The Journal of Physical Chemistty, Vol. 83, No. 7, 1979 805
Reactions in Microemulsions
Reactions in Microemulsions. 4. Kinetics of Chlorophyll Sensitized Photoreduction of Methyl Red and Crystal Violet by Ascorbate C. E. Jones, C. A. Jones, and R. A. Mackay" Department of Chemistry, Drexel University, Philadelphia, Pennsylvanla 19 104 (Received August 14, 1978; Revised Manuscript Received November 16, 1978) Pi16lication costs assisted by the US. Department of Energy
We have investigated the photoreduction of adsorbed dye (methyl red and crystal violet) sensitized by chlorophyll a in an anionic mineral oil in water microemulsion system. The 100-A microdroplets occupy 40% of the total volume, and at the pigment concentrations employed (110 yM) there is on the average less than one chlorophyll molecule per drop. Using ascorbate as the water-soluble reducing agent, the reaction exhibits a pseudo-first-order dependence on crystal violet, but a pseudo-zero-order dependence on methyl red. The chlorophyll fluorescence is quenched by methyl red, but not by crystal violet. The effect of sensitizer and of ascorbate concentration arid light intensity on the quantum yield has also been examined. From the results of these and auxiliary studies, a reaction mechanism is proposed which involves the formation of a chlorophyll-dye triplet exciplex rather than the initial formation of an oxidized or reduced pigment species. Based on this scheme, a number of rate constants have been estimated.
Introduction We recently reported a study of the photodegradation of chlorophyll in a n oil-in-water microemulsion,' a transparent and stable fluid medium of large interfacial area. By a proper choice of oil, the pigrnent will tend to accumulate in the microdroplet surface region where it is available to participate in photosensitized redox reactions between water soluble and oil soluble or adsorbed substances. Previous studies involving porphyrins have shown that otherwise transient species can be stabilized in the droplet inlerphase of micellar emulsions,2 and that chlorophyll mediates the photoreduction of various dyes.l8 Chlorophyll in high concentration is present in an aggregated form in the chloroplast membrane,4 and the aqueous-oil interface in a microdroplet is similar to the protein-lipid interface in many respects. Before the effects of interpigment interactions are investigated, it is necessary to have a good understanding of the role of low concentrations of chlorophyll in photosensitizing redox reactions in this type of colloidal medium. We have therefore undertaken a detailed study of the kinetics of the photoreduction of methyl red and crystal violet by ascorbate in a mineral oil in water microemulsion stabilized by an anionic surfactant and l-pentanol as cosurfactant. Studies of chlorin-sensitized redox reactions have been numerous and, in particular, the photoreduction of methyl red has received considerable a t t e n t i ~ n . ~ The - l ~ kinetics are often complex and a thorough description of the methyl red photoreduction has not appeared. One study attempted to establish the stoichiometry8 while others reported quantum yieldsgJOand determined the reaction order with respect, to the individual reactants.11-13 Both zero- and first-order kinetics with respect to methyl red have been observed. Variation of reaction conditions by different workers, including type of chlorin, its adsorbent, type of reducing agent, solvent, pH, and reactant concentrations, makes rt difficult to compare their results and draw general conclusions. However, the mechanism cited most often is the oxidation of the excited chlorin to the radical cation by the oxidant (methyl red) followed by reaction of the cation with the reducing agent. This scheme predicts a non-zero-order dependence on both 0022-3654/79/2083-0805$01 .OO/O
methyl red and reducing agent.
