Photorkernirfry and Phoiobiology 1975. Vol. 2 2 , pp. 221-229. Pergamon Press.

Printed in Great Britain

INFLUENCE OF THE ENVIRONMENT ON THE EXCITATION WAVELENGTH DEPENDENCE OF THE FLUORESCENCE QUANTUM YIELD OF INDOLE I. TATISCHEFF and R. KLEIN Institut du Radium, Laboratoire Curie. Laboratoire associe au C.N.R.S. N” 198, 1 I , rue P. et M. Curie, 75231 Paris Cedex 05. France (Received 10 March 1975; accepted 27 June 1975) Abstract-The influence of excitation wavelength, pH, oxygen and solvents, upon fluorescence quantum yields, were measured for i n L k Indole in neutral aqueous solution exhibits the same wavelength dependence as tryptophan, which indicates that COO- absorption is not responsible for the effect. Parameters such as pH and oxygen influence only the absolute fluorescence quantum yields but not their relative variation with wavelength, indicating competition with fluorescence emission for S, deactivation. without any influence upon deactivation of higher excited states. In contrast, solvents exhibit ii specific influence upon the wavelength dependence; for indole. the decrease of fluorescence yield excited around 215 nm, compared with the yield in the first absorption band is about 40% in water. 10% in acetonitrile, 70% in n-hexane and cyclohexane, whereas no appreciable decrease occurs in methanol, ethanol or n-butanol. These observations, together with the Stokes shifts of the emission spectra, may be well correlated with Kosower’s Z-values, expressing microscopic solvent-solute interactions. INTRODUCTION

Dependence of the fluorescence quantum yields on excitation wavelength of aromatic amino acids was recently shown in neutral, aerobic dilute aqueous solutions at room temperature (Tatischeff and Klein, 1974). A quite similar experimental approach for observing excitation wavelength dependence upon fluorescence quantum yield in dilute solutions has been developed independently by Getoff et al. (Kaler and Getoff, 1974; Kohler and Getoff, 1974). The above observations were in disagreement both with Vavilov’s “law” (see Birks, 1970) and with a widely accepted earlier experimental verification (Teale and Weber, 1957). However, many deviations from Vavilov’s “law” have already appeared in literature (Birks. 1970). The observation for benzene was extended to many derivatives (Braun et al., 1963; Birks, 1969). Naphthalene and derivatives were also shown to be exceptions (Birks et a!.. 1968) and further studies (Fuchs, 1972; see Cundall and Palmer, 1973) confirmed the suggestion that deviation from Vavilov’s law is an inherent property of the benzene molecular structure (Birks, 1970). Although these observations indicate that intramolecular radiationless transitions from higher excited electronic states to the fluorescence emitting state are not effected with unit efficiency, many different suggestions have been given to explain the observed drop of fluorescence yield with decreasing excitation wavelength. Internal conversion to the ground state, singlet -+ triplet conversion, photodissociation. quenching by a photoproduct, excimer formation have been suggested (Birks, 1970). Recently Steen (1974b), after obtaining results in accordance with ours concerning the wavelength dependence of fluor-

escence yield for tryptophan and indole, put forward the hypothesis in terms of competition between internal conversion to s, and electron ejection for the decay of higher excited electronic states. Aside from the theoretical interest of the deviation from Vavilov’s law, the drop of fluorescence yield with decreasing wavelength observed with the aromatic monomers may help to understand similar behaviour already well known for proteins below 260 nm, but lacking adequate explanation (see Longworth, 1971). The present work, concerning indole, deals mainly with experiments on the influence of pH, oxygen and solvents on the wavelength dependence of fluorescence quantum yields. MATERIALS AND METHODS

