Effect of Solvent Hydrogen Bonding on Excited-State Properties of

Nov 12, 2009 - Department of Chemistry, North-Eastern Hill UniVersity, Shillong 793 022, India. ReceiVed: August 18, 2009; ReVised Manuscript ReceiVed...
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J. Phys. Chem. A 2010, 114, 60–67

Effect of Solvent Hydrogen Bonding on Excited-State Properties of Luminol: A Combined Fluorescence 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 fluorescence spectroscopy. The fluorescence spectral behavior of luminol is markedly different in polar protic solvents compared to that in aprotic solvents. A quantitative estimation of the contribution from different solvatochromic parameters, like solvent polarizibility (π*), hydrogen-bond donor (R), and hydrogen-bond acceptor (β), was made using the linear free energy relationship based on the Kamlet-Taft equation. The analysis reveals that the hydrogen-bond-donating ability (acidity) of the solvent is the most important parameter to characterize the 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 degrees of hydration. In the excited state, charge localization at specific points of the luminol phthalhydrazide moiety causes the solvent to interact primarily through hydrogen-bond donation. 1. Introduction Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, LH2) is a versatile chemical that shows striking blue chemiluminescence in the presence of certain metal ions when treated with an appropriate oxidizing agent like hydrogen peroxide. This unique feature of LH2 is often exploited by forensic investigators to detect trace amount of blood left in the crime scene. LH2 is also used by biologists as a cellular assay to detect copper, iron, and cyanides, etc.1-5 Further, LH2-enhanced chemiluminescent probes have been used to characterize and quantify the secretion of oxygen by phagocytozing cells.6 The use of LH2 chemiluminescence has also been reported recently for facile detection of proteins,7 cancer biomarkers,8 as well as reactive oxygen species produced by human neutrophils.9 The ultrahigh sensitivity 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 aspect of LH2 chemiluminescence is its different degrees of sensitivity from one substance to another. LH2 shows higher sensitivity to animal or human blood, organic tissues, and fluids than to other compounds containing metal ions, such as paints, metallic surfaces, household products, or vegetable enzymes. Therefore, the light emitted by LH2 has different intensities and time duration depending on the material of contact, making it an efficient forensic marker. The solution-phase spectroscopic properties of LH2 have drawn enormous interest in recent times due to the biochemical relevance of its photoactivity. The photophysical properties of LH2 in different solvents and solvent mixtures as well as its interaction with several biological molecules were reported in the literature.11-15 LH2 exhibits two principle absorption bands in the 300 and 350 nm region, whereas a single broad fluorescence emission appears in the 400 nm region. Interestingly, the fluorescence emission peak shows a large spectral shift toward longer wavelengths in hydrogen-bonding solvents. * To whom correspondence should be addressed. Phone: (91)-3642722634. Fax: (91)-364-2550076. E-mail:[email protected], sivaprasadm@ yahoo.com.

This shift is believed to be due to the stabilization of the chargetransfer excited state of the intermolecular hydrogen-bonded complex of LH2 with solvent.11 The intermolecular hydrogen bonding and solubility of organic systems are known to play crucial roles in determining the biological activity as well as their application in forensic science.16-19 In general, formation of an intermolecular hydrogen bond between the solute and the solvent results in a decrease in the Gibb’s free energy and thus promotes mixing. Hydrogen bonding can occur in different modes, depending on the structure of the solute and solvent. The situation becomes more complicated when a solute molecule possesses multiple hydrogenbonding sites and the solvent molecule can act both as a proton donor as well as a proton acceptor. Under this condition, the competition among different molecular species resulting from a hydrogen-bonding interaction between the solute and the solvent molecules becomes inevitable. LH2 provides a unique example to study the hydrogen-bonding effect because the molecule itself can exist in more than one prototropic species (Scheme 1) having multiple hydrogen-bonding sites. The keto-amine (I) structure can go to the enol-imine (III) form in a single step or through intermediate structures IIa and/or IIb, respectively. These interconversion and associated spectroscopic properties will depend strongly on the relative abundance of several species as well as their hydrogen-bonding mode with the solvent. Furthermore, the efficacy of hydrogenbond formation in the excited state may change due to charge redistribution after excitation. Also, the relatively strong and unstructured fluorescence of LH2 is an additional advantage to use the spectroscopic ruler for quantitative estimation of the effect of different solvent parameters. The chemiluminescence and fluorescence bands of LH2 in water appear in the same wavelength region (425-430 nm);13 thus, quantitative characterization of this band on different solvent parameters is indispensable. In this paper, we use the steady-state spectral properties of LH2 in a series of pure solvents with varying polarity as well as the hydrogen-bond donor and acceptor abilities to find quantitative information about their relative contribution. Fur-

