Production of Reactive Oxygen Species on Photolysis of Dilute Aqueous Quinone Solutions

Production of Reactive Oxygen Species on Photolysis ofDilute Aqueous Quinone Solutions

Shikha Garg1, Andrew L. Rose2 and T. David Waite*1,2

1School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW, Australia2Centre for Water and Waste Technology, The University of New South Wales, Sydney, NSW, Australia

Received 7 September 2006; accepted 16 January 2007; DOI: 10.1111 ⁄ j.1751-1097.2007.00075.x

ABSTRACT

We have examined the generation of the reactive oxygen species

(ROS) superoxide and hydrogen peroxide (H2O2) by irradiation

of dilute aqueous solutions of disodium anthraquinone-2-6-

disulfonate (AQDS) with simulated sunlight. Irradiating a

solution of AQDS in 2 mM NaHCO3 and 0.01 M NaCl

produced superoxide and H2O2 at nanomolar concentrations.

Experiments in which initial concentrations of dioxygen, H2O2,

the superoxide radical trap nitroblue tetrazolium and the

electron donor dimethyl sulfoxide were varied suggested that

the interaction of solvent water with photo-excited quinone

moieties produces dioxygen-reducing radicals, and that these are

the primary source of ROS in the system. A kinetic model for

ROS production is proposed based on our experimental data.

INTRODUCTION

A variety of transient reactive oxygen species (ROS), includingthe hydroxyl radical (OH•), superoxide (O��2 ) and hydrogenperoxide (H2O2), are produced in sunlit surface waters (1–3).

These ROS are highly redox-active due to the presence of anunpaired electron and ⁄ or their redox potential and thus playan important role in geochemical cycling of trace metals and

degradation of organic pollutants in natural environments.The kinetics of ROS formation in natural systems is an activearea of research, with specific attention being given to the

pathways by which particular ROS are generated (1,4–6).Earlier reports have provided evidence for the presence ofquinones in natural organic matter (NOM) (7–9) and anumber of investigators have postulated that quinone moieties

present in NOM may be responsible for production of ROS insunlit surface waters (6,10,11).

Quinones possess both potent photodynamic action and the

ability to undergo redox cycling. In the process of photo-chemical redox cycling, quinones can produce ROS via eitheroxidation of water or reduction of dioxygen (12–14). Quinones

and their reduced derivatives act as electron donors andacceptors in photosynthesis (15) and it is possible that they actas electron shuttles in sunlit surface waters as well. Figure 1shows the overall flow of electrons on photolysis of quinones

and provides a key to the abbreviations used throughout thisarticle. The reactions involved in formation of the major long-

lived intermediates are shown in Fig. 2, and the possiblesubsequent reactions of these intermediates to yield the final

products of photolysis are shown in Fig. 3. These reactionswill be referred to by number in square brackets hereafter.

As shown in Fig. 2, on absorption of a photon of light,

ground state molecules of quinone (Q) are first excited to thesinglet state [1] which undergoes rapid intersystem crossing toform the excited triplet state (3Q�) (16). 3Q� can be quenched

by triplet dioxygen (3O2), regenerating Q and producingexcited singlet oxygen (1O2) (17) [2], or can undergo naturaldecay to ground state Q [3]. Alternatively, 3Q� can interact

with water producing a relatively long-lived oxidizing inter-mediate that is probably a quinone-water exciplex, (Q-H2O)*(12,18,19) [4]. In the event that Q is a good electron acceptor,e.g. chlorinated benzoquinones, substantial amounts of free

hydroxyl radical may escape the (Q-H2O)* exciplex (20).However, formation of free hydroxyl radicals has been rejectedin the case of anthraquinone derivatives (19,21) and for

benzoquinone and most of its derivatives (17,18,20,22) andthus it appears that the formation of free hydroxyl radicals israther exceptional in quinone photochemistry.

The major differences between mechanisms proposed inexisting reports on quinone photochemistry relate to the fateof (Q-H2O)*. Gorner (22) and Von Sonntag et al. (17)

suggested that (Q-H2O)* formed on photolysis of 1,4-benzo-quinone (BQ) enolizes to form benzene-1,2,4-triol, (Q(OH)3),also known as hydroxy-hydroquinone [5]. In contrast, studieson anthraquinone and its derivatives (12,19) have suggested

that (Q-H2O)* reacts with the parent Q to produce semiqui-none (Q��) and a hydroxyquinone radical (QOH�) [6]. Inaddition to reactions [5] and [6], (Q-H2O)* can undergo

natural decay to yield parent quinone [7]; however, the yield ofthe final product of quinone photolysis will be governed byeither reaction [5] or [6]. Although there is no evidence for

enolization of anthraquinone, the existence of Q(OH)3 hasbeen shown for naphthoquinone (NQ; whose structure isreasonably similar to AQDS) and is suggested to have similarproperties to that formed from a BQ parent molecule (23). At

low concentrations of Q (<10 lM) and assuming a diffusion-controlled rate constant (�1010 M)1 s)1) for reaction [6], thelifetime of (Q-H2O)* would exceed 100 ls if reaction [6]

governs the fate of (Q-H2O)*. As such a long lifetime is notexpected for an exciplex and there will be high steric hindranceof bimolecular reactions of (Q-H2O)*, the occurrence of

reaction [6] is highly unlikely. Thus the enolization reaction [5]*Corresponding author email: [email protected] (T. David Waite)� 2007TheAuthors. JournalCompilation.TheAmericanSociety ofPhotobiology 0031-8655/07

Photochemistry and Photobiology, 2007, 83: 904–913

904

is likely to govern the formation of quinone photoproducts atlow Q concentrations.

