P2

EMa

b

Ac

d

ARRAA

K2PESNF

1

it(ftatrmbni

b

1h

Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40

Contents lists available at SciVerse ScienceDirect

Journal of Photochemistry and Photobiology A:Chemistry

journa l h om epa ge: www.elsev ier .com/ locate / jphotochem

hotophysical characterization of the plant growth regulator-(1-naphthyl) acetamide

liana Sousa Da Silvaa,b,∗, Pascal Wong-Wah-Chungb,c,d,ohamed Sarakhab,d, Hugh D. Burrowsa,∗

Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, PortugalClermont Université, Université Blaise Pascal, Institut Chimie de Clermont Ferrand (ICCF), UMR CNRS 6296, Équipe Photochimie, BP 80026, F-63171ubière Cedex, FranceClermont Université, ENSCCF, BP 10448, F-63000 Clermont-Ferrand, FranceCNRS, UMR 6505, F-63173 Aubière, France

a r t i c l e i n f o

rticle history:eceived 3 March 2013eceived in revised form 20 May 2013ccepted 22 May 2013vailable online 30 May 2013

eywords:-(1-Naphthyl) acetamideesticidesxcited statesolvent polarity

a b s t r a c t

The photophysical properties of the widely used plant growth regulator 2-(1-naphthyl) acetamide(NAD) were studied in water and representative organic solvents (ethanol, ethylene glycol, acetonitrile,chloroform, 1,4-dioxane) employing steady-state and time-resolved spectroscopy. Quantum yields andlifetimes of fluorescence, phosphorescence and triplet formation and triplet–triplet absorption spectrawere obtained. From these, all radiative and radiationless rate constants have been determined, togetherwith singlet and triplet excited state energies (4.00 and 2.69 eV, respectively). The fluorescence quan-tum yield and lifetime increased on going from water (˚F 0.066, �F 35.0 ns) to non-hydrogen bondingsolvents (˚F 0.357, �F 51.0 ns in 1,4-dioxane), probably due to decreased internal conversion. Fluores-cence was quenched by several anions through an electron transfer process. A limit on the reductionpotential of 1NAD* of E0 2.1 ± 0.2 V was estimated. The attribution of the transient absorption seen in

3

anosecond laser flash photolysisluorescence

nanosecond laser flash photolysis to NAD* was confirmed by energy transfer and oxygen quenching.Quenching of triplet states leads to singlet oxygen formation, with quantum yields varying from 0.097in water to 0.396 in chloroform. However, these are lower than the triplet state quantum yields, par-ticularly in water (˚T 0.424), indicating competing quenching pathways, probably involving electrontransfer. The relevance of these results to the photoreactivity of NAD under environmental conditions

is discussed.

. Introduction

Naphthalene derivatives are of environmental and biologicalnterest, and have widespread application in fields such as agricul-ure, pharmaceutics and industry. 2-(1-Naphthyl) acetamide (NAD)Fig. 1) is a synthetic plant growth regulator whose behavior resultsrom its structural similarity to the natural plant growth regula-or hormone indole auxin [1]. It has been used for many years ingriculture to promote the growth of numerous fruits, for root cut-ings and as a fruit thinning agent [2,3]. However, NAD and itsesidues have been the object of studies by the European Com-ission and other regulatory bodies, and a risk assessment has

een presented [4]. In addition, analytical methods such as lumi-escence [5,6] and chromatography [7,8] have been developed for

ts quantification in food residues and water. According to the U.S.

∗ Corresponding authors. Tel.: +351 239 854482; fax: +351 239 827703.E-mail addresses: elianas@qui.uc.pt (E.S. Da Silva),

urrows@ci.uc.pt (H.D. Burrows).

010-6030/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jphotochem.2013.05.009

© 2013 Elsevier B.V. All rights reserved.

Environmental Protection Agency (EPA) approximately 20,000 lbs(9000 kg) of naphthalene acetate active ingredients such as NADand 1-naphthyl acetic acid (1-NAA), are applied annually in theUSA [9].

Unfortunately, in contrast with the full photophysical charac-terization available for naphthalene [10], there is a lack of detailedinformation in the literature on the excited state properties ofNAD. Detailed study of photophysical properties of such moleculesis of fundamental importance [11], normally requiring informa-tion on the effect of solvents on the ground and excited states ofmolecules [12–14], and, in particular, how such interactions affectthe nature and energy of the electronic excited states. This is par-ticularly relevant to environmentally relevant compounds, such asNAD, as their photodegradation may take place both in water andin the more hydrophobic environments occurring in plants andsoils. Changing solvent polarity and hydrogen bond ability with

these systems has been shown to lead to shifts in electronic absorp-tion and luminescence spectra [15–20]. In addition, although morelimited information is available, they may also cause changes intriplet excited state behavior [21,22].

30 E.S. Da Silva et al. / Journal of Photochemistry and

200 22 5 25 0 27 5 30 0 32 5 35 0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

O

NH2

Ab

so

rba

nc

e

Wavelengt h/nm

Fi

dcpspitcuos(ctavdhstNird

2

2

ptreSP

wps

d

ig. 1. UV absorption spectrum of aqueous NAD solution (1.0 × 10−4 mol L−1) andts chemical structure.

We have recently reported a detailed study of the photodegra-ation of NAD, both under UV light and simulated solar irradiationonditions [23], including identification of major degradationroducts and toxicity studies. The primary photoproducts lead to aignificant increase in toxicity, which subsequently decreases uponrolonged irradiation. Mechanistic studies of the effect of oxygen

ndicate involvement of both triplet and singlet excited states inhe photoreactivity of NAD, together with a major role of the radi-al cation. However, the lack of photophysical data on NAD limitsnderstanding its photoreactivity. To rectify this, we have carriedut a detailed study, involving complete characterization of excitedtates, including the effects of solvent properties such as polaritydielectric constant), hydrogen bonding, refractive index and vis-osity on the NAD ground and excited state behavior. Solvents ofhree different types were selected: water, ethanol, ethylene glycolnd chloroform were used as polar hydrogen-bond donating sol-ents, acetonitrile was used as polar aprotic non-hydrogen-bondonating solvent and 1,4-dioxane was used as a non-polar and non-ydrogen-bond donating solvent. Detailed results are presented oninglet and triplet state properties and of the overall set of deac-ivation rate constants: kF, kP, kic and kisc. Quenching studies ofAD excited states by oxygen and of fluorescence by a variety of

norganic anions have also been studied to obtain insights into theelative importance of electron and energy transfer in excited stateeactivation.

. Materials and methods

.1. Materials

2-(1-Naphthyl) acetamide (NAD), biphenyl, benzophenone andhenalenone (97%) were purchased from Sigma–Aldrich and naph-halene from J.K. Baker Chemical Co. All products were used aseceived. Acetonitrile was purchased from Merck, ethanol, ethyl-ne glycol, deuterium oxide, methylcyclohexane and glycerol fromigma–Aldrich, and 1,4-dioxane, chloroform and cyclohexane fromanreac. All solvents were spectroscopic grade.

An aqueous NAD stock solution was prepared with deionisedater (Millipore Milli-Q; resistivity of 18.2 M� cm−1), at naturalH. Fresh daily solutions were prepared from dilution of this stock

olution. Solutions in organic solvents were prepared daily.

Samples were bubbled for 20 min with argon and oxygen to havee-aerated and oxygenated samples, respectively.

