2018
P. CORDIER AND L. I. GROSSWEINER
Pulse Radiolysis of Aqueous Fluorescein’” by P. Cordierlb and L. I. Grossweiner10 Department of Radiation Therapy, Michael Reeae Hospital and Medical Center, Chicago, Illinoia 60616 (Received October 11, 1967)
A pulse-radiolysis investigation of aqueous fluorescein has shown that the initial reaction products are the semiquinone from the reduction of the dye by cas- and H atoms, the semioxidized radical from the attack of OH, and a long-lived “red product” attributed to OH and H atom adducts. Rate-constant measurements are reported of the dye-radical formation and decay reactions, based on analog computer analysis. Other new results obtained in this work include the formation of an unstable complex between the semiquinone and the dye which leads to long semiquinone lifetime at high initial dye concentrations and decay of the OH adduct by water elimination to form the semioxidized radical species. The pulse-radiolysis mechanism is in general agreement with cobalt-60 irradiation results.
Introduction The pulse radiolysis of aqueous dyes aids in the identification of the short-lived intermediates involved in their radiation chemistry. A previous investigation of eosin Y (the disodium salt of 2’,4’,5’,7’-tetrabromofluorescein) has shown that the dye is half-reduced by cas- to a semiquinone intermediate (R) and half-oxidized by OH to a radical species (X) which probably has the phenoxy1 structure.2J These dye radicals are obtained also by flash photolysis, in which case the triplet state is oxidized or reduced by reacting with appropriate substrates including the dye i t ~ e l f . ~ -I~n addition to R and X, a long-lived intermediate absorbing above the eosin visible band was obtained (the “red product”) which has not been observed in photochemical work. Although the structure of the red product was unknown, it was shown that it results from the attack of OH (and possibly H atoms) on the dye. One of the most interesting aspects of the eosin radiation chemistry is the high reactivity with both the reducing and oxidizing products of water radiolysis. However, this factor complicates the determination of accurate rate constants, particularly in that the strong visible absorptions of the dye and its radical intermediates preclude the use of competition techniques, and one must rely on analysis of the kinetics which requires accurate values of extinction coefficients and G values. I n the present pulse-radiolysis investigation of the parent dye fluorescein, an electronic analog computer has been used to assist in fitting the data to the proposed kinetics scheme. Some of the data obtained with eosin have been recalculated with the analog computer and the results are summarized in the Discussion. I n general, the results with fluorescein support the earlier work with eosin, particularly in the identification of the intermediates and the reactions by which they are formed. Experimental Methods The irradiation source was a microwave linear acThe Journal of Physical Chemistry
celerator which provided 1-Fsec pulses of 33-MeV electrons at a maximum dose of approximately 1600 rads/pulse. The experimental arrangement and general pulse-radiolysis procedures used are described in ref 3. The only significant changes from the previous work are that all spectral data were obtained with the photoelectric-monitoring method and the actual dose was measured for each pulse with a silicon photodiode located behind the irradiation cell, which was calibrated with the modified Fricke d ~ s i m e t e r . ~Commercial fluorescein (Allied Chemical) was purified by dissolving the powder in ammonium hydroxide at pH 11, precipitating as acid dye with glacial acetic acid, filtering, washing with distilled water, and drying. The extinction coefficient after repeating this procedure a second time was (8.2 f: 0.1) X lo4 M-’ cm-l at 491 mp from pH 8 to 11. The other chemicals were CP grade, and all solutions were made with triply distilled water. The analog computer used for data analysis was a Type TR-20 supplied by Electronic Associates, Inc. The unit was equipped with 16 operational amplifiers, 6 integrators, 14 variable potentiometers, and 3 (1) (a) This work was supported by U. 6 . Public Health Service Grant No. GM-12716 from the Xational Institute of General Medical Sciences; (b) Laboratorie de Chimie Physique, Facult6 de Sciences, Orsay, France. (c) Department of Physics, Illinois Institute of Technology, Chicago, Ill. 60616. (2) L. I. Grossweiner, A . F. Rodde, Jr., G. Sandberg, and J. Chrysochoos, Nature, 210, 1154 (1966). (3) J. Chrysochoos, J. Ovadia, and L. I. Grossweiner, J . Phys. Chem., 71, 1629 (1967). (4) L. I. Grossweiner and E. F. Zwicker, J . Chem. Phys., 31, 1141 (1959); 34, 1411 (1961). (5) L. Lindqvist, Ark. Kemi, 16, 79 (1960). (6) E. F. Zicker and L. I. Grossweiner, J . Phys. Chem., 6 7 , 549 (1963). (7) V. Kasche and L. Lindqvist, Photochem. Photobiol., 4, 923 (1965). (8) T. Ohno, S. Kato, and M. Koizumi, Bull. C h e w Soc. Jap., 39, 232 (1966). (9) L. iM.Dorfman and M. S. Matheson, Progr. Reaction Kinetics, 3, 237 (1965).
