Electron Paramagnetic Resonance Volume 22

A Review of the Literature Published Between 2008 and 2009

By B. C. Gilbert, V. Chechik, D. M. Murphy

The Royal Society of Chemistry

Copyright © 2011 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84755-061-3

Contents

Preface Victor Chechik, Bruce Gilbert and Damien Murphy, v,
Recent developments and applications of the coupled EPR/Spin trapping technique (EPR/ST) Olivier Ouari, Micaël Hardy, Hakim Karoui and Paul Tordo, 1,
EPR investigations of organic non-covalent assemblies with spin labels and spin probes Marco Lucarini and Elisabetta Mezzina, 41,
Spin labels and spin probes for measurements of local pH and electrostatics by EPR Maxim A. Voinov and Alex I. Smirnov, 71,
High-field EPR of bioorganic radicals Stefan Stoll, 107,
Dynamic nuclear polarization in liquids M. Bennati, I. Tkach and M.-T. Türke, 155,

CHAPTER 1

Recent developments and applications of the coupled EPR/Spin trapping technique (EPR/ST)

Olivier Ouari, Micaël Hardy, Hakim Karoui and Paul Tordo

DOI: 10.1039/9781849730877-00001

1 Introduction

The trapping of a short-lived free radical with a diamagnetic spin trap to generate a persistent spin adduct which could be characterized by its EPR spectrum constitutes the well known spin trapping technique, hereafter abbreviated EPR/ST (Scheme 1).

EPR/ST was introduced in the late 1960s and since then it has been widely used and its advantages and drawbacks largely debated. In spite of the enormous progress made in four decades, EPR/ST is still faced with limitations particularly for the investigation of in vivo free radical processes. During the last five years, about 2 000 papers appeared containing references to the concept spin trapping. This important literature illustrates the wide scope of applications of EPR/ST and the continuing efforts to improve its efficiency and reliability. Some examples illustrating the range of applications of EPR/ST are described hereafter. Within the limited pages of this chapter the list could not be exhaustive, then, our goal was to give the reader the highlights on the considerable potential of the method.

2 New spin traps

Efforts continue to be devoted to the development of new spin traps especially suited to characterize free radicals involved in biological processes. A variety of substituents have been introduced around the nitronyl function of linear or cyclic nitrones to monitor their spin trapping properties, and in the last five years the synthesis and the use in EPR/ST of around hundred new nitrone spin traps (Table 1) have been described. The structures of the most popular spin traps used today and mentioned herein are shown in Scheme 2.

2.1 Influence of nitrone substituents on the spin trapping properties

A series of linear phosphorylated nitrones (50, 65–69) were synthesized and the half life time (t1/2) of their superoxide adducts was shown to range from 7 to 9 min., thus confirming that the introduction of an electron-withdrawing group on the quaternary carbon bound to the nitronyl function results in a significant improvement of the spin adduct lifetime. However, due to the limitations of linear nitrones to allow the identification of the trapped radicals the development of new spin traps has mainly concerned pyrroline N-oxide derivatives.

Various substituents have been introduced on the ring of pyrroline N-oxides to examine their influence on the spin trapping properties, especially concerning O2•- radicals in buffers. Stolze et al. synthesized a series of AMPO (Scheme 2) spin traps (1–4). The EPR spectra obtained during the trapping of O2•- correspond to the superimposition of the signals of many species, the estimated t1/2 for the superoxide adducts ranged from 10 to 20 min.

Han et al. synthesized the CPCOMPO (20), a spirolactonyl derivative of EMPO. A rate constant value of 60 M-1s-1 was measured for the trapping of superoxide, and the resulting spin adduct exhibited a t1/2 of 2.4 min.

When a substituent is introduced on the C4 of DEPMPO, in a cis position with the phosphoryl group, the half life time of the corresponding superoxide spin adduct is not significantly affected. Furthermore, the EPR pattern is simplified and the trapping reaction is almost stereospecific. Thus, NHS-DEPMPO (23), a DEPMPO analogue bearing a N-hydroxysuccinimide (NHS) active ester group on C4 was prepared. NHSDEPMPO is a very versatile building block which allows facile and straightforward synthesis of a large variety of bifunctional spin traps (22-26). Depending on the introduced substituent, the half life times of the superoxide adducts of these bifunctional spin traps were evaluated in between 21 and 40 min.. Their ability to trap oxygen-, sulfur- and carboncentered radicals was also investigated.