Photobiology A: Chemistry 265 (2013) 29– 40

2.2. Absorption and fluorescence studies

UV–visible absorption spectra measurements were acquiredeither on a Cary 3 double-beam (Varian) or on a Shimadzu UV-2010 double-beam spectrometer with 1 cm quartz cuvettes overthe range 200–800 nm.

Corrected steady state fluorescence emission and excita-tion spectra were measured using a 1 cm quartz cuvette ona Horiba–Jobin–Yvon SPEX Fluorolog 3–22 spectrofluorometerequipped with a 300 W xenon lamp. Fluorescence emission andexcitation spectra were registered at absorption and emissionmaxima wavelength, respectively, with 1 mm slits. Fluorescencequantum yields were measured using naphthalene in ethanol asstandard (˚F = 0.21) [10], according to Eq. (1):

˚F,S = ˚F,RISARn2

S

IRASn2R

fSfR

(1)

where

fS =ISN2

ISO2

and fR =IRN2

IRO2

˚F represents the fluorescence quantum yield, I is the integratedarea under the respective fluorescence emission spectra, A is therespective absorbance, n is the refractive index of the solvents usedand f represents the de-aerating factor, which is given by the ratioof the integrated area under the respective fluorescence spectrain absence and presence of oxygen, for the sample and the ref-erence. The subscripts S and R refer to the sample and reference,respectively. Both the sample and reference were excited at thesame wavelength. The absorbance of the solutions at the excitationwavelength was maintained around 0.1 and 1 mm slits were used.

The energy of singlet state, ES, was determined from the cross-ing point of the normalized absorption and fluorescence emissionspectra.

Fluorescence lifetimes, �F, were measured using the methodof time correlated single photon counting (TCSPC) with a Horiba-JI-IBH NanoLED (282 nm) as excitation source, a Philips XP2020Qphotomultiplier, and Canberra instruments TAC and MCA. Mea-surements were performed in de-aerated samples at the maximumemission wavelength. The fluorescence decays were analyzed usingthe modulating functions method of Striker [24] with automaticcorrection for the photomultiplier “wavelength shift”. The decaywas fitted by using FluoFit Pro version 4.

Fluorescence quenching studies were performed in water byadding different concentrations (range 10−4 to 10−2 mol L−1)of aqueous KBr, KI, KCl, KSCN, NaN3 solutions, and in ace-tonitrile using triethylamine solution in the same solvent, toNAD solution at a constant concentration (1.0 × 10−5 mol L−1).Stern–Volmer plots were constructed from relative integrated flu-orescence emission intensities and the Stern–Volmer quenchingcoefficients, KSV, were obtained by linear regression according toexpression (2):

I0I

= 1 − KSV [Q ] (2)

where I0 and I are the fluorescence intensities in absence andpresence of quencher, respectively, [Q] represents the quencherconcentration and the slope of the curve is equal to the Stern–Volmer constant KSV.

2.3. Phosphorescence studies

The phosphorescence measurements used the Horiba–Jobin–Ivon SPEX Fluorolog 3-22 spectrofluorometer equipped with a Spex1934D phosphorimeter accessory having pulsed excitation lamp

y and

acfog4

atbc

˚

wits

2l

nt3∼tasdl

ttte

e(Apmm

oaoaal(

�

wett

tm

E.S. Da Silva et al. / Journal of Photochemistr

nd appropriate software. Phosphorescence studies of NAD werearried out in ethanol, acetonitrile and ethylene glycol solutionsrozen at 77 K in order to avoid quenching by molecular oxygen orther impurities dissolved in these solvents. The NAD containinglass was excited at the maximum absorption wavelength. Slits of.0 mm were used for excitation and emission measurements.

The emission and excitation spectra were recorded as wells decay studies in order to determine phosphorescence life-imes. Phosphorescence quantum yields were determined usingenzophenone in ethanol as reference (˚P = 0.74) [10], and are cal-ulated using the following equation:

P,S = ˚P,RISAR

IRAS(3)

here ˚P represents the phosphorescence quantum yield, I is thentegrated area under the respective phosphorescence spectra, A ishe respective absorbance and the subscripts S and R refer to theample and reference, respectively.

.4. Transient absorption spectra, triplet absorption coefficient,ifetimes and quantum yields

Transient absorption experiments were carried out on aanosecond laser flash photolysis spectrometer from Applied Pho-ophysics (LKS 60). Excitation was achieved at 266 nm and/or55 nm from the Quanta Ray GCR130-1 Nd:YAG laser (pulse width5 ns). The transient absorbance was monitored by a detection sys-

em consisting in a pulsed xenon lamp (150 W), monochromatornd a IP28 photomultiplier Housing (Applied Photophysics). Theignal from the photomultiplier was digitized by a programmableigital oscilloscope TDS 30528 (Tektronix) and the signal was ana-

yzed with a 32-bit RISC-processor kinetic spectrometer.Triplet lifetimes, �T, were calculated from kinetic analysis of the

ransient decays. The rate constants for oxygen quenching of theriplet state kO2

T were determined by measuring the decay of theriplet–triplet absorption at the absorption maximum for NAD inach solvent in the presence and absence of air.

The triplet absorption coefficient, εT, was determined by thenergy transfer method [25] using benzophenone in acetonitrileε520 nm = 6500 L mol−1 cm−1; ˚T = 1.0) [10] as triplet energy donor.ll solutions were de-aerated by purging with argon, and sealedrior to measurement. The excitation wavelength was 355 nm. Theolar triplet–triplet absorption coefficients [26] were then deter-ined from Eq. (4):

εDTT

εATT

= �ODD

�ODA(4)

where εDTT and εA

TT are the triplet molar absorption coefficientsf donor and acceptor, respectively, �ODD is the maximumbsorbance from the transient triplet–triplet absorption spectraf the donor in the absence of acceptor; �ODA is the maximumbsorbance of the acceptor triplet when the acceptor and donorre present. When the acceptor decay rate constant (k3) is not neg-igible, a correction is necessary to calculate the �ODA, given by Eq.5):

ODAobs = �ODA exp

[− ln(k2/k3)

k2/k3 − 1

](5)

here k2 is the decay rate constant of the donor in the pres-nce of the acceptor and �ODA

obs is the maximum obtained in theriplet–singlet difference spectra of the acceptor in the presence of

he donor.

The triplet quantum yields of NAD, ˚T, were obtained byhe comparative actinometry method using naphthalene in

ethylcyclohexane as reference (ε = 13,200 L mol−1 cm−1;

Photobiology A: Chemistry 265 (2013) 29– 40 31

˚T = 0.75) [10]. Optically matched samples of NAD and naph-thalene around 0.2 at the excitation wavelength were irradiatedwith the 266 nm laser in the nitrogen-saturated solvent. Thequantum yield of triplet formation of NAD is given according toEq. (6):

˚ST = εnapththalene

TT

εSTT

�ODSmax

�ODnapththalenemax

˚napththaleneT (6)

where S and R refer to the sample and reference, respectively. Allmeasurements were conducted at room temperature.