Experimental Section Actinometry. The intensity of the light was measured with aqueous Reinecke's salt, KCr(NH3)2(NCS)4, which is an actinometric standard used for the long wavelength visible region as described by Adamson.14 The actinometric measurements of the xenon lamp intensity were performed periodically, always in dim red light. The experimental setup was the same as that used in the kinetic runs. Additional light intensity measurements were made with a Scientech power meter (Model 362). The meter and the chemical actinometry gave a power output of 3.6 X lo4 einstein s-l from the xenon lamp. Microemulsion Preparation. The oil-in-water (o/w) microemulsion was prepared by mixing sodium cetyl sulfate (SCS)(12.4% w/w), l-pentanol (19.2%),mineral oil (8.8%),and water (59.6%). Agitation was not required, but the mixture was stirred to speed formation of the micellar solution. The four components could be added in any order and the microemulsion would form. A psuedo three component phase map of this system may be found in ref 15. In the kinetic runs the H 2 0 component was a p H 7.00 phosphate buffer (Fischer certified). Chlorophyll Solutions. The procedure followed for the separation of chlorophyll a was similar to that of Anderson and Calvin.16 For convenience of handling and to speed dissolution of the chlorophyll in the microemulsion, it was first dissolved in ether. Aliquots of the ether solution were placed in volumetric flasks, the ether was carefully evaporated, and the microemulsion added to the mark. The concentration of chlorophyll was determined from its absorption spectrum.l Beer's law is obeyed, at least up to concentrations of M. Kinetic Experiments. The irradiation source was a 1000-W xenon lamp fitted with a water (infrared) filter and an interference filter centered at 667.0 nm with a halt-band width of 8.0 nm. The well-stirred 3-mL sample was held in a 1.0-cm quartz fluorescence cell. The hollow brass cell holder was thermostated a t 25.0 " C by water circulated from a constant temperature bath. The samples were purged with nitrogen for 10 min just prior to each run, and 0 1979 American
Chemical Society
806
C. E. Jones, C.A. Jones, and R. A. Mackay
The Journal of Physical Chemistry, Vol. 83, No. 7, 1979
TABLE I: Dependence of Relative Quantum Yield @R on Light Intensity (I,,)for Chlorophyll Mediated Photoreduction of Methvl Red bv Ascorbatea
I,& (einstein sec-') x
108
@RC
5.75 0.72 5.00 1.00 3.20 0.93 0.57 1.07 a 3.3 x lom6 M chlorophyll, 1.06 X M ascorbate, 8 X l o m 5M initial methyl red. Measured a t 6670 A ; 80 A half-band width. Quantum yield, relative to that of 5 X lo-' einsteins/s, +16%. Average value = 0.93 i 0.11.
TABLE 11: Relative Quantum Yield Vs. Initial Methyl Red Concentrationa @R~ M x lo5 @Itb 0.81 9.7 1.00 0.81 11.0 0.92 1.11 13.0 1.02 0.90 14.0 1.20 0.84 a 1.1 x M chlorophyll a, 1.0 X M ascorbate. Quantum yield *15%, relative to the value at M. M X 105
2.7 4.1 5.6 7.0 8.4
the cell compartment flushed with nitrogen during the run. The disappearance of dye was monitored by a Gilford spectrophotometer Model 240, connected to a chart recorder. The methyl red and crystal violet were followed at 500 and 600 nm, respectively. Physical Measurements. UV-visible spectra were obtained on a Cary 14 spectrophotometer. Fluorescence measurements employed an Aminco-Bowman spectrophotofluorimeter with a frontface attachment in addition to the regular cell holder. Polarographic measurements utilized a Beckman Electroscan 30 in conjunction with a three-electrode system.
