Chemiluminescence of Some Luminol-Like Molecules
The Journal of Physical Chemistry, Vol. 83, No. 21, 1979 2687
A Pulse Radiolysis Study of the Chemiluminescence of Some Luminol-Like Molecules Elhanan Wurzbergt and Yehuda Haas” Deparfment of fhyscial Chemistty, The Hebrew University, Jerusalem, Israel (Received March 20, 1978; Revised Manuscript Received April 2, 1979)
An electron-beam-initiatedchemiluminescencestudy on luminol and some similar molecules is presented. The mechanism previously suggested for luminol is found to be consistent with results obtained with other molecules. Substitution effects on the mechanism and on the related rate constants are quantitatively evaluated. The higher chemiluminescence yields observed for molecules bearing electron-donating substituents is found to be due not only to higher fluorescence efficiencies of the product, but also to a more favorable reaction scheme. In particular, it is suggested that competition between a light-producing sequence and a “dark”,nonlight producing sequence is one of the main causes for the overall poor light yield in aqueous solutions. Comparison of the results with those obtained by other workers, using methods such as mixing with oxidants and electrochemiluminescence, indicates that the mechanism revealed in the pulse radiolysis study may also apply for the other experimental methods.
Introduction The oxidation of luminol and related compounds (hereafter referred to as hydrazides) in aqueous basic solution is a well-known example of chemiluminescence in solution.lP2 Structural effects on the light yield have been studied in great detail, and it was found that the basic 2,3-dihydro-l,4-phthalanzindione(DPD, I) structure is
U
Ia, R = NH, Ib,R = OH IC, R = CH, Id, R = NO, Ie, R = C(=O)CH,Cl
essential for high efficiency. Drew3i4has shown that, for a given substituent, substitution is position 5 leads to brighter luminescence than in position 6. Also, he has shown that electron-donating groups substituted at position 5 tend to increase the yield, while electron-attracting groups decrease it. Thus, chemiluminescence decreases along the sequence NH2 > NHCH, > OH > CH, > C1> NOz. The emitting species in the case of luminol (Ia) is believed to be 3-aminophthalic acid (3-APA). The assignment is due mainly to the similarity between the chemiluminescence spectrum of luminol and the fluorescence spectrum of the acida5The acid was actually isolated in high yield by carrying out the reaction in dimethyl sulfoxidee6 It is generally accepted that the corresponding acids are the emitting species for other derivatives of I a l s ~ . ’Recently, ~~ Rusin et aL7questioned this assignment, as they could not reconcile it with their energy transfer data. They suggest some unspecified intermediate as the possible emitter. We used the pulse radiolysis technique to study the same phenomenons and concluded also that energy transfer to, e.g., fluorescein, is not compatible with 3-APA as the emitter. As detailed in ref 8, one can interpret our experimental results as well as those of ref 7 by assuming a chemical mechanism of dye electronic excitation, rather than energy transfer from Department of Chemistry, Cornell University, Ithaca, N.Y. 14853. 0022-3654/79/ 2083-2687$01.OO/O
3-MA. With this interpretation the identity of the emitter as a phthalic acid derivative may be retained. The mechanism of the reaction has not been elucidated to this day. This is due to the complexity of the reaction and the existence of a large number of secondary and side reactions. It has been shown that, in the absence of oxygen, luminol is consumed under normal reaction conditions: thus proving that the system is open to nonlight producing reaction channels. Recently, the technique of e-beam irradiation was applied to the study of the system.lOJ1 In these experiments, OH radicals produced upon dissociation of water molecules by high-energy electrons initiate a radical reaction which eventually leads to oxidative chemiluminescence. Thus, for the first time, a fast, submicrosecond method is used to study the mechanism of the reaction. Indeed, BaxendalelO was able to suggest a detailed mechanism, based on the observation of transient species in the reaction solution. He also measured the rate constants of the different stages and gave tentative assignment of the intermediates. His mechanism may be represented by the following sequence (LH represents for luminol): LH
+ OH- -%R + H 2 0
(1)
k2
R+02-S
(2)
k3
S-T
T
+T
k4
(3) Z*
+Y
(4)
According to this mechanism, one can give the chemiluminescence intensity, I , byl0J1 with To the initial concentration of T and y a proportionality constant. Equation 5 was used to verify the proposed mechanism, as a plot of I-lI2 vs. t does indeed yield a straight line. A more quantitative analysis showed that by using k4 as derived from light absorption studies, Towas found to be significantlysmaller than [OH],,.l’ This discrepancy was interpreted as indicating the existence of dark reactions, intercepting some of the OH radicals initially formed. In fact, in the absence of independent measurement of e, the molar extinction coefficient, absorption data yield only the rate klc. The values reported 0 1979 American Chemical Society
2688
The Journal of Physical Chemistry, Vol. 83, No. 21, 1979
0.77650
t
I VERT. DIV ~ 1 . 2 - 1
.. ...”. ...
