The fine phenomena in the EPR spectra of the hole-like paramagnetic centre formed in cytosine(thiocytosine)-HCl crystals upon irradiation are studied with EPR spectroscopy. The spin Hamiltonian including the electronic Zeeman term, hyperfine coupling, quadrupole coupling and the nuclear Zeeman term has been used in the second-order perturbation treatment for the interpretation of the experimental observations. It is shown that the quadrupolar term causes a detectable shift of the EPR lines and also induces the "forbidden\u27\u27 transitions. From the shift of the "allowed\u27\u27 transitions and the positions of the ``forbidden\u27\u27 transitions, the chlorine quadrupolar coupling tensor [P], has been determined.Proučavane su slabo izražene, neredovite pojave u EPR spektrima paramagnetičnog centra sličnog šupljini u ozračenim kristalima citozin hidroklorida s primjesama tiocitozina. Za objašnjenje ponašanja upotrijebljen je spinski Hamiltonijan koji se sastoji od elektronskog Zeemanovog člana, nuklearnog Zeemanovog člana, člana hiperfine i člana kvadrupolne sprege jezgre s elektronom. Pokazano je da kvadrupolni član koji potječe od klora ne samo unosi pomak EPR spektralnih linija, nego uzrokuje i pojavu dodatnih, “zabranjenih” prijelaza. Iz mjerenja slabo izraženih pomaka linija i položaja “zabranjenih” linija izračunat je kvadrupolni tenzor

Herak, Janko N.

Krilov, Dubravka

Pranjić-Petrović, Tatjana

Sanković, Krešimir

English

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ISSN1330–0008CODENFIZAE4FINE PHENOMENA IN THE EPR SPECTRA OF THE HOLE-LIKEPARAMAGNETIC CENTRE IN CYTOSINE HYDROCHLORIDEDUBRAVKA KRILOVa, KREŠIMIR SANKOVIĆc, TATJANA PRANJIĆ-PETROVIĆband JANKO N. HERAKcaSchool of Medicine and cFaculty of Pharmacy and Biochemistry, University of Zagreb,Zagreb, CroatiabSchool of Medicine, University of Rijeka, Rijeka, CroatiaDedicated to Professor Mladen Paić on the occasion of his 90th birthdayReceived 24 July 1995UDC 538.955PACS 76.30.-vThe fine phenomena in the EPR spectra of the hole-like paramagnetic centre formedin cytosine(thiocytosine)–HCl crystals upon irradiation are studied with EPR spec-troscopy. The spin Hamiltonian including the electronic Zeeman term, hyperfinecoupling, quadrupole coupling and the nuclear Zeeman term has been used in thesecond-order perturbation treatment for the interpretation of the experimental ob-servations. It is shown that the quadrupolar term causes a detectable shift of theEPR lines and also induces the “forbidden” transitions. From the shift of the “al-lowed” transitions and the positions of the “forbidden” transitions, the chlorinequadrupolar coupling tensor P̂ , has been determined.1. IntroductionIt has been well established that the transfer and trapping of the excess energydeposited by ionizing radiation in nucleic acids and the related model systems,strongly depend on the presence of thioanalogs as substitutional impurities in theirradiated systems [1-6]. Thiocytosine and thioguanine, present in concentrationsFIZIKA A 4 (1995) 3, 637–646 637krilov et al.: fine phenomena in the epr spectra . . .less than 1% of that of cytosine or guanine, stabilize the damage predominantly in aform of sulfur-centered radicals. The crystals composed of hydrochlorinated bases,cytosine or guanine, with traces of thiocytosine or thioguanine as substitutionalimpurities, when irradiated with γ– or X-rays, exhibit the paramagnetic specieswith the unpaired electron shared by sulfur and chlorine. It has been shown thatthe paramagnetic centre is an electron-deficient, hole-like species [6].The dominating spectral features are determined by an anisotropic g-factorand an anisotropic quartet hyperfine splitting, due to the coupling of a 35Cl nu-cleus (I = 3/2), with natural abundance of about 75%, along with a less intensiveEPR spectrum of the same characteristics, brought about by the less abundant Clisotope, 37Cl (I = 3/2, 25% natural abundance). The spectra are quite well under-stood in terms of the spin Hamiltonian consisting of the large electronic Zeemanterm and a smaller, perturbing hyperfine coupling term. The first-order perturba-tion treatment is adequate for understanding of most