A. F. RODDE, JR.,AND L. I. GBOSSWEINER
764
he Chmiilurninescent Reaction of Hydrated Electrons with xcited Fluorescein Dyesla y A. F. Rodde, Jr.,Ib and L. I. Grossweiner*l* Department of Radiation Therapy, Michael Reese Hospital and iMedical Center, Chicago, Illinois 60615 (Received October 16, 1970)
Pub1ication costs assisted by the National Institutes of Health
It has been shown with a flash photolysis-pulse radiolysis technique that hydrated electrons react with a photochemical product of aqueous eosin or fluorescein in a chemiluminescent process. The emission has been identified with the fluorescent state, where the intensity varies linearly with the electron pulse dose and with the square of the initial triplet concentration. It is proposed that triplet-triplet interactions lead to a loosely bound triplet-singlet complex which reacts with esq- to generate the excited singlet state of the monomer and the dye semiquinone.
ntroduction Early w3rk of Sommermeyer, et C Z Z . , ~ ~reported that X-ray irradiation of several aromatics and dyes in aqueous solution induces light emission with the spectrum of the uv-exciited fluorescence. However, the yield was enhanced by low concentrations of iodide ion and quenched by nitrate ion and fructose, which do not influence the fluorescence. Sommermeyer and Priitz2b proposed that the hydrated electron eaq- is involved in the emission procciss, which was confirmed by pulse , ~ found radiolysis measurements of Prutz, et C Z ~ .who that eaq- scavengers quench the radiation-induced luminescence However, the emission is quenched also by hydroxyl radical scavengers and high iodide ion concentrations, which indicates that an OH. reaction product of the dye is volved as well. Prutz and oxidizes the dye and then Land4 proposed that O eaq- reduces the oxidized dye product to the fluorescent state Bo
-
+ OH. +Sox
80,
+
eaq-
S1"
(1) (2)
Pulse radiolysis studies of eosin and have shown that O W * reacts to form both the ring adduct SOH. and a phenoxy1 type semioxidized radical X. An attempt was made to distinguish between these two oxidation products in earlier work by generating the X radical with a xenon light flash prior to irradiation with a, 35-MeV electron pulse.8 The triplet dye T1 formed by intersystem crossing reacts with itself and unexcited dye via known quenching and electron transfer processes
3" TI
+ hv +SI*
(3)
Si" --+ Ti
(4)
+ TI
+Ti
+ So
The Journal of Physical Chemistry, Vol. 76,No.6, 1971
(5)
+
TI TI--j- R Ti
+X
+ So --+ 2So
Ti+So*R+X
(5') (6) (6')
where R is the dye s e m i q u i n ~ n e . ~ - ~Itl was found that prior application of the light flash has no effect on the luminescence yield and it was concluded that the OH. adduct of fluorescein or eosin (formed only by the electron pulse) is responsible for the emission
SOH. i eaq- --+SI" 4- OH-
(2')
A different type of emission was observed in the work of ref 8 that requires optical excitation prior to the electron pulse. I n this case scavenging of OH- has no quenching effect and it was proposed that eaq- reacts with the triplet state of the dye to form the excited state of the dye semiquinone. The present work was
(1) (a) Supported by NIH Grant GM-12716. (b) Based in part on the thesis of A.F.R. submitted in partial fulfillment of the requirements for the Ph.D. degree in physics to Illinois Institute of Technology. (c) Physics Department,, Illinois Institute of Technology, Chicago, Ill. 60616. (2) (a) K. Sommermeyer, V. K. Birkwald, and W. Prutz, Strahlentherapde, 116, 354 (1961); (b) K. Sommermeyer and W. Prtits, 2. Naturforsch. A, 21, 1081 (1966). (3) W. Prtitz, K. Sommermeyer, and E. J. Land, Nature, 212, 1043 (1966). (4) W. Prtitz and E. J. Land, Biophysik, 3, 349 (1967). (5) J. Chrysochoos, J. Ovadia, and L. I. Grossweiner, J . Phys. Chem., 71, 1629 (1967). (6) P. Cordier and L. I. Grossweiner, {bid., 72, 2018 (1968). (7) L. I. Grossweiner, Aduan. Chem. Ser., No. 81,277 (1968). (8) L. I. Grossweiner and A. F. Rodde, Jr., J. Phvs. Chem., 72, 756 (1968). (9) L. Lindqvist, Arkiv Kemi, 16, 79 (1960). (10) V. Kasche and L. Lindqvist, Photochem. Pholobiol., 5 , 507 (1965). (11) T. Ohno, S. Kato, and M. Koizumi, BuEZ. Chem. Soc. Jap., 39, 232 (1966).