E. Wurzberg and Y. Haas 086104
I VERT. DIV.121844-I
0.17619
H
500 JI sec
SOON sec
r
.. .......
I VERT. DIV.4.6316-2
0,3304
I
050592
1
I
u 500
,
p sec
Figure 1. A typical oscilloscope trace with computer analysis: upper part, second-order kinetics; lower part, first-order kinetics,
in ref 10 and 11 were based on the assumption that all of the hydroxyl radicals end up as T. The lower values of Toobtained from eq 5 were thus interpreted as indicating the breakdown of this assumption. Writing the overall chemiluminescence yield as ~ C = L
4~4~x4~
with 4c the chemical yield of the immediate precursor, 4EX the yield of the excited state of the emitting molecule, and q5F the fluorescence quantum yield, one finds that this is equivalent to stating that 4c < 1. Assuming 4EX = 1 for HDP, 4~ values were derived in ref 11. In the absence of independent measurements of 4EXor of the true rate constants, the values reported must be regarded with caution. In the hope of shedding some more light on factors involved in the chemiluminescence of hydrazides, we extended the e-beam excitation study to the related compounds listed above. It was found that the results correlate fairly well with chemiluminescence properties studied by conventional methods. Real time monitoring of intermediates, easily achieved in the pulse radiolysis study, allows further insight as to parameters governing light-producing efficiencies. From the previous discussion, it is obvious that absolute rate constants cannot be derived from the absorption studies. Nevertheless, we continue to calculate the rate constants by using the method of ref 10, thus allowing comparison with previous results. It should be borne in mind that the relative values are more significant than the absolute ones. Experimental Section Pulse Radiolysis Measurements. The e-beam source was a Varian V-7715B linear accelerator, operated at 5 MeV with a constant current of 200 mA. Radiation dose was controlled by varying the pulse length between 0.05 and 1.5 ps. The sample was contained in a Spectrosil cell, 1 cm long for emission measurements and 4 cm long for absorption. When operated in the absorption mode, mirrors were used to increase the optical path of the probing light by passing it through the cell three times. The light (either from the luminescent sample or from the
0 0988
I
1
I
1
I
1
200 y sec
Figure 2. Dose effect on the kinetics: Chemiluminescence decay analyzed for second-order kinetics: top, 700-rd shot; bottom, 2500-rd shot.