of the observed phenomena[4]. However, at certain orientations of the crystal in the magnetic field of the spec-trometer, not only the quartet pattern behaves as predicted, but also additional,“forbidden” transitions occur. In order to understand such phenomena, a morecomplete spin Hamiltonian and a more precise treatment should be used.In the present work, we describe the fine phenomena for the hole-like param-agnetic centre in irradiated cytosine hydrochloride, with thiocytosine as impurity.It will be shown that some of the spectroscopic features could not be explained ifquadrupolar interaction and the nuclear Zeeman term are ignored, in spite of thefact that these terms are very small.2. ExperimentalThe crystals of cytosine hydrochloride, with thiocytosine as a substitutionalimpurity, designated as C(TC)HCl, were grown by evaporation of the solutionof cytosine (with the traces of thiocytosine) in hydrochloric acid. Typically, 1%of thiocytosine is added to cytosine before dissolving the powder in HCl. Actualconcentrations of thiocytosine were measured by particle-induced X-ray emission(PIXE) [7]. The ratio of thiocytosine to cytosine was found to be 0.006 (±30%).Single crystal of cytosine hydrochloride (and also of C(TC)HCl) is monoclinic withthe space group P21/n, with a = 832, b = 683, c = 1104 pm and β = 96.7◦ [8].There are four molecules in the unit cell, two of them magnetically distinct. Thecrystal planes were identified by the reflection of the laser beam from a crystalmounted on a goniometer.For irradiation and EPR measurements, the crystals were mounted on metalrods in desired orientations. A weak EPR signal originating from irradiated gluecould not be avoided. The crystals were irradiated at 77 K, using a strong 60Cosource. The dose rate was about 10 kGy h−1 and the total dose about 40 kGy. Themeasurements were made at 77 K with a Varian E-109 spectrometer, equipped witha unit for digital signal recording (Scientific Software Services). For the analysis ofthe data, the orthogonal a∗, b, c crystallographic system of axes was used.638 FIZIKA A 4 (1995) 3, 637–646krilov et al.: fine phenomena in the epr spectra . . .3. ResultsThe representative EPR spectra of the hole-like paramagnetic centre inC(TC)HCl are shown in Fig. 1. Spectrum a) is recorded for the magnetic field,B, in the c direction, B||c, and spectrum b) for B ⊥ b, 12◦ from the B||b direc-tion.Fig. 1. EPR spectra (first derivative curves) of the irradiated single crystal ofC(TC)HCl, for two different orientations in the B ⊥ b plane (for B||c and B ⊥b, 12◦ from the c axis, respectively). The crystals were irradiated at 77 K, andmeasured at 77 K at microwave frequency of 9.250 GHz, with the microwave powerof 5 mW. The solid bars under the spectra indicate the positions of the first-orderresonance lines for species A containing the 35Cl isotope. The dashed bars indicatethe positions of the second-order resonance lines.In both of these crystal orientations the crystal symmetry makes all themolecules magnetically equivalent. The EPR spectra clearly indicate the presenceof more than one paramagnetic species. It is already known that there are twodistinct hole-like centres, named A and B, and a signal at g ≈ 2.003 brought aboutby the defects in the host lattice (not associated with thio-impurities) and the un-avoidable radicals associated with the irradiated glue. In the present report we dealonly with the quartet patterns of centre A, indicated by the bars in Fig. 1. Forthe orientations where the hyperfine splittings are large, the coupling of both Clisotopes, 35Cl and 37Cl, is clearly discriminated, the latter isotope giving smallerFIZIKA A 4 (1995) 3, 637–646 639krilov et al.: fine phenomena in the epr spectra . . .intensity. The spectra differ in the line splittings and in the total line spread byabout 20%, as a consequence of the different nuclear magnetic moments of the twochlorine isotopes. In the crystal orientations where the hyperfine coupling is small,as depicted in both tracings in Fig. 1, the weaker signal, associated with 37Cl is notclearly discriminated, because the differences between the two spectra are smallerthan the line-width. In the