10.1021/jp907970b  2010 American Chemical Society Published on Web 11/12/2009

Excited-State Properties of Luminol SCHEME 1: Possible Tautomeric Structures of Luminol (LH2)a

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()

Fi 1 - 10-A ni i Fs 1 - 10-A ns s

φif ) φsf

2

(1)

where Ai and As are the optical density of the sample and standard, respectively, and ni is the refractive index of the solvent at 293 K. The relative experimental error of the measured quantum yield was estimated within (5%. The pH variation experiments were carried out in a Systronics µ-pH system (type 361, resolution 0.01 pH) at constant temperature (293 K). 2.3. Estimating the Relative Contribution of Solvent Parameters on LH2 Spectral Properties. The effects of solvent polarity and hydrogen bonding on the steady-state spectral properties of LH2 are interpreted by means of the linear solvation energy relationship (LSER) concept using Kamlet-Taft eq 222

P ) P0 + sπ* + aR + bβ

a

Numbering used in calculation is also shown.

thermore, the effects of specific LH2-water hydrogen bonding on the solution-phase spectral properties were theoretically modeled by using the density functional approach. 2. Materials and Methods 2.1. Chemicals. Luminol (LH2, 97%) was received from Sigma-Aldrich Chemical Pvt. Ltd. and used without any further purification. The organic solvents used were of spectroscopic grade (>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 water purification system (Millipore India Pvt. Ltd.). The chromophore concentration (∼1.2 × 10-5 mol dm-3) was very low to avoid any aggregation and kept constant during spectral measurements in different solvents. 2.2. Experimental Procedure. Steady-state absorption spectra were recorded on a Perkin-Elmer model Lambda25 absorption spectrophotometer. Fluorescence spectra were taken in a Hitachi model FL4500 spectrofluorimeter, and all spectra were corrected for the instrument response function. Quartz cuvettes of 10 mm optical path length received from PerkinElmer, USA (part no. B0831009), and Hellma, Germany (type 111-QS), were used for measuring absorption and fluorescence spectra, respectively. For fluorescence emission, the sample was excited at 360 nm unless otherwise mentioned, whereas excitation spectra were obtained by monitoring at the respective emission maximum. In all cases, a 5 nm band pass was used in the excitation and emission side. Fluorescence quantum yields (φf) were calculated by comparing the total fluorescence intensity under the whole fluorescence spectral range with that of a standard (quinine bisulfate in 0.5 M H2SO4 solution, φfs ) 0.54620) with the following equation using adequate correction for the solvent refractive index (n)21

(2)

where P is the value of the solvent-dependent property to be modeled and P0, s, a, and b are the coefficients determined from the LSER analysis. The term π* indicates the measure of solvent dipolarity/polarizibility,23 whereas R and β are the scale of hydrogen-bond donation acidity and acceptance basicity of the solvent, respectively.19 The corresponding parameters for 14 solvents were taken from the literature25,26 and are given in Table 1. The correlations of the spectroscopic data were carried out by multiple linear regression analysis as implemented in the ORIGIN 6.0 (Microcal Inc.) program package. 2.4. Theoretical Calculations. Density functional theory (DFT) has successfully been applied to study the hydrogen bonding in various systems including organic as well as biological model systems.27-30 Pan et al. used the B3LYP functional along with the 6-31++G(d,p) basis set to verify the extent of specific solvation of formic acid or formate anion model systems with few water molecules,31 whereas in a recent paper the effectiveness of DFT calculations to predict the hydrogen-bonding strength between different drug molecules and corresponding receptors was investigated.32 The success of the DFT method in elucidating the complex phenomenon like hydrogen-bonding prompts us to use it further in studying the energetic parameters of isolated LH2 and its complex with water molecules. Several conformers of LH2 are possible. To elucidate the lowest energy structure, full geometry optimization was performed for all possible conformers of LH2 both in isolated as well as in their mono- and dihydrated complexes using the B3LYP method and 6-311++G(d,p) basis set as implemented in the Gaussian03 program package.33 Frequency calculations were done in each stationary point to characterize the minimum energy equilibrium structure. Vertical transition energies up to the first 10 singlet excited states and corresponding oscillator strengths for all structures were estimated using the timedependent DFT method (TD-DFT) at the same level of calculation. A similar methodology was successfully applied recently for characterizing the spectroscopic properties of large organic heterocyclic systems.34-36 3. Results and Discussion 3.1. Steady-State Spectral Properties in Pure Solvents. Figure 1 shows some representative absorption and emission spectra of LH2 in aqueous medium, and Table 2 summarizes the steady-state spectral behavior of LH2 in solvents with varying polarity and hydrogen-bonding parameters. In homogeneous solvents, LH2 shows two absorption maxima. One relatively