In the case where BQ is the parent quinone, Q(OH)3 has

then been reported to either react with another Q molecule toproduce one molecule each of hydroquinone (QH2) andhydroxyquinone (QOH) [8], or oxidize in the presence of 3O2

to form QOH only [9] (20). The mechanism of reaction [8] hasnot been described in detail, and may well proceed via a one-electron transfer to initially form Q�� and QOH� radicals [10](23). Reaction [9] is spin restricted (due to the biradicalcharacter of 3O2) and is expected to be slow under theconditions used here. The mechanism of this reaction isunclear at this stage but may occur in two steps via formation

of O��2 and QOH� as suggested earlier (23) and shown inFig. 2.

Thus, the possible intermediate products of the reaction of

Q(OH)3 with Q are Q��, QOH�, QH2 and QOH. Of these,QOH is considered to be relatively stable. The remainingintermediates, due to their radical nature and ⁄or low thermo-

dynamic stability in alkaline oxygenated solutions, undergo avariety of reactions with each other, 3O2 and other principalnon-radical species in solution (namely Q and Q(OH)3) as

shown in Fig. 3 (reactions [11]–[24]), with a number of thesereactions leading to formation of O��2 . The superoxide radicalpossesses an intermediate redox state between dioxygen andH2O2 and therefore undergoes both oxidation and reduction

reactions, with itself and all other major intermediates presentin the solution, ultimately resulting in formation of therelatively stable H2O2. All possible reactions of O��2 are shown

in Fig. 3 ([25]–[28]).The above review shows that quinones undergo a variety of

reactions, finally resulting in the formation of stable QOH and

H2O2 in oxygenated solution. Although Fig.3 represents all

Figure 1. Reaction scheme representing the overall flow of electrons from AQDS to the final products during photolysis. The general class of thestructures and abbreviations are: (A) quinone, Q; (B) semiquinone radical, Q��; (C) hydroquinone, QH2; (D) quinone-hydroxyl adduct, Q�OH�;(E) excited hydrated triplet, ðQ�H2OÞ�; (F) hydroxyquinone, QOH; (G) hydroxyquinone radical, QOH�; (H) hydroxyhydroquinone, QðOHÞ3.

H O O2

O2

Figure 2. Mechanism for production of the various primary interme-diates from photolysis of quinones in aqueous solution.

Figure 3. Various possible reactions of the primary intermediatesformed from quinone photolysis, as shown in Fig. 2.

Photochemistry and Photobiology, 2007, 83 905

possible reactions that can occur between the major interme-diates formed on photolysis of quinones, the feasibility andrelative importance of these reactions depend on the type ofquinone and the particular experimental conditions used

(including pH, quinone concentration, dioxygen concentrationand experimental matrix).

In the present study, we progress our understanding of the

photochemistry of quinones and the related production of ROSby examining the production of superoxide and H2O2 onphotolysis of disodiumanthraquinone-2,6-disulfonate (AQDS).

AQDS has been chosen for our study because of both its highsolubility and its stability toward irreversible side-reactions inalkaline solutions. Overall, the aims of this study are to:

(i) measure the kinetics of superoxide and H2O2 productionfrom irradiation of various concentrations of AQDS incircumneutral, carbonate-buffered solutions;

(ii) verify critical aspects of the mechanism for quinone

photolysis and associated ROS production through semi-quantitative comparative experiments; and

(iii) develop a kinetic model of ROS formation and identifythe importance of the various reactions involved in thephotolysis of AQDS under these conditions.

MATERIALS AND METHODS

Reagents. All reagent solutions were prepared using 18 MWÆcm resis-tivity Milli-Q water unless otherwise stated. All experiments werecarried out in 2 mM NaHCO3 and 0.01 M NaCl solution at pH 8.1(referred to as NaHCO3 ⁄NaCl hereafter). To avoid interference frommetal-catalyzed reactions (10), 15 lM diethylenetriaminepentaacetate(Fluka) was added to the NaHCO3 ⁄NaCl solution and allowed toequilibrate overnight. A solution of 0.1 M HCl for flushing of theexperimental apparatus was prepared from 30% w ⁄w HCl (reagentgrade; Sigma). A 10 mM stock solution of AQDS (>98.0%; Sigma)was prepared daily and stored in the dark at 4�C prior to use.Nitroblue tetrazolium (NBT) used for comparative experiments wasobtained from Sigma.

Xanthine (Sigma) and xanthine oxidase (XO; Sigma), used forcalibration of the superoxide measurements, were prepared as des-cribed in Supplemental Materials. A stock solution of 1 mM H2O2,prepared by dilution of a nominal 30% w ⁄w H2O2 solution (reagentgrade; Univar), was used for calibration of the H2O2 measurements.The nominal 30% w ⁄w solution was standardized by UV spectropho-tometry at 240 nm (24). Stock solutions of Cypridina luciferinmethoxy-analogue (MCLA; Sigma), amplex red (Invitrogen) andhorseradish peroxidase (Sigma) were prepared as described in Supple-mental Materials.