Materials. All chemicals were of the purest grade available. L-Tryptophan was purchased from Cyclo-Chemical Corporation. Indole was from Eastman or Calbiochem; further purification by recrystallization from water or methanol was tried for indole. Solvents were purchased from Matheson, Coleman and Bell (cyclohexane, acetonitrile). Merck (n-hexane, cyclohexane, methanol, n-butanol, H,SO,, NaOH), and Prolabo (ethanol). Further purifimtion was achieved for ethanol (twice distilled) and n-hexane and cyclohexane (passed through a column of activated silica). Water was triply distilled. The influence of oxygen was studied by degassing solutions and solvents by 5 successive “freeze-thaw” cycles. Solute concentrations were or 2 x 10-’M. Measurements were performed at room temperature (OA = +24”C). Quantum yield. The absolute fluorescence quantum yield at a given excitation wavelength, A,, for a substance in solution, is defined as the ratio of the number of emitted photons to the number of quanta absorbed

221

222

I. TATISCHEFF and R. KLEIN

The relative yield as a function of excitation wavelength is

where represents the yield in the first absorption band. Detailed calculations of I,,(Lo), Ikl,) and @(lo), methods of measurements of @,X&) and @ were given in our earlier work (Tatischeff and Klein, 1974). Absorption spectra. The values oft, the molar extinction coefficient of the substance of concentration c, were measured by means of an absorption spectrophotometer (Beckman DK 2A or Cary 17). The fractional absorption spectrum was then calculated by

(3) with a = e(/IO)c/ (/ = 0.2cm) and /3 = k(l,)G, which accounts for solvent absorption Fluorescence excitation spectra. The analysing monochromator was set at the maximum, A,,, of the emission spectrum and the fluorescence intensity was measured as the excitation wavelength Lo was varied. The measured excitation spectrum is then

where K is a constant, I,,,(lo) is the light intensity measured by a monitor, which consists of a fluorite beam splitter and a sodium salicylate quantum counter associated with a PM tube; f ( l o ) represents a correction factor between the measured intensity I,,,(&) and the number of photons that elt'ectively reach the sample Iex(Ao). The corrected excitation spectrum is given by

RESULTS

Fractional absorption, corrected excitation spectrum and relative fluorescence quantum yield for indole in aqueous unbuffered neutral solution (24°C) are presented in Fig. 1. Since indole shows the same behaviour as tryptophan (Fig. 3 from Tatischeff and Klein, 1974), COO- absorption is not responsible for the observed wavelength dependence of the fluorescence quantum yield for tryptophan. Similarly, this is also true for tyrosine as Kohler and Getoff (1974) found a 30% drop of fluorescence for an aqueous solution of phenol excited in the second electronic band. Comparison of tryptophan and indole was also carried out at pH 10.8 (NaOH 10-3M) and pH 2.5 (H~SO 3.5~ x 10-3 N ) . were found to The fluorescence quantum yields be 0.20 and 0.08 for tryptophan and 0.26 and 0.20 for indole, at pH 10.8 and 2.5, respectively. Relative variation of indole fluorescence yield versus wavelength was not affected by pH, contrary to phenol; the phenolate ion shows a higher drop of fluorescence yield [$(A,)/$, = 0.40 instead of 0.701 with a strong red-shift equivalent to the absorption shift (Kohler and Getoff, 1974). This means that for indole, pH does influence SI deactivation but not deactivation of the higher excited states.

Fluorescence emission spectra. The excitation monochromator was set at a chosen excitation wavelength A,, and the wavelength Ai of the analysing monochromator was varied. The total number of emitted photons is then

where G IS a geometry factor, 7'(Ai) is the transmission characteristic of the analysing spectrometer, obtained by a method similar to Melhuish (1962) and Parker (1962). The variation of relative quantum yield vs wavelength, @&i0), was obtained by comparing corrected excitation spectrum with fractional absorption spectrum using the relative values of the correction factor f(A0), determined by 2 independent methods (Tatischeff and Klein, 1974). The yield @(Ao) was obtained by the relative quantum yield method in optically dilute solutions (Parker and Rees, 1960; see Demas and Crosby, 1971). z-Tryptophan, 2x M , in neutral unbuffered aqueous solution, at 24°C was taken as a standard, with a,,( = 0.13, at 280 nm (Tatischeff and Klein, 1974). The unknown quantum yields, measured at do = 280 nm, are related to the corrected emission spectra C[dQ/dV] by

(7)

where prime refers to the standard. The last term accounts for the refractive index correction, when measurements are effected in solvents of different refractive index n and n'.