Other DEPMPO analogues with different phosphoryl groups on C5, 27 (CYPMPO) and 28 (DPPMPO), were prepared and tested. The spin trapping properties of CYPMPO (27) and DPPMPO (28) were compared to those of DEPMPO. Concerning the superoxide adducts, t1/2 was 15 min. for CYPMPO-OOH and 8 min. for DPPMPO-OOH. DPPMPO was used to detect superoxide radicals in activated neutrophils.

2.2 Use of cyclodextrins in EPR/ST

The ability of cyclodextrins to form inclusion complexes by noncovalent bonding with a variety of guests has become an exciting field of research. When β-cyclodextrins (β-CD) are used to encapsulate superoxide adducts of PBN, DMPO and DEPMPO, a seven-fold enhancement in adduct stability and a partial protection against glutathione peroxidase- and ascorbate anion-induced reduction was reported by Karoui et al.

Spulber et al. reported the use of cyclodextrins to encapsulate oxygenand carbon-centred radical adducts formed from DMPO, PBN and 2-methyl-2-nitroso-propane (MNP). They showed that the presence of β-cyclodextrin resulted in a significant increase (factor 23) of the lifetime of DMPO-OH and PBN-OH spin adducts.

Bardelang et al. have studied the association of a series of EMPO analogues (6–12) bearing alkyl groups which modulate the affinity of the nitrone moiety for the β-CD cavity. The influence of the association constant on the trapping properties was evaluated as well as the supramolecular protection of the superoxide adducts towards reduction.

Sulfur trioxide radical anion, SO3•-, was trapped with DEPMPO, DPPMPO and CYMPO in the presence of glucosylated β-CD (Gβ-CD). The influence of inclusion of the traps and spin adducts on the kinetics of radical trappings and spin adduct decays was investigated.

The first grafting of a nitrone spin trap with a β-cyclodextrin was performed by Bardelang et al. who prepared Me2CD-PBN (40) and Me3CDPBN (41). NMR studies showed that the nitrone moieties are included in the cyclodextrin cavity. Nevertheless, the formation of self-inclusion complexes does not prevent the spin trapping. The half life time of the superoxide spin adducts were increased although they remain modest due to the very short half-lifetime of PBN-OOH. Polovyanenko et al. used 40 and 41 to trap glutathiyl radicals (GS•), t1/2 for 40-SG and 41-SG increases by a factor of 6.8 and 5.5 respectively, compared to that of the PBN-SG adduct.

Pyrroline N-oxides covalently bound to β-CD were also prepared (17–19, 26). With CD-NMPO (17)31 and CD-DEPMPO (26),19 both the rate of trapping of superoxide and the t1/2 of the corresponding spin adducts were increased. Moreover, partial protection of the CD-DEPMPO-OOH adduct against bioreductant agents was observed even in blood samples. The lipophilic nitrones 18 and 19 were prepared by Han et al., and the trapping of superoxide was investigated in DMSO/water solutions.

2.3 Vectorized spin traps

In mitochondria, leakage of electrons from the respiratory chain (ETC) is an important side reaction generating superoxide radical (2 to 5% of the total amount of breathed oxygen). In healthy cells the concentration of superoxide is controlled by an appropriate pool of antioxidants, however, during mitochondria dysfunction, superoxide production may increase dramatically and worsen the cell disorders. It is now well established, that chemical probes bearing a triphenylphosphonium group can be accumulated into the mitochondrial compartment. Thus, to improve the detection of Reactive Oxygen Species (ROS) within mitochondria, various mitochondria-targeted spin traps bearing a triphenylphosphonium or a pyridinium group were synthesized (5, 25, 29, 42, 43, 45).

Mito-DEPMPO (25)18 allowed for the first time the detection of superoxide radicals generated from isolated and intact mitochondria using EPR/ ST (Scheme 3).

Mito-Spin (29) was shown to accumulate within mitochondria and its ability to reduce the concentration of oxidizing species was established. However, due to its facile oxidation to nitroxide MitoSpinox (Scheme 4), Mito-Spin is useless as spin trap to distinguish between different radicals in mitochondria.

Lipid peroxidation plays a pivotal role in several diseases associated with oxidative stress. To study the implication of ROS in lipid peroxidation processes, different EMPO derivatives (13–16, 21) and PBN derivatives (46–49, 52–64) that accumulate in lipophilic compartments were developed.

