756 appear to be correlated with the dielectric constant of the medium, in that the separation of peaks increased with decreasing dielectric constant and has been interpreted in terms of solvent-induced conformational population changes. Except for cyclohexane, the gross behavior in the present system at any one temperature is similar; the chemical shift difference is greatest for the solvent of lowest dielectric constant. Consideration of the variation of the dielectric constant with temperature and the corresponding changes in the separation of peaks in three solvents, however, casts some doubt that the above correlation holds for Ia. While the dielectric constant of chloroform varies about 27% between -20 and 6Ool1lonly a 6% change in the dielectric constant of m-xylene is reported over the same temperature range.12 It can be estimated from published datala that the dielectric constant of liquid cyclohexane will change about 7y0 over this interval. The relative changes in separation of methyl resonances over this temperature range, however, are practically the same for the three solvents. Moreover, in many compounds studied by Snyder, l 4 vicinal coupling constants appear to be affected little by changes in the dielectric of the medium and suggest little change in the population of conformers. We suggest, instead, that the enhanced separation of methyl resonances in m-xylene (and other solvents) is a result of oriented solvent molecules weakly bonded to a specific part of the a-phenylethyl group or alternatively, due to the average orientation of all collisions
COMMUNICATIONS TO THE EDITOR between magnetically anisotropic and unsymmetrical solvent and solute molecule^.'^ The effect of temperature can be rationalized with this picture. Between -20 and 60°, the molar volume of mxylene and chloroform increases 8 and 11%, respectively.16J1 Assuming a linear extrapolation, the molar volume of liquid cyclohexane can be expected to increase 10% over this same temperature range.” It is commonly accepted that thermal expansion of liquids is accompanied by the formation of holes in the medium. Consequently, the distance between solvent molecules or between solvent and solute molecules must increase. Therefore, local magnetic fields caused by the proximity of anisotropic solvent or solute molecules will tend to diminish a t higher temperatures. The separation of nonequivalent proton resonances due to intrinsic asymmetry, however, will still remain a t higher temperature. (11) 8 . 0 . Morgan and H. H. Lowry, J . Phys. Chem., 34,2383 (1930). (12) L. M. Heil, Phys. Rev., 39, 666 (1932). (13) W. M. Heston, Jr., and C. P. Smyth, J . Am. Chem. SOC.,72, 99 (1950). (14) E.I. Snyder, {bid., 88, 1155 (1966). (15) (a) A. D. Buckingham, T. Schaefer, and W. G. Schneider, J . Chem. Phys., 32, 1227 (1960); (b) J. W. Emsley, J. Feeney, and L. H. Sutcliffe, “High Resolution Nuclear Magnetic Resonance Spectroscopy,” Vol. I, Pergamon Press, New York, N. Y.,1965, pp 93,94. (16) F. D. Rossini, “Selected Values of Physical and Thermodynamic Properties of Hydrocarbons and Related Compounds,” Carnegie Press, Carnegie Institute of Technology, Pittsburgh, Pa., 1953,p 297. (17) E.Kuss, 2.Angew. Physik, 7 , 372 (1955).
C O M M U N I C A T I O N S T O THE E D I T O R Chemiluminescent Reactions of Fluorescein Dyes in Aqueous Solution
Sir: It was reported recently that electron pulse irradiation of several dyes in deaerated aqueous solution (rhodamine B, acriflavin, fluorescein, and eosin) induces visible light emission which builds up and decays over a period of microseconds.‘ The emission was quenched by either eaq- or OH scavengers, which led to a proposed mechanism based on the chemiluminescent reaction of ea,,- with the oxidized dye radical.2 Pulseradiolysis studies of the fluorescein^^*^ have shown that OH reacts with the dye via two paths, leading to both semioxidized dye (X) which is presumed to have the phenoxy1 structure5t6 and a long-lived “red product,” which has been attributed to an OH a d d ~ c t . I~n an attempt to identify the chemiluminescent species, we The Journal of Physical Chemistry
performed a combined flash photolysis-pulse radiolysis experiment, in which the deaerated dye solution was irradiated first with a 15-psec flash of visible light (>400 mp) followed by a 1-psec pulse of 33-MeV electrons (-150 rads). Optical emission and absorption measurements were made during the process. The flash illumination of 2-5 pM fluorescein or eosin (S) led to the excitation of triple dye (T) which decays (1) W.Prtitz, K.Sommermeyer, and E. J. Land, Nature, 212, 1043 (1966). (2) W. Pmtz and E. J. Land, Biophyeik, 3, 349 (1967). (3) J. Chrysochoos, J. Ovadia, and L. I. Grossweiner, J . Phys‘ Chem., 71, 1629 (1967). (4) P. Cordier and L. I. Grossweiner, submitted for publication in J . Phys. Chem. (5) L. Lindqvist, Arkiv Kemi, 16, 79 (1960). (6) V. Kasche and L. Lindqvist, Photochem. Photobiol., 4, 923 (1965).