Experimental apparatus. All experiments were carried out in awater-jacketed 1 L glass reactor equipped with a quartz window onone side. The reactor was covered with aluminum foil to avoidinterference from outside light and a gastight lid was fitted to preventgas exchange with the atmosphere. The temperature of the reactionsolution was maintained at 25 ± 0.5�C with a recirculating waterbath. A ThermoOriel 150 W Xe lamp equipped with AM1 direct filterwas used to simulate the solar spectrum at the Earth’s surface. (Whilethis broadband irradiation source may not be ideal for detailedmechanistic insights, use of a narrowband light source would severelylimit the applicability of the results to natural systems and thus is notsuited to the motivation or objectives of this study.) The lamp waspositioned horizontally adjacent to the quartz window, illuminating a5 cm deep cross section of the sample. For superoxide detection,samples were continuously withdrawn from the reactor and loadedinto the sample column of a 10-port injection valve by a peristalticpump operating at 30 rpm. Periodically, samples were automaticallyinjected into a FeLume flow injection chemiluminescence system(Waterville Analytical) for analysis. The valve was configured suchthat the sample volume used was replaced by an equivalent volume ofNaHCO3 ⁄NaCl solution to maintain a constant volume of sample in

the reactor. For H2O2 production, 2 mL of the sample was manuallywithdrawn every 3 min for analysis. For experiments conducted at lowdioxygen concentration, the reaction solution was sparged continu-ously with argon containing 300 p.p.m. CO2 (BOC gases) at a flow rateof 0.5–1 L min)1.

Superoxide determination. Superoxide was measured using theMCLA chemiluminescence method (26), employing 1 lM MCLA in50 mM acetate buffer at pH 6.0 as the chemiluminescence reagent.A 450 lL sample volume was injected into the FeLume system at2 min intervals using a 10-port Valco injection valve. The voltage ofthe photomultipler tube detector was )1200 V. For semi-quantita-tive experiments, 500 mL of 1 lM AQDS solution was photolyzedand sample was pumped continuously through the flow cell for30 min (i.e. the injection valve was omitted). For experimentsperformed at low dioxygen concentration, the MCLA reagentsolution was also sparged with high purity argon (BOC gases) toprevent reaction of dioxygen present in the reagent with quinoneradicals in the sample to produce superoxide, which results in anerror in measurement.

The system was calibrated immediately after the experiment asfollows. Superoxide was generated for calibration purposes in freshlyprepared NaHCO3 ⁄NaCl and AQDS matrix by the xanthine–XOmethod (25). In a previous study (26), the production rate ofsuperoxide by xanthine ⁄XO at pH 8.1 for different concentrationsof xanthine were found to be 27 and 61 pM s)1 for 5 and 50 lM ofxanthine and 1 UÆL)1 XO, respectively. The steady-state concentra-tion of superoxide was calculated as the ratio of production rateand disproportionation rate at pH 8.1. In the calibration systemsuperoxide can reduce AQDS to semiquinone radical (12,27).However, the steady-state concentration of superoxide was unaffec-ted due to regeneration of superoxide via reaction of semiquinoneradical with dioxygen (see Supplemental Materials). The PMT signalassociated with three concentrations of superoxide (generated using0, 5 and 50 lM xanthine), each in duplicate, was determined and acalibration curve constructed by linear regression of calibrationdata.

Hydrogen peroxide determination. Hydrogen peroxide producedfrom photolysis of AQDS was measured by a fluorescence techniqueusing 2.0 lM amplex red (AR) with 1 kUÆL)1 horseradish peroxi-dase (HRP) as the reagent (28). H2O2 in the presence of HRP reactswith amplex red with 1:1 stoichiometry to produce fluorescentresorufin which has absorption and fluorescence emission maxima at563 and 587 nm respectively. For H2O2 measurement, a stocksolution of AR (100 lM) and HRP (50 kUÆL)1) mix was preparedin Milli-Q water and stored at )86�C when not in use. A detaileddescription of the preparation of AR and HRP mix is provided inSupplemental Materials. For measurement of H2O2 generation rate,a 1 L solution of AQDS in NaHCO3 ⁄NaCl was photolyzed and2 mL of sample was removed for analysis every 3 min for 1 h in thepresence of light and, subsequently, for 15 min with the lampextinguished. The loss in sample volume was only 5% during theduration of the experiment and thus did not affect the AQDSphotochemistry significantly.

For semi-quantitative experiments in which the effect of deoxy-genation and NBT addition on H2O2 production was examined,3.5 mL of a solution of AQDS in NaHCO3 ⁄NaCl in a 1 cm quartzcuvette was photolyzed for 5 min. Immediately after photolysis,70 lL of the mixed AR and HRP stock solution was added and thefluorescence was measured. For experiments conducted in theabsence of dioxygen, 3.5 mL of AQDS solution in a quartz cuvettewas sparged continuously for 10 min with high purity argoncontaining 300 p.p.m. CO2. The stopper cap of the cuvette was leftpartially open to allow for escape of dioxygen. After 10 min, thestopper cap was quickly closed and the experiments were performedas described above.

