1622
D. E. HOLMES, N. B. NAZHAT, AND J. J. WEISS
and 3
Wb(C1,Hi)= - $ 3
%=1
[i
cos28
+ 11 3
consequently
= -0.0087
Wb(C1,Hi) i=l
The total interaction energy involved in the hydrogen atom induced dipoles is less than 1% of the total interaction energy bettween the carbon atom C1 dipole and the three hydrogen atoms.
Effects of Dissolved Oxygen on the Electron Spin Resonance Signal Intensities of Trapped Hydrogen Atoms and Some of Their Reactions in Acidic Ice Matrices by D. E. Holmes,l N. B. Nazhat, and J. J. Weiss Laboratory of Radiation Chemistry, School of Chemistry, T h e University, Arewcastle u p o n T y n e , England (Receiued J a n u a r y 6 , 1060)
Electron spin resonance spectra of irradiated acidic ices saturated with oxygen and nitrogen show that the formation and the yields of trapped hydrogen atoms a t 77°K are practically independent of dissolved oxygen while their esr signal intensities are dependent, under certain conditions, on the oxygen content and the applied microwave power. During annealing of irradiated ices containing thymine and oxygen a t 100”K, hydrogen atoms react to form thymine hydrogen atom adduct radicals and hydroperoxide radicals. Upon annealing to 175”K, oxygen reacts with the thymine hydrogen atom adduct radicals to form 5,B-dihydrothymyl peroside radicals. These reactions of hydrogen atoms and oxygen, in irradiated ices a t 77-195”K1 are discussed in relation to similar reactions in the liquid phase.
Introduction Hydrogen atoms are formed and trapped in y-irradiated, frozen, acidic solutions (ices). 2 - 4 The hydrogen atom yields reportedly are not affected by dissolved oxygen.2 Upon annealing and release of the hydrogen atoms from their traps, the atoms can add to the 5,6 double bond of dissolved thymine to form 5,6dihydrothymyl-5-yl radicals (thH ) .6--8 This communication describes the increase in the “apparent” yield of trapped hydrogen atoms (H,) and the decrease in the yield of dihydrothymyl radicals when nitrogensaturated acidic ices containing thymine are saturated w i t h oxygen before freezing and irradiation.
-
Methods Reagent grade H2S04, 0 2 , K2, and thymine (th) were used with triply distilled water to make the solutions. The samples were 1 L V in H,S04 and were bubbled with the appropriate gas for 15 min before freezing into small ice cylinders as previously described. These cylinders were then irradiated in sealed, nitrogen-flushed, Pyrex T h e Journal of Physical Chemistry
tubes at 77°K with 6oCoy rays to a total dose of approximately 3 Mrads at a dose rate of 0.15 RIrad/hr. After irradiation, the samples were transferred, without allowing to warm up, into a standard electron spin reso(1) Fellow in Radiological Research of the James Picker Foundation, 1966-1968. Present address: Department of Biochemistry 8: Biophysics, University of Hawaii, Honolulu, Hawaii 96822. (2) H. Zeldes and R. Livingston, P h y s . Ret., 96, 1702 (1954).
(3) (a) L. Kevan, P. K. Moorthy, and J. J. Weiss, ,Vatwe, 199,’689 (1963); (b) J. Zimbrick and L. Kevan, J . Chem. P h y s . , 47, 5000 (1967). (4) P. N.Moorthy and J. J . Weiss in “Solvated Electron,” Advances
in Chemistry Series, No. 50, American Chemical Society, Washington, D. C., 1965, p 180. (5) R. Livingston and A. J. Weinberger, J . Chem. P h y s . , 33, 499 (1960). (6) C . Elston, Ph.D. Dissertation, The University, Newcastle upon Tyne, 1967. (7) D. E. Holmes and J. J. Weiss, I n t . J . Radiat. Biol., 14, 187 (1968). (8) D. E. Holmes, B. N. Nashat, and J. J. TTeiss, Radiat. Res., 35, 510 (1968). (9) L. Kevan, P. N. Moorthy, and J. J. Weiss, J . A m e r . Chem. SOC., 86, 771 (1964).