Results The stirring rate must be rapid in relation to the reaction rate so as to maintain a uniform instantaneous concentration of species throughout the cell. It was determined that using a stirring rate of 0.03 mL s-l and an absorbance of less than 1.0 for the chlorophyll red band (667 nm), the maximum usable reaction rate was about 0.3 pM s-l. In general, the rates were maintained around 0.1 pM s-l, and none were in excess of 0.2 pM s-l. Since the ascorbate and dye concentrations were on the order of 1 and 10 pM, respectively, the reductant concentration was essentially constant during the entire reaction. Even though the reaction mixtures were purged with nitrogen, an induction period due to dissolved oxygen was observed. The oxygen caused no loss of methyl red or chlorophyll but did result in a net consumption of ascorbate. During reduction, methyl red, an azo dye, is transformed to the partially reduced hydrazo derivative which may be oxidized back to the azo form by oxygen. The only net result is the reaction of some ascorbate. Introducing known small amounts of ascorbate revealed M. this residual oxygen content to be about 6-8 X After consumption of the oxygen the methyl red reaction rate was constant and independent of the amount of oxygen initially present, as long as the initial ascorbate concentration was relatively high (21mM). Interruption of the reaction by shuttering the xenon lamp and addition of small aliquots of methyl red or ascorbate produced no further induction period after the initial removal of residual oxygen. In this way the reaction stoichiometry was determined. A small amount of ascorbate was allowed to react entirely, thus removing all the oxygen but leaving most of the methyl red unreacted. An additional known amount of ascorbate was then added and again reacted completely. The change in methyl red absorbance yielded the corresponding moles of methyl red reacted. The ratio of moles of ascorbate reacted to moles of methyl red reacted was 2.09 f 0.20. Since the ascorbate undergoes a two-electron oxidation to dehydroascorbate, the methyl red undergoes a four-electron reduction. The reaction products in the microemulsion have not been identified but may correspond to the chemical reduction products,
#
0 00 0
ASCORBATE
CONCENTRATION (mM)
Figure 1. Dependence of the quantum yield for methyl red reduction on ascorbate concentration at 25.0 OC.
anthranilic acid and N,N-dimethyl-p-phenylenediamine.l0 The effect of the concentration of chlorophyll on the reaction rate was also determined,3 and the quantum yield remains essentially constant over a chlorophyll concentration range of two orders of magnitude (0.1-10 pM). It is therefore not surprising that since the quantum yield is independent of the chlorophyll a concentration, it is also independent of the light intensity, a t least over an order of magnitude as shown in Table I. Generally speaking, the chlorophyll concentration was less than 4 pM, and no photobleaching of the red band was observed. The disappearance of methyl red is zero order in dye, and goes to completion. Not surprisingly therefore, the quantum yield is found to be independent of the initial methyl red concentration over the range 100-10 pM (Table 11). However, at very high initial concentrations of oxidant (e.g., 1 mM), the apparent quantum yield decrease^.^ A t this concentration methyl red quenches the chlorophyll fluorescence significantly ( l l % ) , with a Stern-Volmer constant of 106 M-l. A value of 114 M-' has been reported in methan01.l~ From this observation it is concluded that the quenching of fluorescence is not part of the reaction pathway, but competes with it. Apparently, removal of excited singlet chlorophyll by quenching does not lead to a reactive state of methyl red which in turn implicates the chlorophyll triplet state as the precursor of reaction. The ascorbate exhibits saturation kinetics in the reaction with methyl red in the microemulsion. A plot of the quantum yield vs. ascorbate concentration is shown in Figure 1. The points represent experimental values and the line is a least-squares fit of the data to the equation
d = - a(D) b
+ (D)
where 4 is the quantum yield, ( D ) is the ascorbate con-
The Journal of Physical Chemistry, Vol. 83, No. 7: 1979 807
Reactions in Microernulsions
TABLE 111: Relative Quantum Yield as a Function of the Chlorin Used in the Methyl Red-Ascorbate Reaction' -. concn, M chlorin
chlorin
chloroDhvlla 2.8 x chlorophyll a 2.8 X 1.0 x chlorophyll b 2.8 X chlorophyll a pheophytin a 1.0 x pheophytin a 1.0 x 10-5 1.0 x dieophvtin a pheophytin a 1.0 X chlorophyll a 3.0 X lo-'} pheophytin a 3.0 X lo-' a In all cases the reaction is zero initial concentration 8 X M. relative to pure chlorophyll a.