The system was calibrated by standard addition of H2O2 stock to afreshly prepared aliquot of the sample matrix. In general four H2O2

concentrations were determined, and a calibration curve was con-structed by linear regression of the calibration data. For experiments inwhich the effect of NBT and DMSO on H2O2 production wasexamined, calibration was performed in the presence of NBT andDMSO (at all concentrations used in the experiments) to account forany interference in measurement of H2O2 due to the addition of NBTand DMSO (though none was observed).

906 Shikha Garg et al.

In order to ensure that superoxide and H2O2 formation in oursystem was not caused by thermal reactions of AQDS, both superoxideand H2O2 measurements were performed in the dark. Dark productionrates of H2O2 and superoxide were small and contributed less than 5%to the total production rates measured on photolysis of AQDS.

AQDS measurement. The initial and final AQDS concentration inthe reaction solution (before and after 30 min irradiation) wasmeasured by high-performance liquid chromatography (HPLC) usinga 10 min linear gradient elution starting with 15% acetonitrile and85% Milli-Q water and ranging up to 35% acetonitrile and 65%Milli-Q water at a flow rate of 0.7 mL min)1. The decay in AQDS wasmonitored at 325 nm referenced with 800 nm using a diode array UV-VIS spectrophotometer. We chose 325 nm for AQDS measurement inorder to minimize interference from hydroxylated-AQDS (whichexhibits strong absorption bands at 256 and 491 nm [29]). The systemwas calibrated by standard additions of AQDS in NaHCO3 ⁄NaClsolution.

RESULTS AND DISCUSSION

Photo-induced production of superoxide and H2O2 from AQDS

Irradiation of AQDS with simulated sunlight produced nano-molar concentrations of superoxide and H2O2 (Fig. 4). In all

experiments, the superoxide concentration reached a peakafter several minutes and then decreased slightly over time,whereas the H2O2 concentration increased almost linearly over

the duration of irradiation. The rate of H2O2 productionincreased almost linearly with increasing AQDS concentration.The rate of superoxide generation also increased with increas-ing AQDS concentration, but in a more complex, nonlinear

manner.In the dark, superoxide decayed rapidly. The observed

decay rate of superoxide was higher than that expected from

uncatalyzed disproportionation, with the observed half-life afactor of 2.8 smaller than the value calculated using thereported superoxide disproportionation rate constant of

4.8 · 104 M)1 s)1 at pH 8.1 (27). This suggests that there issome additional loss of superoxide due to its scavenging by Qor its photo-products. H2O2 concentrations remained steady in

the dark, suggesting that there are no major decay reactionsfor H2O2 in this system. However, it may be possible thatH2O2 could potentially react with short-lived species that areonly present during irradiation. If this were the case, then the

net rate of H2O2 production should depend on its initialconcentration, as its rate of consumption would increase withincreasing H2O2 concentration, while its rate of production

should be concentration independent. We did not observe anydifference in the net production rate of H2O2 on photolysis of1 lM AQDS when an initial concentration of 100 nM H2O2

was added compared to no added H2O2 (Fig. 5), suggestingthat the interaction of H2O2 with short-lived species is notimportant in our system.

Effect of dioxygen on ROS production from AQDS

In order to test the intermediacy of dioxygen in the productionof ROS, we measured the production of superoxide in argon-sparged solution (Fig. 6). Deoxygenation was found to have

only a slight effect (P > 0.02, using an unpaired single tailedStudent’s t-test) on superoxide generation. However, completeremoval of dioxygen was not achieved, with the presence of

residual dioxygen (�10–30 lM) in the system confirmed bymeasurement of ferrous oxidation kinetics (see SupplementalMaterials for method details).

At low concentrations of Q, (Q-H2O)* most likely enolizesto yield Q(OH)3 [5] which then reacts to form QOH and QH2,

(a)

(b)

Figure 4. Production of (A) superoxide and (B) hydrogen peroxide onphotolysis of 1 lM (closed squares), 2 lM (open squares) and 3 lM(closed circles) AQDS for 1 h followed by 15 min in the dark. Symbolsrepresent experimentally measured values and lines represent modelvalues. Error bars represent the standard deviation of duplicateexperiments.

Figure 5. Linear production rate of hydrogen peroxide on irradiationof 1 lM AQDS with either no H2O2 or 100 nM H2O2 added initially.Error bars represent the standard deviation of triplicate experiments.


either via the intermediate radicals Q�� and ⁄ or QOH� (reac-tions [9] and [10]) or a two electron transfer (reaction [8]).

However in the event that reaction [8] occurs to a significantextent, QH2 will undergo comproportionation with Q to yieldQ�� [20]. Thus the ultimate products formed from the decay of

Q(OH)3 are the radicals Q�� and ⁄or QOH�. At low Qconcentrations in air-saturated solutions, it is highly likelythat the subsequent reactions of Q�� and ⁄ or QOH� withdioxygen to yield superoxide (reactions [17] and [18]) will

dominate as the concentration of dioxygen will be muchgreater than that of any other competing species. In this case,we can write the rate law for superoxide as

d½O��2 �dt

¼ kformation½3O2�½QðOHÞ3� � kdisp½O��2 �2 � kloss½O��2 �

ð1Þ

where kformation is the apparent second-order rate constant forthe reaction between Q(OH)3 and

3O2 and kloss and kdisp rep-

resent the apparent rate constants for first- and second-ordersuperoxide decay.