Gamliel et al. synthesized a large series of molecules (13–16, 52–64) and determined by 13C NMR their localisation within liposomal bilayers. Then, the ability of various radicals, generated by a Fenton reaction, to penetrate the lipid bilayer was determined by EPR/ST.

Hay et al. designed a series of PBN (46–49) to trap radicals at a pre- determined depth within biological membranes. Large unilamellar vesicles (LUV) were used as biological membrane models; after incorporation of the traps into the membrane, lipidyl radicals were generated by reduction of t-BuOOH by a membrane permeable CuI complex.

Durand et al. prepared an AMPO analogue (FAMPO (21)) bearing a fluorinated amphiphilic carrier conjugates. The spin trapping properties were explored as well as the cytoprotective properties against hydrogen peroxide, HNE and SIN-1 (3-morpholinosynonimine hydrochloride) in bovine aortic endothelial cells.

2.4 Miscellaneous spin traps

A series of heteroarylnitrones (83–88) designed to combine neuroprotective as well as spin trapping properties was developed. These heteroarynitrones protect cells from death induced by exposure to hydrogen peroxide. The spin trapping of oxygen-, carbon- and sulfur- centered radicals with these nitrones was performed.

N-Aryl-ketonitrone PBN like spin traps (76–82) were synthesized; their spin trapping properties were found to be limited to the trapping of carbon- and alkoxy-centered radicals.

The development of Hydrazyl PBNs (70–75) that contain in the same molecule a stable hydrazyl radical moiety and a PBN like moiety was described by Ionita. These molecules were used as conventional spin traps of short-lived radicals, particularly hydroxyl radicals, and they were also used to simultaneously generate and trap dPPh2 radicals (Scheme 5).

A dual sensor spin trap (89) was prepared by Caldwell et al. to detect and distinguish iron (III) ions from hydroxyl and methyl radicals. Typically, iron (III) reacts with the phenol unit inducing opening of the cyclopropane ring and cyclisation to yield a stable nitroxide (Scheme 6).

Benzazepine nitrones (30–39) were synthesized; they were evaluated as protectants against oxidative stress induced in rat brain mitochondria by 6-hydroxydopamine, a neurotoxin producing experimental model of Parkinson’s disease. The inhibition of hydroxyl radicals, lipid peroxidation and protein carbonylation were evaluated, and all the compounds tested were more efficient than PBN. No spin trapping experiments using these nitrones were reported.

3 Applications of EPR/ST in biological systems

In the following paragraph, we mention a few recent papers using EPR/ST to characterize free radical species such as O2•- and nitric oxide (•NO) involved in physiological processes. The characterization of these species in cigarette smoke will be also emphasized.

3.1 EPR/ST of superoxide anion radical

O2•- is produced by one electron reduction of molecular oxygen during mitochondrial respiration. It constitutes the main source of various reactive oxygen species in vivo, like peroxynitrite (ONOO-), hydrogen peroxide (H2O2) and hydroxyl radical (HOd). Since the early years of EPR/ST development, it has been a challenge to detect superoxide spin adduct particularly in biological systems. Numerous spin trapping agents have been developed and the most recent reported nitrones devoted to superoxide detection are mentioned in Table 1.

Shi et al. evaluated the abilities of several nitrones to trap cell-generated superoxide induced by 1,6-benzo[a]pyrene quinone in a human epithelial cell line. Considering the superoxide spin adduct stability, among the different nitrones they used, DEPMPO and BMPO appeared as the best candidates.

During EPR/ST experiments in aqueous media, using DMPO as spin trap, spontaneous conversion of the superoxide spin adduct to the hydroxyl spin adduct is observed. In biological systems, this conversion can be mediated by endogenous reducing agent or catalyzed by glutathione peroxidase using glutathione. By using DEPMPO as spin trap, this spontaneous conversion is hardly observed when a low flux of superoxide is used.

Mojovic et al. showed that the conversion of DEPMPO-OOH to DEPMPO-OH depends on the oxygen concentration and they claimed that the conversion mechanism is independent on hypoxanthine (HX) and xanthine oxidase (XO) concentrations. However, these results must be considered with caution because, during the trapping of superoxide with DEPMPO, Tordo et al. showed that increasing XO concentration from 0.04 to 0.4 U mL-1 increased dramatically the formation of DEPMPO-OH as observed on the ESR signals (Fig 1). This observation suggests that O2•- should play a significant role in the conversion of DEPMPO-OOH to DEPMPO.