.,A

nm

Fig. 1. Relative fluorescence yield of Indole (2 x M) in aqueous solution as a function of excitation wavelength. Ac(Ao):corrected fluorescence excitation spectrum in arbitrary units; B(IZo): fractional absorption spectrum; [@(Ao)/@ : relative fluorescence yield; (bandwidths: excitation A l o = 2.6 nm-emission M = 3.2 nrn).

Fluorescence quantum yield of indole

223

Table 1. Solvent systems used, macroscopic parameters, Z values and indole emission maxima

1 2 3 4 5 6 7 8 9 10 11

12 13

water acetonitrile methanol n-butanol cyclohexane n-hexane n-butanol-water : 99-1 ethanol-water : 0.95-99.05 19-81 47.5-52.5 95-5 ethunol-(wuter)-cyclohexane : 9.5- (0.5) -90 0.9y0.05t99

Dielectric constant, D (25°C) (c)

Refractive index, tiD (20°C)

18.4 36.2 32.6 17.1 2.02 (20") 1.88 (28")

(24.3)

"I (cm-')

(4

Z (kcal/mol) (d)

1.3325 (25") 1.3441 1.3288 1.3993 1.4264 1.3749

94.6 71.3 83.6 11.7 (59.8) 60. I

28,650 3 1,250 30,500 31.000 33,300 33,250

(79.6)

30,600

94.5, 92.7 89.5, 81.2

28,400 28.750 29.250 30,400

(74.8) (65.6)

3 1,250 32,500

(1.3576) (25")

(4

(a) Solvent systems as indicated in Figs. 5 and 6; (b) for solvent mixtures, numbers indicate percentage by volume of each component; (c) D and n values from Kosower (1968). Handbook of Chemistry and Physics (1957) (Edited by C. D. Hodgman), Chemical Rubber Co.. Cleveland, Ohio. Handbook of Biochemistry (1968) (Edited by H. A. Sober), Chemical Rubber Co.; (d) for Z values see Kosower (1958, 1968), values in brackets are calculated from Fig. 5(b); (e) measured maxima for indole corrected emission spectra in each solvent system, degree of confidence 100 cm-'.

In view of the poor solubility of tryptophan in nonpolar solvents, the influence of solvents was studied upon indole fluorescence in 6 solvents and I mixed solvents indicated in Table 1. Figure 2 compares corrected excitation spectra and fractional absorption of indole in 957; ethanol, acetonitrile and cyclohexane. It is easy to see that although both spectra are properly fitted in the first absorption band, excitation spectra show great variations in the second absorption band; this means that the relative fluorescence yield variation vs wavelength is influenced by the nature of the solvents, as shown in Fig. 3; the decrease of the fluorescence yield excited around

5

-

I

0

x

t

. {

,&+.+++~+hA

a"

-.------.-.La.

. ' . ..

*

MeOH

t'

A,.

nrn

A,,

EtOH

nm

3. Relative fluorescence yields of indole 2 x loe5k!in various solvents as a function of excitation wavelength. Arrows indicate the 'B,,strong electronic absorption maximum; bars correspond to the mean measured deviation for all the measurements. Figure

Figure 2. Corrected excitation spectrum A,(&) (---) comfor indole pared to fractional absorption B(&) (-) 2x M in (a) (95%) ethanol, (b) acetonitrile, (c) cyclohexane.

