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redox reactions of methylene blue: A pulse radiolysis study
0146-5724/89 $3.00+0.00 Copyright © 1989PergamonPress plc
Radiat. Phys. Chem. Vol. 34, No. 4, pp. 721-727, 1989 Int. J. Radiat. Appl. lnstrum., Part C Printed in Great Britain. All rights reserved
REDOX REACTIONS OF M E T H Y L E N E BLUE: A PULSE RADIOLYSIS S T U D Y KAMALKISHORE, S. N. GUHA, J. MAHADEVAN, P. N. MOORTHY and J. P. MITTAL Chemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India Abstract~ne-electron oxidation of methylene blue (MB + has been studied using specific oxidizing radicals such as Clf, l~rf, Iq3 and TI(II) in acidic and neutral aqueous solutions). The transient spectrum showed absorption maxima at 525 nm (E. = 40,000 dm3mol- t cm- t) and 360 nm (E = 9700 dm3mol- tcrn- t) in the acidic pH region. At neutral pH also the absorption maxima were at 525 and 360 nm but the extinction coefficients were lower by 30%. A pKo of ~4.3 was observed for the equilibrium MBH3+~MB2++H +. In the case of lq3 radical as the oxidant, the equilibrium: lq3+ MB + ~ N3 + MB2+ was observed for which an equilibrium constant of 120 was estimated from the experimental data. From this as well as from cyclic voltammetric experiments, the redox potential for the MB2+/MB+ couple was calculated as 1.25V vs NHE. The transient species produced by the reaction of OH radicals with methylene blue gave a very different spectrum with 2~, = 400 nm and a pKa of ~ 8.6, and hence it is inferred that OH radicals do not bring about one-electron oxidation of the molecule.
INTRODUCTION
photolysis study of this compound (Kamat and Lichtin, 1981) semioxidized methylene blue was suggested to be formed by quenching of triplet methylene blue by the ground state molecule. Keene et al. (1965) have investigated the reactions of OH radicals with MB + and reported that the semioxidized species has an absorption band with 2m at 520 nm. However OH is not a specific oxidizing radical as it can react through other modes also such as addition and abstraction reactions. Also, under the conditions employed by Keene et al. both OH radical and eaqwould react with MB + and hence the results could not be quite conclusive. We have studied the one-electron oxidation of MB + by specific oxidizing radicals such as C1z, l~r2, lq3, TI(II) and compared the spectra and other characteristics of the semioxidized species with those of the transient produced by OH radical reaction with MB +. We have also determined the one electron reduction potentials for oxidation and reduction of MB +. The results of these investigations are reported here.
Phenothiazine compounds find applications in diverse areas wherein their redox chemistry plays a key role. Thus, for example, the thiazine dye thionine, is a well known sensitizer used in photogalvanic cells for the conversion of light to electrical energy. The key process involved here is the reduction of photoproduced triplet thionine by a reductant to form semithionine which then undergoes rapid disproportionation to the fully reduced form, leucothionine. Discharge of leucothionine at the anode and of the concomitantly formed oxidized form of the reducant at the cathode generates an electric current in the external circuit (Kamat et al., 1979). On the other hand phenothiazine drugs such as chlorpromazine, promethazine, trimeprazine etc. have shown considerable promise as effective radiation sensitizers in the treatment of cancer (Bahnemann et al., 1981). Here too, the key intermediate is believed to be a semioxidized species formed by reaction of the drug with OH radicals resulting from the interaction of ionizing radiation with the major component viz. water in the living cell. In order to gain further insight into the EXPERIMENTAL nature of the semireduced and oxidized species from Methylene blue used in the study was from E. phenothiazine compounds we have undertaken detailed investigation of the one electron redox pro- Merck and was purified by dissolving it in water and cesses involving these compounds in aqueous removing impurities by repeated solvent extraction solution by the technique of nanosecond pulse radiol- with chloroform. The aqueous solution was then ysis. The results of our investigations on thionine evaporated in vacuum oven and the residue obtained have been reported earlier (Guha e t al., 1987; Kishore was pure methylene blue. The extinction coefficient at et al., 1987). In the case of the related compound 660 nm (80,000 dma mol- ~crn- 1) agrees well with the methylene blue (MB*), there are reports of its one literature values (Bergmann and Konski, 1963). All electron reduction in the literature (Keene et al., other chemicals used were either BDH "AnalaR' 1965), but no further detailed studies have been grade or E. Merck "G.R." grade. Solutions were reported on its one electron oxidation. In a laser flash prepared in water obtained from a Barnstead 721
722
KAMALKISHOREet al. 0.20
0.16
0.12
0.08
6
0.04
0.00 --0.02
--0.06
--0.10 500
400
500
600
700
800
"~ (nm)
Fig. 1. Absorption spectra of the transients formed by the reaction of MB ÷ with CI~ (×--2ps, 0--6/is, A--18/~s after the e-pulse) and TI2÷ (0--20 ps after the e-pulse), pH = 1.7. "Nanopure" water system. Gases employed to saturate the solutions viz. N2, 02 and N20 were Iolar grade (Indian Oxygen Ltd). Phosphate and borate buffers as well as plain H2SO4 and NaOH were used for adjusting pHs in appropriate regions. A 7-MeV electron accelerator (Ray Technology, England), giving single pulses of 25 ns duration, was used for irradiating the samples, taken in 1 cm square "spectrosil" cuvettes. Pulse radiolysis set up employed has been described in detail earlier (Guha et al., 1987). Electron pulse dose was measured using aerated 0 . 0 5 m o l d m 3 KCNS solutions taking GE for (CNS)E species to be 21,522dm3mol-Jcm ~ per 100eV at 500nm. Average dose per pulse was 1.5 x 1017eV cm -3, but for estimating extinction coefficients and decay analysis a lower dose ( ~ 4 x 10L6eV) was employed. Spectral and kinetic data acquisition and analysis was done with an IBM-PC-XT using program "ABIWAT" (Panajkar et al., 1988). A Bruker E-310 modular polarograph with AC sweep module E-310/050 was used for carrying out cyclic voltametry at a glassy carbon electrode (Metrohm EA-276/2) with SCE as the reference and Pt-wire as the counter electrode. Electrolyte used was 0.2 mol dm -3 Na2SO 4 and the solutions were purged with N 2. MB ÷ concentration employed was 10-4moldm 3 and pH was adjusted to 6.8 using phosphate buffer. The glassy carbon electrode was cleaned after every run. RESULTS AND DISCUSSION
