Effect of Solvent Hydrogen Bonding on Excited-State Properties of Luminol: A CombinedFluorescence and DFT Study

N. Shaemningwar Moyon, Asit Kumar Chandra, and Sivaprasad Mitra*Department of Chemistry, North-Eastern Hill UniVersity, Shillong 793 022, India

ReceiVed: August 18, 2009; ReVised Manuscript ReceiVed: October 29, 2009

The effect of solvent on the photoluminescence behavior of luminol was studied by steady-state fluorescencespectroscopy. The fluorescence spectral behavior of luminol is markedly different in polar protic solventscompared to that in aprotic solvents. A quantitative estimation of the contribution from different solvatochromicparameters, like solvent polarizibility (π*), hydrogen-bond donor (R), and hydrogen-bond acceptor (�), wasmade using the linear free energy relationship based on the Kamlet-Taft equation. The analysis reveals thatthe hydrogen-bond-donating ability (acidity) of the solvent is the most important parameter to characterizethe excited-state behavior of luminol. Quantum mechanical calculations using density functional theory (DFT)predict the most stable structure, out of several possible tautomeric conformers of luminol with varying degreesof hydration. In the excited state, charge localization at specific points of the luminol phthalhydrazide moietycauses the solvent to interact primarily through hydrogen-bond donation.

1. Introduction

Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, LH2) isa versatile chemical that shows striking blue chemiluminescencein the presence of certain metal ions when treated with anappropriate oxidizing agent like hydrogen peroxide. This uniquefeature of LH2 is often exploited by forensic investigators todetect trace amount of blood left in the crime scene. LH2 isalso used by biologists as a cellular assay to detect copper, iron,and cyanides, etc.1-5 Further, LH2-enhanced chemiluminescentprobes have been used to characterize and quantify the secretionof oxygen by phagocytozing cells.6 The use of LH2 chemilu-minescence has also been reported recently for facile detectionof proteins,7 cancer biomarkers,8 as well as reactive oxygenspecies produced by human neutrophils.9 The ultrahigh sensitiv-ity of the time-resolved chemiluminescence behavior of LH2

can be used to measure OH/O2- radical species concentrations

as low as 2 × 10-7 mol dm-3 in water.10 An important aspectof LH2 chemiluminescence is its different degrees of sensitivityfrom one substance to another. LH2 shows higher sensitivity toanimal or human blood, organic tissues, and fluids than to othercompounds containing metal ions, such as paints, metallicsurfaces, household products, or vegetable enzymes. Therefore,the light emitted by LH2 has different intensities and timeduration depending on the material of contact, making it anefficient forensic marker.

The solution-phase spectroscopic properties of LH2 havedrawn enormous interest in recent times due to the biochemicalrelevance of its photoactivity. The photophysical properties ofLH2 in different solvents and solvent mixtures as well as itsinteraction with several biological molecules were reported inthe literature.11-15 LH2 exhibits two principle absorption bandsin the 300 and 350 nm region, whereas a single broadfluorescence emission appears in the 400 nm region. Interest-ingly, the fluorescence emission peak shows a large spectralshift toward longer wavelengths in hydrogen-bonding solvents.

This shift is believed to be due to the stabilization of the charge-transfer excited state of the intermolecular hydrogen-bondedcomplex of LH2 with solvent.11

The intermolecular hydrogen bonding and solubility oforganic systems are known to play crucial roles in determiningthe biological activity as well as their application in forensicscience.16-19 In general, formation of an intermolecular hydrogenbond between the solute and the solvent results in a decreasein the Gibb’s free energy and thus promotes mixing. Hydrogenbonding can occur in different modes, depending on the structureof the solute and solvent. The situation becomes more compli-cated when a solute molecule possesses multiple hydrogen-bonding sites and the solvent molecule can act both as a protondonor as well as a proton acceptor. Under this condition, thecompetition among different molecular species resulting froma hydrogen-bonding interaction between the solute and thesolvent molecules becomes inevitable. LH2 provides a uniqueexample to study the hydrogen-bonding effect because themolecule itself can exist in more than one prototropic species(Scheme 1) having multiple hydrogen-bonding sites. Theketo-amine (I) structure can go to the enol-imine (III) formin a single step or through intermediate structures IIa and/orIIb, respectively. These interconversion and associated spec-troscopic properties will depend strongly on the relativeabundance of several species as well as their hydrogen-bondingmode with the solvent. Furthermore, the efficacy of hydrogen-bond formation in the excited state may change due to chargeredistribution after excitation. Also, the relatively strong andunstructured fluorescence of LH2 is an additional advantage touse the spectroscopic ruler for quantitative estimation of theeffect of different solvent parameters. The chemiluminescenceand fluorescence bands of LH2 in water appear in the samewavelength region (425-430 nm);13 thus, quantitative charac-terization of this band on different solvent parameters isindispensable.