CHEMILUMINESCENT RI~ACTIQN OF HYDRATED ELECTRONS undertaken to explore the new emission process under improved experimental conditions. I n particular, new optics and circiiitry made it possible to measure the emission at low electron pulse dose where the bimolecular eaq- reactions do not complicate the kinetics analysis. ’The results prove that the earlier identification of the luminescence precursor with the dye triplet state is incorrect and indicate that a new dye species is involved with the properties of a loosely bound complex laetween the triplet state and ground state dye.
Experimental Detaiis The experiments were performed with a modified linear accelerator pultie radiolysis arrangement which permits optical sorpltion and emission measurements after irradiation of the sample with a flash lamp and subsequent application 0f the 35-MeV electron pulse. The electron pul;se duration used was 0.2 psec a t 10-500 rads per pulse, tis measured for each run with a pulse current transformer calibrated with the modified Fricke dosimeter.12 An E.G. & 6. FX-33 flashtube operated at 36 J input provided a 16) psec (l/e) light flash. The timing circuits made it possible to trigger the electron pulse at any time delay after the light flash and display both the emission signal and the optical absorption transient on appwpriate time scales with a Tektronix Type 556 dual beam oscill~scope.~~ The sample was contained in a hi em long, 25 mm o.d. cell of Supracil Torr by quartz and could be evacuated to 5 X pumping and shaking. Beam profile studies showed a uniform dose distribution over the cell face. Two-channe1 optical absorption measurements were made by focusing the monjtor’ing light from an Osram XBO 450-W xenon arc through the cell in two passes with front surfaced mimore, and dividing the beam between a Jarrell Ash 0.25-m grating monochromator and the Hilger E 498 quartz prism spectrograph with the E 720 photoelectric scanning unit, followed by RCA 1P28 photomultipliers a t their exit slits. A separate monitoring channel using a University Labs Model 240 He-Ne laser with solid state detector was available at 633 nm. The flash lamp was located immediately adjacent and parallel to the irmdiat ion cell. Emission measurements were made with the same pulse radiolysis optical system by blocking the xenc)~.arc. Commercial Eosin Y was purified with the chromatographic method of Koch14 to give emax(51Snm) = 9.7 X lo4 M-I cm-l, and the disodium salt cf fluorescein was crystallized twice to give emax(491 r i m > =.I 8.2 X lo4 M-l cm-l. Other chemicals were reagent grade and triply distilled water was used for all solutions.
esultei and Analysis Typical data showing the chemiluminescent process investigated in this work are reproduced on a single time base in Figure K for the case of 10 pM eosin (pH 9.0) in
765 I I
I
Time, psec.
Figure 1. Typical transient signals from deaerated 10 p i ? eosin (pH 9.0) in the presence of 0.01 M formate. The flash lamp was triggered a t t = 0 and the 35-MeV electron pulse applied a t t = 250 psec: curve a, optical density a t 405 nm; curve b, optical density at 633 nm; curve c, luminescence at 550 nm.
the presence of 0.01 M formatc to scavenge OH. radicals. The application of the xenon light flash at zero time (A >350 nm) generates the dye triplet state measured by its absorption at 633 nm (curve b). The decay of the triplet is accompanied by the growth of absorption at 405 nm (curve a) attributed to the dye semiquinone dianion (R) formed by the T-S and T-T electron-transfer reactions 5‘ and 6’. The application of a 0.2-psec electron pulse (520 rads) a t 250-psec time delay after the light flash leads to a fast increase in the R concentration accompanied by strong light emission at 550 nm (curve c). The absorptivity at 633 nm shows a spike due to eaq- followed by the continuation of triplet decay. However, the 405 nm R absorption continues to grow slowly from additional dye reduction by COZ-, produced by the scavenging of OH. by formate.5 Measurements of the emission decay on a faster time scale show that it is exponential in time with a decreasing lifetime at, higher dye concentrations and parallels the disappearance of eaq-. However, the luminescence buildup is “instantaneous” for the 0.1 psec time resolution of the apparatus. (12) L. M. Dorfman and M. S. Matheson, Progr. React. Kind., 3, 237 (1965). (13) A. F. Rodde, Jr., Ph.D. Thesis, Illinois Institute of Technology, Dec 1970. (14) L. Koch, J. Assoc. Ofic.Agr. Chem., 39, 397 (1956).