t t
ascorbate
hRb
1.0 x 1.0 x 10-3
1.00 1.27
1.0 x 10-3 1.0 x 10-3 3.0 X lo-$
1.36 0.98 2.02 2.09 2-27
6.0X 6.0
10-3
order in methyl red of Quantum yield i15%,
TABLE IV: Summary of Crystal Violet Kinetics' lob 3.9 3.9 2.2 3.9
(ascorbate) X lo3 @/(CV)' 1.2 39 0.4 13 37 1.2 1.2d 23
' 2.19 x M initial crystal violet, 1.45 x M chlorophyll a. X LO8 einstein s-', CV = crystal violet. Quantum yield i: 15%. Phenylhydrazine used as reducing agent. centration (M), a =: 0.122, and b = 1.08 X There are two important features to note about the reductant dependence of the quantum yield. First, at low ascorbate concentrations (> ( D ) ,and there is a first-order dependence of on ( D ) ,with slope a / b = 113. Second, at high ascorbate concentration where ( D ) >> b, the quantum yield is approximately constant with a value of 0.12. Some preliminary results were obtained using pheophytin a and chlorophyll b as photosensitizing agents in place of the chlorophyll a and are shown in Table 111. It was discovered that, these two alone and in combination with the chlorophyll a gave kinetics and quantum yields similar to those of chlorophyll a. The fact that the quantum yield is not changing significantly indicates that the methyl red-ascorbate reaction is not necessarily dependent on the particular structure of chlorophyll a, and that the central magnesium atom is not necessary for the reaction to occur. The crystal violet-ascorbate system, in contrast to the methyl red-ascorbate system, exhibits kinetics which are first order with respect to dye. In addition, crystal violet does not quench chlorophyll fluorescence in the microemulsion. A summary of the crystal violet-ascorbate system studies are given in Table IV. Once again, as in the methyl red-ascorbate case, the reaction is independent of the chlorophyll concentration and the light intensity. The quantum yield, of course, is dependent on the dye concentration, since the reaction is first order; hence the third column gives the value of the quotient of 4 and the crystal violet concentration. It will be noted, however, that for a crystal violet concentration of about 10 FM, the quantum yield is two orders of magnitude lower than that for methyl red. The reaction once again is first order in ascorbate over the range tested. I t was not possible to determine if the ascorbate dependence of the quantum yield also exhibited saturation, because the ascorbate concentration would have to be above its solubility limit in the microemulsion. For the SCS microemulsion composition employed here the droplet diameter is about 10 nm, corresponding to a
droplet concentration on the order of M.15 For a chlorophyll concentration of M there is only one pigment molcule per hundred drops on the average, and the probability of a droplet containing more than one is negligible. Therefore, at the concentrationsemployed, each chlorophyll molecule is isolated and there should be no effects due to any form of aggregation or other interaction. The dyes are adsorbed by the microdroplet as shown by spectral and electrochemicals t u d i e ~ .The ~ dye reductions do not proceed in the dark.