In this paper, we use the steady-state spectral properties ofLH2 in a series of pure solvents with varying polarity as wellas the hydrogen-bond donor and acceptor abilities to findquantitative information about their relative contribution. Fur-

* To whom correspondence should be addressed. Phone: (91)-364-2722634. Fax: (91)-364-2550076. E-mail:smitra@nehu.ac.in, sivaprasadm@yahoo.com.

J. Phys. Chem. A 2010, 114, 60–6760

10.1021/jp907970b 2010 American Chemical SocietyPublished on Web 11/12/2009

thermore, the effects of specific LH2-water hydrogen bondingon the solution-phase spectral properties were theoreticallymodeled by using the density functional approach.

2. Materials and Methods

2.1. Chemicals. Luminol (LH2, 97%) was received fromSigma-Aldrich Chemical Pvt. Ltd. and used without any furtherpurification. The organic solvents used were of spectroscopicgrade (>99.5%) as received from Alfa Aesar and, in some cases,from Aldrich Chemical Co. The analytical-grade type-II water,also used as solvent, was obtained from an Elix 10 waterpurification system (Millipore India Pvt. Ltd.). The chromophoreconcentration (∼1.2 × 10-5 mol dm-3) was very low to avoidany aggregation and kept constant during spectral measurementsin different solvents.

2.2. Experimental Procedure. Steady-state absorption spec-tra were recorded on a Perkin-Elmer model Lambda25 absorp-tion spectrophotometer. Fluorescence spectra were taken in aHitachi model FL4500 spectrofluorimeter, and all spectra werecorrected for the instrument response function. Quartz cuvettesof 10 mm optical path length received from PerkinElmer, USA(part no. B0831009), and Hellma, Germany (type 111-QS), wereused for measuring absorption and fluorescence spectra, respec-tively. For fluorescence emission, the sample was excited at360 nm unless otherwise mentioned, whereas excitation spectrawere obtained by monitoring at the respective emission maxi-mum. In all cases, a 5 nm band pass was used in the excitationand emission side. Fluorescence quantum yields (φf) werecalculated by comparing the total fluorescence intensity underthe whole fluorescence spectral range with that of a standard(quinine bisulfate in 0.5 M H2SO4 solution, φf

s ) 0.54620) withthe following equation using adequate correction for the solventrefractive index (n)21

where Ai and As are the optical density of the sample andstandard, respectively, and ni is the refractive index of the solventat 293 K. The relative experimental error of the measuredquantum yield was estimated within (5%. The pH variationexperiments were carried out in a Systronics µ-pH system (type361, resolution 0.01 pH) at constant temperature (293 K).

2.3. Estimating the Relative Contribution of SolventParameters on LH2 Spectral Properties. The effects of solventpolarity and hydrogen bonding on the steady-state spectralproperties of LH2 are interpreted by means of the linear solvationenergy relationship (LSER) concept using Kamlet-Taft eq 222

where P is the value of the solvent-dependent property to bemodeled and P0, s, a, and b are the coefficients determined fromthe LSER analysis. The term π* indicates the measure of solventdipolarity/polarizibility,23 whereas R and � are the scale ofhydrogen-bond donation acidity and acceptance basicity of thesolvent, respectively.19 The corresponding parameters for 14solvents were taken from the literature25,26 and are given in Table1. The correlations of the spectroscopic data were carried outby multiple linear regression analysis as implemented in theORIGIN 6.0 (Microcal Inc.) program package.