lamp after traversing the sample) was dispersed with a monochromator with 5-A resolution and detected with a photomultiplier tube. The resulting photovoltage was displayed on oscilloscope, or fed into a transient digitizer (Biomation 8100). The output from the digitizer was analyzed with an on-line Nova 1200 minicomputer. A typical trace is shown in Figure 1, along with the corresponding computer output. It is evident that a secondorder plot fits the experimental decay curve much better than a first-order one. In the early experiments it was found that repeated irradiation of the sample caused severe changes in the kinetics. Consequently,the solution was thereafter replaced after each pulse. Apart from this “long term” instability, it was found that, on increasing the pulse dose, deviations from pure second-order kinetics are observed. Figure 2 shows the effect for 700- and 2500-rd doses. Accordingly, work was usually restricted to radiation doses under 1000 rd. Other Experimental Details. Fluorescence spectra were measured with a Model 210 Spectro spectrofluorimeter made by G. K. Turner. Quantum yields were determined by using quinine sulfate as standard (+F = 0.54 at 25 OC).13 Lifetimes in the nanosecond range were obtained with the apparatus described in ref 14. Solutions were always made up less than 12 h before the experiments and kept in a cool dark room until used. M ethanol solution in Dosimetry was done by using a water as a standard [G(e,;) = 2.75 at pH 10,c(A = 578) = 1.06 X M-l cm-l]. The materials used in this study were as follows: Luminol, 5-amino-2,3-dihydro-l,4-phthalazindione (ADP), was obtained from Fluka and purified by recrystallization.lg 5-Nitro-2,3-dihydro-1,4-phthalazindione (NDP) and 5-hydroxy-2,3-dihydro-1,4-phthalazindione (HDP) were prepared according to ref 15. 5-Chloroacetamide 2,3-dihydro-l,.i-phthalazindione(CDP) and N,N-bis(phtha1azine-4,4-dionyl-5’)2,5-diketopiperazine (PDD) were prepared according to ref 16. 5-Methyl-2,3-dihydro-1,4phthalazendione (MDP) was prepared according to ref 17.
The Journal of Physical Chemistry, Vol. 83, No. 2 1, 7979 2089
Chemiluminescence of Some Luminol-Like Molecules
TABLE I: Rate Constants, Quantum Yields, and Maximum Emission Wavelength for Some Hydrazides under e-Beam Excitationa hydrazide k , , M-ls-I k , , M-ls-’ k , , s-l k , , M - l s - ’ @ c L , ~% $IF, % ,,A, ADP (luminol) HDP MDP CDP NDP PDD a
9.8 X 10’
9 x 109 1.2 x 1 O ‘ O 9~ 1 0 9 3
See text for notation.
1.1 x 109 4x
loa
not obsd
Measured by the method of ref 11.
X X X X
lo8
lo8 lo8 lo8 not obsd
0.35 c
1.4 X
lo8
0.15
3.9 5.1 2.5 3.6
-5x 104
1.7 X 10’ not obsd
x 1OlU
2.5 X l o 4
1 x io5
2.4
0.3
1.1
0.085 -10-3
nm 425 415 420
HDP > MDP > CDP, which happens to be the same order as that of light emission efficiency. It is, also, the order of the electron releasing power of the substituent groups. It thus appears that reaction 2 is intimately connected with the light emission species, while kl may in fact represent a rather nonselective attachment of OH radicals to the hydrazide, possibly at several different sites. Note that the value of kl is similar to that measured for OH addition to simple aromatic molecules,z6
The Journal of Physical Chemistry, Vol. 83, No. 21, 1979 2691
One may conclude that the well-known fact that electron-releasing substituents in position 5 enhance light yields is connected with step 2 in the mechanism, i.e., addition of oxygen. The most striking evidence supporting this suggestion is provided by luminol: NH2 is an electron-repelling group and kz is correspondingly observed to be quite large at high pH values. Lowering the pH converts NH2 to NH3+,an electron-attracting group, and no reaction with oxygen is observed! This is to be contrasted with the case of HDP: at high pH values the substituent is 0-, at lower ones, OH. Both are electronrepelling groups, and indeed oxygen addition is observed at all pH values. The connection between step 2 and the overall efficiency requires further study, but it may provide one explanation for the relative efficient light emission observed when electron-repelling substituents are placed in position 5 . The overall yield is of course dependent on more than a single factor, and Figure 7 is one indication of the complex situation. Chemiluminescence is evidently strongly quenched as the temperature is raised; the figure shows total yield. It should be noted that initial intensity was not as strongly affected, and much of the decrease is due to a faster decay (larger k4). The data could not be fitted by an Arrhenius plot. We tentatively suggest that this effect is