2019
PULSE RADIOLYSIS OF AQUEOUS FLUORESCEIN “quarter-square” multipliers, Approximate fitting was done with the solutions displayed in repeat operation on a Tektronix 532 oscilloscope and final results were read out on a Moseley Model 25 X-Y plotter. The general method consisted of simulating the solutions to the differential rate equations by appropriate integrating circuits, in which the concentration of each component is represented by a time-varying voltage. The fitting procedure requires the matching of the computer-generated solutions to the experimental data by appropriate adjustments of the coefficient-setting potentiometers. The computer could solve up to 5 simultaneous nonlinear, differential equations with a readout accuracy of at least 1%. Other details on the fitting procedures are given below.
Results 1. Transient Spectra. Figure 1 shows typical transient spectra obtained by pulse irradiation of 20 p M fluorescein a t pH 9.0 a t a dose of 1500 rads. A summary of results for other conditions is given in Table I. The strong band that shifts from 355 to 395 mp in going from neutral to alkaline solutions corresponds with the monoanion and dianion of the fluorescein semiquinone, respectively, with pK, = 9.5 as determined by flash photolysis.6 The quenching of this transient by oxygen or HzOz shows that it is a reduction product of cas-. The band near 415 mp that is unchanged by p H is identified with a product of OH attack. It is suppressed by formate but not by oxygen or H20z. This species is believed to be the semioxidized radical X, which was located at 428 mp by flash photolysis.6 The diffuse absorption above the fluorescein 491-mp band is much longer lived and decays by a first-order process (see Figure 2). This complex transient was designated the red product in the eosin inve~tigation.~The present work also shows the occurrence of at least two components. The weak absorption and overlap with the strong fluorescein band make an accurate determination of the spectral locations difficult. The bands near 570 =k 20 mp are quenched by formate but not by oxygen and are attributed to OH adducts. Another component near 650 m p is quenched by oxygen or 10 mM formate but not by 1 m M H2.02, and is probably due to H atom adducts. The slow growth of the semiquinone when both NzO and formate are present (see Pigure 3) is attributed to fluorescein reduction by GOz-. The absence of transients in air-saturated solutions containing formate indicates that oxygen oxidizes the semiquinone to the dye. 2. Formation of the Dye Intermediates. The initial yields of R and X from the pulse radiolysis of 5-50 p M deaerated fluorescein solutions at 1530 rads/pulse are given as the points in Figure 4. These values were obtained from the saturation optical density changes 25-50 psec after the irradiation pulse, based on BR
WAVELENQTH- mfi
Figure 1. Transient spectra from the pulse radiolysis of 20 p M fluorescein: (a) deaerated, pH 8.7, 60-psec delay; (b) air-saturated, pH 9.1, 60-psec delay.
3.0 a 2.0.-
1.0 * -
d d
Q
0.6.. 0.4-
0.3z
TIME
-
mlllluc.
Figure 2. (a) Return of optical density a t 491 mp after pulse radiolysis of deaerated 40 p M fluorescein a t pH 10.5; (b) decay of optical density at 600 mp after pulse radiolysis of deaerated 40 p M fluorescein at pH 10.5; ( c ) decay of optical density at 600 mp after pulse radiolysis of deaerated 100 g M fluorescein a t pH 10.5.
0.4-
TIME-pne.