We can also write the rate law equations for the excited

species Q(OH)3 and3Q* as

d½QðOHÞ3�dt

¼ k5½ðQ�H2OÞ�� kformation½O2�½QðOHÞ3� ð2Þ

d½3Q��dt

¼ k1½Q� � k2½3O2�½3Q�� k3½3Q�� k4½3Q�� ð3Þ

d½ðQ�H2OÞ��dt

¼ k4½3Q�� k5½ðQ�H2OÞ�� k7½ðQ�H2OÞ��

ð4Þ

Assuming that the concentrations of 3Q* and Q(OH3) rapidly

reach steady-state, such that

d½3Q��dt

¼ d½QðOHÞ3�dt

¼ d½ðQ�H2OÞ�

dt¼ 0

on the timescale of superoxide generation, then

½3Q�� ¼ k1½Q�k2½3O2� þ k3 þ k4

ð5Þ

½ðQ�H2OÞ�� ¼k4½3Q��k5 þ k7

½QðOH3Þ� ¼k5½ðQ�H2OÞ��kformation½3O2�

¼ k4k5k1½Q�kformation½3O2� k2½3O2� þ k3 þ k4ð Þðk5 þ k7Þ

ð6Þ

The net superoxide production rate is then given by

d½O��2 �dt

¼ kformation½3O2�

� k4k1k5½Q�kformation½3O2� k2½3O2� þ k3 þ k4ð Þðk5 þ k7Þ� kdisp½O��2 �

2 � kloss½O��2 � ð7Þ

The reaction of the quinone triplet with solvent water toform the exciplex (reaction [4]) is expected to be faster than itsquenching by dioxygen (reaction [2]) or natural decay(k4 k2½O2� þ k3) which simplifies Eq. (7) to

d½O��2 �dt

¼ kformation½3O2�k4k1½Q�kformation½3O2�k4

� kdisp½O��2 �2 � kloss½O��2 �

¼ k1½Q� � kdisp½O��2 �2 � kloss½O��2 � ð8Þ

This is independent of dioxygen concentration providedthere is stoichiometrically sufficient dioxygen for reactions [2],

[17] and [18] to occur, which is the case here as the dioxygenremaining in the system (�10–30 lM) is greater than theconcentration of Q (1–3 lM). It thus appears that, despite

deoxygenation of the solution, superoxide production byreduction of dioxygen could still occur at a comparable rateto that in dioxygen-saturated solution. This result is, therefore,not incompatible with reduction of dioxygen being the primary

source of superoxide in this system.Deoxygenation was also observed to have no significant

effect (P > 0.03, using unpaired single-tailed Student’s t-test)

on H2O2 formation (Fig. 6), which is expected if it is formedprimarily from superoxide disproportionation.

Effect of addition of NBT on H2O2 formation from AQDS

To verify that superoxide is a precursor for H2O2 formation,we measured H2O2 production in the presence of the super-oxide radical trap NBT. Superoxide reacts with NBT toproduce diformazan (30) via a two-step process involving

formation of partially reduced monoformazan as a stableintermediate.

Addition of increasing NBT concentrations decreased

production of H2O2, with nearly complete inhibition ofH2O2 formation on addition of 133 lM NBT (Fig. 7). Thisobservation suggests that superoxide is the major precursor for

H2O2 formation. It is to be noted that other reducing species

Figure 6. Superoxide and hydrogen peroxide generation rates fromphotolysis of 1 lM AQDS in an oxygenated solution in equilibriumwith the atmosphere (open bars) and in a deoxygenated solution thatcontains �10–30 lM dioxygen after sparging with argon (closed bars).Error bars represent the standard deviation of triplicate experiments.


present (e.g. Q�� and QOH�) could also reduce NBT. Inparticular, there may be a competition between NBT anddioxygen to react with Q�� and ⁄ or QOH�, which may affectthe superoxide production rate. Even so, a decrease in H2O2

formation rate due to a decrease in superoxide concentrationfor any reason still supports a mechanism where superoxidedisproportionation is the main source of H2O2 formation.

Thus we conclude that superoxide disproportionation is theprimary source of H2O2 during photolysis of AQDS at pH 8.1.

Relative rates of AQDS degradation and H2O2 production

The rate of degradation of AQDS on irradiation of aqueoussolutions containing different concentrations of AQDS isshown in Fig. 8. The H2O2 production rate under similar

irradiation conditions is also shown. The average ratio ofH2O2 production rate to AQDS decay rate over all AQDS

concentration was 1.29 ± 0.05, which is reasonably close tothe ratio of 1 reported by other investigators (29).