also due to competition between “dark” and “light” reactions, with the former becoming faster at higher temperatures. Concentration dependence of luminol and HDP,discussed in ref 11, is further evidence for the existence of side reactions. T h e Light Emitting S t e p and the Nature of the E m itter. The possibility of a dioxetane intermediate in the chemiluminescence of hydrazides has been raisedS2After dioxetanes were actually isolated in 1969, it became even more interesting to explore their role. Our analysis shows definitely that reaction 4,leading to the excited molecule, is second order, and, therefore, is not compatible with the existence of a dioxetane intermediate, unless the latter is very short lived. We cannot completely rule out a sequence such as 2T k4 D, D k5 products, with D a dioxetane and k,[D] >> k4[TI2. However, since the assumed D carries excess energy, it should dissociate quickly, so that this sequence is not experimentally distinguishable from reaction 4 as described above. As mentioned above, Rusin et al.7 raised an objection to the assignment of 3-APA as the emitting species. They based their arguments on the incompatability of 3-APA with Forster type energy transfer to some fluorescent dyes. As a possible alternative they suggest an intermediate. Our results are consistent with the assignment of phthalic acid derivatives as the emitters in hydrazide chemiluminescence. We observe the typical emission of the acid (characterized by the emission spectrum) as soon as any emission develops at all within our time resolution of about 2 ps. It appears extremely unlikely that for three different molecules (luminol, HDP, and MDP) the emission spectrum of an intermediate would coincide so well with that of the acid. Our pulse radiolysis data thus indicate that the proposed intermediate is formed as fast as the acid and has a very similar spectrum. As shown in ref 8, the “energy transfer” results may be reconciled with the acid as the emitter, and our present data further support this otherwise well-established proposition. T h e Prospects of Designing a More Efficient Chemiluminescent System. Much effort has been expended at obtaining higher light yields1*2mostly by some substitution. Even though hydrazides with higher yields than luminol -+
-
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The Journal of Physical Chemistry, Vol. 83, No. 21, 1979
have been reported, luminol still ranks as one of the most efficient systems. Limiting our discussion to reactions in aqueous solutions only and assuming that Baxendale’s mechanism holds also for conventional (not pulse radiolytic) reaction conditions, one may understand substitution effects in the following way. Unwanted “dark” reactions are governed to a large extent by the nature of the substituent in position 5 . An electron-attracting one causes radical attack in a “wrong” place, and no reaction with oxygen is obtained at all. A strongly electron-donating group is thus required. This, in turn, allows the “right” intermediate to be formed, but also may lead to subsequent oxidation on the aromatic ring itself. Increasing the electron density on the ring by further substitution in position 8 apparently further enhances this trend.27 Use of compounds containing more than one aromatic ring was expected to increase light yields, as the quantum efficiency of fluorescence of the corresponding acids is larger. In practice, these larger molecules appear to be more vulnerable to unwanted side reactions, and the overall yield decreases, compared to luminol. This trend appears in our study in the case of PDD.
Conclusion The main results of this work may be summarized as follows: (1)The mechanism suggested by Baxendale for luminol chemiluminescence under e-beam excitation holds also for other hydrazides. (2) The emitting species appears to be the corresponding phthalic acid, and in any case its characteristic spectrum is observed a few microseconds after excitation. (3) The mechanism suggested by Baxendale may be the one governing reaction initiated by other methods, such as mixing with oxidants, electrochemiluminescence, etc. (4) A dioxetane intermediate is not revealed in our study. ( 5 ) The reason for low chemiluminescence yields of hydrazides in aqueous solution appears to be the presence
Y.
Haas and E.
Wurzberg
of many side reactions, competing with the multi-stage light-producing sequence. (6) Substitution at position 5 with strongly electronattracting groups leads to OH radical attachment in a “wrong” position, and the resulting radical does not follow the light-producing sequence.