The Journal of Physical Chemistry, VoE. 76, No. 6, 19rl
A. F. RODDE, JR.,AND lid. I. GROSSWEINER
766 I .o
)n
2
c .-
s .-
0.5
c VI
P)
c
.-C c
0 .VI .-VI
--
E
W
, A 25
50
75
20 40 60 Benzoote concentration, JIM’,
Electron pulse dose, rads.
80
Figure 2. Dependence of emission intensity at 110 psec delay on electron pulse dose: deaerated PO p M eosin in the presence of 0.01 M formate (pH 9.0).
Figure 3. Quenching of emission by benzoate: deaerated 5 p M eosin in the presence of 0.01 M formate (pH 9.0), 30 rads/pulse.
It was deduced in the earlier report8 that eaq- is responsible for the emission because He and OH. are scavenged by the added formate. This conclusion is supported b:7 the data in Figure 2 showing that the emission intensity is a linear function of the pulse dose, which argues against the participation of more than one type of water radiolysis radical. Additional evidence has been obtained by adding sodium benzoate to compete with this fast reaction between eaq- and the dye. Although OH. reacts rapidly with benzoate [Ic(OH benzoate) = (3.2 i 0.5) X IO9M-I sec-’ (average of values in ref 15) ] the scavenging by 0.01 M sodium formate [k(OH formate) = 2 X lo9J4-l sec-’ 16] controls at the low benzoate concentrations employed. The measiirementf were made by observing the effect of added benzoate on the emission decay lifetime, with all others factors held constant, Le., the flash lamp energy, electron pulse dose, dye concentration, and delay time. A semiempirical fit to the data obtains by assuming that eaq- reacts with the dye, benzoate, and the unknown dye precursor of the emission process P with the second-order rate constants k ~ k ,g , and kp, respectively. The assumption that the lifetime of P is much longer than that of the actual emitting state (shown below to be the first excited singlet state) leads immediately to the following relationship for the decay ol luminescence 1(t) IC, [etLq-I0 [plave- (kD[Dl+kB [ B I + k p P l + k o ] t (I)
emission lifetime against the benzoate concentration in Figure 3 follows the form of eq I. The slope leads to kg = 3.6 X lo9 M-l sec-I in good agreement with the average of published values: (3.3 f 0.3) X lo9 M-’ sec-’.17 The intercept gives I ~ D7 2.3 X 101OM-lsec-l for the reaction of eaa- with eosin, in good agreement with the pulse radiolysis result of 2.2 X 1010M-l sec-l,’ noting that JCO N 6 X loa sec-’ is much smaller than kD[D] and P must be present in quite low concentrations (see below). Although this analysis confirms that eaq- is a principal reactant in the luminescent process, the identification of the coproduct P has proven to be more difficult. It was believed at first that P is the dye triplet state, in which case the emitting species must be the excited state of the dye semiquinone. However, Figure 4 shows that the luminescence emission spectra for eosin and fluorescein (points) are identical with the usual fluorescence excited by visible light (A > 450 nm) under the pulse radiolysis spectrophotometric conditions. mate correction for photomultiplier sensitivity indicates that emission yield from eosin is 1.5 i 0.5 times as high as fluorescein. Ruling out the triplet state itself, information about the precursor P has been obtained by measuring the emission intensity as a function of the delay time between the light flash and electron pulse for dye concentrations from 2.5 to 15 pM. A typical result is shown in Figure 5 for 10 p M eosin. The time delay
+
+
PV
where [eaq-jOis the initial eaq- concentration produced by the electron pulse, [PIByis the average concentration of the precursor during the emission process, and k, is the eaq- decay constant in the buffer solution under the experimental con&tions. The linear plot of reciprocal The Journal of Physical Chemistry, Val. 76, No. 6 , 1971
(1.5) M. Anbar, D. Meyerstein, and P. Nett, J . Phys. Chem., 70, 2660 (1966). (16) M. S. Matheson, Ann. Rev. Phys. Chem., 113, 77 (1962). (17) M. S. Matheson and L. M. Dorfman, “Pulse Radiolysis,” M. I. T. Press, Cambridge, Mass., 1969, p 121.
