Marina Brustolon

Analysis of the Spatial Distribution of Free Radicals in Ammonium Tartrate by Pulse EPR Techniques

Abstract Marrale, M., Brai, M., Barbon, A. and Brustolon, M. Analysis of the Spatial Distribution of Free Radicals in Ammonium Tartrate by Pulse EPR Techniques. Radiat. Res. 171, 349–359 (2009). Using pulse electron paramagnetic resonance (EPR) on a series of l(+)-ammonium tartrate (AT) dosimeters exposed to radiations with different linear energy transfer (LET), we assessed the ability of pulse EPR spectroscopy to discriminate the quality of various radiation beams such as 60Co γ-ray photons, protons and thermal neutrons at various doses by analyzing the local radical distributions produced by the different beams. We performed two types of pulse EPR investigations: two-pulse electron spin echo decay obtained by varying the microwave power, and a double electron-electron resonance (DEER) study. Both methods provide information about the dipolar interactions among the free radicals and about their spatial distributions. The first method provided information on the instantaneous diffusion and hence the microscopic concentration of the radicals that is compared with the macroscopic one obtained by CW-EPR. The DEER spectra yielded the distributions of distances between pairs of radicals two to five crystal cells apart produced by the same radiation event, a result reported here for the first time. The inter-radical distributions given by the DEER results have been simulated by modeling the radical distributions according to the details of the matter-radiation interactions for the various beams. The results of both types of pulse experiments are strongly dependent on the radiation quality. This was also observed for samples giving indistinguishable CW-EPR spectral profiles. We conclude that the pulse EPR measurements can be valuable tools for distinguishing the LET of the radiation beams, an important parameter for radiobiological considerations.

E.P.R. and ENDOR investigation of tempone nitroxide radical in a single crystal of tetramethyl-1,3-cyclobutanedione

The E.P.R. and ENDOR spectra of the free nitroxide radical tempone diluted in a single crystal of 2,2,4,4-tetramethylcyclobutanedione (TMCB) have been analysed and the g tensor, nitrogen hyperfine and quadrupole interaction tensors, and methyl protons tensors have been determined. It is found that the principal axes of the g tensor and of the tensors A N and Q N do not fully coincide. The negative sign of the quadrupole interaction tensor element Qzz where Z is along the direction of the nitrogen p orbital agrees with previous measurements on similar systems.

Triplet states of the nonlinear optical chromophore DCM in single crystals of potassium hydrogen phthalate and their relationship to single-molecule dark states

Single-molecule dark states are often attributed to photoexcited triplets with scant evidence of the participation of paramagnetic molecules. The photodynamics of blinking single molecules of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) in crystals of potassium hydrogen phthalate (KAP) were compared with the lifetimes of DCM triplet states, likewise in KAP, whose zero-field splitting (ZFS) tensors were fully characterized by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. Luminescent mixed crystals of KAP were grown from solutions containing 10(-4) -10(-9) M DCM, a model optically nonlinear chromophore. The luminescent dye was localized in the {111} crystalline growth sectors. The photoexcited triplets states of DCM in the heavily dyed (10(-4) M) crystals were analyzed by TR-EPR spectroscopy. The photoexcited singlet states of DCM in lightly dyed crystals (10(-9) M) were analyzed by single-molecule microscopy. Large blue shifts in the absorption and emission spectra of DCM in KAP were interpreted as a consequence of protonation at the dimethylamino nitrogen atom, an assignment supported by calculations of the zero-field splitting (ZFS) tensors of molecules in their triplet states. Experimental ZFS tensors with eigenvalues comparable to those of the computed tensors were determined from the angular dependence of the EPR spectra of DCMH(+) triplets within KAP single crystals with respect to the applied magnetic field. Data from individual growth sectors failed to show magnetically equivalent site occupancies, evidence of the kinetic ordering during growth. The intermittent fluorescence of individual chromophores was analyzed. The distributions of on(off) times were characterized by distributed rates fit to power laws. The lifetime of the triplet states was analyzed from the time decay of the EPR signals between 100 and 165 K. The data were well fit with a single time constant for the signal decay, a result wholly inconsistent with the blinking of single molecules with off times commonly of tens of seconds. Triplet decay was extrapolated to approximately 25 micros at room temperature. Therefore, the assumption that single-molecule dark states originate with triplet excited states is not sustainable for single DCM molecules in KAP.

Interaction between Starburst Dendrimers and SDS micelles studied by continuous-wave and pulsed electron spin resonances

A computer aided analysis of both cw-and pulsed-Electron Paramagnetic Resonance (EPR) spectra of 5doxylstearic acid (5DXSA) as a probe was carried out to compare the aggregation process of Sodium Dodecyl Sulfate (SDS) surfactants in the absence and in the presence of Starburst Dendrimers (SBDs), and to provide information on the interactions between SDS and SBDs. Mobility and polarity parameters were extracted from the cw-EPR analysis whereas the analysis of the Electron Spin Echo Envelope Modulation (ESEEM) signal provided details about the doxyl environment in the SDS micelles. In the absence of SBDs, the formation of SDS micelles was revealed by the decrease in mobility of the probes inserted in the micelles. The high packing of SDS chains in the micelles prevented the water permeability at the doxyl site. In the presence of the dendrimers, the analysis of the EPR spectra suggested the formation of SDS aggregates at the dendrimer surface (cooperative interaction). The larger the size of the dendrimers and the protonation of their surface, the stronger the interactions resulted between the SDS surfactants and the SBD surface. The analysis of the ESEEM pattern indicated that the cooperative interaction of the surfactant with the SBD surface led to a less packed structure of the aggregates. A schematic view was proposed to describe the local structure of the doxyl group and its environment in the absence and in the presence of the dendrimers.