References and Notes (1) (2) (3) (4)
K. D. Gunderman, Top. Curr. Chem., 46, 61 (1974). E. H. White and D. F. Roswell, Acc. Chem. Res., 3, 54 (1970). H. D. K. Drew and R. F. Garwood, J . Chem. Soc., 35 (1939). H. D. K. Drew and R. F. Garwood, Trans. Faraday SOC.,35, 207 (1939). (5) E. H. White and M. M. Bursey, J . Am. Chem. Soc., 86, 41 (1964). (6) E. H. White, 0. C. Zafiriou, H. M. Kagi, and J. H. M. Hill, J. Am. Chem. Soc., 86, 940 (1964). (7) B. A. Rusin, V. N. Emokhonov, E. L. Frankevich, and V. L. Tal’rose, High Energy Chem., 10, 77, 81, 85 (1976). (8) Y. Haas and E. Wurzberg, following paper in this issue. (9) P. B. Shevlin and H. H. Neufeld, J . Org. Chem., 35, 2178 (1970). (10) J. H. Baxendale, Trans. Faraday Soc., 69, 1665 (1973). (11) E. Wurzberg and Y. Haas, Chem. Phys. Lett., 55, 250 (1978). (12) R. B. Brundrett and E. H. White, J. Am. Chem. Soc., 96, 7497 (1974). (13) W. H. Melhuish, J. Phys. Chem., 65, 229 (1961). (14) N. Lasser and J. Feitelson, J . Phys. Chem., 77, 1011 (1973). (15) H. D. K. Drew and F. H. Pearman, J. Chem. SOC., 26 (1937). (16) E. Domagalina and J. Orhynska, Pol. J. Pharmacol. Pharm., 26, 473 (1974). (17) H. D. K. Drew and R. F. Garwood, J . Chem. Soc., 836 (1939). (18) E. D. Amstutz, E. A. Fehnel, and C. R. Neumoyer, J . Am. Chem. Sac., 68, 352 (1946). (19) J. Lee and H. H. Seliger, Photochem. Photobiol., 15, 227 (1972). (20) J. Nikokavours and G. Vassilopoulas, Chem. Chron. New Ser., 1, 115 (1972). (21) Y. Haas and G. Stein, J. Phys. Chem., 75,3677 (1971). (22) V. N. Emokhonov, B. A. Rusin, Yu. B. Koltishin, A. L. Poshchin, E. L. Frankevich, and V. L. Tal’rose, HghEnergy Chem., 11, 342 (1977). (23) J. Nikokavours, A. E. Mantaka, D.G. Marketos, N. Th. Rakintz’s and G. Vassilopoulos, Z. phys. Chem. (Frankfurtam Main),78, 76 (1972). (24) H. D. K. Drew, Trans. Faraday Soc., 35, 207 (1939). (25) J. D. Gorsuch and D. M. Hercules, Photochem. Photobiol., 15, 567 (1972). (26) L. M. Dorfman and G. E. Adams, Natl. Stand. Ref. Data Ser., Natl. Bur. Stand. No. 46 (1973). (27) E. H. White and K. Matsuo, J . Org. Chem., 32, 1921 (1967). (28)D.F. Roswell, V. Paul, and E. H. White, J. Am. Chem. SOC., 89, 3944 (1967).
Chemiluminescence of Luminol and Related Compounds under Electron-Beam Excitation. Enhancement of Light Yields by Addition of Dyes Yehuda Haas” and Elhanan Wurrbergt Deparlment of Physical Chemistry, The Hebrew University, Jerusalem, Israel (Received March 20, 1978; Revised Manuscript Received April 2, 1979)
The emission from mixtures of luminol-like molecules and some dyes is studied by electron-beam excitation. It is found that total light yields in the presence of dyes may be somewhat increased over those obtained with the luminol alone. A possible mechanism leading to dye emission is discussed. It is suggested that a chemical reaction is involved and not energy transfer from 3-aminophthalic acid, widely believed to be the emitting species in luminol chemiluminescence. The intensity and kinetics of dye emission may be explained by a radical reaction mechanism with no net change in dye concentration.
Introduction We have recently used the method of electron-beam excitation to estimate the chemiluminescent quantum yield of luminol and some related compounds. The overall chemiluminescence yields, q5cL,was found to be rather low +Departmentof Chemistry, Cornell University, Ithaca, N.Y. 14853. 0022-3654/79/2083-2692$01 .OO/O
for all the molecules under study.l Wishing to increase the overall light yield, we decided to try to initiate the chemiluminescent reaction in the presence of efficient fluorescent dyes, in the hope that fast energy transfer from the excited species formed chemically will serve to excite the dyes. This may result in a considerable increase in light yields in luminol-dye mixtures, as fluorescence yields @ 1979 American Chemical Society