The quantum yields of H2O2 production and AQDS decaywere calculated using

UH2O2¼ PH2O2

depth-integrated absorbed photon flux

¼ PH2O2P

all kIokð1� 10�ekc½Q�‘Þ ð9Þ

and

UAQDS ¼PAQDS

depth-integrated absorbed photon flux

¼ PAQDSP

all kIokð1� 10�ekc½Q�‘Þ

where Iok is lamp intensity in Ein L)1 s)1 nm)1, �kc is molarabsorptivity of AQDS in M)1 cm)1 , PH2O2

is H2O2 productionrate, PAQDS is the AQDS decay rate and ‘ ¼ 5cm is the

pathlength. The calculated quantum yields for AQDS decayand H2O2 production are shown in Fig. 8. The quantum yieldsof both H2O2 production and AQDS decay decrease with

increasing initial quinone concentration in a nonlinear man-ner, suggesting the involvement of bimolecular reaction ofAQDS in H2O2 production.

As in our system the decay of (Q-H2O)* occurs principally

via the enolization reaction, three possible pathways forformation of H2O2 by reduction of dioxygen may be envis-aged. In mechanism I, Q(OH)3 oxidizes in the presence of

oxygen to form QOH [9]. In this case, for each mole of Qconsumed, one mole of both QOH and H2O2 is produced, i.e.

Mechanism I:

Q�!hm Q� �!H2OðQ�H2OÞ� �! QðOHÞ3�!3O2

O��2 þQOH� ð10Þ

QOH� þ 3O2 �! QOHþO��2 ð11Þ

O��2 þO��2 �! H2O2 þ 3O2 ð12Þ

In mechanism II, Q(OH)3 reacts with Q to yield Q��andQOH� (reaction [10]). In this case, for each mole of Q

consumed, one mole each of QOH and H2O2 is produced, i.e.Mechanism II:

Q�!hm Q� �!H2OðQ�H2OÞ� �! QðOHÞ3�!Q

Q�� þQOH� ð13Þ

Q�� þ 3O2 �! QþO��2 ð14Þ

QOH� þ 3O2 �! QOHþO��2 ð15Þ

O��2 þO��2 �! H2O2 þ 3O2 ð16Þ

In mechanism III, Q(OH)3 reacts with Q to produce QOHand QH2 (reaction [8]), i.e. Mechanism III:

Figure 7. Effect of NBT addition on hydrogen peroxide concentrationafter photolysis of 1 lM AQDS for 5 min. Error bars represent thestandard deviation of triplicate experiments. Calibration of hydrogenperoxide measurements was performed in the presence of NBT toaccount for any interference from NBT in the measurement technique.

Figure 8. Rates of AQDS decay (open squares) and hydrogen perox-ide production (closed squares), and quantum yields of AQDS decay(open circles) and hydrogen peroxide production (closed circles) onirradiation of 0.5 L aqueous solutions of various concentrations ofAQDS at pH 8.1 for 30 min.


Q�!hm Q� �!H2OðQ�H2OÞ� �! QðOHÞ3�!Q

QOHþQH2 ð17Þ

QH2 is unstable in oxygenated alkaline solution and oxidizes

rapidly to yield Q and H2O2 with stoichiometry of QH2 con-sumption, Q production and H2O2 production of 1:1:1 (31).

Thus, the theoretical ratio of H2O2 produced per AQDS

consumed on photolysis is unity irrespective of the mechanism.Our measured ratio of H2O2 production rate and Q degrada-tion rate is close to unity and thus is in reasonable agreement

with the theoretical value. A slightly lower decay rate ofAQDS relative to H2O2 production rate may possibly occurdue to a slight error in AQDS measurement as a result ofinterference from other quinone derivatives present that may

not have been separated completely by HPLC.Based on our experimental data we are not able to

distinguish between mechanisms I, II and III, as the theoretical

yield of the major products and their stoichiometry is similar inall cases. However, we can reject mechanism I as the oxidationof Q(OH)3 by triplet dioxygen is expected to be very slow as

the reaction is spin forbidden. The only difference between theother two mechanisms is the formation of the hydroxyquinoneradical in mechanism II but not in mechanism III. In an earlierinvestigation of the photochemistry of AQDS (19), formation

of the hydroxyquinone radical as the precursor of hydroxy-quinone formation was reported, which supports mechanismII. Thus, in the absence of any further evidence, we have

modeled our experimental data using mechanism II (see below)though it is expected that similar results would be obtained viamechanism III.

Effect of DMSO on ROS production

Addition of DMSO increased the production of both super-oxide and H2O2 during photolysis of AQDS (Fig. 9). This

observation is in accordance with a recent study (14) thatfound conversion of dioxygen to H2O2 (via superoxideformation) on photolysis of 9,10-anthraquinone and itsderivatives increases in the presence of an electron donor. In

an irradiated solution of quinone, DMSO can react with either

(i) the excited triplet, resulting in physical quenching of theexcited triplet quinone (17):

3Q� þDMSO �! Q ð18Þ

(ii) the exciplex (Q-H2O)* to yield Q�� (18,20):

ðQ�H2OÞ� þDMSO �! Q�� þ CH�3 þ CH3SOðOHÞ þHþ

ð19Þ

(iii) the excited triplet, leading to the formation of semi-quinone radicals and methyl radicals (32):

3Q� þDMSO �! Q�� þ CH�3 þ CH3SO ð20Þ

Possibility (i) would clearly produce less ROS in thepresence of DMSO due to a lower yield of dioxygen-reducing

radicals, and thus is not supported by our experimentalobservations. Possibilities (ii) and (iii) are both consistent withour experimental results as 3Q* interacts primarily with H2O to

form (Q-H2O)* and so the apparent formation rates of both3Q* and (Q-H2O)* are approximately equal.