1

CH,CN

I. TATISCHEFF and R. KLEIN

224

Table 2.Fluorescence quantum yields of indole in aerated and/or deaerated solvent systems at excitation wavelength (a) Lo = 280nm

c

1

kli

i

kli

i

Solvent (b) water acetonitrile methanol n-butanol cyclohexane purified cyclohexane n-hexane n-hutanol-water

99-1

0.274 0.00,(7) 0.32 0.23-0.270.32 0.26 0.26,k 0.00,(2) 0.16

0.34 0.33-0.35 0.45i 0.00,(4) 0.49 0.42

2.1 2.1 3.4-2.7-2.1 2.9 2.7

5.3

0.34

I .9

0.24 0.33 0.35 0.28,i 0.00,(2)

3.2 2.0 1.9 2.5

I .9 2.C-1.9 I .2 I .@I

1.4

ethanol-water

0.95-99.05 19-81 41.5-52.5 95-5

0.39

1.6

ethanol+watertcyclohexane

9.5-(0.5) -90 0.9y0.05t99

0.40 0.45

1.5

I .2

M ) in water is taken as standard of fluorescence quantum yield with = 0.13.at (a) Trp (2x lo= 280 nm (Tatischeff and Klein, 1974);(b) as in Table 1; (c) number in brackets indicates the number of measurements and stated accuracy corresponds to the mean measured deviation: without number, measurements are single ones; (d) the yield is related to the ratio of S , non-radiative decay (ZkIi)to radiative decay (k,) by: (C,k,,/k,) = (1 - a#,) (8).

215 nm, compared to the yield in the first absorption band is about 40% in water, 10% in acetonitrile, 70”/, in n-hexane and cyclohexane, whereas no appreciable decrease occurs in alcohols. Results in aqueous solutions were not affected by indole purification. For cyclohexane and hexane, purification of degassed solvents was checked both by absorption and a search for impurity fluorescence (intense 254 nm excitation). The Q1 values were affected

by solvent impurities and oxygen (Table 2). but not the relative fluorescence yield (Table 3). Besides this new effect of solvents upon the relative fluorescence yield variation vs wavelength, the Stokes shifts of the emission maxima were measured for 13 solvent systems studied. Wavelength maxima (in cm-’) of the corrected fluorescence spectra are indicated in Table 1. The absolute fluorescence yields excited at 280 nm in aerated or deaerated solutions

Table 3. Fluorescence quantum yields of indole excited in the electronic absorption band centered about A0 = 215nm

1 water

2 acetonitrile 3 methanol 4 n-butanol 5 cyclohexane 6 n-hexane n-hutanol--water

7

99-1

0.62i: 0.02(5) 0.90 (2) 1 1 2 1

(2) 0.31k 0.03(6) 0.34It 0.02(2) -1

0.17 0.29 0.23-0.21-0.32 0.26 0.08 0.05

0.31 0.3H.35 0.14 0.14

0.61 0.11 0 0 2.23 1.94

0.34

0

0.14

0.39

0.72 0.47 0.15 0

0.42 0.2I

0 1.17

i~rhuiiolwater 8 095-99.05 19-81 10 47.5-52.5

9

I1

95-5

0.58 0.68

0.87 1 5 1

(3)

0.22 0.30 0.285

ethanol (warer~cyclohexane

I2 9 , s(0.5) -90 13 0.9y0.05)-99

1.04 0.46

(a) Solvent systems as indicated in Figs. 5 and 6;(b) as in Table 1; (c) as in Table 2; (d) (@& and (@2)dcaeralcd calculated with Q 2 / @ , above and results of Table 2;(e) relative weight of S2 deactivation not reaching S, to S, - S , internal conversion.

Fluorescence quantum yield of indole A,, 400

L

I

nm 350 I

v,

315 I

290 I

1

pm-'