Nitric Oxide Synthases (NOS) are the enzymes responsible for nitric oxide (•NO) production using L-arginine as substrate. It has been shown that tetrahydrobiopterin (BH4) is a cofactor regulating NO production, and BH4 depletion stimulates endothelial NOS (eNOS) superoxide release causing deficient NO production. Then, O2•- released in the endothelium is thought to be responsible for oxidative stress situations that favour atherosclerosis and hypertension. Druhan et al. studied the effect of several arginine derivatives on O2•- production from eNOS under conditions of BH4 depletion. By trapping superoxide in the presence of L-arginine and endogeneous inhibitors such as asymmetric dimethylarginine and NG-monomethyl-L-arginine, more than 100% increase of eNOS-derived O2•- was evaluated.

Hardy et al. reported the synthesis of a new efficient nitrone-spin trap (Mito-DEPMPO) and the characterization of Mito-DEPMPOO-OH the corresponding superoxide spin adduct. Mito-DEPMPO-OOH was shown to be 2 to 2.5 times more persistent than DEPMPO-OOH in buffer solutions at physiological pH. Using this new nitrone, Hardy et al. detected MitoDEPMPO-OOH spin adduct obtained by trapping O2•- formed from isolated and intact mitochondria. This result constitutes the first EPR/ST characterization of mitochondrial superoxide.

It has been suggested that free radicals generated during the metabolism of acetaldehyde are responsible for initiating alcohol-induced liver injury and furthermore carcinogenic mutations and DNA damage leading to breast cancer. Aldehyde Oxidase (AO) is the major cytosolic enzyme responsible for the metabolism of endogenous aldehydes leading to the production of the corresponding carboxylic acids and reactive oxygen species such as O2•- and H2O2. Using EPR/ST with DMPO as spin trap, Kundu et al. showed that the reaction of AO with 4(dimethylamino)cinnamaldehyde (p-DMAC) in the presence of oxygen produces significant amount of O2•- and H2O2.

(Continues…)Excerpted from Electron Paramagnetic Resonance Volume 22 by B. C. Gilbert, V. Chechik, D. M. Murphy. Copyright © 2011 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

Electron Paramagnetic Resonance Volume 20

By B C Gilbert, M J Davies, D M Murphy

The Royal Society of Chemistry

Copyright © 2007 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-325-5

Contents

ESR Imaging Beyond Phantoms: Application to Polymer Degradation Shulamith Schlick and Mikhail V. Motyakin, 1,
Advances in Spin Trapping J.-L. Clément and P. Tordo, 29,
Site-Directed Spin-Labelling (SDSL) Applications in Biological Systems Jimmy B. Feix and Candice S. Klug, 50,
Quantum Chemical Approaches to Spin-Hamiltonian Parameters Frank Neese, 73,
Getting an Inside View of Nanomaterials with Spin Labels and Spin Probes Victor Chechik and Agneta Caragheorgheopo, 96,
EPR, ENDOR and EPR Imaging of Defects in Diamond M.E. Newton, 131,
EPR of Exchange Coupled Oligomers David Collison and Eric J.L. McInnes, 157,
Biological Free Radicals and Biomedical Applications of EPR Spectroscopy Simon K. Jackson, John T. Hancock and Philip E. James, 192,
Progress in High-Field EPR of Inorganic Materials Peter C. Riedi, 245,

CHAPTER 1

ESR Imaging Beyond Phantoms: Application to Polymer Degradation

BY SHULAMITH SCHLICK AND MIKHAIL V. MOTYAKIN

1 Introduction and Motivation

1.1 Polymer Degradation. – Polymers undergo degradation when exposed to heat, mechanical stress, and ionizing or UV irradiation in the presence of oxygen, due to the formation of reactive intermediates such as free radicals R· and ROO·, and hydroperoxides ROOH. Exposure to environmental factors leads to profound changes in polymer properties, on both molecular and macroscopic levels. The chemical structure is modified due, for example, to chain scission and cross-linking, resulting in changes of the elastic properties and of the degree of crystallinity. The chemistry of degradation is complicated because even small amounts of chromophores, free radicals, and metallic residues from polymerization reactions can introduce additional reaction pathways that usually enhance the rate of degradation. Polymer degradation is equivalent to corrosion in metals, a fundamental problem with important practical ramifications.