Discussion There are two general mechanisms which have been invoked to explain the participation of chlorophyll in photoredox reactions, an oxidative and reductive mechanism.l* In these mechanisms, excited chlorophyll (Chl*) reacts with the oxidant or reductant to produce the radical cation (Chl+) or the reduced form ChlH, respectively. These chlorophyll species then react with the remaining partner of the redox system. An additional modification to either of these mechanisms involves the formation of an exciplex between Chl* and the oxidant or r e d u ~ t a n t . ~ ~ ~ ~ ~ This exciplex then reacts with the remaining partner of the redox system. In this case, there may be no direct intermediacy of Chl' or ChlH. Any mechanism which is proposed must account for all the observed facts. One scheme which does so is presented but is not intended to represent the only possibility. Direct energy transfer from excited triplet chlorophyll to the dye is unlikely on energetic grounds since the lowest energy absorption maximum of chlorophyll (667 nm) is 5000 cm-l lower in energy than that of methyl red (500 nm), and the triplet state of chlorophyll has been reported to be about 3000 cm-l lower than the first excited singlet.21 Similarly, ascorbate has no accessible excited states. It seems reasonable that since the reaction shows saturation with respect to ascorbate, some type of complex is involved. Since methyl red quenches chlorophyll fluorescence while ascorbate does not, the identification of the complex as an ascorbate-excited singlet chlorophyll exciplex seems unlikely. Since quenching of the chlorophyll fluorescence by methyl red appears to compete with the reaction, the complex from which the reaction is proceeding cannot involve the excited singlet state of chlorophyll, but must be a triplet exciplex. Finally, since the rate of quenching of triplet chlorophyll b in ethanol by methyl red is reported to be lo4times faster than by ascorbate,22it appears likely that the exciplex involves the oxidant rather than the reductant. While these considerations have been applied with respect to methyl red, it will be seen that the same basic mechanism can be applied to the crystal violet results. It bears repeating that the basis for the mechanism is the formation of a chlorophyll-dye triplet exciplex from which the reaction takes place rather than direct electron transfer from the chlorophyll to the dye molecule with subsequent ion pair separation. Such a triplet exciplex may well form an ion pair but it is held that such an ion pair remains associated and serves the same reactive purpose. Dissociation of such an ion pair would not be favored in the relatively nonpolar environment of the microdroplet interphase region. In the remainder of this discussion it should be understood that all references to a triplet exciplex allow for the possibility of an associated ion pair and that kinetics explained by one can be equally well explained by the other.
808
The Journal of Physical Chemistry, Vol. 83, No. 7, 1979
C. E. Jones, C. A. Jones, and R. A. Mackay
Scheme I 1p
hu d
Scheme 11. Mechanistic Assumptions for Methyl Red 1. Assumptions
'p*
a. efficient quenching by singlet exciplex
(3) kt 3p* + 1p
k,
>> h-1
b. efficient triplex exciplex formation
h , ( N > h,
(4)
c. rate of triplet exciplex decay to ground state much greater than rate of dissociation ht, k2
3P* + A
3(AP)*+ D
2. Result
F=?
3(AP)*
k-2
-+ -
3(AP)*
kte
k3
>> k-2
A
lP
products
(7)
(8)
+ IP
(9)
The proposed mechanism is shown in Scheme I, where P is a pigment (chlorophyll), D an electron donor (ascorbate), and A an electron acceptor (dye). The first four steps are the photophysical processes involving the formation and decay of excited singlet ('P*) and triplet (3P*) chlorophyll. The fifth step shows the formation and dissociation of a singlet exciplex between chlorophyll and the dye molecule. The sixth step is the decay of the singlet exciplex back to the ground state. There is no spectral evidence for the formation of a complex between ground state chlorophyll and dye. The seventh step is the formation and dissociation of a triplet exciplex. The reducing agent (ascorbate) reacts with the triplet exciplex (step 9), while the singlet exciplex is necessary to explain the quenching. In step 9 it is assumed that the initial oneelectron transfer from D to A is rate controlling. Since the 2:l reaction stoichiometry is maintained, a second oneelectron transfer should rapidly occur yielding dehydroascorbate and the hydrazo dye intermediate which can then react in the dark with another molecule of ascorbate. The eighth step is the decay of the triplet exciplex back to the dye molecule and ground state chlorophyll. Using the mechanism presented in Scheme I, an expression is obtained for the rate of reaction by applying the steady-state approximation to the concentration of the following species: the triplet exciplex, 3(AP)*;the triplet chlorophyll, 3P*; the singlet exciplex, l(AP)*;and the singlet excited chlorophyll, IP*. The resulting expression for the rate in terms of measurable concentrations and the rate constants for the different processes is in eq I.