Figure 4. Correction emission spectra of indole 2x M in various solvents: (a) water, (b) (95%) ethanol, (c) acetonitrile, (d) cyclohexane, excited at 280 nm and related to constant absorption (EcL' = 0.01); (e) standard of fluorescence quantum yield: tryptophan 2 x lo-' M in water. were also measured (Table 2). Figure 4 shows the corrected emission spectra for indole in 4 solvents and for tryptophan in water, all at constant absorption (EL./ = 0.01). The fluorescence yields b2 are calculated from the measured fluorescence yields at 280 nm and the measured fluorescence drop. Results are given in Table 3. The degree of confidence for the corrected emission maxima is estimated to f100cm- for quantum yield measurements, the accuracy should be +_ 10%. No refractive index corrections were made as a function of wavelength, but correction should not exceed 6% between 215 nm and 300 nm for water and it cannot be responsible for the observed variation of the fluorescence yield. The bars in Fig. 3 do not indicate the accuracy of the relative fluorescence yield measurements but only the reproducibility. As absorption is very low around 235nm, the accuracy is much less in this range. The observed trough in acetonitrile, the only solvent sensitive to irradiation in our conditions, is not thought to be significant.

';

DISCUSSION

Injluence of solvents upon jfuorescence quantum yields excited in the first absorption band

Although indoles and tryptophan have received continuing attention for many years, there remain great discrepancies in the published fluorescence yields excited in the first absorption bands, as shown

225

in Table 4. This is not surprising in view of the experimental difficulties in absolute yield measurements. Therefore, relative values of the yields measured as a function of parameters such as solvents, temperature, concentration or excitation wavelength, are likely to be more valuable than a comparison of absolute values obtained in different laboratories. Nevertheless, because of the great number of measurements, such comparison may have a statistical value in order to point out erroneous measurements. In view of recent determinations of the fluorescence quantum yield of tryptophan in aqueous solution (Borresen, 1967; Chen, 1967; Eisinger, 1969; Tatischeff and Klein, 1974), it seems that all measurements relative to a tryptophan yield of 0.20 are too high (Weber and Teale, 1957; White, 1959; Longworth et al., 1966; Cowgill, 1967). It is noteworthy that the fluorescence yield for indole in water (Eisinger and Navon, 1969; Feitelson, 1970; Kirby and Steiner, 1970; De Lauder and Wahl, 1971; Tatischeff and Klein, this work), using different standards (Table 4, our primary standard to measure GTrpwas quinine sulfate in H,SO, 0.1 N , @ = 0.51, Tatischeff and Klein, 1974), agree well with each other. The small difference is probably due to the influence of temperature in part, which is a very sensitive parameter in water (Gally and Edelman, 1962; Walker et a/., 1969; Kirby and Steiner, 1970; Laustriat and Gerard, 1974). In view of the above agreement, Walker's values in water and cyclohexane and Andrews' value (1974) in perfluoromethylcyclohexane seem too high. Our values for indole fluorescence yields in different solvents are in good agreement with those calculated from De Lauder and Wahl (1971). The slight discrepancy between Kirby and Steiner (1970) and De Lauder and Wahl (1971) for indole in methanol is perhaps the result of different hydration; we found that for methanol the initial yield of 0.23 became greater with time, reaching 0.32 (Table 2). Furthermore, the influence of oxygen in methanol was all the more notable the lower the yield. As already pointed out, oxygen has no effect in water but is very sensitive in non-polar solvents (Feitelson, 1970; De Lauder and Wahl, 1971). Therefore, Kirby's values in (apparently non-degassed) non-polar solvents seem too high. In our fluorescence yield measurements for alcohol-water mixtures, we also found, as indicated by Kirby and Steiner (1970), that the yield for mixtures reaches a maximum higher than yields in either one of these solvents (Table 2). The general tendency of indole fluorescence yield in pure solvents is to decrease as the polarity of the solvent increases. One explanation may be a solventinduced increase in the rate of internal conversion from S1 to So, as already suggested for other solutes showing the same behaviour (Forster and Rokos, 1967; Cundall and Pereira, 1973; Werner and Hoffman, 1973). However, in view of the variation of the results, fluorescence yield is not, in our opinion, a good parameter for the study of solvent effects.