Electron spin resonance (ESR) methods have been used extensively for detecting and identifying the radicals formed, clarifying the degradation mechanism, and simulating the variation of the ESR spectra with temperature. Simulations of ESR line shapes for specific models of dynamics have been developed for the study of oxidative degradation of polymers due to ionizing radiation. Irradiation in vacuo has enabled the study of the type and mobility of alkyl radicals R· derived from the polymer. These studies were initially performed on polytetrafluoroethylene (Teflon) and other perfluorinated polymers, because perfluoroalkyl radicals can be stabilized even at ambient temperature; in these polymers mid-chain and end-chain alkyl radicals have been detected. Admission of oxygen led to the formation of the corresponding peroxy radicals, ROO·. The motional mechanism of the peroxy radicals was deduced by simulation of the temperature dependence of the spectra; in this way a correlation between dynamics and reactivity has been established. This approach has also been extended to protiated polymers, for instance polyethylene and polypropylene.

Direct ESR and spin-trapping experiments have identified oxygen radicals as well as membrane-derived fragments in Nafion, a perfluorinated ionomer used as a proton exchange membrane (PEM) in fuel cells, exposed to oxygen radicals produced in the Fenton reaction or by UV irradiation of hydrogen peroxide. Identification of the radicals was possible by variation of sample preparation methods, temperature used for spectra acquisition, and annealing conditions.

1.2 Polymer Stabilization by Hindered Amines. – Recent research efforts on the effects of radiation and thermal treatment on polymeric materials have two main goals: understanding the degradation mechanism and predicting polymer lifetimes, and development of protective additives. Hindered amine stabilizers (HAS) rank among the most important recent developments for stabilization of polymeric materials. Nitroxides and amino ethers are major products of reactions involving HAS. The HAS-derived nitroxides (HAS-NO) are thermally stable, but can scavenge free radicals to yield diamagnetic species; the amino ethers can regenerate the original nitroxide, thus resulting in an efficient protective effect. Some of these events are shown in Figure 1, where >NH denotes the amine, >NO· the nitroxide, and R· ROO· and ROOH the reactive intermediates derived from polymer chains exposed to oxygen and irradiation or heat. The intermediate radical N-peroxy radical >NOO· has been detected by ESR. The most stable is >NO·. Though stable, however, HAS-NO can react with alkyl radicals derived from polymeric precursors, and stabilize the polymer. In spite of numerous studies, important information on the degradation steps and kinetics is still incomplete or missing altogether.

1.3 Motivation for ESRI Studies of Polymer Degradation. – The concept of diffusion-limited oxidation (DLO) has greatly contributed to the understanding of the mechanism for polymer degradation: if oxygen diffusion is slow compared to the rate of degradation, as in accelerated degradation in the laboratory, only thin surface layers in contact with air are degraded, while the sample interior is little, if at all, affected; this is the DLO regime.

The presence of oxygen is crucial for oxidative degradation of polymers. In the diffusion limited oxidation regime, the distribution of oxygen in the polymer can be described by equation (1),

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (1)

where the first term on the right describes the rate of oxygen diffusion, and D is the oxygen diffusion coefficient; the second term describes the rate of oxygen consumption due to degradation, assuming a first order reaction, and k is the rate constant for oxygen consumption. For steady state conditions, the rate of oxygen consumption is equal to the oxygen supply by diffusion:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (2)

For the boundary conditions [O2] = [O2] at x = 0 and [partial derivative][O2]/[partial derivative]x = 0 at x = 1, the solution of equation (2) is (l is half sample thickness):

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (3)

Assuming that all oxygen in the polymer reacts and the oxygen-containing products are not lost by diffusion, equation (3) describes the distribution of oxygen-containing products such as hydroxyl, carbonyl, and nitroxide. For large values of x equation (3) can be approximated as

C/C0 = exp [-(k/D)1/2 x] (4)

where C0 and C are the concentrations of oxidation products on the surface and at depth x, respectively.

The parameter a = (D/k)1/2, known in the literature as the degradation depth, shows the depth where most (90%) of oxygen-containing products are located. The degradation profile for two values of a, and a sample depth of 4 mm are shown in Figure 2. For a degradation depth comparable with the sample depth, a = 3.4 mm, the degradation profile is essentially homogeneous; this is the case of low k, low oxygen consumption, and low degradation rates. For a degradation depth smaller than the sample depth, a = 0.4 mm in Figure 2, the profile is heterogeneous; this is the DLO regime, with high k, high oxygen consumption, and high degradation rates.