Electron spin-lattice relaxation time and spectral diffusion in γ-irradiated l-alanine

We performed continuous (CW) wave and pulsed ESR experiments to obtain information on the relaxation behavior of the l-alanine radical in an irradiated single crystal. The analysis of the CW saturation behavior gives a relaxation time of 2.8 μs. The echo detected saturation recovery was obtained for a number of different experimental conditions. In any case only a portion of the 120 G wide ESR spectrum can be affected by the microwave (MW) pulses, spectral diffusion is active and a multi-exponential decay is therefore obtained. We measured characteristic spectral diffusion times of 1–10 and 20–50 μs. We found that a long time of about 200 μs can be measured only by using a train of long selective saturating pulses and short detecting pulses. The stimulated echo decay is bi-exponential, and the characteristic times are very short. A variable temperature investigation in the range 200 to 290 K showed that the decay is governed by the spectral diffusion and by the transverse nuclear spin relaxation timeT2n of the methyl protons.

Crystal violet: Study of the photo-fading of an early synthetic dye in aqueous solution and on paper with HPLC-PDA, LC-MS and FORS

The photo-fading of crystal violet (CV), one of the earliest synthetic dyes and an ink component, is examined both in solution and on paper. Aqueous solutions of CV were exposed to UV light (365nm) and samples were taken at constant time intervals and analysed with a High Performance Liquid Chromatography-Photo Diode Array (HPLC-PDA) and Liquid Chromatography-Mass Spectroscopy (LC-MS). Demethylation products were positively identified. Also, deamination probably occurred. The oxidation at the central carbon likely generates Michlers ketone (MK) or its derivatives, but still needs confirmation. To study CV on paper, Whatman paper was immersed in CV and exposed to UV light. Before and after different irradiation periods, reflectance spectra were recorded with Fibre Optic Reflectance Spectrophotometry (FORS). A decrease in CV concentration and a change in aggregation type for CV molecules upon irradiation was observed. Colorimetric L*a*b* values before and during irradiation were also measured. Also, CV was extracted from paper before and after different irradiation periods and analysed with HPLC-PDA. Photo-fading of CV on paper produced the same products as in solution, at least within the first 100 hours of irradiation. Finally, a photo-fading of CV in the presence of MK on Whatman paper was performed. It was demonstrated that MK both accelerates CV degradation and is consumed during the reaction. The degradation pathway identified in this work is suitable for explaining the photo/fading of other dyes belonging to the triarylmethane group.

Endor and eseem study of the radical obtained by γ irradiation of a single crystal of ammonium tartrate

The radical obtained by γ irradiation of a single crystal of ammonium tartrate has been identified by paramagnetic resonance spectroscopies. The proton hyperfine coupling tensors have been obtained, from the angular dependence of the ENDOR transition frequencies. ESEEM spectra of the radical have been also obtained. The intensities of the ESEEM lines are related to the depth of the modulation, which has a dramatic angular dependence.

ESR and ENDOR studies of γ-irradiated single crystals of potassium hydrogen malonate☆

The −OOC-ĊH-COO− radical trapped in a γ-irradiated single crystal of potassium hydrogen (KH) malonate is investigated at room temperature by ESR and ENDOR spectroscopy. The α-proton and 13C hyperfine tensors show that the free radical is oriented in the monoclinic crystal lattice with its Ċ-H bond along the twofold symmetry axis b. ENDOR spectra allow the determination of the superhyperfine interactions of the protons of neighboring molecules and hydrogen-bonded protons. The covalent character of the “very short” hydrogen bond present in the KH malonate crystal structure is discussed. An analysis of the factors affecting the intensity ratios of the two ENDOR transitions of the a proton is performed.

Discrimination of Radiation Quality Through Second Harmonic Out-of- Phase cw-ESR Detection

The ability to discriminate the quality of ionizing radiation is important because the biological effects produced in tissue strongly depends on both absorbed dose and linear energy transfer (LET) of ionizing particles. Here we present an experimental electron spin resonance (ESR) analysis aimed at discriminating the effective LETs of various radiation beams (e.g., 19.3 MeV protons, 60Co photons and thermal neutrons). The measurement of the intensities of the continuous wave spectrometer signal channel first harmonic in-phase and the second harmonic out-of-phase components are used to distinguish the radiation quality. A computational analysis, was carried out to evaluate the dependence of the first harmonic in-phase and second harmonic out-of-phase components on microwave power, modulation amplitude and relaxation times, and highlights that these components could be used to point out differences in the relaxation times. On the basis of this numerical analysis the experimental results are discussed. The methodology described in this study has the potential to provide information on radiation quality.

ENDOR spectra of nitroxide radicals in disordered matrices

An ENDOR study on several nitroxide radicals included in polycrystalline and glassy matrices is reported. A calculation program has been developed to simulate ENDOR spectra of radicals in disordered matrices. The shape of the computed spectra can be changed from crystal-like to powder-like type through the intermediate cases. The principal values of the AH hyperfine tensors have been obtained for tempone, tempol, tempyo and di-t-butyl-nitroxide. The influence of the host matrix on radical conformations is also discussed.