I. TATISCHEFF and R. KLEIN

226

Table 4. Literature values of indole fluorescence quantum yields excited in the first electronic absorption band, at room temperature (a)

Water

0.45 ( I ) (0.24) (3) 0.40 (2) (0.46) (7) 0.40 (4) 0.40 (6) 0.23 (8) 0.28 (1 1) 0.24 (12) 0.24 (12) 0.28 (10)

0.45 (9)

Formamide

0.23 (1 1)

Dimethylsulfoxide

0.42 (5)

p-Dioxane

0.62 (6) 0.42 (11) 0.29 (12)

0.24(12) 2-Methylpentane

Deuterium oxide

0.39 (1 1)

Methanol

0.32 (1 1) 0.24 (12) 0.27 (12)

,I-Butanol

0.58 (6)

Propylene-glycol

0.38 (1 1)

0.59 (15)

0.63 (9) n-Hexane

Ethanol

0.30, (12) O.3Os (12)

0.29, (12)

0.19, (12) 0.40 (12) 0.44 (12)

0.30 (12) Cyclohexane

0.34 (12) 0.37 (13)

0.41 (11) 0.59 (14) 0.32 (12) 0.43 (12) 0.49 (12)

Me thylcyclohexane Perfluoromethylcyclohexane ~~~

0.36 (10) 0.03 (15) ~

~~

(a) Numbers in brackets indicate references gathered below: References Standard 1. Weber and Teale (1957) 2. White (1959) Tyrosine in water = 0.21 3. Drushel et al. (1963) Quinine sulfate in water (H,SO,) = 0.55 4. Gladchenko et a\. (1965) 5. Longworth et a/. (1966) Tryptophan in water = 0.19 6. Cowgill (1 967) Tryptophan in water = 0.20 7. Bridges and Williams (1968) Quinine bisulfate in 0.1 N H,SO, = 0.55 8. Eisinger and Navon (1969) p-Terphenyl in aerated cyclohexane = 0.87 9. Walker et al. (1969) Quinine bisulfate, in 0.1 N H,S04 = 0.55 10. Feitelson (1970) Terphenyl in deoxygenated cyclohexane = 0.93 11. Kirby and Steiner (1970) Tryptophan in water = 0.14 12. De Lauder and Wahl (1971) D,L-Tryptophan in water = 0.14 13. Feitelson (1971) (from Fig. 3) Terphenyl in deoxygenated cyclohexane = 0.93 14. Walker et al. (1971) Quinine bisulfate in 0.1 N H,SO, = 0.55 15. Andrews and Forster (1974) Indole in cyclohexane = 0.59

Influence of solvents on Stokes sh$s of the emission spectra

contribute, the most important interaction affecting the indole excited state has little to do with the l-poThe well-recognized Stokes shift of the indole emis- sition. Therefore, Stryer's explanation of the deutersion with regard to absorption spectra (Van Duuren, ium isotope effect upon fluorescence quantum yield 1961, 1963; Mataga et al., 1964; Konev, 1967; Walker (1966). namely NH deprotonation during the S, et al., 1966. 1967, 1971) is a better choice for study excitcd lifetime, cannot account for solvent effects. of the influence of solvents. Solvents are known to Among remaining suggestions of 1 : 1 or 1 :2 excihave but little influence on indole absorption spectra plexes (Walker et at., 1966, 1967, 1969, 1971; Long(Van Duuren, 1961; Mataga et al., 1964), although worth, 1968), solvent reorientation (Eisinger and more recent work did show an influence, mainly on Navon, 1969), and 2-H isomerisation (Chopin and the 'Loband, correlated with the ability of the solute Wharton, 1969), it is not possible as yet to select the to form N-hydrogen bond with the solvent (Strick- most satisfactory explanation. land et al., 1972; Andrews and Forster, 1972; Auer, Attempts to correlate the Stokes shifts with solvent 1973). macroscopic parameters such as dielectric constant In contrast, the predominant red-shift of emission (D),refractive index (n) and viscosity are known to spectra with increasing polarity of the solvent is not be unavailing (Basu, 1964).More sophisticated theory, at all correlated with N-hydrogen bonding, as sug- taking into account electrostatic interactions (see gested by Mataga and Tsuno (1957), inasmuch as Basu, 1964; Mataga et al., 1956; Lippert. 1957) are such shifts are observed as well for N-methylindole not always satisfactory, given the neglect of crucial (Van Duuren, 1961; Eisinger and Navon, 1969; Long- short range interactions such as charge transfer and worth, 1968; Walker et nl., 1967). This means that hydrogen bonding. This explains the success of emwhatever the mechanism of solute-solvent interaction pirical parameters (Grunwald and Winstein, 1948; is, and although many interfering interactions may Kosower, 1958) which take into account all solute-