2.4. Theoretical Calculations. Density functional theory(DFT) has successfully been applied to study the hydrogenbonding in various systems including organic as well asbiological model systems.27-30 Pan et al. used the B3LYPfunctional along with the 6-31++G(d,p) basis set to verify theextent of specific solvation of formic acid or formate anionmodel systems with few water molecules,31 whereas in a recentpaper the effectiveness of DFT calculations to predict thehydrogen-bonding strength between different drug moleculesand corresponding receptors was investigated.32 The success ofthe DFT method in elucidating the complex phenomenon likehydrogen-bonding prompts us to use it further in studying theenergetic parameters of isolated LH2 and its complex with watermolecules. Several conformers of LH2 are possible. To elucidatethe lowest energy structure, full geometry optimization wasperformed for all possible conformers of LH2 both in isolatedas well as in their mono- and dihydrated complexes using theB3LYP method and 6-311++G(d,p) basis set as implementedin the Gaussian03 program package.33 Frequency calculationswere done in each stationary point to characterize the minimumenergy equilibrium structure. Vertical transition energies up tothe first 10 singlet excited states and corresponding oscillatorstrengths for all structures were estimated using the time-dependent DFT method (TD-DFT) at the same level ofcalculation. A similar methodology was successfully appliedrecently for characterizing the spectroscopic properties of largeorganic heterocyclic systems.34-36

3. Results and Discussion

3.1. Steady-State Spectral Properties in Pure Solvents.Figure 1 shows some representative absorption and emissionspectra of LH2 in aqueous medium, and Table 2 summarizesthe steady-state spectral behavior of LH2 in solvents with varyingpolarity and hydrogen-bonding parameters. In homogeneoussolvents, LH2 shows two absorption maxima. One relatively

SCHEME 1: Possible Tautomeric Structures of Luminol(LH2)a

a Numbering used in calculation is also shown.

φfi ) φf

s Fi

Fs

1 - 10-As

1 - 10-Ai(ni

ns)2

(1)

P ) P0 + sπ* + aR + b� (2)

Excited-State Properties of Luminol J. Phys. Chem. A, Vol. 114, No. 1, 2010 61

structured high-energy peak appears in the 280-320 nm region,whereas the other unstructured, broad low-energy absorptionis in the 330-380 nm region. However, the emission obtainedby exciting at both these absorptions show strongly intense,unstructured, and broad spectra ranging from 375 to 520 nm.The excitation spectra corresponding to this emission again showa broad peak at ∼350 nm. In a recent report,13 Vasilescu et al.proposed an acid-base type of equilibrium to explain the twoabsorption bands of LH2 in highly alkaline (pH ≈ 12.0) DMSOsolution for the formation of the corresponding dianion ofstructure III. This was further confirmed by the concomitantquenching of the LH2 fluorescence and appearance of a newbroad band at 475-480 nm with increasing alkali concentration.However, formation of the dianionic species in the presentexperimental condition of neutral aqueous LH2 solution (pH ≈6.4) can be ruled out. The absence of any new fluorescenceband further supports this hypothesis. The origin of the broadabsorption in the 330-380 nm region can be assigned as theS1(π)r S0(π) transition, whereas the origin of the high-energyabsorption at ∼300 nm may be due to S2r S0 excitation. Fromthe relatively large absorption coefficient (εmax ≈ 22 275 dm3

mol-1 cm-1) of this band, which is comparable to that of the

∼350 nm absorption (εmax ≈ 23 300 dm3 mol-1 cm-1), it canbe concluded that this transition is also π* r π in nature. Theassignment of these absorptions as well as further verificationfor the long-wavelength absorption at 475-480 nm to beoriginated from the anionic species is confirmed from thetheoretical calculations described in the following sections.

Although the results in Table 2 do not show any regularvariation of the steady-state spectral properties, careful observa-tion reveals several interesting trends. For example, the fluo-rescence maxima (λem) show an appreciable shift in proticsolvents along with an almost 2-fold increase in the fluorescencequantum yield (φf) when compared with their aprotic counter-part. Furthermore, the full width at half maxima (fwhm) forboth the absorption and the emission peaks are much higher inwater compared to the other solvents. All these results indicatethat consideration of the hydrogen-bonding interaction is veryimportant to describe LH2 photophysics, more particularly inaqueous medium.

