powder changes significantly during the first hours after X irradiation and that the stable state is only reached 80 - 100 hours after irradiation. Desrosiers et al. [119] repeated this study more accurately in 2006 and confirmed the general time-dependent trend. The most plausible explanation are slow radical processes: transformation of unstable radicals into stable radicals or decay of unstable radicals into diamagnetic products. An explanation for the EPR spectral changes in terms of the individual radical components and their time- dependent behaviour can be of value for establishing dosimetric protocols and yield important insights in the underlying radical processes. E.g., the spectra of the immediate precursors of the stable radicals could be isolated. Therefore, we studied the evolution of the EPR and ENDOR spectrum of X-irradiated solid sucrose during the first days after irradiation, with the primary aim of understanding the nature of the observed changes (radical transformation or decay, the number of species involved, . . . ).
5.3.2.1 Experimental procedures
The crystals were X irradiated at RT for 5 minutes (estimated total dose of 5 kGy). After the irradiation, the samples were put in a Q-band EPR/ENDOR sample tube for the EMR measurements. The time-dependence of the EPR spectra was recorded at RT. For these experiments a modulation amplitude of 0.1 mT and a total recording time for each spectrum of 41 s were used and the magnetic field was measured using a Bruker ER035 NMR Gauss meter and calibrated with a standard DPPH field marker (g = 2.0036). All spectra (of this
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time series) were normalised to a microwave frequency of 33.9935 GHz. The field-frequency ENDOR (FF-ENDOR) measurements (see Section 2.2.6) were performed at 80 K and for the measurements ’immediately after ir- radiation’ the cavity was precooled to 80 K to minimise further radical transformations after sample insertion. The total time between the start of the irradiation and the stabilisation of the sample temperature at 80 K is estimated to be about 10 minutes. The spectra were normalised to a microwave frequency of 33.9700 GHz.
The samples were always mounted in the tube with the long axis (the rotation axis) approximately parallel to <b>. The orientation of the magnetic field in the plane perpendicular to <b> was determined visually, on the basis of the well defined geometrical features of the single crystals, and checked more accurately afterwards using the well known stable radical ENDOR signals. The actual orientation may still deviate by about 5◦from the reported one. 5.3.2.2 Results and discussion
Immediately after irradiation at RT, following observations can be made • when the sample is kept at RT, the EPR spectrum continuously changes.
After approximately 4 hours the spectrum becomes essentially stable and during the subsequent days only minor changes were observed.
• when the sample is kept at 80 K, the spectrum is stable for at least 6 hours. This indicates that the radical processes observed at RT do not proceed at 80 K – the radicals are trapped.
• the measuring temperature (80 K or RT) has no significant influence on the shape of the EPR spectrum.
In Figure 5.6 FF-ENDOR scans are presented at different stages of the EPR- spectrum evolution for approximately Bk<c>. This is achieved by cooling the sample to 80 K after different time intervals of RT annealing. Note that
• several ENDOR transitions have decreased in intensity after 3 hours and have virtually disappeared after 10 hours of RT annealing.
• no new ENDOR transitions emerge upon RT annealing.
• the ENDOR transitions due to the stable radicals T1, T2 and T3 do not gain noticeably in intensity during 10 hours of RT annealing.
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Figure 5.6: FF-ENDOR spectra of sucrose single crystals with B approximately parallel to <c> after (a) 10 minutes, (b) 3 hours and (c) 10 hours of RT annealing. T1, T2 and T3 are the stable radicals characterised and identified in Papers I-III. U1, U2 and the features indicated with dashed arrows belong to six different semistable species. The spectra were normalised to a microwave frequency of 33.9700 GHz.
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Figure 5.7: Top: first-derivative Q-band EPR spectra (B approximately parallel to <c>) recorded on sucrose single crystals at RT, starting from 10 minutes after irradiation (blue) and ending 6 hours after irradiation (red). Bottom: field integration of the spectrum obtained by substracting the red from the blue spectrum (black) and the EIE spectrum of U1, reconstructed from the FF-ENDOR spectrum of Figure 5.6 (green).
These observations suggest that the transformation of the EPR spectrum is the result of the decay of several semistable radicals into diamagnetic species. At least six different semistable radical species can be observed (Figure 5.6a). However, Figure 5.7 indicates that a single species (U1) dominates the EPR spectrum immediately after irradiation and, consequently, that the EPR spectrum changes can be attributed mainly to its decay. In this figure the first derivative EPR spectra are shown in a time series covering approximately the first 6 hours after the start of the irradiation. The transformation corresponds to a gradual fading of a component dominated by a broad-line triplet, which can be attributed to U1. This time series also indicates that the final (stable) components are already present immediately after irradiation, in accordance with the FF-ENDOR-spectrum evolution. The time dependence of the total signal intensity (Figure 5.8) then indicates that the stable radicals account for less than half of the total radical yield immediately after irradiation.
For Bk<c>, U1 exhibits a broad-triplet EIE spectrum arising from two HFC tensors whose ENDOR transitions practically coincide at∼73 MHz (Figures 5.6 and 5.7). One of the other (minor) semistable radical species (U2), whose
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Figure 5.8: Time dependence of the total intensity (double integration) of the first- derivative Q-band EPR spectrum of sucrose single crystals recorded at RT and with B approximately parallel to <c>, during roughly the first 3 hours after irradiation (cf. Figure 5.7).
EI-EPR spectrum is a wider-split triplet and whose ENDOR transitions appear in the 95 - 98 MHz range, may account for some of the weaker EPR signals at the left- and right-hand sides of the spectrum recorded 10 minutes after irradiation. FF-ENDOR spectra were recorded at a number of other magnetic field orientations (all in the plane perpendicular to <b>). Although these did not allow structural identification of the metastable radicals, they indicate that radical U1 has an α and a β proton (the latter with an isotropic HFC value in the range 40 - 50 MHz), whereas U2 has two β protons with large isotropic HFC values (probably in the range 85 - 95 MHz).
5.3.2.3 Conclusion
The EPR spectrum of RT irradiated sucrose single crystals undergoes drastic changes during the first 3-4 hours after irradiation, which can to a large extent be attributed to the decay of a metastable radical species (U1) into a diamagnetic species. Structural characterisation of U1 would be interesting from a fundamental point of view. There is, however, no (significant) conversion of this metastable radical species into stable radicals so that
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such knowledge would not yield information on the processes of glycosidic bond cleavage and carbonyl group formation, which characterize the stable radicals (T1, T2 and T3). If we are to obtain information on those formation mechanisms, we must therefore perform EMR measurements after in-situ irradiation at temperatures below RT to stabilise the precursors. We have carried out such measurements at 10 K after X irradiation at 10 K in the EPR lab in Oslo. The results of this study are discussed in the next section.
Furthermore, FF-ENDOR spectroscopy proofs to be a very powerful tool for mapping spectral changes (and, consequently, radical processes) of multicom- posite EPR spectra.
5.3.3 Radicals present after in-situ X-ray irradiation at 10 K