This general rate expression will be applied to the kinetics of both the methyl red and crystal violet reactions. The use of physically reasonable and consistent assumptions in the rate equation leads to expressions for the quantum yield which agree with the experimental results for both dyes. The kinetic scheme, as it applies to methyl red, is summarized in Scheme 11. The first assumption is to
For &,(A) > k-z 2. Result
4/(A) = a ( D ) / ( b + (D)) where a = k2kisc/kt(ks+ kist), b = kt,/k, inefficient singlet exciplex formation is required to explain the lack of quenching of chlorophyll a fluorescence by crystal violet. The second assumption is that the corresponding formation of the triplet exciplex is also not favored, which results in a first-order dependence of quantum yield on concentration. The third assumption is the same as for the methyl red case. The resulting expression for the quantum yield is first order with respect to the dye concentration and, once again at low ascorbate concentrations, the reaction is first-order with respect to the reducing agent. The predicted saturation kinetics were not observed because of the low quantum yield. Projected ascorbate concentrations required to observe a leveling off of @ would cause the microemulsion to “break’ (phase separation). Once again the quantum yield is independent of light intensity and chlorophyll a concentration. The a value for crystal violet is equal to kz&/kt, where the value of @t is the a value for methyl red (0.12). However, only the value of a / b (3.3 X lo4M-2) can be obtained for crystal violet, which is equal to 4tkzks/ktekt. Although it is not necessary that the value of kte/k3for both crystal violet and methyl red be the same, they will be assumed equal in order to estimate k2/kt. The value for k 2 / k t then becomes 300 M-l. The value of kt for chlorophyll a in pyridine at room temperature is reported to be about lo3 This results in a value for k z of 3 X lo5M-l s-l. This may be compared with a value of lo9M-l
s-l for a diffusion-controlled reaction in water. Based on the above mechanism, the order of magnitude of a number of other rate constants may be estimated. Using a singlet lifetime of 10 ns18330and a value of @t about 0.1 from the methyl red kinetic data, k , and k,, are lo8 and lo7 s-l, respectively. In this mechanism the Stern-Volmer quenching constant K,, = k l / ( k , + k,,,) kl/k,. Using the measured K,, for methyl red of lo2 M-l, k l has the value 1O1O M-l s-l. If the rate of singlet exciplex formation is diffusion controlled, then k l would normally be on the order of lo9 M-I s-l in water. However, the effective dye concentration in the droplet surface layer is about one order of magnitude higher than the overall concentration, which apparently manifests itself in a k l value of 1O1O M-’ S-1.
For methyl red, if k 2 is also a diffusion-limited rate constant, it will have a value comparable to that of k l (1O’O M-l s-l). For a low methyl red concentration of M, kz(A) is equal to lo4 s-l. In order to meet the condition kz(A) > kt, kt must be less than lo4 s-l. Thus the triplet lifetime must be greater than 0.1 ms. This may be compared with reported values of 121 and 1.1-0.62 msa31 It should be noted that this study has demonstrated the utility of microemulsions for the investigation of photochemical reactions between species located in various environments in the vicinity of a microscopic oil-water interface. The chlorin head group of the pigment is located in the interphase region of the microdroplet, while the dye is adsorbed at or near the droplet ~ u r f a c e .The ~ formation of a pigment-dye exciplex may be enhanced by the effect of both the proximity of the reactants and possibly the structure and intermiate polarity of the interfacial region.
Summary The chlorophyll sensitized photoreduction of methyl red and crystal violet by ascorbate is found to be zero and first order in dye, respectively. The kinetic data are consistent with the formation, a t room temperature, of an oxidant-pigment exciplex which is subsequently reduced. The scheme predicts a triplet lifetime in the microemulsion of greater than 100 ps. Acknowledgment. This work was support by the Division of Basic Sciences of the Department of Energy.