Methylene blue has no pKa between pH 0-14 and it is in the form MB + in this pH region (Keene et al., 1965). The oxidizing radicals used in the study viz. t~l~-, l~lr~-,lq 3 and TI(II) were produced by the reaction of OH radicals with the solutes CI-, Br-, N3,
and TI ÷ respectively at the appropriate pHs. These radicals react by electron transfer giving rise to a one-electron oxidized species according to: MB + + O x ~ M B 2+ + Red-;
(1)
where Ox is an oxidizing radical and Red- its reduced form. Reaction o f C I : with M B ÷
This reaction can be studied only at a pH below 3 because at higher pH the reactivity of CI- with OH is very low. We have studied its reaction with MB + at pH 1.7 by irradiating 02 saturated 10-4 mol dm -3 MB + solutions containing 10 -2 mol dm 3 C1 . The decay of CI{ was found to be faster in the presence of MB ÷ indicating reaction between the two. Time resolved spectra of the transient produced as a result of this reaction are shown in Fig. 1. In these solutions 02 has been used as a scavenger for H-atoms which would otherwise react with MB ÷ giving a reduced species. It was first established that HO2 radicals do not react with MB ÷ by irradiating 02 saturated methylene blue solution containing 0 . 1 m o l d m -3 tert-butanol as OH scavenger. In these solutions no transient absorption was observed in the wavelength region 300-850 nm. The time resolved spectra given in Fig. 1 show that while absorbance at 340 nm due to CI{ decreases with time, the absorbance at 525 nm due to the product species from MB ÷ increases and reaches a maximum after ~ 6 p s. The product transient species shows two absorption bands, one around 525nm (E = 4 0 , 0 0 0 d m 3 m o l - l c m -1) and a weaker one at ~360 nm (e = 9700 dm 3mol -~ cm-~). The traces for build up of absorbance at 520 nm and the growth of bleaching at 580 nm are shown in Fig. 2(a). From these, the rate constant for the formation of the transient by C1/- reaction was determined to be
Redox reactions of methylene blue
(a)
~ " - - -
--
~
520 nm
(i,)
723
500p=
OtltV
/ i o. v
5 2 0 nm
0 mV 580nm
5 8 0 am
Fig. 2. (a) Build up of transient absorption and dye bleaching in the CI~- reaction with MB + at pH 1.7. (b) Decay of transient absorption and recovery of dye bleaching in the same system at pH 1.7.
8 +__0.5 x 109 dm a mo1-1 s -1. Similarly, the traces for the decay of the absorption at 520 nm and the recovery of bleaching at 580nm are shown in Fig. 2(b). From these traces it was found that the transient species decayed by second order kinetics with a rate constant of 4 _+ 0.4 x 109dm 3 mol -t s -~. The extent of bleaching recovery is about 50% which indicates that the decay of the transient could be due to disproportionation.
Reaction of TI(II) with MB + TI(II) has two pKas at 4.6 and 7.7: OH-
TI2+ .°H-4.6TI(OH)+ ~ .
TI(OH)2.
(2)
We have studied the reaction of Tl(II) with MB + at pH 1.7 and 6.7. In the first case oxygen saturated solutions of 10-4mol dm -3 MB + containing 2 x 10 -3 mol dm -3 Tl + were subjected to electron pulse radiolysis. O2 was used to scavenge H-atoms, while OH reacted with Tl + to give Tl 2+. The transient spectrum obtained was very similar to the one obtained in the case of reaction with (~l2 and was recorded 20/~s after the electron pulse (the time taken to reach maximum signal height). It is shown in Fig. 1 along with the transient spectrum obtained by using CI~- as the oxidant. From this it is obvious that CI~and TI 2+ give rise to the same transient species. The rate constant for the reaction of T1=+ with MB + was determined from the formation trace obtained at 525 nm and was found to be 2.4 x 109 dm3 mol -~ s -~. The 2k/d value for the decay of the transient species was determined to be 1.05x105s -t ( 2 k = 4 . 2 x 109 dm 3 mo1-1 s-'). The reaction of TI(OH) + with MB + was studied at pH 6.7 using N20 saturated solutions of 10 -4 mol dm -3 MB + containing 2 x 10 -3 tool dm -3 TI +. In this case although the spectrum of the transient species (Fig. 3) was similar to the one obtained at lower pH (1.7), the extinction coefficient at 525nm was lower b y ~ 30% (28,000dm 3 tool -~ cm-1). This result suggests that the transient proRPC 34t4--T
duced by Tl(II) reaction has a pKa between pH 1.7 and 6.7. The rate constant for the reaction of TI(OH) + with MB + was determined by following the formation kinetics of the transient absorption at 525 nm and found to be the same as that for the reaction of TI2+ with MB + at the acidic pH. This indicates that the efficiencies of the reaction of TI(OH) + and Tl 2+ with MB + are comparable. The 2k/d value for the transient decay was estimated to be 3 . 8 x 1 0 4 S - 1 (2k = 1 x l09 dm 3 mol -l s -l) which is lower than the value in acidic solutions and again indicates the existence of a pK.
Reaction of f~r7 with MB + The reaction of ]~r~- radicals with MB + was studied at pH6.8 by pulse irradiating N 2 0 saturated 10 -4 mol dm -3 MB + solutions containing 0.02mol dm -a KBr. The spectrum of the transient species was identical to the one obtained in the case of TI(OH) + reaction with MB + at this pH and is shown in Fig. 3. The extinction coefficients at 525 nm in the two cases agreed with each other. From the formation traces at 525 nm, the rate constant for the reaction of l~r~- with MB + was determined to be 1.6 + 0.1 x 109dm3mol -l s - ' . 2 k / d for the second order decay of the transient was determined to be 2 x l(Ps -~ in presence of 0 . 0 2 m o l d m - a K B r ; and was found to increase with decreasing Br- concentration, being 1.6 x 104 s -l and 2.3 x 104 s -l respectively at 0.1 and 0.01 mol dm -3 Br- concentrations. Thus at very low concentration of KBr, 2kid value may approach that observed with TI(OH) + as the oxidant, viz. 3.8 × l(Ps -~, so that the decay rate constant can be inferred to be the same in the two systems. There was a small decrease in the yield of the transient with increasing concentration of Br-, e.g. in one experiment changing Br- concentration from 0.02 mol dm -3 to 0.1 mol dm -3, resulted in a decrease in the absorbance of the transient at 525 nm from 0.078 to 0.071. This indicates the existence of an
724
KAMALK]SHOREet al.
