Radiation Physics and Chemistry 64 (2002) 13–18

Radiation-induced reduction of ditetrazolium salt in aqueoussolutions

Ali Sadeghia, Mahnaz Chaychiana, Mohamad Al-Sheikhlya,*, W.L. McLaughlinb

aDepartment of Materials and Nuclear Engineering, University of Maryland, College Park, MD 20742, USAb Ionizing Radiation Division, Physics Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

Received 16 October 2000; received in revised form 22 March 2001; accepted 3 May 2001

Abstract

Color formation in aqueous solutions of the ditetrazolium salt blue tetrazolium (BT2+) in the absence or presence ofoxygen is a complex radiation chemical reaction. The final stable product is the poorly soluble diformazan violet to bluepigment having a broad spectral absorption band (lmax ¼ 552 nm). The reaction of BT2+ with the hydrated electronproceeds by rapid reduction of BT2+ followed by protonation at the nitrogen closest to the unsubstituted phenyl group,

via the two intermediate tetrazolinyl radicals shared by the ditetrazole ring nitrogens. The effect of solution pH, N2Osaturation, and the presence of the reducing agent dextrose are examined. The system serves as a radiochromic sensorand a dosimeter of ionizing radiations. Solutions of 5mmol l�1 BT2+ at pH 7.3 serve as dosimeters over an absorbed-

dose range of approximately 0.2–6 kGy (dearated, with a range of 1–8mmol l�1 dextrose) and of about 1–15 kGy(aerated, with 0.1mol l�1 sodium formate and 5mmol l�1 dextrose). r 2002 Published by Elsevier Science Ltd.

Keywords: Blue tetrazolium; Diformazan; Ditetrazolium salts; Dosimetry; Radiochromic solutions; Radiolytic reduction

1. Introduction

The ditetrazolium salt blue tetrazolium (BT2+) wasfirst synthesized and used as an ultraviolet-light sensitivebiological stain in histochemical demonstration of

succinate dehydrogenase in liver and kidney (Rutenburget al., 1950) and later used for studying the histochem-istry of NAD-linked oxidative enzymes (Farber and

Bueding, 1956). The BT2+ salt is pale yellowish inaqueous solution, but, upon irradiation, it yields a deep-blue hydrophobic diformazan pigment by reductive

ring-opening.The mechanism of reduction of nitro blue tetrazolium

(NBT2+), a structural analog of BT2+, was previouslyinvestigated by the stopped-flow and the pulse-radiolysis

techniques (Bielski et al., 1980; Kovacs et al., 1999a).A similar reduction mechanism can be proposed for

BT2+, with the stepwise addition of four electrons andthe formation of the transient tetrazole ring-shared free

radicals (BT+ � , MF � ), and one stable intermediateconsisting of one tetrazolium center and one formazancenter (MF+): Scheme 1.

Pulse radiolysis studies (Kovacs et al., 1999a) ofNBT2+ in aqueous solutions have shown that the red-colored monoformazan is produced under reducing

conditions whereas under oxidizing conditions (N2O-saturation) unstable OH adducts, absorbing in the nearultraviolet, were formed. Kovacs et al. (1999b) have

developed the radiochromic NBT2+ solution in aqueousethanol for high-dose gamma-ray dosimetry over thedose ranges 0.1–1 and 1–30 kGy, using the monoforma-zan (MF) and diformazan (DF) absorption maxima of

522 and 612 nm wavelengths, respectively.The aim of the present work is to examine the role

of dextrose (a-D-glucose) in enhancing the reduction

of BT2+ and to assess the suitability of BT2+ inliquid-phase radiochromic sensor systems using water assolvent. Dextrose was used before in solid film dosimeter

*Corresponding author.Tel.: +1-301-314-5214; fax: +1-301-

314-9467.

E-mail address: mohamad@eng.umd.edu (M. Al-Sheikhly).

0969-806X/02/$ - see front matter r 2002 Published by Elsevier Science Ltd.

PII: S 0 9 6 9 - 8 0 6 X ( 0 1 ) 0 0 4 4 1 - 8

formulation (Al-Sheikhly et al., 1999) and we havedecided to use it in the present study in aqueous

solutions. Dextrose does not react with e�aq and canscavenge the dOH radicals to produce some reducingradicals. In addition to evaluating the dosimetricresponse and useful dose range for oxygen-free aqueous

solutions containing dextrose, the influence of the

following parameters on the radiation chemical yieldwere studied: BT2+ concentration, dextrose concentra-

tion, oxygen, N2O-saturation and pH.