227

Fluorescence quantum yield of indole solvent interactions at a microscopic level (Kosower, 1968). In view of the poor correlation of the indole Stokes shifts with the function D-1 n2-1 F(D,n) = -- 2 0 1 2nZ 1 '

+

i

+

taking into account the dipolar interaction (Mataga et al., 1964; Walker et ul., 1967; this work Fig. 5a), we chose to correlate our v, - vf measurements with Kosower's Z values, taking v, = 3.48 x lo4 cm- ', corresponding to the 'L,state of indole. This represents a mean value with f 150cm-' deviation for the different solvents studied. Figure 5b shows that a good correlation is obtained for 9 solvents, whence the possibility of calculating Z-values for 4 other solvent systems used (5, 7, 12 and 13). The good 2-correlation for indole Stokes shifts was already noted by Walker et a/. (1966) as unpublished results. Indeed, the observed correlation only means that the alkyl-pyridinium iodide used to define Z values (Kosower, 1958) is a "good model" for indole solutesolvent interaction, without implying a specific molecular mechanism, unless a charge-transfer process is likely (Kosower, 1968). It is noteworthy that Z (or equivalent ET (30) values, Kosower, 1968), defined from absorption of charge-transfer complexes, also give good correlations with Stokes shifts or fluorescence quantum yields for different molecules such as 9-methylanthroate (Werner and Hofhan, 1973) and substituted naphthalene sulfonates (Forster and Rokos, 1967; Kosower and Tanizawa, 1972). It seems that the model should be a good one for molecules for which, as noticed by Forster and Rokos (1967),

Kcol/ml

Figure 6. Ratio of indole S, deactivation not reaching S, to S, - S , internal conversion correlated with Z values. Arabic numerals refer to solvent systems as indicated in Tables 1 and 3. the fluoresence yield decrease is correlated with increasing fluorescence red-shift. Influence of solvents on the relative fluorescence quantum yield variation with wavelength

Whatever its molecular origin, the observed correlation between v, - vs and 2 enables one to search for a general correlation between the observed drop [l - (4z/41)]of fluorescence quantum yield d2 excited in the ' B b band compared to 4' ('La or ' L , excitation). The relative weight of Sz deactivation not reaching S, via internal conversion is given by (9)

Figure 6 shows the correlation between (42/$1) - 1 and 2,to wit the relative weight of S2 deactivation not reaching S1 via internal conversion. The grcat influence of low alcohol concentrations in cyclohexaneethanol mixtures (Walker et al., 1966) is also obvious in Figs. 5 and 6. To explain the correlation in Fig. 6, it is tempting to suggest that excited solutesolvent interaction is more efficient to induce internal conversion from S, to S, as the polarity of the solvent increases (range I of Fig. 6) until unit efficiency is reached (Z 2 72 kcal/mol). Solvent-induced increase in the rate of internal conversion has already been postulated for S, (Forster and Rokos, 1967; Cundall and Pereira, 1973; Werner and Hoffman, 1973) but our results indicate that such a mechanism could also influence higher excited state deactivation, which imI plies a very fast solutesolvent interaction. Indeed, Struve et al. (1973, 1974)recently asserted that solvent Kcal /mol reorientation around a dipole can be very fast Figure 5. Stokes shifts for indole fluorescence emission in 4 x 10- l 1 s) so that solvent reorientation around different solvents: (a) correlation with S, can not be excluded. However, solvent-relaxationF(D,n) = (D - 1/20 + I ) - (n2 - 1/2n2 + 1) (Mataga et a/., 1964);(b) correlation with Z values (Kosower, 1958, 1968); facilitated intersystem crossing to the triplet state, as numbers refer to solvent systems as indicated in Tables suggested by Seliskar and Brand (1971), can not yet 1 and 3. be excluded. ( 5