0.10 0.08, 0.08 o
0.06
-~v
0 04
f
0.04
0.02
I 0
0.02
I 4
I
I 8 I~H
I
I 12
0.0
--0.02
--0.04
--0.06
--0.081 300
I 400
I 500
I 600
I 700
I 800
(rim)
Fig. 3. Absorption spectra of the transients formed by the reaction of MB+ with TI(OH)+ (A) and ])r2(O) at pH 6.7 and 6.8 respectively (inset: plot ofA O.D. vs pH at 520 nm).
equilibrium reaction but the change was not large enough for evaluating the equilibrium constant. (212, l)r{, TI2÷ and TI(OH) ÷ are all specific oxidizing radicals and cause one electron oxidation of the solutes. The spectra of the transients obtained by the reaction of (::12, l~r~- and T12+ with MB + at pH 1.7 were identical. Similarly, the spectra of the transients obtained at neutral pH by the reaction of l~r2 and TI(OH) ÷ with MB ÷ were also identical. The decay rates of the transients produced by the reaction of different oxidizing radicals with MB + also agree with each other when compared at the same pH. Kamat and Lichtin (1981) have also reported ~'m for the semioxidized MB ÷ to be at 520 nm. All these results indicate that the transient species is the one-electron oxidation product of MB ÷. Thionine which has a very similar structure also reacts with these oxidizing species to give a transient spectrum with 2m at 480 nm (Kishore et al., 1987). In fact, in the case of many phenothiazines the one-electron oxidation products have 2m in the 500 nm region, indicating common site for oxidation which is possibly loss of electron from the heterocyclic thiazine ring. Thus MB ÷ on oxidation would give rise initially to MB 2+, which may exist in different conjugate acid-base forms depending on the pH. p K , o f the semioxidized methylene blue
Br2 radicals were employed over the entire pH range 1-14 to oxidize methylene blue to determine the p K a of the semioxidized species. The solutions were adjusted to the required pH and saturated with 02 so
that eaq and H-atoms are exclusively scavenged by 02, and the transient species is only due to the reaction of l~r2 . O.D. vs pH plot is shown in Fig. 3 (inset) from which pK, value of 4.3 was estimated. The nearest pKa of the ground state methylene blue is ~ 0 corresponding to the equilibrium: MBH 2+.
~0
•MB ++H+;
(3)
MB ÷ has no ionizable protons (see Scheme 1). Thus the pKa of 4.3 for the semioxidized species could be for the equilibrium: MBH3 +
4.3 MB2+ +H+"
(4)
It was also observed that the yield of the semioxidized methylene blue at pH 1.7 produced by the reactions of ~12, T12+ and t~r{ was the same under similar conditions indicating that the efficiencies of (~12, TF + and l~r~- for oxidation of MB + are the same. This was also borne out by the extent of bleaching in these cases. As the spectra of the transient species obtained through the reactions of I~lr; and TI(OH) ÷ with MB ÷ at pH 6.8 are identical at the 2m and also in the bleaching region, it is clear that the efficiency of TI(OH) ÷ is equal to that of flr~- and thus equal to that of TI2+ also.
(H3C)2 N ' ~ / S ~ N ( C H a ) :
Scheme 1
+
Redox reactions of methylene blue
5 5 1 2 5 l 2
× x x x x x x
10 -4 10 -3 10-2 10 -2 10 -2 l0 -* 10 -*
2k/d
Ep = E1/2 - 0.90(R T /nF)
(103s -I) 1.0 2.2 3.2 9.7 11.5 13.0 33.5
+ (RT/3nF) In(2kCoRT/3nF" v);
(6)
with 2k = 109dm3mol - I s -1, Co = 1 0 - 4 m o l d m -3 and v (sweep rate) = 1 V/s is 1.27 V vs N H E and is close to the E ° value determined earlier from the equilibrium experiments. Thus an average value of 1.25 ___0.02 vs N H E can be deduced for the reduction potential of MB2+/MB ÷ couple.
Reaction of N3 with M B ÷
Azide catalyzed decay of semioxidized methylene blue
lq 3 radicals were also found to react with MB ÷ at neutral pH to give a transient spectrum with 2m = 525 nm. However in this case, the yield of semioxidized methylene blue was strongly dependent on the concentration of N 3 . As the N3 concentration was changed from 10 -3 to 10 -2 mol dm -3, the transient absorbance at 525 n m (for the same dose) decreased by ~ 5 0 % , whereas the pseudo first order rate constant for the reaction of lq 3 with MB ÷ increased. The extent of bleaching was also found to be less at higher N3 concentrations indicating that the extent of electron transfer was less at higher ['N3 ]. All these results suggest the existence of an equilibrium reaction of the type: MB + + lq3 ~ - MB 2+ + N 3 .
E~/2 as calculated using
tO be 1.3V vs NHE, equation (6):
Table 1. Effect o f [N 3 ] on the decay rate constant of semioxidized methylene blue ([MB + ] = 5x 10-Smoldm 3;pH=6.8) IN3] ( m o l d m 3)
725
(5)
In such a case the observed rate of attainment of equilibrium is given by:
kob~ = kf[MB + ] + kr[N3 ] where kf and k b are respectively the rate constants for the forward and reverse reactions (Alfassi et al., 1987). Thus a plot of kobs/[N; ]vs [MB+]/[N;] would be a straight line with slope equal to kf and intercept equal to k,. The equilibrium constant (K = kf/kr) was thus determined to be ~ 120. K was also determined by plotting [MB 2+] x [ N ; ] vs [MB +] x [lq3]. The plot is shown in Fig. 5 and it gave a value of ~ 1 l0 for K. Assuming the redox potential for t h e / q / N ; couple to be 1.35 vs N H E (Alfassi et al., 1987), the value for the MB2+/MB + couple was calculated to be ~ 1.22 V vs N H E at neutral pH.