2. Experimental

The blue tetrazolium chloride salt (C40H32N8O2Cl2;

MW 727.66) (BT2+) was used as received from Aldrich.Dextrose (C6H12O6; MW 180.162) was received fromFisher and used as the precursor to the reducing radicalsin the reduction of BT2+. Water was purified by

Millipore Milli-Q-system. The solutions were stored inthe dark at room temperature (E231C). Solutions werepurged with pure dry N2 or N2O for at least 20min prior

to irradiation. For O2 saturation, solutions were purgedwith pure dry gas for 15min prior to irradiation as wellas during the course of irradiation, in order to maintain

constant saturation conditions.For irradiation, the solutions were held in 25ml

volumetric flasks with parafilm-lined caps. Each flaskwas fitted into a cylindrical polystyrene holder with

5mm wall thickness, which served to supply approx-imate electron-equilibrium conditions for 1.25MeVgamma rays. This assembly was irradiated isotropically

with gamma radiation in an annular geometry by athermostated arrangement of twelve 60Co sources(Gammacell Model 220; Nordion International Inc.,

Ontario, Canada) at an absorbed dose rate of 3.1Gy s�1.O2-saturated solutions were irradiated in 100ml flasksfitted into a cylindrical Styrofoam holder. The absorp-

tion spectra and optical absorbances at specific wave-lengths (at 1 nm band pass) were measured with adouble-beam Cary Model 3E spectrophotometer (Var-ian Australia Pty. Ltd) using 10mm path-length

standard glass cuvettes. The reference-beam cell wasfilled with deionized water.

3. Results and discussion

3.1. Deoxygenated solutions

Radiolysis of water generates known concentrations

of free radicals, radical ions, and stable products (Spinksand Woods, 1990):

H2O-dOH;Hd; e�aq;H2;H2O2;H3Oþ ð1Þ

with the following yields in mol J�1: G(dOH)=0.29;G(e�aq)=0.29; G(Hd)=0.062; G(H2)=0.042; G(H2O2)=0.082. In order to elucidate the role of dextrose in the

reduction of BT2+, the concentration effects of BT2+

and dextrose on the reduction yield of BT2+ wereinvestigated.

To study the effect of [BT2+] on the sensitivity of theliquid-phase radiochromic sensor, gamma radiation

Scheme 1. Reduction mechanism of di-tetrazolium.

A. Sadeghi et al. / Radiation Physics and Chemistry 64 (2002) 13–1814

responses for five sensor solutions, all containing5mmol l�1 dextrose, and a variable concentration of

BT2+, ranging from 0.2mmol l�1 to 5mmol l�1, weremeasured. No adjustment in pH was made at this pointin the study. Fig. 1a shows the responses to gamma

radiation of BT2+ in terms of absorbance at 552 nm as afunction of absorbed dose in water (10mm optical pathlength). Fig. 1b shows the dose-dependent absorptionspectra of the unirradiated and irradiated 3mmol l�1

solutions to several different doses. The maximum of theradiation-induced absorption band is at 552 nm. As isevident from Fig. 1a, the lower concentration solutions

have the greater initial sensitivities to gamma radiation,but show a tendency to saturate at higher doses. Thegamma radiation sensitivity decreases with increasing

BT2+ concentration above 0.5mmol l�1 with no sig-nificant saturation at the highest concentrations (3 and5mmol l�1 up to a dose of 4 kGy). At a BT2+

concentration of 0.2mmol l�1, the initial rate of

gamma-induced reduction is high but saturation begins

at a relatively low dose (E1.5 kGy). Table 1 shows

the increase in the G (diformazan) with decreasinginitial BT2+ concentration at an absorbed dose of1 kGy. The G values are expected to reach higher

levels if each hydrated electron reduces BT2+ [G(e�aq)/4=0.07 mmol J�1]. The finding that the G values arelower may be attributed to back reactions, i.e. oxidationof the reduced species with dOH or other radicals. The

results also suggest that solutions of 3 and 5mmol l�1

BT2+ at pH 7.3 (dearated, with 5mmol l�1 dextrose) canserve as dosimeters over an absorbed-dose range of

approximately 0.2–6 kGy.To study the effect of dextrose concentration on the

sensitivity of the liquid-phase sensor, gamma radiation

responses were evaluated for five sensor solutions allcontaining 5mmol l�1 BT2+ and a variable concentra-tion of dextrose, ranging from 1mmol l�1 to 8mmol l�1

(Fig. 2). The response shows an approximately linear

function for all dextrose concentrations, with higherconcentration solutions having the greater sensitivity togamma radiation. Table 2 shows the increase in

G(diformazan) with increasing initial dextrose concen-tration at an absorbed dose of 2 kGy. No evidence ofsaturation was seen, even at the highest dextrose

concentration (8.0mmol l�1), for doses as high as8.0 kGy. The results show that a solution of 5mmol l�1