Figure 3. Growth of semireduced fluorescein in the presence of 10 mM sodium formate and 24 mM nitrous oxide a t p H 10.4: circles, experimental data at different dye concentrations: solid lines, analog computer fit. The rate constants are given in the text. V0Eurn.e7d, Number 6 June 1068
P. CORDIERAND L. I. GROSSWEINER
2020
Table I: Transient Spectra from the Pulse Radiolysis of Aqueous 20 pM Fluorescein Solutions
PH
Delay time, Naeo
7.2 8.7 10.6 7.2 9.1 10.5 9.2 9.4 10.6
80 60 60 GO 60 40 120 80 1000
10.5
80
,
Additive
None None None Satd air Satd air Satd air 10 mM formate 1 mM HZOZ 10 mM formate satd NzO 10 mM formate satd air a
+ +
-355 vs 355 s
... .. ... ... ... ...
...
... 395 8 395 vs
...
... .
a
-
Transient band maximaa--------
.
415 w 415 s 415 w 415 s 415 s 415 s
580 w 570 w 590 w =550 w 550 w 550 w
=650 vw =G50 w =G50 w
...
415 s
550 s
=G50 w
395 s
...
..
...
.,.
...
...
...
395 vs
... ...
...
...
.*.
...
Wavelengths in mp. Approximate band strength: vs (very strong), s (strong), w (weak), and vw (very weak).
(2')
---t
(4)
ees- +products
(4')
----t
2.4-L 2.2.. 2.01.0.-
-*
+ eeq- +R S + OH. X + OHS + OH. +SOH. OH. + OH. +HzOz eeq- + eeq20H- + Hz S
2..+
1.61.4-
8 0 " 1.2.1.0.-
OH.
0.8.0.6
--
0.2.IO
2'0
30
io
(2)
(3)
(5)
The error from the neglect of H atom, Hz, and HzOz reactions is discussed below. The rate constant of reaction' was determined from the dependence of the pseudo-first-order eaq- lifetime on fluorescein concentration at low dose (-100 rads), in the presence of 10 m M formate to suppress the overlapping red-product absorption. The results give kl = (1.4 i 0.2) X 1O'O M-' sec-I. I n the absence of dye, the actual eaq- half-time was 27
0.4.-
0
+ eeq- +OH-
(1)
so
*
(394 mp) = 5 X lo4 M-l cm-l and ex(428 mp) = 5 X lo4 M-' ~ m - ' , with ~ a correction for the overlapping dye absorption. A plot of reciprocal G vs. reciprocal dye concentration gives surprisingly good straight lines (Figure 5 ) which facilitates the extrapolation to infinitely high fluorescein concentration. The maximum initial yields are G(R)* = 3.3 0.3 and G(X)* = 1.4 f 0.2. (Straight lines would be expected if eaq- and OH disappear in pseudo-first-order reactions in competition with their reactions with the dye). The value of G(X)* compared to GOH = 2.65* indicates that approximately half of the initial OH reacts to give the red product. Based on GH = 0.559 and G, = 2.8 0.1,10 it appears that H atoms contribute to the formation of R, but the low reduction yield cannot be estimated from these data. The analysis of dye-radical formation was based on the set of reactions
4
*
*
The Journal of Physical Chemistry
01 0
Ob
1.b io+x
V[s]
Ih
2:O
Figure 5. The data in Figure 4 plotted as reciprocal G tr8. reciprocal fluorescein concentration, which gives G(R)* = 3.3 rf 0.3 and G(X)* = 1.4 rf 0.2. (10) J. K. Thomas, private communication.