At neutral pH, when lq 3 radical was used for oxidizing MB ÷, it was observed that the decay rate of the semioxidized methylene blue (MB 2+) increased with increasing N ; concentration (Table 1). This increase was much more than what is expected on the basis of increased ionic strength. Complex formation with N ; was ruled out on two grounds, viz. (i) no such behaviour was observed on addition of C1- or SO 2- to the M B + / N ; system containing low N ; concentration. The 2 k i d value for the second order decay of the transient was unaffected by the presence of these ions, (ii) as pointed out earlier the extent of bleaching decreased with increasing N ; concentration and it was attribfited to formation of the oxidized species to a lesser extent. If complex formation had taken place then the extent of bleaching would not have decreased. The faster decay in presence of N3- ions could be attributed to an azide catalysed decay of MB 2+. To test this hypothesis, the oxidation of MB ÷ was brought about using Bf£ radicals in the presence and absence of very low concentration of N [ (5 x 10 - s m o l d m - 3 ) . It was seen that 2 k / d value increased from 1.35 to 1.5 x 104s - ' in presence of 5 x 10 -5 mol dm -3 N 3 , a 10% increase even at this low concentration.
6
0
Cyclic voltametry The redox potential for MB2+/MB + couple was also determined by cyclic voltametry using glassy carbon as working electrode. Voltage was scanned between 0.6 and 1.6 V vs SCE at different sweep rates varying from 0.1 to 1 V/s. Only one peak on the anodic scan was seen and no cathodic peak was observed. This is because the one-electron oxidized species reacts quickly (faster than the sweep time) to give products which are not reducible in this potential range. The plot of peak current vs square root of the sweep rate (V/s) was linear indicating the oxidation process to be diffusion controlled (Alfassi et aL, 1987). Ep value at sweep rate l V/s was determined
%
4:
x I
,J
x = +
%
Oi 0
I I
I 2
I 3
I 4
I 5
6
,o,O(.o, 0o- : Fig. 4. Plot of [MB 2+1 [N31 vs [MB +1 [lq31, pH = 6.8.
]~.MAL IfdSHOREet
726
al.
0.03
;"-' Z
r-1
5x108
Ira "D
oE 0.02
~
c~
-
~ / f
o
5 T 4
E
o
108
O.Ol
0.05 EiB+]/ EN;]
o.1o
O.Ol
,~.
.~ -2 -1
Fig. 5. Plot of kob,/[N;] vs [MB+]/[N;-], pH = 6.8. I 0.2
Reaction o f O H with M B ÷
The spectra of the transients formed by OH radical reaction with MB ÷ at pHs 6.8 and 10.1 are shown in Fig. 6. These were recorded in 02 saturated solutions of 10-4mol dm -~ MB ÷. It is seen that the 390 nm peak present at neutral pH shifts to 420nm at pH 10.1 while the shoulder at 500 nm is unaffected by pH change. The spectrum at pH 1.7 was exactly similar to the one obtained at pH 6.8. It is seen from these spectra that the 525 nm peak observed in the case of semioxidized methylene blue is almost absent in these spectra or its contribution is very small, indicating that OH reacts through other modes such as addition or abstraction rather than by electron transfer. The shift in 2m as we go from neutral to alkaline pHs suggests the presence of a pK, for the transient. O.D. vs pH plot at 390 nm is shown in Fig. 6 (inset) and it indicates a pKa value o f ~ 8.7, which is very different from the one obtained in the case of the semioxidized methylene blue. The rate constant for the reaction of OH radicals with MB ÷ was estimated from the pseudo first order
~1 I I I [ I 0.0 -0.2 -0.4 -0.6 -0.8 -10.0 E ol
Fig. 7. Redox titration curve for semiquinone.
methylene blue
formation traces at 390nm, the bimolecular rate constant obtained being 1.15 x 10~°dm3mol-~s-1. Decay rate constant for the transient species could not be determined due to interference from the final product absorption in the wavelength region of interest. One-electron reduction potential o f M B ÷
Various organic radicals derived from alcohols, glucose, dioxane etc. by their reaction with OH radicals (Guha et al., 1987) were utilized to reduce MB ÷ in neutral solutions. For this purpose N : O saturated solutions of 10-4mol dm -3 MB + containing appropriate concentration of the organic solute were subjected to pulse radiolysis. The absorbance of the methylene blue semiquinone formed due to various organic radicals was monitored at 400 nm. The rate constant for the reaction of MB + with the
8.6
E
0.I
0,04
O e~
d
6
0.03
<~ 0 . 0 5
0,0 300
I 6
I 7
I 8
I I I 9 I0 II
I~H
12
I
400
500
"~ (nm)
0
600
Fig. 6. Absorption spectra of transient products formed by the reaction of OH with MB + at different pH (inset: A O.D. vs pH plot).
Redox reactions of methylene blue organic radical was also determined. The plots o f O.D.400 and k M B + O r s . r a d ' VS E °l of the organic radicals are shown in Fig. 7. F r o m both the plots, the value of reduction potential of the couple MB+/MB" was estimated to be 0.04 V vs N H E , which is close to the value reported earlier in the case of related c o m p o u n d thionine (Guha et al., 1987).
REFERENCES
Alfassi Z. B., Harriman A., Huie R. E., Mosseri S. E. and Neta P. (1987) J. Phys. Chem. 91, 212. Bergmann B. K. and Konski C. T. O. (1963) J. Phys. Chem. 67, 2169.
727
Bahnemann D., Asmus K. D. and Wilson R. L. (1981) J. Chem. Soc. Perkin Trans. 11, 890. Ouha S. N., Moorthy P. N., Kishore K., Naik D. B. and Rao K. N. (1987) Proc. Ind. Acad. Sci. (Chem. Sci.) 99, 261. Kamat P. V., Karkhanavala M. D. and Moorthy P. N. (1979) Ind. J. Chem. 18A, 206 and 210. Kamat P. V. and Lichtin N. N. (1981) J. Photochem. 84, 814. Keene J. P., Land E. J. and Swallow A. J. (1965) In Pulse Radiolysis (Edited by Ebert M., Keene J. P., Swallow A. J. and Baxendale J. H.), p. 227. Academic Press, New York. Kishore K., Guha S. N. and Moorthy P. N. (1987) Proc. Ind. Acad. Sci. (Chem. Sci.) 99, 351. Panajkar M. S., Moorthy P. N. and Shirke N. D. (1988) Syrup. Personal Computers in Science and Engineering, BARC, Bombay, February 1988.