2.5. Singlet oxygen measurements

Singlet oxygen quantum yield, ˚�, was detected using time-resolved phosphorescence [27] by monitoring room temperaturephosphorescence decay centered at 1270 nm using a HamamatsuR5509-42 photomultiplier, cooled at 193 K in a liquid nitrogenchamber, following laser excitation of aerated NAD solutions indifferent solvents at 266 nm, with an Applied Photophysics flashkinetic spectrometer. Decays were then extrapolated to time zeroand the initial phosphorescence signal studied as a function oflaser intensity. This was then compared with a standard, biphenylin cyclohexane (˚� = 0.73) [28], excited at 266 nm. Studies werecarried out in optically matched solutions (ABS = 0.2) in the abovesolvents. The singlet oxygen quantum yield sensitized by NAD wascalculated from Eq. (7):

˚S� = IS

IR= ABSR

ABSS

n2S

n2R

˚R� (7)

where I is the intensity of the phosphorescence decay measured at1270 nm, ABS is the absorbance of the solutions at 266 nm, n is therefractive index for each solvent, ˚� is the singlet oxygen quantumyield and S and R represent sample and reference, respectively. Wenote that the radiative lifetime of singlet oxygen shows a solventdependence [29]. However, although the use of different solventsfor sample and reference in these measurements may increase theuncertainty of the singlet oxygen yield in water, we do not feelthe error is significant, and in the absence of a reliable singlet oxy-gen standard for measurements in water with excitation at 266 nmprefer to use a well characterized standard.

Photosensitized formation of singlet oxygen, 1O2, was achievedby using phenalenone as sensitizer in chloroform (˚� = 0.98) [30].Aerated solutions of phenalenone with constant concentration(3.0 × 10−5 mol L−1) were excited with a pulse laser at 355 nmin the presence of NAD concentrations ranging from 1.0 × 10−4

to 1.0 × 10−2 mol L−1. The singlet oxygen phosphorescence decaywas measured at 1270 nm for each solution and by studying thepseudo-first-order rate constant as a function of quencher concen-tration it was possible to obtain the quenching rate constant of 1O2by NAD.

3. Results and discussion

To simplify the presentation in this paper, all the figures pre-sented in the text will refer to NAD in water, which is of mostinterest for environmental considerations. However, results aregiven for all solvents in the tables.

3.1. Absorption spectroscopy

The UV absorption spectrum of NAD was recorded in water,

D2O, ethanol, ethylene glycol, acetonitrile, chloroform and in 1,4-dioxane. As shown in Fig. 1, the UV absorption spectrum of NAD inaqueous solution presents two main bands: the short wavelengthabsorption has a maximum around 223 nm while the lowest energy

32 E.S. Da Silva et al. / Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40

Table 1Maximum absorption wavelength (�max) and molar absorption coefficient (ε), for NAD singlet ground state as function of solvent polarity. The refractive index (nD), [10]dielectric constant (�) [10] and viscosity () [10] of solvents are given for better clarity of discussion.

Solvent Abs. (�max/nm) εa (L mol−1 cm−1) nD � (×10−3 Pa s)

Water 280 6540 1.333 80.1 1.00D2O 280 – 1.328 – –Ethylene glycol 283 6443 1.431 37.7 19.9Ethanol 282 7477 1.361 24.5 1.20Acetonitrile 282 7090 1.344 35.9 0.34Chloroform 283 4740 1.445 4.81 0.581,4-Dioxane 283 7250 1.422 2.20 1.43

a Estimated errors ±5%.

Table 2Maximum emission wavelength (�max), Stokes shift, singlet excited state energy (ES), determined fluorescence quantum yields (˚F) and measured lifetimes (�F), chi squaredfactor (2) and calculated radiative rate constant (kR = ˚F/�F) for the emission decay of NAD in water, D2O and organic solvents.

Solvent Em. (�max/nm) Stokes shift (nm) ES (eV) ˚Fa �F (ns) 2 kR (×106/s−1)

Water 324 44.0 3.99 0.066 35.0 1.09 1.89D2O 324 44.0 3.99 0.068 34.1 1.05 1.99Ethylene glycol 325 42.0 3.99 0.096 52.9 0.98 1.81Ethanol 325 43.0 4.09 0.096 49.0 1.05 1.96Acetonitrile 325 43.0 4.00 0.186 54.0 1.08 3.44

4.093.95

bsa

valaatNtε(Afsc

3e

st(TeeahflswaworsNm

non-hydrogen bonding solvents. These values are similar to thosereported in literature for naphthalene derivatives. For instance,Tamaki et al. reported a quantum yield of 0.14 for naphthyl aceticacid derivatives in acetonitrile [34]. The fact that the fluorescence

260 28 0 30 0 32 0 34 0 36 0 38 0 40 00.00

0.15

0.30

0.45

0.60

0.75

0.90

1.05

No

rma

lize

d i

nte

ns

ity

/a.u

.

Chloroform 325 42.0

1,4-Dioxane 326 43.0

a Estimated errors ±10%.

and within the wavelength range 250–320 nm shows vibrationaltructure with maximum centered at 280 nm. Both of these bandsre characteristic of �–�* transitions of the aromatic ring.

Neither the shape of the low energy absorption band nor itsibronic structure was influenced by change in solvent. However,

slight red shift was observed in the absorption maximum wave-ength when passing from water to a non-polar organic solvent,s can be seen from the results presented in Table 1. This is ingreement with the polarizing effect of the solvent medium onhe �–�* transition [20,31,19]. The molar absorption coefficients ofAD, ε, were determined for each solvent at the maximum absorp-

ion wavelength band, and the results are reported in Table 1. The2 8 0 value in water is in good agreement with previous reports6747 L mol−1 cm−1) [4], and is a typical value for a �–�* transition.

slight increase in ε with decrease of solvent polarity was observedor all solvents except chloroform. The low value obtained in thisolvent, 4740 L mol−1 cm−1, may well be associated with contactharge transfer contributions in this medium [32].

.2. Fluorescence spectra, quantum yields, lifetimes and singletxcited state energy

NAD fluorescence emission and excitation spectra were mea-ured in water, D2O and in the studied organic solvents. Fig. 2 showshe fluorescence emission (�ex = 280 nm) and excitation spectra�em = 324 nm) of aqueous NAD solution (1.0 × 10−5 mol L−1) whileable 2 summarizes the results in all solvents. The fluorescencemission spectrum shows a broad band with vibronic structurextending from 300 to 400 nm. In aqueous solution, two maximat 324 and 338 nm were observed together with two shoulders atigher wavelengths. This is consistent with previous reports on theuorescence spectra of NAD [33]. For all the solvents, the emissionpectra were not affected when excitation was made at differentavelengths, confirming its origin from a single emitting species

nd that NAD does not contain any fluorescent impurities. No effectas observed on the shape of the emission or excitation spectra

n going from H2O to the other solvents, although, as seen in the

esults presented in Table 2, there is a slight red shift in the emis-ion maximum wavelength with solvent polarity. In all solvents,AD excitation and absorption spectra were identical, and wereirror images of the emission spectrum indicating that there is no

0.022 6.77 1.10 3.25 0.357 51.0 1.01 7.00

significant change in the structure of NAD chromophore betweenthe ground and excited state.

The Stokes shift data, given by the difference between the maxi-mum peak of normalized absorption and emission spectra, and theenergy of the lowest singlet excited state, ES, which is estimatedfrom the intersection of the normalized absorption and emissionspectra, are also included in Table 2. No significant changes areobserved on the Stokes shifts when replacing water by organic sol-vents. Moreover, ES is independent on solvent, with an energy of4.00 eV.

The fluorescence quantum yields, ˚F, as well as the singlet life-times, �F, were determined in de-aerated solutions (see Table 2).As will be discussed later, molecular oxygen quenches the singletexcited state. The results indicate that NAD fluoresces with reason-able quantum yields (≤0.357), with ˚F increasing as one goes to

Wavelength/nm

Fig. 2. Normalized excitation (�) and emission (©) fluorescence spectra of aqueousNAD solution (1.0 × 10−5 mol L−1).