BT2+ with a wide range of dextrose concentrations

(1–8mmol l�1) is a good dosimeter in the range of about0.2–6 kGy.These experiments were conducted in the absence of

oxygen so that all hydrated electrons are expected to

react with BT2+. Dextrose does not react with e�aq(Buxton et al., 1988). The hydroxyl radicals can reactwith dextrose (k ¼ 1:5� 109 lmol�1 s�1, Buxton et al.,

1988) by abstracting H-atoms from the variouspositions, with minimal selectivity, although thereappears to be a slight preference for attack at C(1)

and C(6) (von Sonntag, 1987a). H-atoms also reactwith dextrose, though more slowly than dOH(k ¼ 6:1� 107 lmol�1 s�1, Buxton et al., 1988), to

produce the same radicals. Some of the radicals formedfrom dextrose are reducing agents (von Sonntag, 1987a)

Fig. 1. Gamma-ray response curves for blue tetrazolium

chloride (BT2+) in N2-saturated aqueous solutions containing

5mmol l�1 dextrose. Absorbance measured at 522 nm (optical

path length 10mm). Temperature of irradiation 231C. (a)

[BT2+] in mmol l�1: 0.2 (~), 0.5 (&), 1.0 (m), 3.0 (K), 5.0 (J).

(b) Dose dependent absorption spectra of the unirradiated and

irradiated 3mmol l�1 BT2+ solution.

Table 1

G (diformazan) as a function of the initial [BT2+] at an

absorbed dose of 1 kGy. (Calculated by taking a molar

absorption coefficient of 35,000 lmol�1 cm�1, Kovacs et al.,

1999b)

[BT2+], mmol l�1 G (diformazan), mmol J�1

0.2 0.0454

0.5 0.0483

1 0.0432

3 0.0236

5 0.0114

A. Sadeghi et al. / Radiation Physics and Chemistry 64 (2002) 13–18 15

and may take part in the reduction of BT2+. However, ifany of the dextrose primary radicals undergo water

elimination, the secondary radicals generated may beoxidizing species (Steenken, 1979) and may contribute tothe back reactions by oxidizing the formazan.

Although Hd and dOH can react with dextrose, theycan also react with BT2+. The rate constants have notbeen measured but are expected to be similar to those ofmany other aromatic compounds (Buxton et al., 1988),

i.e. close to 1� 1010 lmol�1 s�1 for dOH and 2–4 times

lower for Hd. Therefore, for 0.2mmol l�1 BT2+ solu-tions containing 5mmol l�1 dextrose about 20% of dOH

will react with BT2+ and for solution containing5mmol l�1 BT2+ and 5mmol l�1 dextrose about 85%of dOH will react with BT2+. This competition is

expressed as a decrease in yield with increasing [BT2+](Fig. 1) and an increase in yield with increasing[dextrose] (Fig. 2).Further study of the role of dextrose as the precursor

for the reducing radicals involved in the reduction ofBT2+ was carried out with N2O-saturated solutions,where e�aq is rapidly converted into dOH (Dainton and

Peterson, 1962),

H2Oþ e�aqþN2O-dOHþN2þOH� ð2Þ

with k ¼ 9:1� 109 lmol�1 s�1 (Janata and Schuler,

1982). Fig. 3 shows that the increase in the absorbanceat 522 nm with dose in N2O-saturated aqueous solutionof 0.5mmol l�1 BT2+ in the presence of dextrose is

smaller than that in N2-saturated solution. G(diforma-zan) in N2-saturated solution at 1 kGy absorbed dose is0.077 mmol J�1 while G(diformazan) in N2O-saturated

solution at the same absorbed dose is 0.026 mmol J�1. Inthe absence of dextrose there was no production ofdiformazan, indicating that dOH radicals are notinvolved in the process. Instead, an unknown reddish-

brown product was formed. Hence, the addition ofdextrose increases the reduction yield mainly by scaven-ging dOH radicals and only partly by producing

reducing radicals.All the above experiments were carried out in neutral

unbuffered solutions. Experiments with N2-saturated

solutions containing 3mmol l�1 BT2+ and 5mmol l�1

dextrose, irradiated with a dose of 1.0 kGy, showed thatreduction yield was practically independent of pHbetween pH 6 and pH 10. The measured absorbance

decreased at lower pH, probably due to competition ofH+ for the solvated electrons and the instability of thedye. The absorbance increased at pH>10, probably due

to increased molar absorption coefficient of the dye.Because of these changes with pH, the present dosimetersolutions are to be used only between pH 6 and pH 10.