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TABLE 1: Solvent Parameters no. of solvent

solvent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

acetonitrile ethyl acetate benzene tetrahydrofuran 1,4-dioxane toluene DMSO DMF dichloromethane 1-butanol methanol 1-propanol 1-pentyl alcohol water acetone isopropanol

∆f(ε,n)a ET(30)b 0.30 0.19 0.0 0.21 0.03 0.02 0.26 0.27 0.22 0.26 0.31 0.27 0.25 0.32 0.28 0.27

45.6 38.1 34.3 37.4 36.0 33.9 45.1 43.2 40.7 49.7 55.4 50.7 49.1 63.1 42.2 48.4

Rc

βc

πc

0.19 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.84 0.98 0.84

0.40 0.45 0.10 0.55 0.37 0.11 0.76 0.69 0.1 0.84 0.66 0.90

0.75 0.55 0.59 0.58 0.55 0.54 0.1 0.88 0.81 0.47 0.60 0.52

1.17 0.47 1.09 0.08 0.48 0.71 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 are represented 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.

structured high-energy peak appears in the 280-320 nm region, whereas the other unstructured, broad low-energy absorption is in the 330-380 nm region. However, the emission obtained by 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 show a broad peak at ∼350 nm. In a recent report,13 Vasilescu et al. proposed an acid-base type of equilibrium to explain the two absorption bands of LH2 in highly alkaline (pH ≈ 12.0) DMSO solution for the formation of the corresponding dianion of structure III. This was further confirmed by the concomitant quenching of the LH2 fluorescence and appearance of a new broad band at 475-480 nm with increasing alkali concentration. However, formation of the dianionic species in the present experimental condition of neutral aqueous LH2 solution (pH ≈ 6.4) can be ruled out. The absence of any new fluorescence band further supports this hypothesis. The origin of the broad absorption in the 330-380 nm region can be assigned as the S1(π) r S0(π) transition, whereas the origin of the high-energy absorption at ∼300 nm may be due to S2 r S0 excitation. From the 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 can be concluded that this transition is also π* r π in nature. The assignment of these absorptions as well as further verification for the long-wavelength absorption at 475-480 nm to be originated from the anionic species is confirmed from the theoretical calculations described in the following sections. Although the results in Table 2 do not show any regular variation of the steady-state spectral properties, careful observation reveals several interesting trends. For example, the fluorescence maxima (λem) show an appreciable shift in protic solvents along with an almost 2-fold increase in the fluorescence quantum yield (φf) when compared with their aprotic counterpart. Furthermore, the full width at half maxima (fwhm) for both the absorption and the emission peaks are much higher in water compared to the other solvents. All these results indicate that consideration of the hydrogen-bonding interaction is very important to describe LH2 photophysics, more particularly in aqueous medium. 3.2. Solvatochromism of LH2 Photophysics: Estimation of Contributions from Solvent Parameters. To verify the effect of solvent polarity, several steady-state spectral parameters of LH2 in a variety of solvents mentioned in Table 1 were plotted against the solvent polarity parameter ∆f(ε, n). From the results given in Figure 1S in the Supporting Information, it is clear that the spectroscopic properties of LH2 do not show any regular solvatochromism behavior on the solvent polarity parameter. This observation points to the existence of specific solute-solvent interactions. As a trial, the empirical solvent polarity scale, ET(30), built with a betaine dye, was used as it is the most popular choice to correlate several solvent-dependent spectral properties. The uniparametric scale depends on both the solvent dielectric properties and the hydrogen-bonding acidity, but it does not take care of the solvent hydrogen-bonding acceptor basicity.37 The specificity of Lewis acid base interactions in the ET(30) parameter arises from the negative charge localized on the phenolic oxygen of the betaine molecule. As it is seen in the Supporting Information (Figure 2S) again, there is no linear correlation of either the LH2 absorption/emission energies or the Stokes shift even with this parameter. A break point, mostly influenced by LH2 emission properties like the fluorescence maxima (νem) and Stokes shift (∆νss), is obtained around ET(30) ) 38 kcal mol-1. This clearly indicates that apart from solvent polarity, LH2 solvatochromism is strongly modulated by both solvent hydrogen-bond donor acidity and solvent hydrogen-bond acceptor basicity parameters also. In view of this situation, one must look at a multiparametric approach, as devised by Kamlet and Taft and mentioned in eq 2, to assess the contribution of different solvent parameters on LH2 solvatochromism. The s, a, and b coefficients in eq 2 were all obtained by multiple linear regression analysis, and the results are given in Tables 3 and 4. A few representative correlation diagrams of the experimental values with those calculated from multiple regression analysis using eq 2 are shown in Figure 2. A close look into the tables reveals several interesting features for LH2 solvatochromism. (i) In general, the contributions from a as well as b parameters are significant relative to the s parameter, indicating the importance of solvent hydrogen bonding in LH2 spectroscopy. (ii) The excited-state spectral properties like fluorescence maxima, Stokes shift, quantum yield, etc., are mostly controlled by the solvent hydrogen-bond acidity function (a parameter), whereas both a and b contribute almost equally in the absorption property. This indicates an efficient charge localization in LH2 upon excitation (see DFT calculation results in the following section). (iii) The charge localization