Radiat. Phys. Chem. Vol. 34, No. 4, pp. 721-727, 1989 Int. J. Radiat. Appl. lnstrum., Part C Printed in Great Britain. All rights reserved
REDOX REACTIONS OF M E T H Y L E N E BLUE: A PULSE RADIOLYSIS S T U D Y KAMALKISHORE, S. N. GUHA, J. MAHADEVAN, P. N. MOORTHY and J. P. MITTAL Chemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India Abstract~ne-electron oxidation of methylene blue (MB + has been studied using specific oxidizing radicals such as Clf, l~rf, Iq3 and TI(II) in acidic and neutral aqueous solutions). The transient spectrum showed absorption maxima at 525 nm (E. = 40,000 dm3mol- t cm- t) and 360 nm (E = 9700 dm3mol- tcrn- t) in the acidic pH region. At neutral pH also the absorption maxima were at 525 and 360 nm but the extinction coefficients were lower by 30%. A pKo of ~4.3 was observed for the equilibrium MBH3+~MB2++H +. In the case of lq3 radical as the oxidant, the equilibrium: lq3+ MB + ~ N3 + MB2+ was observed for which an equilibrium constant of 120 was estimated from the experimental data. From this as well as from cyclic voltammetric experiments, the redox potential for the MB2+/MB+ couple was calculated as 1.25V vs NHE. The transient species produced by the reaction of OH radicals with methylene blue gave a very different spectrum with 2~, = 400 nm and a pKa of ~ 8.6, and hence it is inferred that OH radicals do not bring about one-electron oxidation of the molecule.
INTRODUCTION
photolysis study of this compound (Kamat and Lichtin, 1981) semioxidized methylene blue was suggested to be formed by quenching of triplet methylene blue by the ground state molecule. Keene et al. (1965) have investigated the reactions of OH radicals with MB + and reported that the semioxidized species has an absorption band with 2m at 520 nm. However OH is not a specific oxidizing radical as it can react through other modes also such as addition and abstraction reactions. Also, under the conditions employed by Keene et al. both OH radical and eaqwould react with MB + and hence the results could not be quite conclusive. We have studied the one-electron oxidation of MB + by specific oxidizing radicals such as C1z, l~r2, lq3, TI(II) and compared the spectra and other characteristics of the semioxidized species with those of the transient produced by OH radical reaction with MB +. We have also determined the one electron reduction potentials for oxidation and reduction of MB +. The results of these investigations are reported here.
Phenothiazine compounds find applications in diverse areas wherein their redox chemistry plays a key role. Thus, for example, the thiazine dye thionine, is a well known sensitizer used in photogalvanic cells for the conversion of light to electrical energy. The key process involved here is the reduction of photoproduced triplet thionine by a reductant to form semithionine which then undergoes rapid disproportionation to the fully reduced form, leucothionine. Discharge of leucothionine at the anode and of the concomitantly formed oxidized form of the reducant at the cathode generates an electric current in the external circuit (Kamat et al., 1979). On the other hand phenothiazine drugs such as chlorpromazine, promethazine, trimeprazine etc. have shown considerable promise as effective radiation sensitizers in the treatment of cancer (Bahnemann et al., 1981). Here too, the key intermediate is believed to be a semioxidized species formed by reaction of the drug with OH radicals resulting from the interaction of ionizing radiation with the major component viz. water in the living cell. In order to gain further insight into the EXPERIMENTAL nature of the semireduced and oxidized species from Methylene blue used in the study was from E. phenothiazine compounds we have undertaken detailed investigation of the one electron redox pro- Merck and was purified by dissolving it in water and cesses involving these compounds in aqueous removing impurities by repeated solvent extraction solution by the technique of nanosecond pulse radiol- with chloroform. The aqueous solution was then ysis. The results of our investigations on thionine evaporated in vacuum oven and the residue obtained have been reported earlier (Guha e t al., 1987; Kishore was pure methylene blue. The extinction coefficient at et al., 1987). In the case of the related compound 660 nm (80,000 dma mol- ~crn- 1) agrees well with the methylene blue (MB*), there are reports of its one literature values (Bergmann and Konski, 1963). All electron reduction in the literature (Keene et al., other chemicals used were either BDH "AnalaR' 1965), but no further detailed studies have been grade or E. Merck "G.R." grade. Solutions were reported on its one electron oxidation. In a laser flash prepared in water obtained from a Barnstead 721
722
KAMALKISHOREet al. 0.20
0.16
0.12
0.08
6
0.04
0.00 --0.02
--0.06
--0.10 500
400
500
600
700
800
"~ (nm)
Fig. 1. Absorption spectra of the transients formed by the reaction of MB ÷ with CI~ (×--2ps, 0--6/is, A--18/~s after the e-pulse) and TI2÷ (0--20 ps after the e-pulse), pH = 1.7. "Nanopure" water system. Gases employed to saturate the solutions viz. N2, 02 and N20 were Iolar grade (Indian Oxygen Ltd). Phosphate and borate buffers as well as plain H2SO4 and NaOH were used for adjusting pHs in appropriate regions. A 7-MeV electron accelerator (Ray Technology, England), giving single pulses of 25 ns duration, was used for irradiating the samples, taken in 1 cm square "spectrosil" cuvettes. Pulse radiolysis set up employed has been described in detail earlier (Guha et al., 1987). Electron pulse dose was measured using aerated 0 . 0 5 m o l d m 3 KCNS solutions taking GE for (CNS)E species to be 21,522dm3mol-Jcm ~ per 100eV at 500nm. Average dose per pulse was 1.5 x 1017eV cm -3, but for estimating extinction coefficients and decay analysis a lower dose ( ~ 4 x 10L6eV) was employed. Spectral and kinetic data acquisition and analysis was done with an IBM-PC-XT using program "ABIWAT" (Panajkar et al., 1988). A Bruker E-310 modular polarograph with AC sweep module E-310/050 was used for carrying out cyclic voltametry at a glassy carbon electrode (Metrohm EA-276/2) with SCE as the reference and Pt-wire as the counter electrode. Electrolyte used was 0.2 mol dm -3 Na2SO 4 and the solutions were purged with N 2. MB ÷ concentration employed was 10-4moldm 3 and pH was adjusted to 6.8 using phosphate buffer. The glassy carbon electrode was cleaned after every run. RESULTS AND DISCUSSION