The DLO concept implies that lifetimes of polymeric materials deduced from the study of average properties of samples involved in accelerated degradation cannot be used to estimate the durability of polymers in normal exposure; profiling methods, which determine the variation of the extent of degradation with sample depth, are needed.

The primary motivation for ESR imaging (ESRI) studies was to develop methods for spatially-resolved degradation: to visualize degradation profiles and variation of degradation processes within sample depth. We have developed 1D and 2D spectra-spatial ESRI for the study of heterophasic systems such as poly(acrylonitrile-butadiene-styrene) (ABS) and heterophasic propylene-ethylene copolymers (HPEC) containing bis(2,2,6,6-tetramethyl-4 -piperidinyl) sebacate (Tinuvin 770) as the stabilizer, and exposed to thermal treatment and UV irradiation. The HAS-NO provided the contrast necessary in the imaging experiments. The major objectives were to examine polymer degradation under different conditions; to assess the effect of rubber phase, polybutadiene in ABS and ethylene-propylene rubber (EPR) in HPEC, on the extent of degradation; and to evaluate the extent of stabilization by HAS. The repeat units in ABS and the formula of Tinuvin 770 are shown in Figure 3.

Recent advances in the ESRI field, for instance the choice of pulsed vs continuous-wave (CW) experiments, combined ESR/NMR imaging, progress in resonators, and applications in various disciplines have been described in detail. The focus of this Chapter is on ESRI experiments and applications that have been used for, and are relevant to, polymeric systems. The experimental details reflect the imaging system in the Detroit laboratory.

This Chapter is organized as follows. In Section 2 we describe ESR spectra in the presence of magnetic field gradients, review applications of ESRI to polymeric systems, and describe crucial experiments that led to the development of ESRI methods in our laboratory. Section 3 describes selected experimental details on sample preparation and treatment, and on the determination of the nitroxide intensity profile (by 1D ESRI) and spectral profile (by 2D spectral-spatial ESRI). The ESR spectra of HAS-NO in the heterophasic polymers are described in Section 4. 1D and 2D ESRI experiments are described in Section 5, which includes results for the ABS and HPEC systems, a comparison of ESRI and FTIR methods, and our experience with the effect of microtoming on crystalline polymers. Conclusions and prospects are presented in Section 6.

2 ESR Imaging and Applications to Polymer Systems

2.1 ESR Spectra in the Presence of Magnetic Field Gradients. – ESR spectroscopy can be transformed into an imaging method, ESRI, by measuring ESR spectra in the presence of magnetic-field gradients. In ESR imaging experiments the microwave power is absorbed by the unpaired electrons located at point x when the resonance condition is fulfilled:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (5)

In equation (5), n is the microwave frequency, Bres is the resonant magnetic field, Gx is the linear magnetic field gradient (in Gauss cm-1) at x, and the other parameters have their usual meaning. As in NMR imaging, the field gradients produce a correspondence between spin location and Bres and allow the encoding of spatial information in the ESR spectra. If the sample consists of two point samples, for example, the distance between the samples along the gradient direction can be deduced if the field gradient is known. In this way it is possible not only to verify the existence of the paramagnetic species in a sample, but also “to tell exactly where the signal came from”. The ESRI methodology is similar to that of NMR imaging (NMRI, or magnetic resonance imaging, MRI).

The challenges in the application of the gradient approach to ESRI are numerous. First, higher gradients are needed compared to NMRI, usually 100-1000 times larger. Second, the ESR spectra are often complex, with multiple lines due to hyperfine interactions and g-value anisotropy; these signals complicate the imaging experiments but in most cases do not add additional information. Third, most systems do not contain stable paramagnetic species on which imaging is based. ESRI experiments are usually performed on paramagnetic transition metal ions, radicals produced by irradiation, or stable nitroxide radicals as dopants; in some experiments triarylmethyl (“trityl”) radicals are used as probes, because their ESR spectra consists of a single narrow line, of width typically ≈ 50 mG in the absence of oxygen.