Excited-State Properties of Luminol

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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 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

28 169 28 172 28 170 28 165 28 170 28 169 27 933 28 090 28 011 28 410 28 169 28 090 27 933 28 572 28 328 28 011

25 126 25 253 24 876 25 316 25 126 24 876 24 876 25 063 24 876 24 331 24 450 24 390 24 450 23 474 25 063 24 510

28 490 28 490 28 249 28 410 28 490 28 410 28 090 28 249 28 490 28 011 28 329 28 090 28 090 28 410 28 090 28 169

3043 2919 3294 2849 3044 3293 3057 3027 3135 4079 3719 3700 3483 5098 3265 3501

0.26 0.21 0.20 0.24 0.25 0.21 0.33 0.26 0.29 0.52 0.42 0.58 0.58 0.78 0.31 0.50

3053 2902 3053 2902 2853 3158 2902 2902 3106 3004 3057 3106 3057 3211 2902 3057

3333 3333 3099 3263 3712 4010 3263 3310 3358 3590 4201 3768 3793 4388 3001 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 P0

s

a.

b

R2

SD

28 183.14 25 362.49 28 492.90 2820.65 0.074 3061.69 3566.69

70.06 -652.76 25.60 722.80 0.225 28.84 -30.11

260.90 -1448.0 26.92 1347.85 0.33 233.77 851.47

-267.79 -391.26 -424.06 -657.84 0.05 -277.26 -491.86

0.90 0.94 0.92 0.88 0.89 0.85 0.89

140.3 166.7 147.2 223.3 0.06 71.7 281.3

property (P) -1

νabs/cm νem/cm-1 νexc/cm-1 ∆νss/cm-1 φf δem/cm-1 δexc/cm-1

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

TABLE 4: Relative Values (in percentage) of the Solvatochromic Parameters Toward LH2 Steady-State Spectral Properties property (P)

Ps(%)

Pa(%)

Pb(%)