Methylene blue has no pKa between pH 0-14 and it is in the form MB + in this pH region (Keene et al., 1965). The oxidizing radicals used in the study viz. t~l~-, l~lr~-,lq 3 and TI(II) were produced by the reaction of OH radicals with the solutes CI-, Br-, N3,
and TI ÷ respectively at the appropriate pHs. These radicals react by electron transfer giving rise to a one-electron oxidized species according to: MB + + O x ~ M B 2+ + Red-;
(1)
where Ox is an oxidizing radical and Red- its reduced form. Reaction o f C I : with M B ÷
This reaction can be studied only at a pH below 3 because at higher pH the reactivity of CI- with OH is very low. We have studied its reaction with MB + at pH 1.7 by irradiating 02 saturated 10-4 mol dm -3 MB + solutions containing 10 -2 mol dm 3 C1 . The decay of CI{ was found to be faster in the presence of MB ÷ indicating reaction between the two. Time resolved spectra of the transient produced as a result of this reaction are shown in Fig. 1. In these solutions 02 has been used as a scavenger for H-atoms which would otherwise react with MB ÷ giving a reduced species. It was first established that HO2 radicals do not react with MB ÷ by irradiating 02 saturated methylene blue solution containing 0 . 1 m o l d m -3 tert-butanol as OH scavenger. In these solutions no transient absorption was observed in the wavelength region 300-850 nm. The time resolved spectra given in Fig. 1 show that while absorbance at 340 nm due to CI{ decreases with time, the absorbance at 525 nm due to the product species from MB ÷ increases and reaches a maximum after ~ 6 p s. The product transient species shows two absorption bands, one around 525nm (E = 4 0 , 0 0 0 d m 3 m o l - l c m -1) and a weaker one at ~360 nm (e = 9700 dm 3mol -~ cm-~). The traces for build up of absorbance at 520 nm and the growth of bleaching at 580 nm are shown in Fig. 2(a). From these, the rate constant for the formation of the transient by C1/- reaction was determined to be
Redox reactions of methylene blue
(a)
~ " - - -
--
~
520 nm
(i,)
723
500p=
OtltV
/ i o. v
5 2 0 nm
0 mV 580nm
5 8 0 am
Fig. 2. (a) Build up of transient absorption and dye bleaching in the CI~- reaction with MB + at pH 1.7. (b) Decay of transient absorption and recovery of dye bleaching in the same system at pH 1.7.
8 +__0.5 x 109 dm a mo1-1 s -1. Similarly, the traces for the decay of the absorption at 520 nm and the recovery of bleaching at 580nm are shown in Fig. 2(b). From these traces it was found that the transient species decayed by second order kinetics with a rate constant of 4 _+ 0.4 x 109dm 3 mol -t s -~. The extent of bleaching recovery is about 50% which indicates that the decay of the transient could be due to disproportionation.
Reaction of TI(II) with MB + TI(II) has two pKas at 4.6 and 7.7: OH-
TI2+ .°H-4.6TI(OH)+ ~ .
TI(OH)2.
(2)
We have studied the reaction of Tl(II) with MB + at pH 1.7 and 6.7. In the first case oxygen saturated solutions of 10-4mol dm -3 MB + containing 2 x 10 -3 mol dm -3 Tl + were subjected to electron pulse radiolysis. O2 was used to scavenge H-atoms, while OH reacted with Tl + to give Tl 2+. The transient spectrum obtained was very similar to the one obtained in the case of reaction with (~l2 and was recorded 20/~s after the electron pulse (the time taken to reach maximum signal height). It is shown in Fig. 1 along with the transient spectrum obtained by using CI~- as the oxidant. From this it is obvious that CI~and TI 2+ give rise to the same transient species. The rate constant for the reaction of T1=+ with MB + was determined from the formation trace obtained at 525 nm and was found to be 2.4 x 109 dm3 mol -~ s -~. The 2k/d value for the decay of the transient species was determined to be 1.05x105s -t ( 2 k = 4 . 2 x 109 dm 3 mo1-1 s-'). The reaction of TI(OH) + with MB + was studied at pH 6.7 using N20 saturated solutions of 10 -4 mol dm -3 MB + containing 2 x 10 -3 tool dm -3 TI +. In this case although the spectrum of the transient species (Fig. 3) was similar to the one obtained at lower pH (1.7), the extinction coefficient at 525nm was lower b y ~ 30% (28,000dm 3 tool -~ cm-1). This result suggests that the transient proRPC 34t4--T
duced by Tl(II) reaction has a pKa between pH 1.7 and 6.7. The rate constant for the reaction of TI(OH) + with MB + was determined by following the formation kinetics of the transient absorption at 525 nm and found to be the same as that for the reaction of TI2+ with MB + at the acidic pH. This indicates that the efficiencies of the reaction of TI(OH) + and Tl 2+ with MB + are comparable. The 2k/d value for the transient decay was estimated to be 3 . 8 x 1 0 4 S - 1 (2k = 1 x l09 dm 3 mol -l s -l) which is lower than the value in acidic solutions and again indicates the existence of a pK.
Reaction of f~r7 with MB + The reaction of ]~r~- radicals with MB + was studied at pH6.8 by pulse irradiating N 2 0 saturated 10 -4 mol dm -3 MB + solutions containing 0.02mol dm -a KBr. The spectrum of the transient species was identical to the one obtained in the case of TI(OH) + reaction with MB + at this pH and is shown in Fig. 3. The extinction coefficients at 525 nm in the two cases agreed with each other. From the formation traces at 525 nm, the rate constant for the reaction of l~r~- with MB + was determined to be 1.6 + 0.1 x 109dm3mol -l s - ' . 2 k / d for the second order decay of the transient was determined to be 2 x l(Ps -~ in presence of 0 . 0 2 m o l d m - a K B r ; and was found to increase with decreasing Br- concentration, being 1.6 x 104 s -l and 2.3 x 104 s -l respectively at 0.1 and 0.01 mol dm -3 Br- concentrations. Thus at very low concentration of KBr, 2kid value may approach that observed with TI(OH) + as the oxidant, viz. 3.8 × l(Ps -~, so that the decay rate constant can be inferred to be the same in the two systems. There was a small decrease in the yield of the transient with increasing concentration of Br-, e.g. in one experiment changing Br- concentration from 0.02 mol dm -3 to 0.1 mol dm -3, resulted in a decrease in the absorbance of the transient at 525 nm from 0.078 to 0.071. This indicates the existence of an
724
KAMALK]SHOREet al.