767
CNEMILWMINESC ENT REACTION OF ‘HYDRATED ELECTRONS
0.i
0.2
0.5
Initial triplet absorbance (633nm). Wavelength, nm.
Figure 4. Comparison of electron-pulse induced emission spectra with ordinary fluorescence: curve a, fluorescence from 10 pM fluorescein (pH 11 3 ) (solid line); emission in the presence of 0.01 M formate (A); curve b, fluorescence from 10 pM eosin (pH 9 ~ 0 (solid ) line); emission in the presence of 0.01 M formate (O), and 0.01 M glucose (e).
Figure 6. Dependence of emission intensity (40 rads/pulse) from deaerated 10 p M eosin (pH 9.0) in the presence of 0.01 M formate on the initial triplet absorbance. Line a, 80-psec time delay; line b, 130-psec time delay. The lines correspond to slope 2.0 on the logarithmic plot.
5r
i
I
I I
0 200 400 Time delay, psec.
600
Figure 5 . Dependence of emission intensity from deaerat,ed 10 p M eosin (pH 9.0) in the presence of 0.01 M formate on delay time between light flash and 40-rad electron pulse.
required for mriximurn emission decreases from 160 psec dye to 95 psec a t 15 MLM dye. These data at 2.5 provide st direct measurement of the P growth and decay rate a t the different dye concentrations. I n another series of experiments the initial triplet yield generated by the light flash was varied with neutral density filters and it was found that the emission intensity at, constant delay time depends on the squaye of the initial triplet concentration, Figure 6. This result effoctively rules out X as the precursor because the T-81 electron-transfer reaction 6’ makes the major contribution to X formation compared to the T-T reaction 5’, particularly at the initial triplet concentrations less than 2 p M . Furthermore, the dependence of
0
IO Eosin concentration,
15
5
yM.
Figure 7. Dependence of precursor lifetime on initial eosin concentration.
the luminescent intensity on delay time shows that the decay of product P is considerably slower than the disappearance of the triplet state and faster than the decay of R and X. (This was shown directly by plotting the emission intensity against the triplet, R,and X eoncentrations at the time of the electron pulse for different dye concentrations.) The decay of V is accelerated by the dye, where the halftime ranges from 400 psec at 2.5 pM eosin to 150 psec at 15 p M eosin. A straightforward analysis based on the assumed decay law -d[P]/dt
=
k’[P]
+ k”[P][D]
(11)
predicts that the decay halftime should vary inversely with the eosin concentrations. The experimental data in Figure 7 lead to k’ ‘v 1300 sec-l and k” ‘u 2 X lo8 M-I sec-l. The Journal of Physical Chemistry, Vol. 76, No. 6, 1071
A. F. RODDE,JR.,AND E. I. GROSSWEINER
768
A careful search was made for the absorption spectrum of P by flash photolysis of aqueous eosin to no avail. It appears that the yield of the T-T reaction responsible for its production must be low compared with reaction 5' or that the P absorption is masked by that of T,R, or X. Apparently the luminescent process is more selective than absorption spectroscopy because of the high exothermicity when P reacts with e&q-. Discussion The experimental results show that triplet eosin (or fluorescein) engages in a T-T reaction leading to the unknown product E' which reacts with eaq- to generate the excited singlet &ate of the dye as the emitting species. The simplest model consistent with these observations is the formation of an unstable TS complex as the product of the 'I?-T quenching reaction. The subsequent lchemiluininescent reaction can be represented as the spin conserving process T(++)
fq+jL) -4-
eaq-($)
+
R(ffJ.1
+ SI*