E.S. Da Silva et al. / Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40 33

Fig. 3. Fluorescence decay of de-aerated aqueous NAD solution (1.0 × 10−5 mol L−1)obtained with �ex. = 282 nm at room temperature. The autocorrelation function(A.C.), weighted residual (W.R.) and chi-squared value (2) are presented as insets.The green line in the decay is the pulse instrumental response. (For interpretation ofto

qt1

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vewwTnrttNpd4fi

300 32 0 34 0 36 0 38 0 40 0 42 0

0.0

8.0x1 06

1.6x1 07

2.4x1 07

3.2x1 07

4.0x1 07

Flu

ore

sc

en

ce

in

ten

sit

y

Wavelength/nm

he references to color in this figure legend, the reader is referred to the web versionf the article.)

uantum yields are less than unity indicates the relevance of radia-ionless pathways in the deactivation of NAD singlet excited state,NAD*. The low value obtained in water, 0.066, may be associatedith hydrogen bonding effects involving the stretching modes of

he hydroxyl group, which could facilitate the radiationless decayo the ground state, and is consistent with quantum yields ofther aromatics, such as benzene [35], that are markedly smallern water than in other common solvents. Although acetonitrilend ethylene glycol have comparable dielectric constants (� ∼= 36),he fluorescence quantum yield in acetonitrile is twice than thatn ethylene glycol. As will be discussed shortly, comparison ofhe photophysical behavior in the two solvents is complicated byhe observation of bi-exponential fluorescence decays in ethylenelycol. However, the results are consistent with hydrogen bond-ng with ethylene glycol enhancing radiationless deactivation andecreasing the fluorescence quantum yield. The lowest value of flu-rescence quantum yield is presented in chloroform, which can bexplained by contact charge transfer [32] as was suggested aboverom the absorption spectra. Moreover, and as can be seen by theesults in Table 2, the replacement of H2O by D2O had no effectn NAD emission properties. With certain systems, notably lan-hanides [36,37], different lifetimes are observed in D2O comparedith H2O due to the effect of vibronic coupling on the non-radiativeecay. However, the extent of coupling decreases with increasingeparation between the electronic states, and the fact that no dif-erences are observed between the fluorescence lifetimes of NADn the two solvents indicates that this effect is insignificant here.

The fluorescence lifetimes, �F, in water, D2O and organic sol-ents were measured with nanosecond time resolution, withxcitation at 282 nm and observation at the maximum emissionavelength. Fig. 3 shows the decay curve of NAD fluorescence inater while the results for all systems are summarized in Table 2.

he fluorescence decay of NAD could be fitted by a single expo-ential for all the solvents, except ethylene glycol and ethanol,evealing a single emitting species. In those two solvents a bet-er fit was obtained by a bi-exponential decay. To attempt to clarifyhe bi-exponential behavior seen in ethylene glycol and ethanol,AD fluorescence decays were studied in three other alcohols, 1-

ropanol, 2-propanol and methanol. In methanol, the fluorescenceecay of NAD followed a mono-exponential fit with a lifetime of9.5 ns while in 1-propanol and 2-propanol the decays were welltted using a bi-exponential, with the major components having

Fig. 4. Quenching of NAD fluorescence (1.0 × 10−5 mol L−1) by KSCN in aqueoussolution. The inset represents the Stern–Volmer plot.

lifetimes of 48.0 and 48.4 ns, respectively. In all the studied alco-hols, except methanol, a small contribution was observed from ashort lifetime component (≈4.5 ns). Although we have not yet man-aged to attribute this, its contribution (5.0%) is always small, andthe lifetimes presented in Table 2 for ethanol and ethylene glycolare those for the major component in the decay.

�F is higher in organic solvents (around 50 ns) than in water(35 ns, ca. 70% less than those in organic solvents). As with the flu-orescence quantum yields this may reflect a strong contribution ofinternal conversion in the decay of the singlet excited state in water.In general, although the values obtained for �F are close to thoseobtained for other naphthalene derivatives [10], they are somewhatsmaller, probably due to the effect of the substituent groups. Thelow value of �F observed in chloroform, 6.77 ns, suggests anotherpathway may be involved in the deactivation, and is in agreementwith the suggestion of contact charge transfer quenching of thesinglet excited state by the solvent. Additionally, the replacementof water by deuterium oxide had no effect on the fluorescencelifetime.

3.3. Fluorescence quenching by inorganic anions

Under environmental conditions, various additional speciesmay contribute to the photochemical degradation of NAD. Manystudies have been published concerning the involvement of elec-tron transfer as a quenching mechanism of fluorescence foraromatic molecules such as naphthalene, anthracene and biphenyl[38–41]. In addition, in our studies of NAD photodegradation, weobserved an effect of azide ion [23]. Although this is commonlyused to identify mechanisms involving singlet oxygen, we sug-gested in our case that the effect involves quenching of NADexcited states. Therefore, we studied the quenching of NAD fluores-cence with some halide ions, thiocyanate, azide, and also with theknown electron-transfer quencher triethylamine (TEA), to obtaininsights on mechanisms that may be involved in its excited statedeactivation. Aqueous solutions of KBr, KI, KCl, KSCN, NaN3 anda triethylamine solution in acetonitrile with different concentra-tions were added to a NAD solution at constant concentration of1.0 × 10−5 mol L−1. In each case, a Stern–Volmer plot of I/I0 vs [Q]was constructed, where [Q] represents the quencher concentration

and the slope of the curve is equal to the Stern–Volmer constantKSV. Fig. 4 shows, as one example, the effect of the quencher KSCNin NAD fluorescence. As can be seen in the figure, the addition ofKSCN did not cause any change in the shape of emission spectra

34 E.S. Da Silva et al. / Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40

Table 3Stern–Volmer constants (KSV) and fluorescence quenching rate constants (kq) forNAD singlet excited state by anions (in water) and triethylamine (in acetonitrile).The redox potentials of the quenchers are also given for better discussion of results.

Quencher KSV (L mol−1) kq (L mol−1 s−1) Reduction potentiala (V)

Cl− ≤0.0038 ≤1.08 × 105 2.41 [42]Br− 18.1 5.18 × 108 1.92 [42]I− 180 5.15 × 109 1.33 [42]SCN− 123 3.52 × 109 1.63 [42]N3

− 102 2.91 × 109 1.33 [42]

orwsal

wbmcqt

5twXasiillmkRelSsrbba[vtaz[utflT

itsTaap

450 50 0 55 0 60 0 65 0

0.00

0.02

0.04

0.06

0.08

0.10

Ph

os

ph

ore

sc

en

ce

in

ten

sit

y/a

.u.

to the literature value for naphthalene (2.66 eV) [10].The ˚P, calculated in ethanol, ethylene glycol and acetonitrile

at 77 K using benzophenone in ethanol at 77 K as reference (0.73)[10] gave similar values (Table 4).

Table 4Phosphorescence properties of NAD in ethanol, ethylene glycol and acetonitrileglasses at 77 K.

Solvent ˚Pa �P (ms) kR (s−1) ˚isc

b (T1–S0)

Ethylene glycol 0.043 210 0.22 0.57

TEA 79.0 1.46 × 109 0.96 [10]

a E0 values for X•/X− vs the normal hydrogen electrode (NHE).

r formation of new bands, but only led to the decrease in fluo-escence intensity due to the quenching process. Similar behavioras observed with the other quenchers. Moreover, the excitation

pectra in the presence of the quenchers resemble the excitationnd absorption spectra in absence of the quenchers, confirming theack of any ground state complexation.