lower spectrum in Fig. 1, the additional lines associatedwith centre A, indicated by the dashed bars under the spectra, are clearly seen.These are the “forbidden” transitions that appear only for certain crystal orienta-tions. We analysed in detail the “regular” and the “extra” EPR lines (the splittingsand relative intensities) only in the B ⊥ b plane. The “forbidden” transitions areexpected to be observable also in the B ⊥ a plane. However, in that plane there aretwo magnetically unequivalent sites and the spectra are too complex for a preciseanalysis. Actually, in the present work only the stronger signal brought about bythe more abundant chlorine isotope, 35Cl, is analysed.For the analysis, we use the following spin Hamiltonian:H = βS̃ĝB + ÂS − gnβnĨB + ĨP̂ I. (1)The first term is the electron Zeeman term, in which β is the Bohr magneton andĝ the “g factor” (actually a tensor). The second term represents the hyperfine cou-pling of the unpaired electron with a Cl nucleus, Â being the hyperfine couplingtensor. The second term is the nuclear Zeeman term with the nuclear “g-factor” gnand the nuclear magneton βn. The last term is the quadrupolar coupling. Here P̂stands for the quadrupolar tensor. The energy states and the transitions could becalculated using the electron Zeeman term as a dominating term and the pertur-bation of the remaining terms to the second order. We essentially use the generalprocedure derived by Iwasaki [9].The energy levels defined by the electron state MS and the nuclear state MIare:E(MS ,MI) = gβBMS +K(MS)MI −12(k̃P̂ k[I(I + 1)− 3M2I ]++12gβB{|A1|2MSM2I −A2[S(S + 1)−M2S ]MI +12A3MS [I(I + 1)V −M2I ]}−−12K(MS){ |P1|2(MS)MI [4I(I + 1)− 8M2I − 1]−14|P2|2(MS)MI [2I(I + 1)− 2M2I − 1]}(2)whereg2 = b̃˜̂gĝbK̂(MS) = (ÂĜ/g)MS − gnβnBE,640 FIZIKA A 4 (1995) 3, 637–646krilov et al.: fine phenomena in the epr spectra . . .K2(MS) = b̃ˆ̃K(MS)K̃(MS)b|P1|2(MS) = k̃(MS)P̂2k(MS)− (k̃(MS)P̂ k(MS))2,|P2|2(MS) = 2TrP̂2+ (k̃(MS)P̂ k(MS))2 − 4(k̃(MS)P̂2k(MS))Thus, the energy for any spin state can be expressed in terms of the ĝ Â and P̂tensors and gnβnB as a function of the external magnetic field in direction b withrespect to the crystal coordinate axes.The fourth term at right in Eq. (2) is neglected, because its contribution is lessthan 0.01 mT. That could not be measured. The nuclear Zeeman term is also small(about 0.05 mT), but it is not neglected because it introduces the asymmetry inthe second-order spectra that could be observed.Fig. 2. Angular variation of the positions of the EPR lines for the three referenceplanes. The dots represent the experimental points and the curves are theoreticallyreproduced within the first-order approximation. The ordinate axis expresses thedeviation of the line positions from a standard position at g = 2.0036. The rect-angle in the middle panel indicates the region most sensitive to the quadrupolarinteractions.The first-order or “allowed” transitions are defined by ∆MS = ±1 and ∆MI =0, giving rise to the quartet pattern with almost equidistant lines for most crystalorientations. In certain orientations the separation between the lines differs signif-icantly. Figure 2 shows the observed angular variation of the quartet lines in thethree mutually perpendicular planes. The full lines in the figure represent the pre-dicted behaviour within the first-order treatment, neglecting the quadrupolar termFIZIKA A 4 (1995) 3, 637–646 641krilov et al.: fine phenomena in the epr spectra . . .and the nuclear Zeeman term. The fit is good, except for the crystal orientationswhere the hyperfine splittings are small. We made a special effort to measure pre-cisely the line positions in the B ⊥ b plane, close to B||c, the region marked bythe rectangle in the middle panel in Fig. 2. The data for that region are separatelyshown in Fig. 3. The irregularities, not predicted in the first-order treatment, arenicely seen. Moreover, the “forbidden” transitions, indicated by the triangles foreach crystal orientation, are present. The “forbidden” transitions are defined by∆MS = ±1, ∆MI = ±1. The intensities of these lines vary significantly whencrystal orientation in the magnetic field is changed.Fig. 3. Angular