1623
ESRSIGNAL INTENSITIES OF TRAPPED HYDROGEN ATOMS nance (esr) dewar with a thin-walled quartz tip which fitted into the esr cavity. Annealing was usually accomplished by transferring the irradiated samples to a container immersed in Dry Ice at 195°K. However, in some cases the samples were annealed by removing the liquid nitrogen from the dewar for predetermined lengths of time which allowed the samples to warm to thermocouple-calibrated temperatures. Esr spectra were recorded with the samples a t 77°K. Most esr measurements were made with a Varian E-3 spectrometer. R!Iicrowave power values were taken directly from a calibrated dial inherent with the spectrometer. A Varian V-4502-13 esr spectrometer in the low-power configuration was used for the Ht measurements below 1-mW rf power. The low-power values were calibrated using an H P 431B power meter. The magnetic field sweep and g-factor calibrations utilized peroxylamine disulfonate and diphenylpicrylhydrazyl (DPPH) , respectively. However, for more accurate measurements of g factors, the klystron frequency of the V-4502 spectrometer was monitored by H P 5253B and H P 2590B frequency converters coupled to an H P 5245L electronic counter while the magnetic field was calibrated using a proton resonance gauss meter coupled to the electronic counter.
Results and Discussion Figure l a shows the first-derivative esr spectrum of nitrogen-saturated, acidic ice (Nz-ice) after irradiation. Figure l b shows a similar spectrum after oxygen saturation (&ice) and irradiation. A comparison of Figure l a with b shows almost no differences in the central regions of the spectra which result from hydroxylfree radicals as well as Sod- radical ions.4 The lines of the Ht doublet are separated by approximately 505 G and have satellite lines as previously reported. lo When thymine M ) is included in the ices (th-ices), esr spectra, Ht ratios, and Ht line widths are observed which are similar to those obtained from samples containing no thymine. Table I gives the relative intensities and line widths of the main Ht line, downfield, a t 1-mW rf power which were observed in each sample under conditions of partial power saturation. The observed Ht signals differ in that the intensities in the 02-ices are larger than in the Nz-ices. This is unusual, since oxygen might be expected to scavenge radiationreleased electrons by reaction 1, which are responsible for hydrogen atom formation4according to reaction 2
e-
--
+ O2 + HS0,-
e-
e-
+ H+
02-
H+ A
Ht
HOz.
+ S042-
H A Ht.
(1) (2) (3)
The concentrations of 0 2 and HS04- are approximately and 1 M , respectively. Although the rate constants of these reactions in ice a t 77°K are not known,
Figure 1. Firstrderivative esr spectra of irradiated acidic (a) Nz-ice and (b) Orice at 77°K and 1-mW microwave power.
Table I: Relative Ht Signal Intensities in Irradiated Ices under Partial Power Saturation Sample
Relative intensity of H t
Relative line width
NZ-ice Orice th-Nz-ice th-ot-ice
4.5 7.9 3.7 7.0
3.4 3.3 3.5 3.4
it has been shown that the ratios of many of the rate constants in frozen aqueous systems are quantitatively parallel to those in aqueous solutions at room temp e r a t ~ r e , Thus, ~ ~ ~ ~consideration of the relevant rate constants in water a t 20” ( k ~= 1.9 X 1Olo M-‘ sec-l, ref 12; kz = lo7M-l sec-’, as estimated from the reaction of e- with H2P04- in ref 13; ka = 3 X 1Olo M-’ sec-l, ref 14) would suggest that if reaction 3 is important, O2 would not be able to capture any appreciable part of the electrons and the Ht intensities should be the same in the presence of oxygen. The failure to observe the esr signal of 02- (gll = 2.175, ref 15) or of HOz. (10) W.Kohnlein and J. H. Venable, Jr., Nature, 215, 618 (1967). (11) L.Kevan, J. Amer. Chem. Soc., 89,4238 (1967). (12) E.J. Hart and E. M. Fielden in “Solvated Electron,” Advances in Chemistry Series, No.50,American Chemical Society, Washington, D. C., 1965,p 253. (13) M.Halmann and I. Platzner, J. Phya. Chem., 70, 2281 (1966). (14) 8. Gordon, E. J. Hart, M. S. Matheson, J. Rabani, and J. K. Thomas, J. Amer. Chem. Soc., 85, 1375 (1963). (15) J. E.Bennett, D. J. E. Ingram, M. C. R. Symons, P. George, and J. S. Griffith, Phil. Mag., 46,443 (1955). Volume 74, Number 7 April 8 , 1970
1624
D. E. HOLMES,K.B. NAZHAT, ASD J. J. WEISS
““i
0.0’
‘
0.0 04
08
IO
rf Power
(rnwb-
I5
10
Figure 2. Relationship between applied microwave power and esr spectral intensity of the downfield, main Ht line in irradiated acidic 02-saturated ( 0 ) and N2-saturated ( 8 )ice at 77°K.