Good straight lines were observed in the Stern–Volmer plotsithin the concentration range 10−4 to 10−2 mol L−1, as can be seen

y the inset plot in Fig. 4, strongly supporting a dynamic quenchingechanism. From the KSV values reported in Table 3, it is possible to

alculate the fluorescence quenching rate constant, kq, for dynamicuenching, from the relationship KSV = kq�F, where �F values areaken from Table 2. These results are also included in Table 3.

In aqueous solution, kq varies in the range 5.18 × 108 to.15 × 109 L mol−1 s−1, indicating that there is a marked effect ofhe type of anion in the quenching of the fluorescence. From Table 3e observe that this is related to the reduction potential of the•/X− couple, with I− having the highest kq, 5.15 × 109 L mol−1 s−1,nd Cl− the lowest. The low kq value observed with Cl− is a limitince the fluorescence intensity of NAD was almost constant withncreasing concentrations of KCl, showing that Cl− is not effectiven quenching NAD fluorescence. Furthermore, N3

− has a very simi-ar efficiency in quenching the fluorescence of NAD to SCN−, but isess effective than I− . Both electron transfer and heavy atom effects

ay be responsible for the quenching. For electron transfer, theq values can be related to the free energy of reaction through theehm–Weller relationship [43]. The free energy depends on thelectrochemical data, and a reasonable linear relationship betweenog kq vs redox potential can be drawn for the anions I−, Br− andCN− (r = 0.856), with slope 1.6. A similar semilogarithmic relation-hip between the quenching rate and electrochemical data has beeneported for the fluorescence quenching of naphthalene derivativesy halide ions [38,39], and suggests that the mechanism responsi-le for the quenching involves electron (or charge) transfer from thenion to NAD singlet excited state, 1NAD*. We note that in both Ref.39] and our case the quenching efficiencies of N3

− and SCN− areery similar, although their redox potentials are different. However,he recommended value of 1.33 V for the reduction potential of thezide/azidyl couple is for buffered neutral solution [42]. Hydra-oic acid (HN3) is a weak acid (pKa 4.65 at zero ionic strength)44] and it is likely that the reduction potential for N3

•/N3− in our

nbuffered solution will differ from this recommended value. Addi-ional support for an electron transfer mechanism comes from theuorescence quenching of NAD in acetonitrile by the electron donorEA.

To obtain evidence for the fact that electron transfer is involvedn the excited singlet state quenching, aerated and de-aerated solu-ions of NAD in the presence of KI and KSCN, respectively, weretudied by laser flash photolysis with excitation of NAD at 266 nm.

he transient bands corresponding to the formation of the radicalnions I2•− at 380 nm and of (SCN)2

•− at 480 nm [45] were observeds well as the formation of NAD triplet state, confirming parallelrocesses of electron transfer and intersystem crossing (isc). With

Wavelengt h/nm

Fig. 5. Phosphorescence spectrum of NAD (1.0 × 10−5 mol L−1) in acetonitrile at 77 K.

the study by Shizuka et al. in 50% ethanol–water [39], there wasno evidence for formation of the inorganic anion radical. However,this requires a second step in which the initially formed radical pairreacts with halide ions [46], which may be inhibited in the mixedsolvent medium used by Shizuka et al. [39]. A possible mechanismis:

1NAD∗ + X− � {1NAD∗· · ·X−} � {NAD•−X

• } → 3NAD∗ + X−

{NAD•−X

• } + X− → NAD•− + X2

•−

NAD•− + H2O � NADH

• + OH−

where X represents the halide anion. The fact that weak quenchingwas observed with bromide ion and no significant quenching withchloride ion places a limit on the reduction potential of 1NAD* ofE0 2.1 ± 0.2 V.

3.4. Phosphorescence spectroscopy and triplet statecharacterization

The phosphorescence emission spectra, quantum yields, ˚P, andlifetimes, �P, of NAD were measured at 77 K in ethanol, acetonitrileand ethylene glycol glasses. As can be seen in Fig. 5, NAD presentswell-resolved emission spectrum in acetonitrile with three mainvibronic bands, which are comparable to those obtained in fluo-rescence spectrum. Similar trends are observed in ethylene glycoland ethanol glasses, although the spectra are not so well resolveddue to problems with light scattering. The lowest wavelength bandobtained in acetonitrile is assigned to the 0-0 transition betweenthe lowest triplet state (T1) and the ground state (S0), and gives theenergy of the triplet excited state (ET), 2.69 eV. This value is close

Ethanol 0.052 64 0.79 0.50Acetonitrile 0.043 0.12 400 0.65

a Estimated errors ±10%.b Calculated using Eq. (8).

E.S. Da Silva et al. / Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40 35

300 35 0 40 0 45 0 50 0

-0.01 5

0.00 0

0.015

0.030

0.045

0.06 0

0.07 5

0.09 0

ΔAb

s.

Wavelength /nm

Fig. 6. Transient absorption spectra of de-aerated aqueous NAD solution(2a

gtTcwa[apTgdtiw

dccao

˚

sc

3l

obewTNtla3w

Table 5Photophysical parameters for the NAD excited triplet state (lifetimes and quantumyield) as function of solvent polarity and oxygen concentration.

Solvent Abs. (�max/nm) �ArgonT

(�s)a �AirT

(�s)a �O2T

(�s)a ˚Ta

Water 420 68 1.4 0.45 0.424D2O 420 65 1.5 0.39 0.414Ethylene glycol 420 338 3.7 0.93 0.610Ethanol 420 125 0.30 0.10 0.547Acetonitrile 420 57 0.25 0.086 0.691Chloroform 425 42 0.41 – 0.522

3.5 × 10−5 mol L−1) obtained by nanosecond laser flash photolysis with excitation at66 nm, recorded at 500 ns, 6.0, 80, and 120 �s after pulse. The kinetic decay profilest 420 and 330 nm are displayed as insets.

The phosphorescence lifetime, �P, was determined in the abovelasses as the average of at least 10 measurements, with a rela-ively low measurement error. The results are also presented inable 4. The fact that the phosphorescence lifetime in ethylene gly-ol is in the hundreds of milliseconds time range is in agreementith the triplet excited state, corresponding to a (�–�*) transition,

s expected from other reports for naphthalene and its derivatives10]. A comparison of �P in polar protic solvents (ethylene glycolnd ethanol) and in the polar aprotic solvent (acetonitrile) sup-orts the role played by the hydrogen bond on the decay lifetime.he explanation for the decrease in lifetime on going from ethylenelycol to acetonitrile is not clear. However, this is completely repro-ucible. The facts that similar quantum yields are observed withhese two solvents while the lifetime is much shorter in CH3CN,ndicates that the magnitude of the effects involved must be similar

ith both radiative and non-radiative processes.The lowest NAD triplet excited state, 3NAD* (T1) may be

eactivated to the singlet ground state S0 by radiative (phosphores-ence) or radiationless processes (isc). We have calculated the rateonstant for phosphorescence decay kp (=˚p/�P) as well as the radi-tionless quantum yield ˚isc (given in Table 4) for the deactivationf 3NAD* state, according to the expression (8):

isc(S1 − T1) = ˚P + ˚isc(T1 − S0) (8)

The results given in Table 4 show that the deactivation of 3NAD*tate occurs preferentially by isc rather than by the radiative pro-ess of phosphorescence.