νabs νem νexc ∆νss φf δem δexc

11.70 30.65 5.37 26.49 37.19 5.34 2.19

43.57 51.03 5.64 49.38 54.55 43.30 61.99

44.73 18.32 88.89 24.13 8.26 51.36 35.82

in the excited state is further confirmed by the negative values of both a and s.38 (iv) An almost 2-fold increase in the fluorescence quantum yield in protic medium (Table 2) is mainly due to the hydrogen-bond acidity of the solvents with a/s ≈ 1.5. However, solvents like DMSO with a higher hydrogenbonding acceptor (HBA) tendency have very little effect (∼8%) on φf. For example, the φf value of LH2 in water is about 0.79 compared to that in 1,4-dioxane (0.25) and DMSO (0.34). This observation is also in line with our recent finding that the yield of LH2 fluorescence decreases substantially in the presence water-soluble proteins like bovine and human serum albumins. The decreased fluorescence intensity of LH2 on binding to albumins most likely reflects reduced water access to the chromophore in the bound state. Finally, (v) the large spectral shift in water and other hydroxylic solvents, as observed in Table 2, is due to the negative value of the a parameter and its corresponding larger contribution (Table 4). In summary, the solvatochromic analysis reveals that in polar protic solvents, like water, for example, several spectroscopic species may be present due to the hydrogen-bonded donor and acceptor properties of the solvent in the ground state; however, in the excited

sate, the main fluorescing species is originated due to the hydrogen-bonded complex formation of LH2 through the solvent hydrogen-bonding acidity behavior. 3.3. Theoretical Calculation Using Density Functional Theory. 3.3.1. Energetic of Different Conformers in the Ground State. Full geometry optimization of different conformers of LH2 in isolated condition (structures given in Scheme 1 along with the numbering scheme) as well as with different degrees of hydration (some of the fully optimized structures are shown in Figure 3) was done using the B3LYP/6311++G(d,p) methodology. The fully optimized structures of all other conformers with varying degrees of complexation with water molecule(s) are shown in Figure 3S, Supporting Information. The energy parameters, relative to the most stable structure, are given in Table 5. It is clear that the conformer IIb is the most stable structure in the isolated, monohydrated as well as in the dihydrated configuration. However, comparison of the relative energies indicates that LH2 most likely exists in the dihydrated complex structure represented by IIb-S3. The structure represented by I-S3 is about ∼7.6 kJ mol-1 higher in energy than IIb-S3. It may still be possible that this high-energy structure will have relatively less abundance in solution along with the structure IIb-S3 in the ground state and more so, in the excited state (see below). However, the existence of all other conformers represented by IIa and III can be neglected to discuss the spectroscopic behavior of LH2 in water. This is because of their relatively higher energy; they are unlikely to be present in solution mixture. It is to be noted here that only the primary hydrogen-bonded complex with water was considered to give different complexes like S1, S2, and S3 (Figure 3). It is obvious that additional solvent molecules will combine to give a secondary water cluster around LH2, and the actual

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

hydrated structure is far more complex than what is considered for these calculations. However, we restrict ourselves only in the first hydration layer, as the effect of internal hydrogen bonding (IHB) is expected to diminish very fast from the center of origin. Consequently, it is reasonable to believe that any further addition of water structure will have an insignificant contribution toward the relative energy parameters. 3.3.2. TD-DFT Calculation on the Excited State. The excited states of the two most stable ground-state structures of dihydrated LH2 discussed above, i.e., for I-S3 and IIb-S3, were calculated using the TD-DFT procedure. The calculated transition energies and corresponding oscillator strengths for several singlet excitations within the experimentally observed absorption wavelength range of 260-400 nm for both structures are given in Table 6. It is noted that the nature of the transition as well as its energy and oscillator strength is comparable for the both the structures. The calculated S1 r S0 transition wavelength of ∼335 nm is in close agreement with the experimentally observed value of 350 nm and the gas-phase absorption energy of 354 nm (Table 3). The nature of the second lowest energy transition in the experiment cannot be assigned unambiguously because of the close energy separation of the next two calculated values (294 and 281 nm, respectively) and their similar oscillator strengths. However, all of the associated orbitals that might be involved in excitation with a significant contribution in this wavelength range, viz. HOMO-1, HOMO, LUMO, and LUMO+1 (Table 6), are shown to be of π type in nature (Figure 4). Further, to confirm that no proton-dissociated anionic species