(SI, = 2.035, ref 16) from reaction 1 before annealing the irradiated OZ-ice and the observation of Ht intensities converging at low rf powers in Nz-and Orices as discussed later suggest that under the present conditions, reactions 2 and 3 do predominate over reaction 1. The slight lowering of the H t intensity upon the inclusion of thymine (Table I) and the observation of a small yield of thymine hydrogen atom addition radicals before annealing indicate that thymine competes with HSOr- for radiation-produced electrons which yields thH or reacts with radiation-induced hydrogen atoms at 77°K. Microwave power saturation studies at 77°K from 0.4 ,L~W to 8 mW indicate that oxygen provides a mechanism of relaxation” for the trapped hydrogen atoms above about 5 pW and increases the “apparent” Ht yield in OZ-ice relative to Nz-ice. Figure 2 shows the power saturation curve for the main line of the downfieId Ht signal in nitrogen-saturated and oxygen-saturated ices. At powers below about 5 rW, the trapped hydrogen atoms in Nz- and OZ-ices have spin-lattice relaxation times ( T I ) such that the spins can easily dispose of their energy and the Ht signal intensities are the same. As the power is increased to greater than 5 pW, the spin-lattice relaxation in the Nz-ice can no longer adequately dispose of the energy, and the Ht intensity saturates. However, in 02-ice, where TI is larger, which adequately disposes of energies up to 20 FW, this allows the Ht signal intensity to increase. Therefore, in Os-ice, the trapped hydrogen atoms evidently relax through a nearby oxygen molecule which in turn is able to transfer the microwave energy to the surrounding environment. Above 20 pW, the spinlattice relaxation time cannot dispose of the energy, the Ht signal intensity power saturates, and the signal decreases. When the irradiated ices are examined at a The Journal of Physical Chemistry
power above the Ht convergence value, the €It intensity from 02-ice is larger than the Ht intensity obtained from the Nz-ice, although both samples contain approximately the same number of trapped hydrogen atoms. Table I shows that there is very little change in the line width of the Ht main line when nitrogen is replaced by oxygen in the irradiated ices. This indicates that the shape of the line remains constant and inhomogeneous saturation broadening occur^.'^ Thus, the line width of the Ht main resonance is apparently governed by hyperfine interactions with protons of the environment, Livingston and Weinberger5 found no effect of dissolved oxygen on Ht or hydrogen yields in 7-irradiated, frozen HzS04 glasses (0.129 mole fraction). The Ht yields in HzS04 glasses are quite large compared to polycrystalline ices, and the oxygen effect may be masked in comparison to that effect obtained in the ices used in the present experiments. Since other authors3have not observed oxygen effects on Ht signal intensities in irradiated polycrystalline acidic ices, the explanation most probably is that rf powers below the Ht saturation level were previously used with the oxygenated samples and thus no oxygen effect would be observed on Ht. Power saturation of Ht in acidic glasses3”>5and ices3“has been generally observed. Ingalls and Pearson’* observed that the esr peak height from a sample of irradiated terphenyl was dependent on the dissolved oxygen content and the microwave power. They found that small changes in the quantity of dissolved oxygen alter the slope of the power curve in some specific range of power, while the power curve was independent of oxygen content at sufficiently low powers. Esr spectra from petroleum oils show similar oxygen effects with microwave po~ver.’