.5. Triplet–triplet absorption spectra, absorption coefficients,ifetimes and triplet state quantum yields

Nanosecond laser flash photolysis studies were undertaken tobtain more detailed information on the reactive species that maye formed upon NAD photoexcitation. Excitation in the lowestnergy absorption band of NAD (250–320 nm) was accomplishedith frequency quadrupled (266 nm) pulses from a Nd/YAG laser.

he transient absorption spectra were acquired on de-aeratedAD solutions in water, D2O, ethylene glycol, ethanol, acetoni-

rile, chloroform and 1,4-dioxane, at different delays times after

aser excitation. Fig. 6 shows the trends obtained for a de-aeratedqueous NAD solution. Inset is shown NAD decay at 420 and30 nm. Three main transient absorption bands arising from NADere observed: a negative band at 280 nm corresponding to the

1,4-Dioxane 425 67 0.42 – 0.427

a Estimated errors ±15%.

bleaching of the ground state, an intense band with maxima at 420and 390 nm, and a third band with maximum at 330 nm. An addi-tional band with maximum absorption at 720 nm attributed to thesolvated electron was also observed [23]. At the laser energiesused (<18 mJ) the hydrated electron is clearly formed through amonophotonic process.

The transient absorption band at 330 nm shows a decay profiledifferent from the band at 420 nm (inset Fig. 6), with a rate con-stant of 8.5 × 103 s−1 and may be attributed to its radical cation, ashas previously been seen with naphthalene [47]. When water wasreplaced by organic solvents, a 5 nm red shift of the main band at420 nm occurs in 1,4-dioxane and in chloroform (see Table 5). Theband at 330 nm was not observed in any of the organic solvents,within the time scale studied.

The absorption band with maximum around 420 nm can beassigned to the NAD triplet–triplet absorption, and is in good agree-ment to that seen for triplet states of naphthalene and derivatives[10,21,47]. To confirm this assumption, and since it is well knownthat molecular oxygen is a strong quencher of triplet excited statesof aromatic molecules, solutions of NAD in all solvents were excitedat 266 nm as function of oxygen concentration. The decay rate con-stant of NAD absorption band at the maximum wavelength wasmeasured in each solvent, and the triplet lifetimes, �T, calculatedas the inverse of this rate constant. These results are summarizedin Table 5. A marked effect of oxygen was observed in the decayof the transient band at 420–425 nm, as shown for aerated and de-aerated aqueous NAD solution (Fig. 7), where the decay of 3NAD*is faster in the presence of oxygen. This behavior was observed forall the solvents, supporting the idea that this band corresponds to3NAD*.

In aerated and oxygenated solutions, the decay of the tripletstate fitted a mono-exponential rate law. In contrast, in somecases, in de-aerated solutions, a better fit was achieved by a bi-exponential decay, probably due to triplet–triplet annihilation assuggested by �T decreasing with an increase of laser energy. Inwater, �T values of 1.4, 67.7 and 0.454 �s were found in aerated,de-aerated and oxygenated solution, respectively. The replacementof H2O by D2O did not cause any significant changes on the tripletlifetimes, as can been seen by the results given in Table 5.

Further confirmation of the triplet nature of the 420–425 nmtransient species came from sensitization experiments using ben-zophenone (BP) as a high quantum yield triplet donor (˚T = 1.0,ET = 287 kJ mol−1, 2.98 eV) [10]. Fig. 8 shows the transient absorp-tion spectra obtained at different times after excitation by355 nm laser pulse of a de-aerated solution of BP in acetonitrile(2.0 × 10−3 mol L−1) containing 4.0 × 10−4 mol L−1 of NAD. Underthese conditions the laser light is totally absorbed by BP to form itstriplet excited state.

The spectra show three bands with maximum absorption at 320,

420 and 520 nm. The bands at 320 and 520 nm are in good agree-ment with the formation of benzophenone triplet excited state(3BP*) in acetonitrile [10], while the band at 420 nm correspondsto 3NAD*. This band is identical to that obtained upon excitation

36 E.S. Da Silva et al. / Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40

0 3 6 9

0.000

0.004

0.00 8

0.01 2

0.01 6

ΔA

bs

.

Time/µs

a

0 50 100 150 200 250 300 350

0.000

0.005

0.01 0

0.01 5

0.020

ΔA

bs

.

Time/µs

b

tored

osati(Tw

Nc3

tttitbiw

m

Fsi

Fig. 7. Kinetic decay of aqueous NAD triplet state moni

f de-aerated NAD solution with a 266 nm laser pulse (Fig. 6). Thepectra at successive times show a decrease of the absorption bandt 520 nm and a consequent increase of the band at 420 nm. Addi-ionally, the decay rate of the 3BP* donor at 520 nm (3.65 × 106 s−1)s slower than the apparent acceptor 3NAD* build up rate at 420 nm6.42 × 106 s−1) due to the simultaneous acceptor triplet decay.hese results indicate that energy transfer occurs from 3BP* to NADith formation of 3NAD* according to:

BP + h� → 1BP∗ isc−→3BP∗

3BP∗ + NAD → 3NAD∗ + BP

This experiment was repeated with various concentrations ofAD (3.5 × 10−5 to 4.0 × 10−4 mol L−1) maintaining constant BPoncentration (2.0 × 10−3 mol L−1). The decay rate at 520 nm ofBP* was found to be linear with NAD concentration. A plot ofhe observed decay rates as function of NAD concentration allowshe calculation of the quenching rate constant for the above reac-ion of 1.67 × 1010 L mol−1 s−1, corresponding to diffusion controln a collisional process. From consideration of energetic effects onhe rate [48], this indicates that the triplet energy of NAD muste significantly less than that of benzophenone (2.98 eV), which is

n agreement with results from phosphorescence in this solventhich gives a 3NAD* energy of 2.69 eV.

We also used this experiment to calculate the triplet stateolar absorption coefficient, εT, of NAD through the energy

300 350 400 450 500 550 600

0.00

0.05

0.10

0.15

0.20

0.25

ΔAb

s.

Wavelength/nm

0.14 μμμμμ

s

0.32 s

0.40 s

0.50 s

0.60 s

ig. 8. Transient spectra observed at various times after subjecting a de-aeratedolution of benzophenone (2.0 × 10−3 mol L−1) containing NAD (4.0 × 10−4 mol L−1)n acetonitrile with laser excitation at 355 nm.

at 420 nm in aerated (a) and de-aerated (b) conditions.

transfer method (ε520 nm BP = 6500 L mol−1 cm−1, ˚T = 1.0) [25]. Amolar absorption coefficient of 13,820 L mol−1 cm−1 was obtainedfor 3NAD* in acetonitrile at 420 nm. Assuming that this coeffi-cient is constant and independent of solvent, we have used thisvalue to calculate the intersystem crossing quantum yield (or tripletquantum yield), ˚T, of NAD in the different solvents by the com-parative method [26] using naphthalene in methylcyclohexane(ε = 13,200 L mol−1 cm−1, ˚T = 0.75) [10]. The results obtained of ˚T

for NAD in water, D2O, ethylene glycol, ethanol, acetonitrile, chlo-roform and 1,4-dioxane are given in Table 5. The ˚T is dependent onsolvent polarity, ranging from 0.42 in water to 0.69 in acetonitrile,indicating that the formation of 3NAD* by intersystem crossing isan important decay pathway of 1NAD* and may be relevant to thephotoreactivity. Changing water to deuterated water did not affectthe triplet state properties, as can be seen by the results in Table 5.