3.2. Solvatochromism of LH2 Photophysics: Estimationof Contributions from Solvent Parameters. To verify theeffect of solvent polarity, several steady-state spectral parametersof LH2 in a variety of solvents mentioned in Table 1 were plottedagainst the solvent polarity parameter ∆f(ε, n). From the resultsgiven in Figure 1S in the Supporting Information, it is clearthat the spectroscopic properties of LH2 do not show any regularsolvatochromism behavior on the solvent polarity parameter.This observation points to the existence of specific solute-solventinteractions. As a trial, the empirical solvent polarity scale,ET(30), built with a betaine dye, was used as it is the mostpopular choice to correlate several solvent-dependent spectralproperties. The uniparametric scale depends on both the solventdielectric properties and the hydrogen-bonding acidity, but itdoes not take care of the solvent hydrogen-bonding acceptorbasicity.37 The specificity of Lewis acid base interactions in theET(30) parameter arises from the negative charge localized onthe phenolic oxygen of the betaine molecule. As it is seen inthe Supporting Information (Figure 2S) again, there is no linearcorrelation of either the LH2 absorption/emission energies orthe Stokes shift even with this parameter. A break point, mostlyinfluenced by LH2 emission properties like the fluorescencemaxima (νem) and Stokes shift (∆νss), is obtained around ET(30)) 38 kcal mol-1. This clearly indicates that apart from solventpolarity, LH2 solvatochromism is strongly modulated by bothsolvent hydrogen-bond donor acidity and solvent hydrogen-bondacceptor basicity parameters also.

In view of this situation, one must look at a multiparametricapproach, as devised by Kamlet and Taft and mentioned in eq2, to assess the contribution of different solvent parameters onLH2 solvatochromism. The s, a, and b coefficients in eq 2 wereall obtained by multiple linear regression analysis, and the resultsare given in Tables 3 and 4. A few representative correlationdiagrams of the experimental values with those calculated frommultiple regression analysis using eq 2 are shown in Figure 2.A close look into the tables reveals several interesting featuresfor LH2 solvatochromism. (i) In general, the contributions froma as well as b parameters are significant relative to the sparameter, indicating the importance of solvent hydrogenbonding in LH2 spectroscopy. (ii) The excited-state spectralproperties like fluorescence maxima, Stokes shift, quantum yield,etc., are mostly controlled by the solvent hydrogen-bond acidityfunction (a parameter), whereas both a and b contribute almostequally in the absorption property. This indicates an efficientcharge localization in LH2 upon excitation (see DFT calculationresults in the following section). (iii) The charge localization

TABLE 1: Solvent Parameters

no. of solvent solvent ∆f(ε,n)a ET(30)b Rc �c πc

1 acetonitrile 0.30 45.6 0.19 0.40 0.752 ethyl acetate 0.19 38.1 0.0 0.45 0.553 benzene 0.0 34.3 0.0 0.10 0.594 tetrahydrofuran 0.21 37.4 0.0 0.55 0.585 1,4-dioxane 0.03 36.0 0.0 0.37 0.556 toluene 0.02 33.9 0.0 0.11 0.547 DMSO 0.26 45.1 0.0 0.76 0.18 DMF 0.27 43.2 0.0 0.69 0.889 dichloromethane 0.22 40.7 0.0 0.1 0.8110 1-butanol 0.26 49.7 0.84 0.84 0.4711 methanol 0.31 55.4 0.98 0.66 0.6012 1-propanol 0.27 50.7 0.84 0.90 0.5213 1-pentyl alcohol 0.25 49.114 water 0.32 63.1 1.17 0.47 1.0915 acetone 0.28 42.2 0.08 0.48 0.7116 isopropanol 0.27 48.4 0.76 0.95 0.48

a Polarity parameter ()(ε - 1)/(2ε + 1) - (n2 - 1)/(2n2 + 1)),where the solvent dielectric constant and refractive indices arerepresented by ε and n, respectively. b Reichardt solvent parameter.c Kamlet-Taft solvent parameters.

Figure 1. Absorption (a), fluorescence emission (b and c, at λexc )350 and 290 nm, respectively), and excitation (d, λmon ) 425 nm)spectra of 1.2 × 10-5 mol dm-3 aqueous solution of LH2.

62 J. Phys. Chem. A, Vol. 114, No. 1, 2010 Moyon et al.

in the excited state is further confirmed by the negative valuesof both a and s.38 (iv) An almost 2-fold increase in thefluorescence quantum yield in protic medium (Table 2) is mainlydue to the hydrogen-bond acidity of the solvents with a/s ≈1.5. However, solvents like DMSO with a higher hydrogen-bonding acceptor (HBA) tendency have very little effect (∼8%)on φf. For example, the φf value of LH2 in water is about 0.79compared to that in 1,4-dioxane (0.25) and DMSO (0.34). Thisobservation is also in line with our recent finding that the yieldof LH2 fluorescence decreases substantially in the presencewater-soluble proteins like bovine and human serum albumins.The decreased fluorescence intensity of LH2 on binding toalbumins most likely reflects reduced water access to thechromophore in the bound state. Finally, (v) the large spectralshift in water and other hydroxylic solvents, as observed in Table2, is due to the negative value of the a parameter and itscorresponding larger contribution (Table 4). In summary, thesolvatochromic analysis reveals that in polar protic solvents,like water, for example, several spectroscopic species may bepresent due to the hydrogen-bonded donor and acceptor proper-ties of the solvent in the ground state; however, in the excited

sate, the main fluorescing species is originated due to thehydrogen-bonded complex formation of LH2 through the solventhydrogen-bonding acidity behavior.