2021
PULSE RADIOLYSIS OF AQUEOUS FLUORESCEIN 2 psec, for an effective value k4’ = 2.6 X lo4 sec-’ which was used in the computer analysis. I n lieu of independent rate constant measurements for (2) and (27, values were estimated by fitting the dependence of G(R) and G(X) on dye concentration [SI with the analog computer solution to the system of reactions 1-5. The coefficient-setting potentiometers were determined by the rate constants taken from the literature8 (2k3 = 1.0 X 1010, 2k4 = 1.1 X lolo, and kg = 3.0 X 1Olo J4-l sec-1) and the values obtained in this work (kl = 1.4 X 1O1O M-l sec-l and k4’ = 2.6 X 104 sec-l). The ratio k 2 / ( k 2 k2’) was taken as equal to G(X)*/GOH = 0.53, so that k2 was the only variable used to fit the data. Typical growth curves of R and X (expressed as G values) are shown in Figure 6 for ICz = 1.5 X loBM-l sec-I. The G(R) vs. [SI line in Figure 4 was obtained from the values of G(R) at 25 psec after the electron pulse and gives a reasonably good fit to the experimental points. This line is not sensitive to the values of k2 and k2’ used and supports the independent measurement of kl. For the case of X growth, it required 25-50 psec to attain saturation depending on the dye concentration. The lower and upper solid lines in Figure 4 for G(X) vs. [SI were obtained at these times, respectively, and bracket the experimental points. The entire calculation was repeated allowing for a possible f25% variation in the numerical values of G(X) because of uncertainties in the extinction coefficients and other possible errors. The average results obtained were kz = (1.6 f: 0.3) X lo9 and k2’ = (1.4 f: 0.2) X lo9 M-l sec-l. When fluorescein was irradiated in the presence of N20 to convert ea,- to OH and formate to scavenge H and OH, the only transient observed was the slow growth of R from the reaction
+
C02-
+ S +R + COz
2.0
.c
[SI
IF===-/
.0
2’5
7b
5’0
IO0
TIME-pew.
Figure 6. Analog computer calculation of transient formation after the electron pulse: solid lines, growth of semireduced fluorescein for different initial dye concentrations; dash-dot lines, growth of semioxidized fluorescein for different initial dye concentrations. The values of G(R) at 25 psec give the solid line for G(R) in Figure 4 and the values of G(X) a t 25 psec (lower line) and 50 psec (upper line) give the solid lines for G(X) in Figure 4. The rate constants are given in the text.
(6)
Typical growth data are shown as the points in Figure 3. The computer analysis was made on the assumption that reaction 6 competes with the bimolecular reaction of GO2- according to
coz- + coz- ---t (C02-)2
2.6
(7)
It was found that the published estimate of 2k7 = sec-l does not give a good computer fit to the data. However, the value of kg was insensitive
lo9 M-’
to 2k7 and a variation in the latter from 2.5 X log to 1.0 X 1O’O gave ks = (2.6 f: 0.9) X lo7 M-‘ sec-l. The lines in Figure 3 were drawn by the computer for 2k7 = 5 X lo9 M-’ sec-l which gives the best fit. 3. Disappearance of the Dye Intermediates. The disappearance of R in the absence of X was studied by irradiating deaerated fluorescein solutions in the presence of formate, so that only the reduction reactions 1 and 6 were involved. The decay of R obeys bimolecular kinetics a t low initial fluorescein concentra-
100
TIME
200 rnllllstc.
-
300
Figure 7. Bimolecular decay of semireduced fluorescein after pulse radiolysis in the presence of 10 mM sodium formate a t p H 10.5.
tions (Figure 7) ; however, the rate becomes very much slower at high dye concentrations with the absorption persisting for some seconds a t 15 p M . Although most of the measurements were made with 10 mM formate, several runs at 1, 10, and 50 m M formate with 5 p M dye gave the same decay rate within experimental error, which appears to eliminate possible chain reactions or other complexities involving C02-. The results can be explained by postulating that R forms a long-lived com(11) J. P. Keene, Y . Raef, and A. J. Swallow, “Pulse Radiolysis,” M. Ebert, J. P. Keene, and J. H. Baxendale, Ed., Academic Press Inc., New York, N. Y., 1965, PP 99-106.
Volume 78,Number 6 June 1068
2022
P. CORDIER AND L. I. GROSSWEINER
plex with the dye itself, so that the rate of the disproportionation reaction R+R+L+S (8) where L is the leuco base, is limited by the equilibrium R
+S
K
RS
= [RS]/[R][S]
(9)
where RS is the complex. The actual decay of R can be shown12to be given by -d[R]/dt
[2&/(1 4 K[Sl)l[R12
where it is assumed that the reactions that establish the equilibrium are much faster than decay via reaction 8. The application of this result is complicated by the possible spectral overlap of the complex with R a t higher dye concentrations. The actual optical density changes (for [SI S constant and ES S 0) are given by
where ERS is the extinction coefficient of the complex at the wavelength of measurement. Thus the reciprocal of the slope ( A ) of the l/AOD os. t plots should be a linear function of the dye concentration [S]K [ S ]