0.10 0.08, 0.08 o
0.06
-~v
0 04
f
0.04
0.02
I 0
0.02
I 4
I
I 8 I~H
I
I 12
0.0
--0.02
--0.04
--0.06
--0.081 300
I 400
I 500
I 600
I 700
I 800
(rim)
Fig. 3. Absorption spectra of the transients formed by the reaction of MB+ with TI(OH)+ (A) and ])r2(O) at pH 6.7 and 6.8 respectively (inset: plot ofA O.D. vs pH at 520 nm).
equilibrium reaction but the change was not large enough for evaluating the equilibrium constant. (212, l)r{, TI2÷ and TI(OH) ÷ are all specific oxidizing radicals and cause one electron oxidation of the solutes. The spectra of the transients obtained by the reaction of (::12, l~r~- and T12+ with MB + at pH 1.7 were identical. Similarly, the spectra of the transients obtained at neutral pH by the reaction of l~r2 and TI(OH) ÷ with MB ÷ were also identical. The decay rates of the transients produced by the reaction of different oxidizing radicals with MB + also agree with each other when compared at the same pH. Kamat and Lichtin (1981) have also reported ~'m for the semioxidized MB ÷ to be at 520 nm. All these results indicate that the transient species is the one-electron oxidation product of MB ÷. Thionine which has a very similar structure also reacts with these oxidizing species to give a transient spectrum with 2m at 480 nm (Kishore et al., 1987). In fact, in the case of many phenothiazines the one-electron oxidation products have 2m in the 500 nm region, indicating common site for oxidation which is possibly loss of electron from the heterocyclic thiazine ring. Thus MB ÷ on oxidation would give rise initially to MB 2+, which may exist in different conjugate acid-base forms depending on the pH. p K , o f the semioxidized methylene blue
Br2 radicals were employed over the entire pH range 1-14 to oxidize methylene blue to determine the p K a of the semioxidized species. The solutions were adjusted to the required pH and saturated with 02 so
that eaq and H-atoms are exclusively scavenged by 02, and the transient species is only due to the reaction of l~r2 . O.D. vs pH plot is shown in Fig. 3 (inset) from which pK, value of 4.3 was estimated. The nearest pKa of the ground state methylene blue is ~ 0 corresponding to the equilibrium: MBH 2+.
~0
•MB ++H+;
(3)
MB ÷ has no ionizable protons (see Scheme 1). Thus the pKa of 4.3 for the semioxidized species could be for the equilibrium: MBH3 +
4.3 MB2+ +H+"
(4)
It was also observed that the yield of the semioxidized methylene blue at pH 1.7 produced by the reactions of ~12, T12+ and t~r{ was the same under similar conditions indicating that the efficiencies of (~12, TF + and l~r~- for oxidation of MB + are the same. This was also borne out by the extent of bleaching in these cases. As the spectra of the transient species obtained through the reactions of I~lr; and TI(OH) ÷ with MB ÷ at pH 6.8 are identical at the 2m and also in the bleaching region, it is clear that the efficiency of TI(OH) ÷ is equal to that of flr~- and thus equal to that of TI2+ also.
(H3C)2 N ' ~ / S ~ N ( C H a ) :
Scheme 1
+
Redox reactions of methylene blue
5 5 1 2 5 l 2
× x x x x x x
10 -4 10 -3 10-2 10 -2 10 -2 l0 -* 10 -*
2k/d
Ep = E1/2 - 0.90(R T /nF)
(103s -I) 1.0 2.2 3.2 9.7 11.5 13.0 33.5
+ (RT/3nF) In(2kCoRT/3nF" v);
(6)
with 2k = 109dm3mol - I s -1, Co = 1 0 - 4 m o l d m -3 and v (sweep rate) = 1 V/s is 1.27 V vs N H E and is close to the E ° value determined earlier from the equilibrium experiments. Thus an average value of 1.25 ___0.02 vs N H E can be deduced for the reduction potential of MB2+/MB ÷ couple.
Reaction of N3 with M B ÷
Azide catalyzed decay of semioxidized methylene blue
lq 3 radicals were also found to react with MB ÷ at neutral pH to give a transient spectrum with 2m = 525 nm. However in this case, the yield of semioxidized methylene blue was strongly dependent on the concentration of N 3 . As the N3 concentration was changed from 10 -3 to 10 -2 mol dm -3, the transient absorbance at 525 n m (for the same dose) decreased by ~ 5 0 % , whereas the pseudo first order rate constant for the reaction of lq 3 with MB ÷ increased. The extent of bleaching was also found to be less at higher N3 concentrations indicating that the extent of electron transfer was less at higher ['N3 ]. All these results suggest the existence of an equilibrium reaction of the type: MB + + lq3 ~ - MB 2+ + N 3 .
E~/2 as calculated using
tO be 1.3V vs NHE, equation (6):
Table 1. Effect o f [N 3 ] on the decay rate constant of semioxidized methylene blue ([MB + ] = 5x 10-Smoldm 3;pH=6.8) IN3] ( m o l d m 3)
725
(5)
In such a case the observed rate of attainment of equilibrium is given by:
kob~ = kf[MB + ] + kr[N3 ] where kf and k b are respectively the rate constants for the forward and reverse reactions (Alfassi et al., 1987). Thus a plot of kobs/[N; ]vs [MB+]/[N;] would be a straight line with slope equal to kf and intercept equal to k,. The equilibrium constant (K = kf/kr) was thus determined to be ~ 120. K was also determined by plotting [MB 2+] x [ N ; ] vs [MB +] x [lq3]. The plot is shown in Fig. 5 and it gave a value of ~ 1 l0 for K. Assuming the redox potential for t h e / q / N ; couple to be 1.35 vs N H E (Alfassi et al., 1987), the value for the MB2+/MB + couple was calculated to be ~ 1.22 V vs N H E at neutral pH.