COMMUNICATIONS TO THE EDITOR
757
-
0.5
-
0.2
0.2
n n
i
0 0 W w
0 w W
0
a
E‘ ::
c
:
--
0.1
-
0.05
0.1
4
-.e Y
(L
P
002
-
0 I
I
OS
500
with the formaftionof X and semireduced dye (R) via X; T S -c R X.+’ the reactions: 2 T -t R It was found that the addition of 250 pM formate strongly quenched the emission from both dyes when the electron pulse was applied at a time delay of 1-5 msec. At this time the triplet had decayed but R and X had nat reacted appreciably. Since formate suppresses only that oxidized dye formed by radiolysis, this result proves that the OH adduct is the reacting species. The proposed chemiluminescent process is
+
+
+ SOH. +S* + OHS* +S + light
+
(1)
Further investigation showed that a strong light emission occurs at shorter delay times, even with high formate present to suppress all oxidation of the dye by OH. This second process is attributed to the reaction of triplet dye with eaq- leading to electronically excited semiquinone
+T
I 300
TIME--see
Figure 1. Comparison of electron pulse induced emissior intensity from 5 fiM eosin (deaerated, p H 9.0) with relative triplet Concentration a t time of electron pulse: points, relative emission intensity; line, triplet decay as measured a t 600 rnp (no electron pulse); 0, 250 p M formate; 0, 1 m M formate.
eaa-
200
1000
TIME-psec
eaq-
I
I 100
R* -+ R
+ light
(11) based on the following evidence. (a) The initial emission intensity follows the triplet concentration at the time that the electron pulse is applied, except at short -+
Figure 2. (a) Triplet eosin decay after light flash irradiation of 5 p M dye (deaerated, with 1 mM formate, p H 9.0). (b) Triplet eosin decay when electron pulse was applied 110 psec after light flash. The optical density at 600 mp shown by the dashed line represents the absorption of the hydrated electron plus triplet eosin.
delay times when the eaq- yield is the limiting factor (Figure 1). (b) The triplet concentration decreases abruptly when the electron pulse is applied and continues at the original pseudo-first-order decay rate when light emission terminates (Figure 2). (c) The emission intensity from eosin under these conditions was at least an order of magnitude stronger than from fluorescein, which is consistent with the considerably higher triplet yield in the former as deduced from fluorescence efficiencies.E Reaction I can lead to triplet formation in radiolysis via intersystem crossing in S*, although the yield would be low because of the fast reaction of eaq- with unI1 represents a new type of excited d ~ e . ~Reaction , ~ process in which the reduction potential of the hydrated electron may be transferred to the metastable dye molecule in a highly exothermic reaction. Aromatic triplet states have been generated by pulse radiolysis in aprotic solvent^,^ but not in media which solvate elec(7) T. Ohno, 5. Kato, and M. Koiaumi, Bull. Chen. SOC. Japan, 39, 232 (1966). (8) J. B. Birks and I. H. iMunro, Progr. Reaction Kinetics, 4, 239
(1967). (9) F. S. Dainton, T. J. Kemp, G . A. Salmon, and J. P. Keene, Nature, 203, 1060 (1964). Volume 76, Number 6 February 1968
758
COMMUNICATIONS TO THE EDITOR
trons. However, the investigation of such reactions is feasible with the combined technique in which the triplet states are excited optically while eaq- is generated by the electron pulse.1°
I
I
I
(10) This work was supported by the U. 8. Public Health Service on Grant No. GM-12716 from the National Institute of General Medical Sciences.