References and Notes (1) C. E. Jones and R. A. Mackay, J. Phys. Chem., 82, 63 (1978). (2) K. Letts and R. A. Mackay, Inorg. Chem., 14, 2993 (1978). (3) C. E. Jones and R. A. Mackay in “Porphyrin Chemistry”, F. R. Longo, Ed., Ann Arbor Press, Ann Arbor, Mich., 1979. (4) J. J. Katz, W.Vettmerer, and J. R. Nwris, Phil. Trans. R . Soc. London, Sect. 6, 273, 227 (1976); F. K. Fong, Proc. Natl. Acad. Sci. U.S.A., 71, 3692 (1974). (5) A. A. Krasnovsky and A. V. Umrikhina, Dokl. Akad. Nauk SSSR, 122, 1061 (1958). (6) I. G. Savkina and V. B. Evstigneev, Biokhimiya, 29, 975 (1964). (7) V. D. Semichaevskii, Ukr. Bot. Zh., 31, 380 (1974). (8) L. P. Vernon, Acta Chem. Scand., 15, 1639 (1961). (9) R. Livingston and R. Pariser, J . Am. Chem. Soc., 70, 1510 (1948). (10) G. R. Seely, J . Phys. Chem., 69, 821 (1965). (11) R. Kapler and L. I. Nekrasov, Biofizika, 11, 420 (1966). (12) L. I. Nekrasov, L. V. Chasovnikova, and N. I. Kobozev, Zh. Fir. Khim., 41, 2634 (1967). (13) N. I. Loboda, L. I. Nekrasov, and N. A. Shchegoleva, Zh. Fiz. Khim., 48, 338 (1974). (14) E. E. Wegner and A. W.Adamson, J . Am. Chem. Soc., 88, 394 (1966). (15) R. A. Mackay, K. Letts, and C. Jones, “Micellization, Solubilization and Microemulsion”, Vol. 2, K. L. Mittal, Ed., Plenum Press, New York, 1977, p 807. (16) A. F. H. Anderson and M. Calvin, Nature(London),104, 285 (1962). (17) R. Livingston and C. L. Ke, J . Am. Chem. Soc., 72, 909 (1950). (18) L. P. Vernon and G. R. Seely, Ed., “The Chlorophylls”, Academic Press, New York, 1966. (19) G. Tollin, Bioenergetics, 6, 89 (1974). (20) N. Y. Andreyeva and A. K. Chibisov, Biofizika, 21, 24 (1976).
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(21) J. S.Connaiiy, D. S. Gorman, and G. R. Seeiy, Ann. N.Y. Acad. Sci., 206, 649 (1973). (22) A. K. Chibisov, A. V. Karyakin, and M. E. Zubriiina, b k l . Akad. Nauk SSSR, 170, 198 (1966). (23)P. G. Bowers and G. Porter, R o c . R. SOC.London, Ser. A , 296, 435 (1967). (24) M. N. Usacheva, V. A. Dagaev, and B. Y. Dain, Theor. f x p . Chem., 6, 628 (1970). (25) G. P. Gurinovich, A. I. Patsko, and A. N. Sevchenko, Dokl. Akad. Nauk SSSR, 174, 873 (1967).
(26)B. M. Dzhagarov, Opt. Spectrosc., 28, 33 (1970). (27) 0.M. Petsoid, I. M. Byteva, and G. P. Gurinovich, Opt. Spectrosc., 34, 343 (1973). (28)A. P. Losev, E. I. Zen'kevich, and E. I. Sagun, Zh. frikl. Spektrosk., 27, 244 (1977). (29) A. T. Gradyushko, V. A. Mashenkov, K. N. Soiov'ev, and M. P. Tsvirko, Zh. Prikl. Spektrosk., 9,514 (1968). (30) D. R. Sanadi and L. P. Vernon, Ed., "Current Topics in Bioenergetics", Vol. 7,"Photosynthesis: Part A", Academic Press, New York, 1978. (31) M. K. Bowman, Chem. fhys. Lett., 48, 17 (1977).