~These effects have been interpreted as due to oxygen decreasing the relaxation time and thereby reducing power saturation. The scavenging of some hydrogen atoms by oxygen during annealing can be seen by comparing the yields of 5,6-dihydrothymyl-5-y1 radicals (thH .) in the irradiated acidic ices containing Ai‘ thymine and dissolved oxygen where the following reactions might take place HHa
+ t h thH. + 02 -+- HOz. --it
(4)
(5)
Upon annealing the th-Nz-ice to 100”K, the trapped hydrogen atoms are released and add to position G of the dissolved thymine to yield thH radicals (Figure 3a) according to reaction 4. The large central signal is
-
(16) 8.J.Wyard and R. C. Smith, Sixth International Symposium on Free Radicals, Cambridge, 1963. (17) D. J. E. Ingram, “Free Radicals as Studied by ESR,” Butterworth and Co. Ltd., London, 1968. (18) R. B. Ingalls and G . A. Pearson, Anal. Chim. Acta, 25, 566 (1961). (19) A. J. Saracen0 and D. D. Coggeshall, J . Chem. Phys., 34, 260 (1961).
1625
ESRSIGNAL INTENSITIES OF TRAPPED HYDROGEN ATOMS Table 11: g Tensors 01
01
oa
011
Ol
2.013 2.0130
2.0060 2.0053
2.0059 2.0059
th-02-ice (Figure 6) 0 2 - (ref 15) HnO2-ice (HOI, ref 16) H2OZ-ice(HOs, ref 16) Polypropylene02 (ref 20)
2.040 2.175 2,035 2.061 2,035
2.018
2,0019 2.0046 2.0019 2.0019
2.019 2.0189 2.036 2,040 2.036
2.0095 2,0092
Peak D P (Figure 3b) th-0%-ice (Figure 4b) Orice (Figure 5)
2.019 2.0189 2.036 2,040 2.036
2.0065
2,0023
2.0059
2.0019
Sample
N2-ice (Figure 3a)
904 - (ref 4)
Figure 3. Firsbderivative esr spectra of irradiated acidic (a) thymine-N2-ice and (b) thymine-02-ice at 77°K after annealing to 100°K.
almost identical with the radical identified as SO4. - by Moorthy and Weiss4 (Table 11). When irradiated Orice containing thymine is annealed to 100"K, hydrogen atoms are released, thH * radicals are formed, and a small downfield peak (DP) is observed (Figure 3b). The large signal from SO4.- in the spectral center prevents any positive identification of peak DP as due to H 0 2 radicals. However, a comparison of the g factor of peak D P with gl from HO2. in Table I1 suggests that HOz. radicals may be formed in 02--ices. The g tensors for the radicals in irradiated ices after various annealings and g tensors for related radicals are listed in Table 11. Since most of the experimental spectra appear to result from fully anisotropic g tensors, gl, and g3 are taken at low- and high-field shoulders, respectively, while 92 is taken at the cross-over point near the center of the spectrum.21 The g tensor g, is approximately equivalent to 911 while the average of g2 and 93 is approximately equivalent to gl. The thH intensity in the th-02-ice annealed to 100°K is decreased by about 40% from that in the th-N2-ice. In contrast to the effect of oxygen on the H, intensity
2.0039
2.040 2.175 2,035 2.061 2,035
2.002 2,0044 2,0045 2.0039
Figure 4. First-derivative esr spectra of irradiated acidic (a) thymine-Nz-ice and (b) thymine-02-ice a t 77°K after annealing to 175OK.
