 －5.05 156.25 a 30 ℃, [Thionine]＝6.33×10－5 mol•dm－3, [D-galactose]＝1.42×10－2 mol•dm－3, [NaOH]＝1.27 mol•dm－3.

The reaction was followed by experiments with varying initial concentration of KNO3, at constant con-centrations of thionine and galactose at different tem-peratures (Table 3). The ionic strength was varied from 0.05 to 0.2 mol•dm－3 as tabulated in Table 2. Since ionic strength influences the reaction rate between charged species, rate of bleaching increases with time by varying concentration of KNO3, which may be at-tributed to that the dye reduction is more favorable in the presence of 3NO－ . Table 3 showed that the apparent rate constant of dye decoloration increasd at different ionic strengths at a temperature from 20 to 40 ℃, but higher temperature and increased ionic strength resulted in the decrease of rate constant. This may be due to self

Thionine Chin. J. Chem., 2009 Vol. 27 No. 7 1235

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

quenching of OH radicals by added 3NO－ .

Table 3 Effect of temperature on dye reduction at different ionic strengths

Ionic strength k×102/s－1

µ /(mol•dm－3) 30 ℃ 35 ℃ 40 ℃ 45 ℃ 50 ℃

5.00×10－2 1.49 1.86 3.3 2.55 2.74

8.75×10－2 1.33 2.03 4.95 4.72 2.5

12.50×10－2 1.7 2.56 3.1 4.76 1.73

16.25×10－2 1.59 3.38 3.83 5.11 3.24

20.00×10－2 1.48 4.12 4.31 5.91 3.98

[Thionine]＝6.33×10－5 mol•dm－3, [D-galactose]＝1.42×10－2 mol•dm－3, [NaOH]＝1.27 mol•dm－3.

Effect of temperature

Effect of temperature on dye reduction has been shown in Figure 4 and the results are reported in Tables 3 and 4. Thermodynamics activation parameters like Ea (40.29 kJ•mol－1) support the initially slow reaction process (Figures 1 & 2) as compared to that the litera-ture value 15.78,4 5.7322 kJ•mol－1 reported the rapid decoloration of thiazine dye. The value of entropy of activation (∆S�) for the reduction of thionine with D-galactose (－26.288 J•K－1•mol－1) showed a highly solvated state of activated complex. The negative value of ΔS� indicates a decrease in the degree of freedom due to the formation of a rigid activation complex resulting in an extensive reorientation of solvent molecules or that the solvent molecules are highly held to the OH－

bond which is the site of oxidation. The fairly high val-ues of enthalpy or activation ΔH�, free energy of activa-tion ΔG� and energy of activation (Ea) indicate that the transition state is highly saturated. It also reveals that the rate determining state is less disorderly oriented relative to the reactants. The positive value of ΔH� (56.17 kJ•mol－1) showed that enthalpy was not the driv-ing force for the formation of complex and entropy was responsible for the formation of the complex that in-volves one charged species and solvent molecules for the reduction of dye.

The present value of ΔS� (－26.288 J•K－1•mol－1) is comparable with the literature values of 39.24, 33.7 and 34.6 J•K－1•mol－1.7 These values integrated that the same but slow rate of reduction was operating during the reduction of thionine with galactose as observed by earlier researchers.1-11 The values of other parameters like ΔH� and ΔG� also support the initially slow reac-tion and then immediate bleaching of the dye. The value of ΔG� (62.59 kJ/mol) is compared with the literature 8.18 kJ/mol22 and ΔH� 56.75 kJ/mol with 15.565 and 18.422 kJ/mol.

Mechanism of reduction

Reaction mechanism involves the interaction be-tween the protonated form of thionine and D-glactose molecule.14,22 Since [OH－] is plentiful (6) oxidation of

Figure 4 Effect of temperature on photodecoloration of thion-ine with D-galactose.

Table 4 Thermodynamic activation parameters of reduction of thionine with galactose

Temperature Ea/

(kJ•mol－1) ∆H�/

(kJ•mol－1) ∆G�/

(kJ•mol－1) ∆S�/

(J•K－1•mol－1)

303 K 40.29 56.17 62.59 －26.28

D-galactose (D) may take place at the same time. Pro-tonation of thionine may also take place and leuco thionine is formed.

[ ]1

1Th

D Th [D]

K K←⎯→ ＝ (4)

where K1 is the equilibrium constant between the two forms of thionine. Protonation of the dye may take place in solvent.

2Th H ThHK←⎯→＋ ＋＋ (5)

Protonated form of the thionine may interact with the reductant molecule to form leuco dye

3 22 3

[ThH ][R ]ThH RH ThH R

[ThH ][RH]K K←⎯→

－

＋ －

＋＋ ＋ 　 ＝ (6)

FastRH ThH Th R 2H⎯⎯⎯→＋ ＋

＋ ＋ ＋ (7)

The rate law for the raction

[ ]d Th[ThH ][RH]

dr k

t＋

－

＝ ＝ (8)

For the triplet transition state of the dye

T[Th] [D] [M] [Th]＝ ＋ ＋ (9)

T 1[Th] [M] [ThH ]K ＋＝ ＋ ＋ (10)

1236 Chin. J. Chem., 2009, Vol. 27, No. 7 AHMED, UDDIN & AZMAT

© 2009 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

or in the form of protonated state [ThH＋] of the dye

T1 3

[ThH ] [ThH ][Th] [Th]

[H ] [H ]k K K

＋ ＋

＋ ＋＝ ＋ ＋ (11)

or

1 1 3T

1 3

1 [H ][Th] [Th]

[H ]

K K K

K K

＋

＋

＋ ＋＝ ＋ (12)

Protonated form of dye will take

1 3 T

1 1 3

[H ][Th][ThH ]

1 [H ]

K K

K K K

＋

＋

＋＝

＋ ＋ (13)

the rate law for the above reaction after substitution of [ThH＋] in Eq. 8 takes

1 3 T

1 1 3

d[Th] [H ][Th] [RH]

d 1 [H ]

K kK

t K K K

＋

＋

－＝

＋ ＋ (14)

the rate law shows first order dependence on RH and fractional order with respect to [H＋], which also verified the first order kinetics. This shows the dependence on the concentration of galactose. The above equation can be written for the dimeric form of thionine in solution:

1 3

1 1 3

[H ]

1 [H ]

K kKk''

K K K

＋

＋＝

＋ ＋

where

T

T

d[Th]d

[RH][Th]tk''

－

＝

or inverse of the equation

1 3 3

1 1 1 1 1

[H ]k'' K kK kK k

⎛ ⎞⎜ ⎟⎝ ⎠

＋＝ × ＋ ＋

The plot 1/k" vs. 1/[H＋] was found to be linear with a positive slope having R2

＝0.6251 and value of 1/k＝3.03×10－3 s showing first order kinetics dependence on reductant concentration.22

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