3.2. Oxygenated solutions

Oxygen readily scavenges solvated electrons and H-atoms according to reactions 3 and 4 (von Sonntag,

1987b) to produce O2� and HO2

d, respectively:

e�aqþO2-O�2 ðk¼ 2� 1010 l mol�1 s�1Þ ð3Þ

HdþO2-HOd2 ðk¼ 2� 1010 l mol�1 s�1Þ ð4Þ

HOd2¼ O�

2 þHþ ð5Þ

In water, the pKa value of HO2d is 4.7 (Bielski and

Richter, 1978). The role of superoxide in the reduction

Fig. 2. Gamma-ray response curves for 5mmol l�1 BT2+ in N2-

saturated aqueous solutions. Absorbance measured at 522 nm

(optical path length 10mm). Temperature of irradiation 231C.

(a) [Dextrose] in mmol l�1: 1 (~), 2 (’), 3 (m), 4 (K), 8 (*). (b)

Dose-dependent absorption spectra of the irradiated 2mmol l�1

dextrose solution.

Table 2

G(diformazan) as a function of the initial [dextrose] at an

absorbed dose of 2 kGy

[dextrose], mmol l�1 G (diformazan), mmol J�1

1 0.0092

2 0.0099

3 0.0121

4 0.0142

8 0.0185

A. Sadeghi et al. / Radiation Physics and Chemistry 64 (2002) 13–1816

of BT+2 was studied in the formate system, involving

the reaction of dOH radicals with formate in oxygenatedsolutions (reactions 6 and 7; Bielski et al., 1980).

HCOO� þd OH-CO�2 þH2O ð6Þ

CO�2 þO2-O�

2 þCO2 ð7Þ

Under these conditions, all the water radicals can beconverted into HOd

2=O�2 :

Solutions containing 0.5mmol l�1 BT+2, 0.1mol l�1

sodium formate, and 5mmol l�1 dextrose at pH 7.3 weresaturated with O2 before and during irradiation. Theabsorbance measured after irradiation has a maximum

at 520 nm at low dose and the peak shifts to 552 nm atincreasing doses (Fig. 4). This probably indicates for-mation of the monoformazan at low doses anddiformazan at high doses. G(diformazan) for a solution

irradiated with 1 kGy is about 0.0023 mmol J�1, muchlower than the value (0.0483) found in deoxygenatedsolutions at the same concentration of BT2+. It is clear

from these results that the O�2 radical ions play a much

lesser role in the reduction of BT+2 in oxygenatedsystems, as compared to the solvated electrons in the

deoxygenated systems. Therefore, O2-saturated solu-tions can serve as dosimeter for higher doses than N2-

saturated solutions, i.e. from about 1–15 kGy. It is

important to stress, however, that the solutions have tobe bubbled with O2 during irradiation to preventdepletion of oxygen. If O2 is bubbled only before

irradiation, the initial yield is low but after a dose of1 kGy, when O2 concentration becomes very low, thereduction yield increases and becomes similar to that

observed under N2.It is noted that with O2-saturated solutions the

absorption peak shifts gradually from 520 to 552 nm

(Fig. 4) whereas in deoxygenated solutions such a shift isnot apparent (Figs. 1 and 2). The reason for thisdifference is not clear. This effect has been observedwith aqueous ethanol solutions of nitro blue tetrazolium

(Kovacs et al., 1999b) where it was explained asmonoformazan formation at low doses(lmax ¼ 522 nm) with gradual shift to higher wave-

lengths at higher doses being due to diformazanpredominance (lmax ¼ 612 nm).

4. Conclusion

Addition of dextrose to deaerated BT2+ solutionsincreases the radiolytic yield of diformazan pigment, i.e.

Fig. 3. Gamma-ray response functions for N2-saturated (~)

and N2O-saturated (J) 0.5mmol l�1 BT2+ solution containing

5mmol l�1 dextrose.

Fig. 4. Gamma ray response functions for 0.5mmol l�1

oxygen-saturated BT2+ solution containing 0.1mol l�1 sodium

formate and 5mmol l�1 dextrose at pH 7.3. The inset shows the

dose-dependent shift in the absorption peak from 520–552nm.

A. Sadeghi et al. / Radiation Physics and Chemistry 64 (2002) 13–18 17

it increases the sensitivity of this system to irradiation.Dextrose does not compete with BT2+ for the hydrated

electrons but scavenges dOH radicals. The latterreaction decreases the probability that dOH radicaloxidize the reduced dye and also produces reducing

radicals that enhance the reduction yield of BT2+.G(diformazan) was found to be lower than expected,probably due to back reactions. BT2+ solutions contain-ing a wide range of dextrose concentrations can be used

as a good dosimeter over a wide dose range. Thesolutions are stable before irradiation but are sensitiveto ultraviolet light (Al-Sheikhly et al., 1999). After short-

pulse irradiation, the color develops rapidly (withinmilliseconds) (Kriminskaya et al., 1988; Kovacs et al.,1996). In the case of polymer films of BT2+ dissolved in

polyvinyl alcohol, the color develops slowly for severalhours after irradiation and then is stable for several days(Al-Sheikhly et al., 1999).

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