variation of the positions of the EPR lines in a selected range oforientations in the B ⊥ b plane. The circles indicate the position of the first-ordertransitions and the triangles the measured second-order lines. The full curves showthe angular variation theoretically reproduced with the data in Table 1. The sidepanel shows the relative intensities of the “forbidden” transitions, I/I0 as a functionof the crystal orientation.Probability of the transition between the |MS ,MI(MS) > and |M′S,M ′I(M ′S) >states is:I ∼ | < MS ,MI(MS)|βB1S̃ĝb1|M′S ,M′I(M′S) > |2= β2B21|G|2| < MS |SI |M′S > |2| < MI(MS)|M′I(M′S) > |2 (3)where|G|2 = (b̃1 ˆ̃gĝb1)−1g2(b̃1 ˆ̃gĝb1)2642 FIZIKA A 4 (1995) 3, 637–646krilov et al.: fine phenomena in the epr spectra . . .and 2 B1 and b1 are the strength and the direction of the oscillating magnetic field,respectively.The intensity of the spin “forbidden” transitions, arising from the quadrupoleinteraction, relative to the first-order (“allowed”) transitions, |MS , k ± 1/2 >←→|Ms − 1, k ∓ 1/2 > are:II0=4k2[(I + 12)2− k2]|P1|2[2KMS(MS − 1)]2. (4)Here k takes on the values (I−1/2), (I−3/2),...,−(I−1/2). For simplicity, for thecalculations of intensity, Eq. (4), the nuclear Zeeman term is neglected.There are also ∆MS = ±1, ∆MI = ±2 transitions, not observed in our experi-ments.TABLE 1.Summary of the EPR tensor elements. The hyperfine and quadrupole couplingsare expressed in mT.Tensor Principal Eigenvectorsvalues < a∗ > < b > < c >2.0021 0.975 0.158 –0.155g 2.0174 0.030 0.790 –0.6122.0416 0.216 0.592 0.7737.25 0.999 0.049 –0.004A(35Cl) 0.93 –0.047 0.916 –0.3990.96 –0.015 0.398 0.9170.180 0.999 0.049 –0.004P –0.090 –0.047 0.916 –0.399–0.090 –0.015 0.398 0.917The procedure for the determination of the three tensors, ĝ, Â and P̂ , is asfollows. From Eq. (2), one can see that the g-factor for each crystal orientation canbe deduced directly from the line positions, independently of Â or P̂ . Then theprincipal elements of ĝ are determined in a standard way. In contrast, Â and P̂have to be deduced simultaneously by fitting the theoretically derived curve to theexperimental data. We rather adopted the following, simplified procedure. First,we deduce the Â tensor in the first-order, already standard procedure. Then theP̂ tensor (smaller than Â) is introduced as a trial tensor and Â modified to fitbest the experimental points for the crystal orientations shown in Fig. 3. In thatFIZIKA A 4 (1995) 3, 637–646 643krilov et al.: fine phenomena in the epr spectra . . .procedure, we used the fact that in most cases the Â and P̂ tensors are coaxialand P̂ is axially symmetric [10,11]. The fit was very sensitive to the choice of theparameters. The best fit was attained for the tensor parameters listed in Table1. The values for ĝ and Â are essentially the same as earlier determined [4] in asimpler procedure. The best estimate of the values for P̂ are 0.180, –0.090 and0.090 mT (±0.005 mT), the tensor being coaxial with the Â tensor. With theseparameters, the second-order transitions are predicted theoretically and comparedwith the observed transitions (Fig. 3). The fit is very good.By knowing the three tensors, the relative intensity of the second-order tran-sitions, induced by the quadrupole term, could be calculated from Eq. (3). Thecalculated relative intensities for the indicated section of the B ⊥ b plane are shownon the side panel in Fig. 3. As seen, the “forbidden” transitions are expected to beobserved in the direction close to B||c, but not exactly in that direction. Note thelack of the extra lines for B||c, tracing a) in Fig. 1.4. DiscussionIt has been shown [4-6] that the hole-like paramagnetic centre in hydrochlori-nated purine or pyrimidine systems, with the thioanalogs of the bases as impuritiesis associated with the unpaired electron shared by the S atom of the impurity and aneighbouring Cl. The proposed structure of the centre is shown in Fig. 4. The cou-pling nuclei (35Cl and 37Cl) have spin I > 1/2 and hence an electron quadrupole,that interacts with the unpaired electron via the nuclear spin. The quadrupolarterm introduces very little perturbation in the normal EPR transitions. The effectis observed only