3.6. Quenching of 1NAD* and 3NAD* by molecular oxygen

Molecular oxygen is an important quencher of both singlet andtriplet excited states of molecules [10,11,49,50], producing photo-physical consequences such as fluorescence and triplet quenching,enhanced intersystem crossing and production of singlet oxygen,1O2 (1�g). Hence, we have determined the rate constants forquenching of 1NAD* by molecular oxygen, kO2

S , in the various sol-vents in oxygenated, aerated and de-aerated solutions, througha Stern–Volmer plot of the fluorescence intensity ratios for aer-ated and de-aerated solutions vs oxygen concentration. A linearfit was obtained, with the slope giving the Stern–Volmer coeffi-cient KSV (=kq�F). Since the fluorescence lifetimes of NAD are known(Table 2), values of kO2

S were then calculated (Table 6). In addi-tion, the rate constants for oxygen quenching of NAD triplet excitedstate, kO2

T were calculated from the following equation:

k = k + kO2 [O ] (9)

obs 0 T 2

where kobs and k0 are the observed first-order rate constants for thedecay of NAD triplet excited state at the absorption maximum foreach solvent in the presence and absence of oxygen, respectively,

Table 6Quenching rate constants of NAD singlet and triplet excited states, kO2

S and kO2T ,

respectively, by molecular oxygen, as function of solvent polarity.

Solvent kO2S (L mol−1 s−1)a kO2

T (L mol−1 s−1)a

Water 1.08 × 1010 1.51 × 109

Ethylene glycol 6.50 × 108 1.82 × 109

Ethanol 8.55 × 109 9.46 × 108

Acetonitrile 1.81 × 101◦ 1.21 × 109

Chloroform 1.47 × 1010 1.00 × 109

1.4-Dioxane 1.18 × 1010 1.82 × 109

a Estimated errors ±10%.

y and Photobiology A: Chemistry 265 (2013) 29– 40 37

atgTas

c1atpifavottiselioqmatpdttrss[

3

t(rdas

ta(uartetastfisty

Table 7Singlet oxygen quantum yield (˚�) formation as function of solvent polarity.

Solvent ˚�a

Water 0.097D2O 0.105Ethylene glycol 0.261Ethanol 0.394Acetonitrile 0.269Chloroform 0.3961,4-Dioxane 0.346

E.S. Da Silva et al. / Journal of Photochemistr

nd [O2] is the oxygen concentration in each solvent [10]. The plot ofhe observed triplet rate constants kobs vs the oxygen concentrationives a linear fit where the slope is equal to kO2

T for all solvents.able 6 lists the quenching rate constants obtained for NAD singletnd triplet excited states by molecular oxygen for all the solventstudied.

The results show that the bimolecular quenching rateonstants of NAD excited states vary over a wide range (108 to010 L mol−1 s−1), in agreement with literature data concerningromatic molecules such as naphthalene and naphthalene deriva-ives [10]. The lowest value of kO2

S , presented in ethylene glycol, isrobably due to its high viscosity. Surprisingly, the rate for quench-

ng NAD triplet excited state in this solvent is slightly higher thanor the singlet excited state. Although we do not, at present, haveny explanation for this, the measurement was repeated and thealue is fully reproducible. In general, the quenching rate constantsf 1NAD* by molecular oxygen, kO2

S , approach the diffusion con-rolled limiting rate constant, kdiff, and present higher values thanhose obtained for the deactivation of 3NAD, kO2

T . This differences related with the spin statistical factor that in triplet excitedtate is (1/9) × kdiff while for singlet excited states it is 1. Forxample, in water, the quenching process of 1NAD* by molecu-ar oxygen has a rate of 1.08 × 1010 L mol−1 s−1 while for 3NAD* its 1.51 × 109 L mol−1 s−1, about 1/9th that of 1NAD*, as frequentlybserved in aromatic systems [51]. Although the details of theuenching mechanism are still not clear, there is general agree-ent that the mechanism depends on the nature of the system,

nd involves energy and/or charge transfer leading to the forma-ion of singlet oxygen 1O2 [11,52,53], a very reactive species, andossibly superoxide anions. Since no other transient species wereetected experimentally by laser flash photolysis, we can presumehat energy transfer between 3NAD* and molecular oxygen leadingo 1O2 formation is highly favorable. This idea is supported by theesults presented in the next section concerning the sensitization ofinglet oxygen and also by previous studies on quenching of triplettate of naphthalene derivatives and formation of singlet oxygen54].

.7. Singlet oxygen measurements

Previous studies on naphthalene derivatives demonstrate thathis type of compounds is able to generate singlet oxygen, 1O21�g), with relatively good yields [55–58]. We have used timeesolved phosphorescence with emission monitored at 1270 nm toetermine the singlet oxygen formation quantum yields (˚�). Inddition, photosensitization has been used to study the rate con-tant for singlet oxygen reacting with NAD.

For the determination of ˚�, optically matched aerated solu-ions of NAD in various solvents were irradiated with a laser pulset 266 nm using as reference biphenyl solution in cyclohexane˚� = 0.73) [28]. This was chosen rather than the more commonlysed phenalenone standard because of its stronger absorptiont the excitation wavelength within the accessible concentrationange. The time-resolved phosphorescence emission intensity ofhese samples at 1270 nm was measured at different laser pulsenergies and extrapolated to the start of the decay. Since all solu-ions have the same absorbance at the excitation wavelength, forny given laser energy, the number of photons absorbed by anyolution will be the same. Individual singlet oxygen luminescenceraces were signal averaged and fitted using a single exponentialunction to yield the luminescence intensity. The phosphorescence

ntensity was then plotted against the laser energy [27], which gavetraight lines whose slopes were compared with that obtained fromhe reference biphenyl, yielding relative singlet oxygen quantumields, ˚�.

a Estimated errors ±10%.

The relative ˚� values obtained for NAD (Table 7) show adependence on solvent and vary between 0.097 and 0.396, in waterand chloroform, respectively. Although these values are not high,we can say that they are within the range of values already reportedfor other naphthalene derivatives [59]. Though there may be tech-nical difficulties with measurements in water due to the shortsinglet oxygen lifetime in this solvent (ca. 4 �s) the effect of thisis small since the substitution of water by deuterated water had noeffect on the ˚� value although the lifetime in deuterated water is20 times higher than in H2O [60]. A comparison of the ˚� for NAD inthe various solvents and the corresponding ˚T values (see Table 5)indicates that the former are much lower than the ˚T values, pro-viding evidence of a relatively low efficiency of energy transfer from3NAD* to molecular oxygen to produce 1O2. The case of water (˚T

0.424, ˚� 0.097) is particularly striking. Therefore, it is likely thatquenching of NAD triplet excited state by molecular oxygen in polarsolvents also involves other pathways, such as electron transfer toproduce the superoxide anion radical and NAD radical cation, bothof which are implicated in the photodegradation of this compoundin water [23].