contributes in this wavelength region, TD-DFT calculation was performed 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 obtained value of 475-480 nm reported by Vasilescu et al.13 These authors proposed the existence of a proton-dissociated dianionic structure of conformer III responsible for this absorption. However, from the energy parameters given in Table 5 it is clear that the formation of this conformation itself is very unlikely. Thus, the anionic species responsible for the longwavelength absorption is believed to be originated from conformer IIb. Furthermore, comparison of the nature of the HOMO and LUMO in Figure 4 reveals that on excitation the electron density is more localized on the carbonyl oxygen and imino nitrogen atoms (O12 and N10, respectively, in Scheme 1), thereby increasing the basicity at these points significantly. This confirms the importance of the hydrogen-bond-donating ability (acidity) of the solvent to discuss the spectroscopic behavior of LH2, more particularly in the excited state, as indeed observed from LSER analysis discussed above. The calculated energy difference between I and IIb in the ground 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 that the water-assisted conversion of I to IIb is associated with a large activation barrier of ∼55 kJ mol-1. Thus, in the ground state the relative abundance of the high-energy structure (I-S3) will be much less, from both kinetic and thermodynamic points

Excited-State Properties of Luminol

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

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

I

IIa

IIb

III

isolated monohydrated (S1) monohydrated (S2) mihydrated (S3)

95.504 52.952 50.450 7.635

99.626 58.062 57.507 14.544

81.421 41.553 41.715 0.0

141.022 90.538 93.064 44.182

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

of view. However, TD-DFT calculation results show that the relative energy difference between I-S3 and IIb-S3 is very small in the first excited state (∼3.4 kJ mol-1). Hence, a simple Boltzmann distribution predicts approximately 25% population of the excited state to exist as I-S3 in solution at room temperature. 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 structures is similar to each other. Naturally, it is expected for them to show similar spectroscopic behavior, particularly in noninteracting solvents. However, as these conformers differ considerably in their mode and extent of hydrogen bonding, it is possible to form different hydrogen-bonded clusters in protic solvents with little difference in energy. The broad nature of the emission spectra of LH2 in protic solvents, as discussed before, may be due to the ensemble-averaged spectral properties of all these microstructures. Conclusions The excited-state photophysical behavior of luminol has been studied by steady-state fluorescence spectroscopy and DFT calculations. The observed solvatochromism in the

TABLE 6: Calculated Singlet Excited-State Transitions, Associated Energies, and Oscillator Strength (f) of I-S3 and IIb-S3a I-S3 singlet state 1 2 3 a

transition HfL H-1 f L H-1 f L+1 H f L+1

IIb-S3

energy /nm

f

transition

energy /nm

f

335 294 282

0.13 0.01 0.07

HfL H f L+1 H-1 f L H f L+1

332 294 281

0.14 0.07 0.05

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

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Figure 4. Frontier orbital diagram for the most stable ground-state conformer of LH2 (structure IIb). The arrow mark in the LUMO indicates charge 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/6311++G(d,p) methodology. The energy of the transition-state (TS) structure (given in the right-hand panel, bond lengths and angles are in Angstroms and degrees, respectively) and six points on both the sides are shown.

luminol spectral properties are rationalized by a multiparametric approach using the Kamlet-Taft equation. It has been found that the photoluminescence behavior of luminol is strongly modulated by specific solute-solvent interaction. Quantitative estimation of the relative contribution of several solvatochromic parameters indicates that the hydrogen-bond donor ability of the solvent is the primary factor governing the excited-state properties. These observations have also been supported by DFT calculations on luminol in isolated conditions as well as with varying degrees of hydration. The results predict that the dihydrated luminol-water complex is the most stable structure, where preferential solvent hydrogen-bond donation occurs at the localized electron-rich

centers like the imine nitrogen and carbonyl oxygen at the 2- and 4- positions of the phthalhydrazide ring system. Acknowledgment. Financial support through research project 34-299/2008(SR) from the University Grants Commission (UGC), Government of India, is gratefully acknowledged. The authors thank Mr. T. Sanjoy Singh and Ms. S. Phukan for their help in preparing some of the figures. Thanks are also due to UGC and DST for supporting the Department of Chemistry through DSA-SAP and FIST, respectively. Supporting Information Available: Variation of luminol absorption (νa), emission (νem) energies, and Stokes shift (∆νss) with solvent polarity parameter, ∆f(ε,n) as well as ET(30)

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