DEPARTMENT OF RADIATION THERAPY AND MICHAEL REESEHOSPITAL MEDICAL CENTER CHICAGO, ILLINOIS
L. I. GROSSWEINER A. F. RODDE, JR.
I
RECEIVED OCTOBER26, 1967
I
NoDhfholene sulfonate (mM) I.o
Excited-Molecule Reactions in the Radiolysis
of Peptides in Concentrated Aqueous Solution' Sir: The radiation chemistry of simple peptides such as N-acetylglycine and N-acetylalanine in dilute aqueous solution can be interpreted almost exclusively in terms of the formation and subsequent reaction of the peptide radicals RCONHcRz.2 In neutral solutions such radicals are formed predominantly through OH attack
H20--mrf HzOz, HB,OH, H+, eaq-
+ RCONHCHRz
OH
-+
H20
+ RCONHcR2
(1) (2)
where reaction 1 represents the radiation-induced ~ t e p . a - ~I n the absence of oxygen, the reaction RCONHCRZ 2RCONH6Rz --f
I
(3)
RCONHCRZ leads to the formation of the cr,cu'-diamino succinic acid derivative. A fraction of the RCONHcRz radicals undergo further oxidation through reactions of the type 2RCONHcRz -+ RCON=CRz
+ RCONHcRz
HzOz
+ RCONHCHRZ
(4)
--+
RCONHC(OH)R,
+ OH
(5)
where the Hz02in reaction 5 is derived from the radiation-induced step l. The products of reactions 4 and 5 are labile and readily decompose on mild hydrolysis, e.g.
HSO
+ RCON=CRz
-+
RCOOH
+ NH3 + RzCO
(6)
to yield ammonia and carbonyl products. In the y radiolysis of evacuated 0.05 M acetylalanine solutions a t pH 7, G(NHa) N G(>CO) N 0.5. We find, however, that there is a very marked increase in the ammonia yield (measured after hydrolysis) The Journal of Physical Chemistry
2 .o Acefylalanine ( M )
3.0
Figure 1. Amide-ammonia yield, G(NH3),as a function of acetylalanine concentration in oxygen-free solution a t pH 7 under y rays. Insert: effect of naphthalene sulfonate on G(NH8) from a 2 M acetylalanine solution.
as the concentration of the acetylalanine is increased above 0.1 M . Data6 obtained in the y radiolysis of Orfree solutions of acetylalanine are given in Figure 1. The ammonia yield tends to level off a t a value of G(NH), 3 in the concentration range 2-3 M . This increase in G(NH3) is not accompanied by a corresponding increase in the yield of carbonyl products; G(>CO) 0.7 over the entire concentration range 0.1-3 M . Hence, the increase in G(NH3) cannot be explained in terms of an enhancement in the yields of reactions 2 and 4.5 In fact, the increase in G(NH3) does not appear to be related in any significant way to the reactivity of the OH radical or its precursor HzO+.' We find, for example, that addition of formate ion, which is an effective OH scavenger
-
-
OH
(IC7
+ HCOO-
--it
HzO
+ COO-
(7)
= 2.5 X lo9M-' sec-1)8 a t concentrations as high
(1) This work was done under the auspices of the U. S. Atomic Energy Commission. (2) (a) W. M. Garrison and B. M. Weeks, Radiation Res. Suppl., 4 , 148 (1964); (b) H. L. Atkins, W. Bennett-Corniea, and W. M. Garrison, J . Phys. Chem., 71, 772 (1967). (3) (a) A. 0. Allen, Radiation Res. Suppl., 4, 54 (1964); (b) E. J . Hart and R. L. Plataman, Mech. Radiobiology, 1, 93 (1961). (4) C. J. Hochanadel and R. Casey, Radiation Res., 2 5 , 198 (1965). (5) The relatively small but apparently very real discrepancies in the reported 100-eV yields of the products of reaction 1 have been discussed by Allen (ref 3). Recent measurements by Hochanadel ~ and Casey (ref 4) give GOH= 2.59, Ge.,- = 2.58, GH = 0.55, G H = 0.45, G H ~ o=~0.72. (6). Experimental methods employed in the present work are described in ref 2a and 2b. The vapor phase chromatography was performed by Mr. H. A. Sokol. (7) The apparent yield of reaction 2 is independent of solute concentration, Le., G(RC0NHCRz) = 2.5 = GOH in both dilute and concentrated solution (ref 2). However, since OH arises from HzO --c HsO + OH (ref 3b), we cannot rule out HzO+ via Ha0 + the possibility that a t the higher solute concentrations a fraction of the HzO+ species reacts directly with RCONHCHRz to give RCONHCRz.
+
+