As discussed above, in the absence of DMSO, superoxide

and H2O2 formation occurs via reduction of dioxygen by thereducing radicals Q�� and QOH� which are formed from(Q-H2O)*. An increase in superoxide and H2O2 production

rates on addition of DMSO therefore implies that theproduction rate of dioxygen-reducing radicals increases inthe presence of DMSO. This occurs for the followingreasons:

(i) catalytic production of both superoxide and H2O2

results from oxidation of DMSO, with the quinonebeing continuously cycled between reduced (Q��) andoxidized (Q) forms without formation of stable QOH.

This is in agreement with the measured decay rate ofAQDS, which decreases in the presence of DMSO(Fig. 10).

(ii) the efficiency of conversion of 3Q� to dioxygen-reducing

radicals increases in the presence of DMSO due to thepreferential reaction of 3Q� (or [Q-H2O]*) with DMSOover non-radical producing pathways, such as quench-ing or natural decay.

The latter argument suggests that as the DMSO concentra-

tion increases, the efficiency of conversion of 3Q� to dioxygen-reducing radicals will approach unity. This is in agreement withmeasured values of PH2O2

(Fig. 10), which show an increase in

PH2O2with increasing DMSO concentration towards an asymp-

totic value.At the asymptote, all 3Q� is effectively converted intodioxygen-reducing radicals by reaction with DMSO. Further-more, the rate of AQDS decay approaches zero, implying that

the steady-state concentration of dioxygen reducing radicals isextremely low, and therefore PH2O2

must approach the rate atwhich 3Q� is being converted to dioxygen-reducing radicals.

Based on the measured value of PH2O2, we have therefore

calculated a value for the first-order rate constant for 3Q�

formation in the system of k01 ¼ 4� 10�3s�1.

Kinetic model for ROS production from photolysis of AQDS

The kinetics of generation of superoxide and H2O2 fromphotolysis of AQDS by simulated sunlight has been modeled

Figure 9. Superoxide and hydrogen peroxide generation in the absence(open bars) and presence (closed bars) of DMSO. DMSO was added atconcentrations of 50 and 141 lM for experiments measuring hydrogenperoxide and superoxide, respectively.


according to the reactions and rate constants listed in Table 1.Model fits to experimental data are shown in Figs. 4 and 10.Note that reaction [1] in this model represents the apparent

rate of formation of 3Q*, incorporating the forward excitationstep of Q and the backward natural decay of the 3Q*. The rateconstant for this reaction, represented by k01, was determined

in the previous section. Reaction [2] is the quenching of AQDStriplet by triplet dioxygen, with a second-order rate constant of2 · 108 M)1 s)1 (33). The rate constant for formation of(Q-H2O)* by interaction of 3Q* with water (reaction [4]) is

based on an earlier reported pseudo-first-order rate constant of7 · 105 s)1 for formation of a quinone-water intermediate(represented as ‘‘transient C’’ in that study) by reaction of

anthraquinone sulphonate (AQS) triplet with water (34).The fate of (Q-H2O)* is governed either by the enolization

reaction (reaction [5]) or its relaxation to the ground state

quinone (reaction [7]). A rate constant of 1 · 106 s)1 was usedfor relaxation of a hydroxylating intermediate which is close toreported value of 2.8 · 106 s)1 in AQS system (34). This rate

constant was chosen such that the lifetime of (Q-H2O)* isabout 5 ls as reported earlier (19). A rate constant of

4.5 · 104 s)1 was used for the enolization reaction based onbest fit to our experimental data. As described above, it is

proposed that (Q-H2O)* formed via reaction [4] reduces Q toyield radicals Q�� and QOH� (reaction [6]). The rate offormation of superoxide and H2O2 is insensitive to the value

chosen for the second-order rate constant for this reactionprovided the rate constant is greater than 1 · 105 M)1 s)1. Theradicals Q�� and QOH� formed in reaction [6] quickly reducedioxygen to form superoxide. The rate constants for these

electron transfer reactions are generally very fast (close tothe diffusion-controlled limit) and are inversely related tothe reduction potential of Q (35). A rate constant of

5 · 108 M)1 s)1 for reaction [17] was reported earlier forAQDS (36) and is used in our model. In an earlier study (23)with NQ, it was suggested that addition of an hydroxy group

to the adjacent benzene ring decreases the rate constant forreaction of Q�� with dioxygen by 1 order of magnitude andthus we have used a rate constant of 5 · 107 M)1 s)1 for

reaction [18]; however, the rate of production of superoxideand H2O2 production is insensitive to this rate constant as longas the process is fast. In addition to oxidation in the presenceof dioxygen, Q�� and QOH� may react either with Q or with

themselves to yield QH2 and QOH with a rate constant for thedisproportionation reactions on the order of 5 · 107 M)1 s)1

(35). In our system the dioxygen concentration is much higher

than the quinone concentration, so the oxidation reaction willoutcompete the reaction of Q�� ⁄QOH� with either Q or itself;thus these reactions are not included in our model.