3.3. Theoretical Calculation Using Density FunctionalTheory. 3.3.1. Energetic of Different Conformers in theGround State. Full geometry optimization of different conform-ers of LH2 in isolated condition (structures given in Scheme 1along with the numbering scheme) as well as with differentdegrees of hydration (some of the fully optimized structuresare shown in Figure 3) was done using the B3LYP/6-311++G(d,p) methodology. The fully optimized structures ofall other conformers with varying degrees of complexation withwater molecule(s) are shown in Figure 3S, Supporting Informa-tion. The energy parameters, relative to the most stable structure,are given in Table 5. It is clear that the conformer IIb is themost stable structure in the isolated, monohydrated as well asin the dihydrated configuration. However, comparison of therelative energies indicates that LH2 most likely exists in thedihydrated complex structure represented by IIb-S3. Thestructure represented by I-S3 is about ∼7.6 kJ mol-1 higher inenergy than IIb-S3. It may still be possible that this high-energystructure will have relatively less abundance in solution alongwith the structure IIb-S3 in the ground state and more so, inthe excited state (see below). However, the existence of all otherconformers represented by IIa and III can be neglected todiscuss the spectroscopic behavior of LH2 in water. This isbecause of their relatively higher energy; they are unlikely tobe present in solution mixture. It is to be noted here that onlythe primary hydrogen-bonded complex with water was consid-ered to give different complexes like S1, S2, and S3 (Figure3). It is obvious that additional solvent molecules will combineto give a secondary water cluster around LH2, and the actual

TABLE 2: Steady-State Spectral Properties of LH2 in Homogeneous solventsa

no. of solventb νabs /cm-1 νem /cm-1 νexc /cm-1 ∆νss /cm-1 φf δem /cm-1 δexc /cm-1

1 28 169 25 126 28 490 3043 0.26 3053 33332 28 172 25 253 28 490 2919 0.21 2902 33333 28 170 24 876 28 249 3294 0.20 3053 30994 28 165 25 316 28 410 2849 0.24 2902 32635 28 170 25 126 28 490 3044 0.25 2853 37126 28 169 24 876 28 410 3293 0.21 3158 40107 27 933 24 876 28 090 3057 0.33 2902 32638 28 090 25 063 28 249 3027 0.26 2902 33109 28 011 24 876 28 490 3135 0.29 3106 335810 28 410 24 331 28 011 4079 0.52 3004 359011 28 169 24 450 28 329 3719 0.42 3057 420112 28 090 24 390 28 090 3700 0.58 3106 376813 27 933 24 450 28 090 3483 0.58 3057 379314 28 572 23 474 28 410 5098 0.78 3211 438815 28 328 25 063 28 090 3265 0.31 2902 300116 28 011 24 510 28 169 3501 0.50 3057 3846

a Abbreviations used: ν ) absorption, emission, and excitation energy; ∆νss ) Stokes shift; φf ) fluorescence quantum yield; δ )corresponding full width at half maximum (fwhm). b The name of the solvents are listed in Table 1.

TABLE 3: Regression Fit to Solvatochromic Parameters Toward the Steady-State Spectral Properties of LH2a

property (P) P0 s a. b R2 SD

νabs/cm-1 28 183.14 70.06 260.90 -267.79 0.90 140.3νem/cm-1 25 362.49 -652.76 -1448.0 -391.26 0.94 166.7νexc/cm-1 28 492.90 25.60 26.92 -424.06 0.92 147.2∆νss/cm-1 2820.65 722.80 1347.85 -657.84 0.88 223.3φf 0.074 0.225 0.33 0.05 0.89 0.06δem/cm-1 3061.69 28.84 233.77 -277.26 0.85 71.7δexc/cm-1 3566.69 -30.11 851.47 -491.86 0.89 281.3

a The regression analysis was done using eq 2; R2 and SD indicate the correlation coefficient and standard deviation in the regressionanalysis, respectively.