At neutral pH, when lq 3 radical was used for oxidizing MB ÷, it was observed that the decay rate of the semioxidized methylene blue (MB 2+) increased with increasing N ; concentration (Table 1). This increase was much more than what is expected on the basis of increased ionic strength. Complex formation with N ; was ruled out on two grounds, viz. (i) no such behaviour was observed on addition of C1- or SO 2- to the M B + / N ; system containing low N ; concentration. The 2 k i d value for the second order decay of the transient was unaffected by the presence of these ions, (ii) as pointed out earlier the extent of bleaching decreased with increasing N ; concentration and it was attribfited to formation of the oxidized species to a lesser extent. If complex formation had taken place then the extent of bleaching would not have decreased. The faster decay in presence of N3- ions could be attributed to an azide catalysed decay of MB 2+. To test this hypothesis, the oxidation of MB ÷ was brought about using Bf£ radicals in the presence and absence of very low concentration of N [ (5 x 10 - s m o l d m - 3 ) . It was seen that 2 k / d value increased from 1.35 to 1.5 x 104s - ' in presence of 5 x 10 -5 mol dm -3 N 3 , a 10% increase even at this low concentration.
6
0
Cyclic voltametry The redox potential for MB2+/MB + couple was also determined by cyclic voltametry using glassy carbon as working electrode. Voltage was scanned between 0.6 and 1.6 V vs SCE at different sweep rates varying from 0.1 to 1 V/s. Only one peak on the anodic scan was seen and no cathodic peak was observed. This is because the one-electron oxidized species reacts quickly (faster than the sweep time) to give products which are not reducible in this potential range. The plot of peak current vs square root of the sweep rate (V/s) was linear indicating the oxidation process to be diffusion controlled (Alfassi et aL, 1987). Ep value at sweep rate l V/s was determined
%
4:
x I
,J
x = +
%
Oi 0
I I
I 2
I 3
I 4
I 5
6
,o,O(.o, 0o- : Fig. 4. Plot of [MB 2+1 [N31 vs [MB +1 [lq31, pH = 6.8.
]~.MAL IfdSHOREet
726
al.
0.03
;"-' Z
r-1
5x108
Ira "D
oE 0.02
~
c~
-
~ / f
o
5 T 4
E
o
108
O.Ol
0.05 EiB+]/ EN;]
o.1o
O.Ol
,~.
.~ -2 -1
Fig. 5. Plot of kob,/[N;] vs [MB+]/[N;-], pH = 6.8. I 0.2
Reaction o f O H with M B ÷
The spectra of the transients formed by OH radical reaction with MB ÷ at pHs 6.8 and 10.1 are shown in Fig. 6. These were recorded in 02 saturated solutions of 10-4mol dm -~ MB ÷. It is seen that the 390 nm peak present at neutral pH shifts to 420nm at pH 10.1 while the shoulder at 500 nm is unaffected by pH change. The spectrum at pH 1.7 was exactly similar to the one obtained at pH 6.8. It is seen from these spectra that the 525 nm peak observed in the case of semioxidized methylene blue is almost absent in these spectra or its contribution is very small, indicating that OH reacts through other modes such as addition or abstraction rather than by electron transfer. The shift in 2m as we go from neutral to alkaline pHs suggests the presence of a pK, for the transient. O.D. vs pH plot at 390 nm is shown in Fig. 6 (inset) and it indicates a pKa value o f ~ 8.7, which is very different from the one obtained in the case of the semioxidized methylene blue. The rate constant for the reaction of OH radicals with MB ÷ was estimated from the pseudo first order
~1 I I I [ I 0.0 -0.2 -0.4 -0.6 -0.8 -10.0 E ol
Fig. 7. Redox titration curve for semiquinone.
methylene blue
formation traces at 390nm, the bimolecular rate constant obtained being 1.15 x 10~°dm3mol-~s-1. Decay rate constant for the transient species could not be determined due to interference from the final product absorption in the wavelength region of interest. One-electron reduction potential o f M B ÷
Various organic radicals derived from alcohols, glucose, dioxane etc. by their reaction with OH radicals (Guha et al., 1987) were utilized to reduce MB ÷ in neutral solutions. For this purpose N : O saturated solutions of 10-4mol dm -3 MB + containing appropriate concentration of the organic solute were subjected to pulse radiolysis. The absorbance of the methylene blue semiquinone formed due to various organic radicals was monitored at 400 nm. The rate constant for the reaction of MB + with the
8.6
E
0.I
0,04
O e~
d
6
0.03
<~ 0 . 0 5
0,0 300
I 6
I 7
I 8
I I I 9 I0 II
I~H
12
I
400
500
"~ (nm)
0
600
Fig. 6. Absorption spectra of transient products formed by the reaction of OH with MB + at different pH (inset: A O.D. vs pH plot).
Redox reactions of methylene blue organic radical was also determined. The plots o f O.D.400 and k M B + O r s . r a d ' VS E °l of the organic radicals are shown in Fig. 7. F r o m both the plots, the value of reduction potential of the couple MB+/MB" was estimated to be 0.04 V vs N H E , which is close to the value reported earlier in the case of related c o m p o u n d thionine (Guha et al., 1987).
REFERENCES
Alfassi Z. B., Harriman A., Huie R. E., Mosseri S. E. and Neta P. (1987) J. Phys. Chem. 91, 212. Bergmann B. K. and Konski C. T. O. (1963) J. Phys. Chem. 67, 2169.
727
Bahnemann D., Asmus K. D. and Wilson R. L. (1981) J. Chem. Soc. Perkin Trans. 11, 890. Ouha S. N., Moorthy P. N., Kishore K., Naik D. B. and Rao K. N. (1987) Proc. Ind. Acad. Sci. (Chem. Sci.) 99, 261. Kamat P. V., Karkhanavala M. D. and Moorthy P. N. (1979) Ind. J. Chem. 18A, 206 and 210. Kamat P. V. and Lichtin N. N. (1981) J. Photochem. 84, 814. Keene J. P., Land E. J. and Swallow A. J. (1965) In Pulse Radiolysis (Edited by Ebert M., Keene J. P., Swallow A. J. and Baxendale J. H.), p. 227. Academic Press, New York. Kishore K., Guha S. N. and Moorthy P. N. (1987) Proc. Ind. Acad. Sci. (Chem. Sci.) 99, 351. Panajkar M. S., Moorthy P. N. and Shirke N. D. (1988) Syrup. Personal Computers in Science and Engineering, BARC, Bombay, February 1988.