The feasibility of ESRI was first demonstrated in 1979 by Hoch and Day, who described the distribution of colour centres in natural diamonds. The instrumentation, software, and applications of ESRI have been described in a 1991 monograph and updated in recent reviews. The early efforts described the type and stability of the gradients necessary for specific ESRI experiments and the software necessary for image reconstruction in spatial and spectral dimensions. These studies also investigated the feasibility of ESRI experiments in a variety of “phantom” samples, and discussed and estimated the spatial resolution. Because of the short relaxation times of the electron spins, most ESRI experiments are performed in the CW mode, unlike MRI which is used in the pulsed mode. In vivo studies are usually performed at lower frequencies, typically below 2 GHz, which can accommodate large samples with high water content. Most experiments in materials science are performed at X band, ≈ 9 GHz. Gradients can be applied in the three spatial dimensions, and a spectral dimension can be added by the method of stepped gradients. The widening scope of ESRI studies was highlighted at the 2004 ESR Symposium in Denver, with focus on spatially-resolved degradation and software development for applications to polymers, and in vivo studies at 300 and 700 MHz for the detection of local oxygen profiles and the study of radical involvement in oxidative diseases.

2.2 ESR Imaging of Polymeric Systems. – While most ESRI efforts are directed to biological applications, a small number of studies on polymeric systems have appeared. Information on the spatial distribution of paramagnetic molecules deduced from ESRI experiments has been used for measuring macroscopic translational diffusion. Diffusion coefficients, D, of paramagnetic diffusants can be deduced from an analysis of the time dependence of the concentration profiles along a selected axis of the sample. The determination of D for spin probes in liquid crystals and model membranes, and the effect of polymer polydispersity, have been described in a series of papers by Freed and coworkers. In our laboratory the diffusion coefficients of paramagnetic guests in ion-containing polymers, polymer solutions, cross-linked polymers swollen by solvents and self-assembled polymeric surfactants have been determined by 1D ESRI. These papers represent an effort to extract quantitative information from ESRI experiments. Moreover, in some cases these experiments allow the comparison of macroscopic diffusion coefficients in the presence of a concentration gradient (measured by ESRI) with the microscopic D values (measured by pulsed field-gradient NMR).

Some of these studies have resulted in measurements of diffusion coefficients that can be deduced only by ESRI. An example is the measurement of D at 300 K for nitroxides probes that differ in their hydrophobicity, and were doped in the various phases (micellar, hexagonal, lamellar, and reverse micellar) of the triblock copolymers poly(ethylene oxide)-b -poly(propylene oxide)-b-poly (ethylene oxide), EOm POnEOm, (commercial name Pluronics). The self-assembling is due to the different hydrophobicity of the two blocks, PEO and PPO, in water as solvent. Ionic, neutral and hydrophobic probes select specific sites in the self assembled system, and these sites are reflected in the rate of transport of the probes: in the value of D. The cationic probe 4-(N,N, N-trimethyl)ammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl iodide (CAT 1) and the hydrophobic probe 5DSE, the methyl ester of doxylstearic acid (5 indicates the carbon atom to which the doxyl group is attached) exhibited a contrasting transport behaviour in aqueous solutions of Pluronic L64, EO13PO30EO13. Although the molecular masses M are similar, 340 for CAT1 (213 for the cation) and 414 for 5DSE, the corresponding D values at each polymer content are very different. The ratio DCAT1/D5DSE is 35 in the micellar phase (polymer contents 20 and 35 % w/w), 11 in the hexagonal phase (polymer content 50 % w/w), and 3.1 in the mixed Lα (lamellar) and L2 (reverse micellar) phase (polymer content 80% w/w). The range of D values measured in this study was 1.0 x 10-5 cm2 s-1 – 1.0 x 10-7 cm s-1. These results indicate that ESRI is the method of choice for the determination of diffusion coefficients for guests present in low concentrations and located in various regions of self-assembled systems; this conclusion is relevant for drug delivery systems.

2.3 ESR Imaging of Polymers Stabilized by Hindered Amines. – Degradation processes can be studied by ESRI in polymers containing hindered amine stabilizers (HAS); this approach was originally suggested by Ohno, who presented 2D spectral-spatial ESRI images of radicals in polypropylene (PP) containing two different stabilizers, but no detailed analysis. The method is based on the formation of stable nitroxide radicals derived from HAS, HAS-NO, during UV- or thermal treatment. ESRI based on HAS-NO represents an important step in the development of ESRI beyond phantoms, because the nitroxides are part of the system, and participate in, and reflect, degradation processes.

(Continues…)Excerpted from Electron Paramagnetic Resonance Volume 20 by B C Gilbert, M J Davies, D M Murphy. Copyright © 2007 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.