TABLE 4: Relative Values (in percentage) of theSolvatochromic Parameters Toward LH2 Steady-StateSpectral Properties

property (P) Ps(%) Pa(%) Pb(%)

νabs 11.70 43.57 44.73νem 30.65 51.03 18.32νexc 5.37 5.64 88.89∆νss 26.49 49.38 24.13φf 37.19 54.55 8.26δem 5.34 43.30 51.36δexc 2.19 61.99 35.82

Excited-State Properties of Luminol J. Phys. Chem. A, Vol. 114, No. 1, 2010 63

hydrated structure is far more complex than what is consideredfor these calculations. However, we restrict ourselves only inthe first hydration layer, as the effect of internal hydrogenbonding (IHB) is expected to diminish very fast from the centerof origin. Consequently, it is reasonable to believe that anyfurther addition of water structure will have an insignificantcontribution toward the relative energy parameters.

3.3.2. TD-DFT Calculation on the Excited State. Theexcited states of the two most stable ground-state structures ofdihydrated LH2 discussed above, i.e., for I-S3 and IIb-S3, werecalculated using the TD-DFT procedure. The calculated transi-tion energies and corresponding oscillator strengths for severalsinglet excitations within the experimentally observed absorptionwavelength range of 260-400 nm for both structures are givenin Table 6. It is noted that the nature of the transition as well asits energy and oscillator strength is comparable for the boththe structures. The calculated S1r S0 transition wavelength of∼335 nm is in close agreement with the experimentally observedvalue of 350 nm and the gas-phase absorption energy of 354nm (Table 3). The nature of the second lowest energy transitionin the experiment cannot be assigned unambiguously becauseof the close energy separation of the next two calculated values(294 and 281 nm, respectively) and their similar oscillatorstrengths. However, all of the associated orbitals that might beinvolved in excitation with a significant contribution in thiswavelength range, viz. HOMO-1, HOMO, LUMO, and LU-MO+1 (Table 6), are shown to be of π type in nature (Figure4). Further, to confirm that no proton-dissociated anionic species

contributes in this wavelength region, TD-DFT calculation wasperformed on fully optimized anionic species of conformer IIb.The lowest energy absorption appears in the ∼450 nm region,which is in close agreement with the experimentally obtainedvalue of 475-480 nm reported by Vasilescu et al.13 Theseauthors proposed the existence of a proton-dissociated dianionicstructure of conformer III responsible for this absorption.However, from the energy parameters given in Table 5 it isclear that the formation of this conformation itself is veryunlikely. Thus, the anionic species responsible for the long-wavelength absorption is believed to be originated fromconformer IIb. Furthermore, comparison of the nature of theHOMO and LUMO in Figure 4 reveals that on excitation theelectron density is more localized on the carbonyl oxygen andimino nitrogen atoms (O12 and N10, respectively, in Scheme1), thereby increasing the basicity at these points significantly.This confirms the importance of the hydrogen-bond-donatingability (acidity) of the solvent to discuss the spectroscopicbehavior of LH2, more particularly in the excited state, as indeedobserved from LSER analysis discussed above.

The calculated energy difference between I and IIb in theground state is ∼14.1 kJ mol-1. The potential-energy surface(PES) (Figure 5), constructed by the intrinsic reaction coordinate(IRC) calculation from the transition state (TS), indicates thatthe water-assisted conversion of I to IIb is associated with alarge activation barrier of ∼55 kJ mol-1. Thus, in the groundstate the relative abundance of the high-energy structure (I-S3)will be much less, from both kinetic and thermodynamic points

Figure 2. Correlation diagram of the LH2 emission energy (νem), Stokes shift (∆νss), and fluorescence quantum yield (φf) with predicted valuesfrom eq 2.

64 J. Phys. Chem. A, Vol. 114, No. 1, 2010 Moyon et al.

of view. However, TD-DFT calculation results show that therelative energy difference between I-S3 and IIb-S3 is very smallin the first excited state (∼3.4 kJ mol-1). Hence, a simpleBoltzmann distribution predicts approximately 25% populationof the excited state to exist as I-S3 in solution at roomtemperature. Therefore, the photochemistry of LH2 can be

considered as an average property of both of these structures.As shown in Table 6, the transition energy, nature of excitation,as well as corresponding oscillator strength of both structuresis similar to each other. Naturally, it is expected for them toshow similar spectroscopic behavior, particularly in noninter-acting solvents. However, as these conformers differ consider-ably in their mode and extent of hydrogen bonding, it is possibleto form different hydrogen-bonded clusters in protic solventswith little difference in energy. The broad nature of the emissionspectra of LH2 in protic solvents, as discussed before, may bedue to the ensemble-averaged spectral properties of all thesemicrostructures.