228

I. TATISCHEFF and R. KLEIN

In range I1 (Fig. 6), Vavilov's law is obeyed until another deactivation process appears, which competes with S, internal conversion to S1, for the more polar solvent combinations (range 111 of Fig. 6). As all these latter combinations ( Z > 88 kcal/mol) include water, for which e - ejection is one of the primary photolytic processes (Grossweiner and Joschek, 1965), and given the correlation between e - yields and luminescence quenching (Feitelson, 1971) together with the observed increase of e - photoejection in the tryptophan band (Steen, 1974a), it is not unlikely that e - photoejection could be the new deactivation process, as suggested by Steen (1974b). The fact that the ratio #2/#1 is insensitive to the presence of quenchers (02,H', impurities) brings out again the idea that the decrease of fluorescence yield excited in the 'Bb band is due to a very fast process. It has been shown that photoionization in aqueous solutions can be a very fast process. The solvated electrons appear in 10-'2-10-'1 s from /%naphthol (Klanning et al., 1973) and in 4 x s from ferrocyanide (Rentzepis et al., 1973). Photoionization of indole, if this fast, could therefore be in competition with the internal conversion of S, to S,, a process of similar rate (Birks, 1970). However, it is worth stressing that, in our opinion, this explanation may be valid only for water or binary mixtures with high water content. In the presence of pure isopropanol, there is no photoejection of electrons from indole excited in the ' L , or 'L,, band (Feitelson, 1971). The electron yields measured in non-aqueous solutions of indole seem due to an effect of the high N 2 0 concentrations used, (Hopkins and Lumry. 1972). Concerning the mechanism of solute-solvent interaction, it is worth noting that the carbon in position 3 could be involved, in view of the following arguments: The polar mesomeric form (11) of indole (I) may make a large contribution in the excited state and is more sensitive to the polarity of the surrounding solvents (Van Duuren, 1963). Position 3 was implicated in some instances; indolyl free radical formation, following e- photoejection in water (Pailthorpe

and Nicholls, 1971), fluorescence quenching by carbony1 substituents in hydrogen bonding solvents (Cowgill, 1967) and protonation (Hinman and Whipple, 1962).

4

I

II

III

The main argument is that benzimidazole (structure 111). in neutral aqueous solution, has both a high

fluorescence yield (41= 0.58 excited at 270nm) and good mirror symmetry (v,,,,, = 34,40Om-') (Tatischeff and Klein, unpublished results). For this solute (111), no e- photoejection is measured in contrast to indole (Grossweiner and Joschek, 1965). It is interesting to mention that for benzimidazole in neutral aqueous solution, we did not observe any drop of the fluorescence quantum yield, consistent with the assumption that, for indole, the fluorescence drop in the high Z solvents (range 111 of Fig. 6) may be due to a higher yield for e- photoejection in the ' B , absorption band, compared to the one in 'La or ' L , bands. Furthermore, Adler (1962) noticed no shift in benzimidazole emission in going from water to alcohol, which is expected if a charged-C at the 3 position is essential for solute-solvent interaction. This view is in close agreement with the suggestion of Vander Donckt (1969), i.e., the formation of hydrogen bond between carbon atoms and protic solvents should be the main solute-solvent interaction of indole derivatives. Further work is planned on the influence of environment on benzimidazole fluorescence similar to the present study of indole. Acknowledgements-The authors wish to thank Prof. M. Duquesne for continuous interest and stimulating discussions and Mrs. G. Tham for her valuable technical assistance. We are also grateful to Dr. C. Helene for his comments about the manuscript.

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