The superoxide formed in reactions [17] and [18] eitherdisproportionates to yield H2O2 (reaction [28]) or reduces Q toQ�� (reaction [27]). It was previously suggested that k17 andk27 vary between different quinones in such a way that

k17 · k27 = 2 · 1015 M)1 s)1 (35). Based on this relationshipwe have calculated the rate constant k27 from the published

(a)

(b)

Figure 10. Effect of increasing DMSO concentration on: (A) hydrogenperoxide generation rate from irradiation of 1 L aqueous solutions of1 lM AQDS; and (B) quinone decay rate from irradiation of 1 Laqueous solutions of 5 lM AQDS. Symbols represent experimentallymeasured rates and solid lines represent model predicted rates. Errorbars represent standard deviation of duplicate experiments.

Table 1. Proposed kinetic model for production of ROS from photo-lysis of AQDS.

†First-order rate constant reported (34) for AQS in aqueous solution.‡Rate of this reaction is insensitive to the model output as long theprocess is fast. §Reported values (27) vary from 9 · 108 M)1 s)1 for1,4-benzoquinone to 4 · 106 M)1 s)1 for 2,3-dimethyl naphthoqui-none.


value for k17. The fact that the calculated value of k27 is lowerthan the reported values for BQ and NQ (27) is congruous

with the standard reduction potential of AQDS being less thanthose of BQ and NQ (37).

The rate constants for the majority of these reactions are pH

dependent, as a number of the reactants occur in bothprotonated and deprotonated forms. The literature values ofthe rate constants used in our model are generally reportedaround neutral pH (7–7.4). However as the pKa of the critical

quinone and oxygen radicals in our system are significantlybelow 7 [for Q��=QH�, pKa = 3.2 (19) and for O��2 =HO�2,pKa = 4.7 (27)], these rate constants are likely to be applicable

to our pH 8.1 system, as there will be no remarkable change inspeciation of these quinone and oxygen radicals due to changein pH from 7 to 8.1.

The simple model proposed here provides an excellent fit toour experimental data and is consistent with the observationsof our semi-quantitative experiments. The model shows noeffect of dioxygen removal (up to 95%) on peak superoxide

and H2O2 concentrations (Table 2). Moreover, the modelcorrectly predicts the effect of DMSO addition on H2O2

production rate. We were not able to determine from modeling

alone whether DMSO reacts with 3Q* or (Q-H2O)*, as bothreactions produced a reasonable fit to our experimental data.A model rate constant of 1 · 109 M)1 s)1 was used for

reaction of 3Q* with DMSO, which is close to the value of2 · 109 M)1 s)1 reported earlier (32). The slight discrepancybetween our model value and the reported rate constant

probably occurs due to the difference in the experimentalmatrix in the two studies. If (Q-H2O)*, rather than 3Q*, reactswith DMSO, a rate constant of 4.5 · 109 M)1 s)1 was deter-mined based on the best fit to our experimental data. The

model predicted decay rates of AQDS are slightly higher thanthe measured rates, which may again be due to the likelihoodof a small error in determination of AQDS resulting from

interference of related quinone species in the HPLC method.

CONCLUSIONS

In this study, we have shown that quinones are capable ofproducing ROS on photolysis. The production of ROS mainlyoccurs via reduction of dioxygen, however the electrons for

this process are derived for the most part from water

molecules. The model proposed in this work correctly predictsthe ROS production rate from photolysis of AQDS over arange of conditions in the absence of trace metals such as ironand copper. These metals are important scavengers of ROS

and may also affect the transformation of the quinone. Futurestudies will focus on coupling the insights gained into thegeneration of ROS on photolysis of quinones that we have

developed here with previously reported models describing theinterplay between redox active metals, oxygen and its reduc-tion products. We believe that such analyses will assist in

developing a greater understanding of photochemical proces-ses occurring in natural waters and their impact on both traceelement redox transformations and ROS generation.

SUPPLEMENTAL MATERIALS

The following supplemental materials are available for this

article:PART A: Preparation of reagents.PART B: Confirmation that the presence of AQDS does not

affect the steady state concentration of superoxide in calibra-tion solutions.PART C: Detection of oxygen in the reaction solution.

This material is available as part of the online art-

icle from: http://www.blackwell-synergy.com/doi/full/10.1111 ⁄j.1751-1097.2007.00075.x

Please note: Blackwell Publishing are not responsible for the

content or functionality of any supplementary materials sup-plied by the authors. Any queries (other than missing material)should be directed to the corresponding author for the article.

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Table 2. Measured and model predicted effect of deoxygenation onROS production rate.

ROS

% decrease in ROSproduction ondeoxygenation Dioxygen

concentration usedin modeling (lM)*Measured Model-predicted

Superoxide 15 15 1H2O2 )4 )4 10

*Due to the difference in the experimental setup, the dioxygen levelsare different in the two cases. For superoxide measurement, 0.5 L ofAQDS solution was sparged with argon prior to and during irradi-ation. The MCLA solution was also sparged continuously. For H2O2

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Documents

Production of Reactive Oxygen Species on Photolysis of Dilute Aqueous Quinone Solutions