Conclusions

The excited-state photophysical behavior of luminol hasbeen studied by steady-state fluorescence spectroscopy andDFT calculations. The observed solvatochromism in the

Figure 3. Fully optimized structures of the IIb conformer of LH2 in isolated (i), monohydrated IIb-S1 and IIb-S2 (ii and iii, respectively), anddihydrated IIb-S3 (iv) states. The geometry optimization was done at the B3LYP/6-311++G(d,p) level of calculation. Bond lengths and angles aregiven in Angstroms and degrees, respectively.

TABLE 5: Relative Energy (kJ mol-1) of the FullyOptimized Structures of Different Conformers of LH2 in theGas Phase Calculated at the B3LYP/6-311++G(d,p) Level

structurea I IIa IIb III

isolated 95.504 99.626 81.421 141.022monohydrated (S1) 52.952 58.062 41.553 90.538monohydrated (S2) 50.450 57.507 41.715 93.064mihydrated (S3) 7.635 14.544 0.0 44.182

a See Figures 3 and 3S (Supporting Information) for thestructures of different conformers with varying degrees of hydration.

TABLE 6: Calculated Singlet Excited-State Transitions, Associated Energies, and Oscillator Strength (f) of I-S3 and IIb-S3a

I-S3 IIb-S3

singlet state transition energy /nm f transition energy /nm f

1 Hf L 335 0.13 Hf L 332 0.142 H-1f L 294 0.01 Hf L+1 294 0.073 H-1f L+1 282 0.07 H-1f L 281 0.05

Hf L+1 Hf L+1

a The frontier orbitals of the HOMO and LUMO are designated as H and L, respectively.

Excited-State Properties of Luminol J. Phys. Chem. A, Vol. 114, No. 1, 2010 65

luminol spectral properties are rationalized by a multipara-metric approach using the Kamlet-Taft equation. It has beenfound that the photoluminescence behavior of luminol isstrongly modulated by specific solute-solvent interaction.Quantitative estimation of the relative contribution of severalsolvatochromic parameters indicates that the hydrogen-bonddonor ability of the solvent is the primary factor governingthe excited-state properties. These observations have alsobeen supported by DFT calculations on luminol in isolatedconditions as well as with varying degrees of hydration. Theresults predict that the dihydrated luminol-water complexis the most stable structure, where preferential solventhydrogen-bond donation occurs at the localized electron-rich

centers like the imine nitrogen and carbonyl oxygen at the2- and 4- positions of the phthalhydrazide ring system.

Acknowledgment. Financial support through research project34-299/2008(SR) from the University Grants Commission(UGC), Government of India, is gratefully acknowledged. Theauthors thank Mr. T. Sanjoy Singh and Ms. S. Phukan for theirhelp in preparing some of the figures. Thanks are also due toUGC and DST for supporting the Department of Chemistrythrough DSA-SAP and FIST, respectively.

Supporting Information Available: Variation of luminolabsorption (νa), emission (νem) energies, and Stokes shift (∆νss)with solvent polarity parameter, ∆f(ε,n) as well as ET(30)

Figure 4. Frontier orbital diagram for the most stable ground-state conformer of LH2 (structure IIb). The arrow mark in the LUMO indicatescharge localization on the oxygen and nitrogen atoms on electronic excitation.

Figure 5. Ground-state potential-energy surface for water-mediated conversion of I to IIb obtained from IRC calculation using B3LYP/6-311++G(d,p) methodology. The energy of the transition-state (TS) structure (given in the right-hand panel, bond lengths and angles are in Angstromsand degrees, respectively) and six points on both the sides are shown.

66 J. Phys. Chem. A, Vol. 114, No. 1, 2010 Moyon et al.

parameters and fully optimized geometries of isolated, mono-hydrated (S1 and S2), and dihydrated (S3) structures ofconformers I, IIa,and III. This material is available free ofcharge via the Internet at http://pubs.acs.org.

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