To test the reactivity of NAD with 1O2, this species was preparedby photosensitization [61,62] through energy transfer (ETR) fromthe triplet excited state of a sensitizer (Sens) to molecular oxygen(3O2):

Sensh�−→1Sens∗ → 3Sens∗

3Sens∗ + 3O2ETR−→Sens + 1O2

Phenalenone was chosen as sensitizer due to the high quan-tum yields of 1O2 formation (˚� = 0.98) [30], and chloroform waschosen as a solvent since it gives a sufficiently long singlet oxy-gen lifetime (�� = 2.5 × 10−4 s) [63]. Aerated solutions containinga constant concentration of phenalenone (3.0 × 10−5 mol L−1) andconcentrations of NAD varying within the range 1.0 × 10−4 to1.0 × 10−2 mol L−1 were excited at 355 nm and the 1O2 phospho-rescence decay was measured at 1270 nm. Rate constants of 1O2decay were obtained from the observed phosphorescence lifetimes(kobs = 1/��). A plot of the observed rate constants kobs vs NAD con-centration gave a straight line (Fig. 9) indicating the overall kineticequation (10):

kobs = k0 + kq[NAD] (10)

where k0 is the rate constant in absence of NAD and kq is thequenching rate constant of 1O2 by NAD.

From this, a second-order rate constant of 2.4 × 105 L mol−1 s−1

was obtained for the quenching reaction between 1O2 and NADin chloroform. Although this value of kq is small, it lies betweenthe values already reported for compounds within the same family

such as 2-naphthol in chloroform (7.1 × 104 L mol−1 s−1), dimethyl-naphthalenes (range from 104 to 105 L mol−1 s−1) and various otherhydroxynaphthalenes (range from 106 to 107 mol−1 L s−1) [59,64].

38 E.S. Da Silva et al. / Journal of Photochemistry and Photobiology A: Chemistry 265 (2013) 29– 40

Table 8Radiative rate constants decay (kR), natural radiative lifetimes (�0

nat), radiationless rate constants decay (kNR), radiationless rate constants decay for internal conversion (kic),radiationless rate constants decay for intersystem crossing (kisc), and quantum yield of internal conversion (˚ic) of NAD in water, D2O and in organic solvents.

Solvent kR (s−1) �0nat (ns) kNR (s−1) ˚ic kic (s−1) kisc (s−1)

Water 1.88 × 106 532 2.67 × 107 0.510 1.46 × 107 1.21 × 107

D2O 1.94 × 106 515 2.66 × 107 0.518 1.48 × 107 1.18 × 107

Ethylene glycol 1.81 × 106 552 1.70 × 107 0.294 5.54 × 106 1.15 × 107

Ethanol 1.96 × 106 510 1.84 × 107 0.357 7.28 × 106 1.11 × 107

Acetonitrile 3.44 × 106 291 1.51 × 107 0.123 2.29 × 106 1.28 × 107

Chloroform 3.25 × 106 307 1.4 8 7 7

1,4-Dioxane 6.94 × 106 144 1.2

0.0 2.0x1 0-3 4.0x1 0

-3 6.0x1 0-3 8.0x1 0

-3 1.0x1 0-2

9.5x1 03

1.0x1 04

1.1x1 04

1.1x1 04

1.1x1 04

1.2x1 04

ko

bs

/s-1

3

iTa

[NAD]/mo l L-1

Fig. 9. Plot of rate constant vs NAD concentration.

.8. Deactivation pathways of NAD excited states

From all above data, it has been possible to fully character-ze the photophysical deactivation routes of NAD excited states.hese include the radiative processes of fluorescence (S1–S0)nd phosphorescence (T1–S0), and the radiationless processes of

Φ F 0.066

hννννE

S0

S1

4.00 eV

Φ ic0.510

Φ isc0.65

Φ0

(a)

Φ F 0.186

hννννE

S0

S1

4.00 eV

Φ ic0.123

Φ isc0.65

Φ0

(b)

Fig. 10. Jablonski diagram for NAD de-aerated s

4 × 10 0.456 6.73 × 10 7.71 × 105 × 107 0.216 4.20 × 106 8.30 × 106

internal conversion (ic) (S1–S0), and intersystem crossing (isc)(S1–T1) and (T1–S0). Using NAD fluorescence quantum yields andthe measured fluorescence lifetimes (Table 2), the natural radia-tive rate constant kR (=˚F/�F) and radiationless rate constantskNR (=1 − ˚F/˚F) were determined for NAD in each solvent asshown in Table 8. From these, the natural radiative lifetimes (�0

nat =1/kR) were calculated, which were found to vary in the range of290–530 ns and are considerable greater than the experimental flu-orescence lifetimes, �F, showing the major role that radiationlessprocess plays in deactivation of 1NAD*. Furthermore, the quan-tum yield of internal conversion, ˚ic, can be calculated from therelationship ˚F + ˚T + ˚ic = 1, which assumes that only these threeprocesses jointly deactivate 1NAD* and also the rate constants forthe radiationless processes, kic (=˚ic/�F) and kisc (=˚T/�F), since thevalues of ˚F and of ˚T are known (see Tables 2 and 5). The photo-physical parameters calculated for the deactivation of 1NAD* in thedifferent solvents are presented in Table 8. It can be noted that the˚ic in water, 0.510, is greater than in the other solvents, showingthat this is the preferred deactivation pathway in this solvent. Fromcomparison of ˚F, ˚ic and ˚isc values (see Tables 2, 5 and 8) for theother solvents, ˚ic is greater than ˚F (except in acetonitrile), butthe dominant pathway of 1NAD* deactivation involves intersystemcrossing.

A Jablonski diagram, summarizing all the processes that occurfor NAD in water and in an organic solvent (acetonitrile) is pre-

sented in Fig. 10.

T1

P.04 3

Φ isc0.424

2.69 eV3O2

1O2 (1Δg)

ETR

Φ Δ0.097

T1

P.043

Φ isc0.691

2.69 eV3O2

1O2 (1Δg)

ETR

Φ Δ0.269

olutions in (a) water and (b) acetonitrile.

y and

4

ta(spltadi1

3

lna[ilitNcr

A

F(P

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

E.S. Da Silva et al. / Journal of Photochemistr

. Conclusion

A comprehensive study has been accomplished on NAD pho-ophysics in various solvents. From the detailed results obtainedlong this study, some important conclusions could be drawn:1) the main deactivation pathway for 1NAD* depends on theolvent; in water, the non radiative S1 → S0 internal conversionrocess is predominant while in organic solvents the radiation-

ess S1 → T1 intersystem crossing becomes the major process; (2)he radiative process of fluorescence S1 → S0 is less important inll solvents when compared to the non radiative ones; (3) 3NAD*eactivates mainly by the radiationless T1 → S0 intersystem cross-

ng and the radiative conversion is very low in all solvents; (4)NAD* is quenched more efficiently by molecular oxygen thanNAD*, as expected from spin-statistical factors; (5) NAD excitationeads to 1O2 formation through triplet sensitization, particularly inon-aqueous solvents. However, singlet oxygen reaction does notppear to be the main route for NAD photodegradation in water23], and there are indications that in this solvent oxygen quench-ng may partially occur through electron transfer. This is likely toead to formation of NAD radical cation. Radical cation formations also observed upon flash photolysis of degassed aqueous solu-ions, and this is suggested to be an important intermediate inAD photodegradation [23]. Finally, as with many other aromaticompounds, it has been shown that NAD singlet excited is also aelatively strong photooxidant.

cknowledgments

Eliana Sousa Da Silva acknowledges the Portugueseundac ão para a Ciência e Tecnologia (FCT) for a PhD grantBD/SFRH/BD/43171/2008) under the framework of the PortugueseOPH/FEDER/QREN program.

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