(Figure 2), the thH intensity is not affected by dissolved oxygen from 1to S mW. If the ices are annealed to 175"K, the SO4.- radical almost completely disappears leaving the thH radical in the N2-ice (Figure 4a) and an asymmetric doublet in the OAce (Figure 4b). The thH signal seen in Figure 3b from partially annealed th-02-ice has now disappeared while the downfield peak D P has become part of the asymmetric doublet in Figure 4b. The change in the signal intensity of peak D P during annealing from 100°K (Figure 3b) to 175°K (Figure 4b) cannot be estimated, since the peak D P rides on the tail of a background signal which decays before 156°K. However, during annealing of the th-02-ice from 156 to 175"K, the signal height of peak DP increases by approximately 17%. A comparison of the respective concentrations (02, M ; thymine, M ) and the rates of reaction 4 (h = lo9 M-' sec-l, estimated from He uracil in ref 22) and reaction 5 (ICs = 1.9 X 1O1O M-' sec-', ref
+
(20) M.Iwasaki and Y . Sakai, J . Polym. Sci., Part A-8, 6,265 (1968). (21) P. W.Atkins and M. C. R. Symons, "The Structure of Inorganic Radicals," Elsevier Publishing Co., Amsterdam, 1967,Appendix
SA.3.
Volume 74,Number 7 April 8, 1970
1626
D. E. HOLMES,N.B. NAZHAT, AKD J. J. ~J'EISS
Figure 6. First derivative esr spectrum of irradiated acidic thymine-02-ice at 77°K after annealing to 195°K.
Figure 5. First-derivative esr spectrum of irradiated acidic Orice at 77°K aft'er annealing to 175°K.
23) indicates that oxygen and thymine compete approximately equally for hydrogen atoms. Therefore, a composite spectrum of HOz and thH radicals should result in Figure 3b. Table I1 shows that the gl factor of Figure 4b is larger than the g factor of peak D P in Figure 3b. Thus, upon annealing th-02-ice to 175"K, the disappearance of the thH signal from the ice while thH. is stable in the th-Nz-ice and the formation of an asymmetric signal with a gl factor different from that g factor of peak D P (assumed to be HO2.) suggest that dihydrothymyl peroxide radicals (thH-02 * ) have been formed as in thH*
+ 02 +thH-02.
(6)
The formation of thH-OZ radicals has been observed by Holmesz4where thH radicals formed by exposure of polycrystalline thymine to gaseous hydrogen atoms react with oxygen in a gas-flow system. Although the esr spectrum of thH-02 appeared to be a singlet, an -unambiguous spectrum could not be obtained. Thus, as observed, the doublet in Figure 4b should contain a contribution from the dihydrothymyl peroxide radical. left in oz-ice without Figure shows the thymine after annealing to 175°K. Although this Spectrum may contain some contributions from the
'
The Journal of Physical Chemistry
SO**- radical, the g:tensor values (Table 11) indicate that Figure 5 probably results from HOZ radicals formed in reaction 5. Annealing the thymine-containing 0 A c e from 175 to 195°K (Figure 6) results in a further change in the asymmetric doublet of Figure 4b. Since the change appears to be due to the loss of a radical with a line at a g factor of about 2.018, the SO4.radical is assumed to decay at approximately 195°K. All esr signals disappear from the ices during annealing to 215°K. These scavenging data mag' explain why Holmes and Weiss' could not observe low yields of Ht or hydrogen atom adduct radicals in irradiated deoxyribonucleic acid ices and gels unless air was removed before freezing and irradiation. When the irradiated deoxyribonucleic acid samples containing dissolved air were annealed to 195°K and examined at 77°K in the esr spectrometer, an asymmetric signal was found25 similar to those in Figures 4b and 6. The results reported in this paper indicate that the applied microwave power and the concentration of dissolved oxygen are important parameters to be considered when comparing esr signals in irradiated, frozen solutions. Acknowledgment. We wish to thank 1.k.W. J. Terry for his assistance in measuring the microwave powers and the g tensors'
(22) G . Scholes and hf. J. SimiC, J . PhW. Chem., 68, 1738 (1964). (23) J. P. Sweet and J. K . Thomas, ibid., 68, 1363 (1964). (24) D. E. Holmes, ph.D. Dissertation, University of California, L O S Angeles, Calif., 1966. (25) D. E. Holmes and J. J. Weiss, unpublished results, 1967.