occasionally. More important effect of the quadrupolar term inthe spin Hamiltonian is the induction of the “forbidden” transitions. Very precisemeasurements give the information on both the shifts of the regular, “allowed”transitions and the appearance of the “forbidden” transitions. We have shown thatthe quadrupolar term is small in comparison to the hyperfine coupling term. Thevalues deduced here (0.180, –0.090 and –0.090 mT) are much smaller than the linewidth. However, from the “forbidden” lines, and even the shift of the “regular”lines, P̂ could be evaluated (See Fig. 3). Inclusion of the nuclear Zeeman term isresponsible for the slight asymmetry of the EPR spectra, in particular the differentspacing between the two low-field and the two high-field “forbidden” lines.The deduced values for P̂ are by about a factor of two smaller than the valuesanticipated from the analysis of the powder spectra [4]. It is quite clear that thepresent, single crystal data are more reliable. The present values are also smallerthan the quadrupolar term derived in a related radical with a Cl atom covalentlybound to the carbon atom with the unpaired electron [12]. That difference is notsurprising since the quadrupolar term, H = ÎP̂ I, contains the quadrupolar cou-pling constant eQ∂2V/∂x2 that depends strongly on the gradient of the electricfield, which is obviously different in the two cases.644 FIZIKA A 4 (1995) 3, 637–646krilov et al.: fine phenomena in the epr spectra . . .Fig. 4. The model of the hole-like paramagnetic centre.The present analysis shows that, although in principle it is possible to deter-mine the quadrupolar term arising from the chlorine nuclei from the shift of thefirst-order transition lines, the procedure is much more reliable if the “forbidden”transitions are utilized. The shift of the first-order lines is small, sometimes almostnot detected, particularly if the lines are overlapped by other resonances. On theother hand, the second-order lines are often well expressed and their mere appear-ance gives sufficient information for the P̂ tensor determination. The best situationis if both types of information are used, as in the present study.References1) K. Sanković, D. Krilov and J. N. Herak, J. Molec. Structure 190 (1988) 177;2) J. N. Herak and J. Hüttermann, Int. J. Radiat. Biol. 60 (1991) 423;3) K. Sanković, D. Krilov and J. N. Herak, Radiat. Res. 128 (1991) 119;4) J. N. Herak, K. Sanković and J. Hüttermann, Int. J. Radiat. Biol. 66 (1994) 3;5) J. N. Herak, K. Sanković, D. Krilov and J. Hüttermann, Radiat. Res, (in press);6) J. N. Herak, K. Sanković, M. Jakšić and J. Hüttermann, Radiat. Res. (in press);7) S. A. E. Johansson and J. L. Campbell, PIXE - a Novel Technique for ElementalAnalysis, John Wiley&Sons, Chichester, U.K., 1988;8) N. S. Mandel, Acta Cryst. B33 (1977) 1079;9) M. Iwasaki, J. Magn. Resonance 16 (1974) 417;10) A. Abragam and B. Bleaney, in Electron Paramagnetic Resonance of Transition Ions,Ch. 2, Clarendon Press, Oxford, 1970;11) W. Gordy, Theory and Application of Electron Spin Resonance, John Wiley&Sons,New York, 1980;12) J. Hüttermann, W. A. Bernhard, E. Haindl and G. Schmidt, Molec. Phys. 32 (1976)1111.FIZIKA A 4 (1995) 3, 637–646 645krilov et al.: fine phenomena in the epr spectra . . .SLABO IZRAŽENE POJAVE U EPR SPEKTRU PARAMAGNETIČNOGCENTRA SLIČNOG ŠUPLJINI U CITOZIN HIDROKLORIDUProučavane su slabo izražene, neredovite pojave u EPR spektrima paramag-netičnog centra sličnog šupljini u ozračenim kristalima citozin hidroklorida s prim-jesama tiocitozina. Za objašnjenje ponašanja upotrijebljen je spinski Hamiltonijankoji se sastoji od elektronskog Zeemanovog člana, nuklearnog Zeemanovog člana,člana hiperfine i člana kvadrupolne sprege jezgre s elektronom. Pokazano je dakvadrupolni član koji potječe od klora ne samo unosi pomak EPR spektralnih lin-ija, nego uzrokuje i pojavu dodatnih, “zabranjenih” prijelaza. Iz mjerenja slaboizraženih pomaka linija i položaja “zabranjenih” linija izračunat je kvadrupolnitenzor.646 FIZIKA A 4 (1995) 3, 637–646

Slabo izražene pojave u epr spektru paramagnetičnog centra sličnog šupljini u citozin hidrokloridu

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