Fluorescence Spectra of 3-Cyano-4-methyl-7-hydroxycoumarin and Its Acid-Base Behavior in the Excited State Michio Takakusa Electrotechnical Laboratory, Tanashl-shi, Tokyo, Japan (Received June 30, 1978; Revised Manuscript Received November 15, 1978) Publication costs assisted by the Nectrotechnical Laboratory
Fluorescence spectra of 3-cyano-4-methyl-7-hydroxycoumarin were measured in ethanolic solutions containing various amounts of water and acid. Spectra composed of two bands, one from the undissociated molecule and the other from the phenolate anion, were observed by excitation of the undissociated molecule with most of the solutions studied. From variation of the fluorescence intensities pK,*'s were determined according to Weller's method. The value varied from 2.3 to 0.01 with the variation of water content from 0.2 to 80%. The rate constants of excited-state dissociation and its reverse reaction were also determined from the fluorescence intensity and lifetime data. With highly acidic solutions the fluorescence band due to the excited-state proton adduct at the carbonyl oxygen was observed in the spectral region between the two bands mentioned above. The green fluorescence band characteristic of most 7-hydroxycoumarins, which is attributed to the zwitterionic tautomer, was not observed. This fact was attributed to the effect of the electron-withdrawing cyano group at the 3 position.
Introduction Derivatives of 7-hydroxycoumarin are generally good fluorophores and are used as laser dyes for the blue spectral region. Among them, 4-methyl-7-hydroxycoumarin, usually called 4-MU, is used most frequently. Shank et a1.l obtained a surprisingly wide tunable range of laser emission by use of its ethanolic solution containing a small amount of diluted hydrochloric acid solution. The range covers the violet through yellowish-green regions. The present author and his co-worker2 found that the water content in the solution was a more important factor, and prepared a neutral solution of 4-MU giving a similar tunability by adding a controlled amount of water to the solvent. The fluorescence spectra of our solution and Shank's were both composed of three fluorescence bands (XflmaX 385, 450, and 490 nm). We call them bands I, 11, and I11 in order of their peak wavelengths from short to longa3 After Shank's report, many investigations on its fluorescence, especially for elucidation of band 111, were made.P8 The emitting species for bands I, 11, and I11 have been determined as the undissociated molecule (I), the " O W 0 ' O
W
CH3
I
CH3
O - w 0 6 H c--* 0 CH3
\ O O H
CH3
I1
phenolate anion (II), and the zwitterionic tautomer (111); respectively. Species I11 can exist only in the excited state. 3-Cyano-7-hydroxycoumarin and its 4-methyl derivative are also known to fluoresce in alcoholic and alkaline aqueous media,lOJ1but little has been reported concerning their fluorescence spectra. We found that 3-cyano-40022-3654/79/2083-0810$01 .OO/O
methyl-7-hydroxycoumarin (3-CN-4-MU) was an efficient laser dye which can be used for the blue and blue-violet spectral regions?2 The wavelength of the laser emission depended on the solvent conditions, but features of the spectral change differed from those observed with 4-MU. In the present paper, we report the fluorescence spectra of 3-CN-4-MU in ethanolic solutions containing water or acid, and discuss the behavior of the excited-state molecules of the compound in those solutions.
Experimental Section Materials. 3-Cyano-4-methyl-7-hydroxycoumarin was prepared according to the literature,13 and purified by recrystallization from ethanol; mp 294-295 "C. Ethanol, perchloric acid, and sodium hydroxide were Reagent Grade. Distilled water which had been left in a polyethylene bottle for 1 week was used to prepare neutral aqueous ethanol solution^.'^ Spectroscopic Measurements. Absorption spectra were measured with a Hitachi 323 spectrophotometer, and fluorescence spectra were measured with a Hitachi MPF-4 spectrofluorometer a t a room temperature. Fluorescence spectra reported in the present paper were all corrected. The intensities were related not to the radiation energies but to the number of photons per unit width of wavelength. The concentration of 3-CN-4-MU was 2 X M unless otherwise noted. The fluorescence lifetime was measured with a JASCO FL-10 phase fluorometer. Results and Discussion Acid-Base Equilibria and Absorption Spectra. Hydroxycoumarins are subject to acid dissociation in aqueous 0 1979 American Chemical Society
