Electron Spin Resonance of Radiation Produced Free Radicals

Transcription

1 (-lfaw) Electron Spin Resonance of Radiation Produced Free Radicals Michael D. Sevilla Oakland University, Rochester, Ml Downloaded via on January 5, 019 at 1:40:34 (UTC). See for options on how to legitimately share published articles. Electron Spin Resonance (ESR) is the branch of spectroscopy which detects chemical species with unpaired electrons. The vast majority of all stable chemical entities exists with an even number of electrons which are paired. A few non-metal compounds have an odd number of electrons (doublet state) such as NO and NOj or an even number of electrons, two of which are unpaired (triplet state) such as molecular oxygen O. Transition metal ions often have one or more unpaired electrons due to incomplete filling of c/-sub-levels. If ESR spectroscopy were confined to the study of only stable species it would have only a very limited scope. However, virtually any molecule, ion or atom when suitably attacked chemically, photolytically, or by high energy radiation will produce species with unpaired electrons. These species may he radical ions produced by electron addition or loss, neutral radicals formed by scission of bonds, or excited triplet states produced by excitation. The lifetime of these unstable intermediates can vary from picoseconds to hours. In principle, ESR can detect, radicals with lifetimes greater than l()-8 sec; however, most work is done on species with much longer lifetimes. ESR Theory and Experiment In the following, the theory behind ESR will he sketched in simplified form. Those further interested should consult the excellent texts on the subject which are now available (1-5). The free electron or an unpaired electron in a chemical species such as a free radical has an associated magnetic field called the magnetic moment. (jue). The magnetic moment of the electron can be thought of as interacting with an external magnetic field (H) just as a bar magnet would interact except that while a bar magnet may take any orientation to H, and consequently a variety of energies, the energies (E) of the electron in the magnetic field are quantized to values of the allowed spin of the electron, ms ±% When ms V the electron magnetic moment is parallel (aligned) with the external field (H) and when ms +V the magnetic moment is antiparallel to H. If the external magnetic field is defined as aligned along the z axis then the energies depend on the component of the electron spin magnetic moment (nz). Therefore, since E(m,s) gzli and gz g(3ms we hav.e ( 1/) Vigi8JH and (+V) +llgph where g has the value.003 for the free electron, (3 is the Bohr magneton (9.7 X 10 7 erg/gauss), and H is the external magnetic field. The energy difference between the two energy levels is ~ AE 'tew gw Thus, as is shown in Figure 1, the separation between the energy levels is a linear function of magnetic field. At 3300 Gauss this transition energy is about 0.3 cm-1, consequently, an x band radar wave with a wavelength of 3 cm and a frequency of 9.5 GHz is sufficient to cause the transition from the ms '/a state to the ms +V state. Typically ESR spectrometers are designed to hold the frequency of the radar wave constant and scan the magnetic field until the energy of separation of the energy levels (g/jh) equals the energy of the microwave radiation (hv), i.e., hv g(3h (Fig. 1). This condition (hr1 g(3h) is known as resonance and results in the absorption of microwave energy. The method of detection of this resonance involves a small sinusoidal variation in external field. Thus, only the change in absorption is detected, not the 106 Journal of Chemical Education Figure 1. Energy levels, transitions and ESR spectra for the electron and hydrogen atom. (A) Energy levels for an unpaired electron in a magnetic field (Fy, (8) first derivative of the ESR absorption for the electron, (C) the energy level diagram for a hydrogen atom in a magnetic field showing the allowed ESR transitions, (D) first derivative ESR spectrum ot the hydrogen atom. The separation between the lines is 506 Gauss and is called the hyperfine splitting or hyperfine coupling constant. absorption itself. As a consequence, ESR spectrometers usually plot the first derivative of the absorption. The resonance is characterized by only two parameters, its g value and lineshape (see Fig. IB), g values of most C, H, N, O organic molecules are in a narrow range (.00 to.0060). Consequently, limited information about radical identity is obtained from this parameter. Unpaired spins which interact with heavier nuclei such as transition metals can result in ESR spectra which show large deviations from the free electron g value. This is a consequence of the coupling of spin and orbital angular momentum of the species. Radicals which contain nuclei with nuclear spin such as hydrogen 1, carbon 13, nitrogen 14, will produce additional resonances due to interaction of the magnetic moment of the electron (ge) with that of the nucleus (^n)- This interaction can he divided into two components. One is called the Fermi Contact term which depends on the unpaired electron density at the nucleus. The other is the dipolar interaction which is the interaction of the electron and the nucleus while the electron is away from the nucleus. The dipolar term averages to zero when the unpaired electron is in a symmetric s-orbitai as in the hydrogen atom, or the molecule rotates

2 rapidly as in solution. In other cases this term gives rise to the anisotropic hyperfine interaction. This is important in the analysis of spectra in rigid matrices which will be discussed in more detail later. The first term, the Fermi Contact term, does not average to zero and gives rise to what is usually called the isotropic hyperfine coupling constant (hfc), a.. The hfc is then a measure of the interaction of the unpaired electron with the magnetic nucleus when the electron is at the nucleus. Including the hfc, the energy levels for a radical with a single magnetic nucleus are given by E msg$h + msmia where ms represents the electron spin quantum numbers and mi represents the possible nuclear spin quantum numbers. For example, mi ± V for a spin V nucleus such as hydrogen and mi -1, 0, +1 for a spin 1 nucleus such as nitrogen. With ms ±V and mi ±V as in the case of the hydrogen atom, four levels result as shown in Figure 1C. Not all transitions between the energy levels are allowed. The E.SR selection rules allow for changes in electron spin quantum numbers but do not allow for changes in nuclear spin quantum numbers, that is, Ams 1 but Ami 0. Thus, only two transitions can occur as indicated in the figure. The hyperfine coupling constant is the difference in energy between the resonances due to these two transitions {Fig. ID). Since the magnetic field is varied, not the frequency, the hyperfine splittings are often reported in Gauss or Tesla (1 G 10_4T). When more than one magnetic nucleus of the same splitting is present a number of resonance lines will appear. For protons the intensities of the lines follow the binomial probability distribution pattern as in NMR spectroscopy. Protons 0 Relative Intensities of Resonances 1 I 3G ( Figure. (A) Computer simulated first derivative ESR spectrum of the methyl radical. The spectrum shows the expected 1:3:3:1 ratio of intensities from the 3 equivalent protons. (B) Computer simulated ESR spectrum of the ethyl radical. The spectrum results from two equivalent protons with G splitting and three equivalent protons with 6,9G splitting. Each line of the 1:3:3:1 quartet due to the methyl group protons is split into a 1::1 pattern by the CH group protons I For example, in Figure A a simulation of the spectrum of the methyl radical CH3- is shown. This spectrum shows the expected 1:3:3:1 ratio of intensities due to three equivalent methyl protons with a hyperfine splitting of 3 G. When other nuclei of differing constants are present they split the spectrum further. For example, in Figure B we show a simulation of the spectrum of the ethyl radical CH3CH- with a 6.9 G splitting for the 3 beta protons in the CH3 group and a. G splitting for the alpha protons in the CH group (6). The importance of the hfc constants are that they give information about the number and type (H, N, F etc.) of magnetic nuclei which can often identify the radical species. In addition, the magnitude of the coupling constants is linearly related to the amount of impaired spin on the atom. The fraction of unpaired spin on an atom is called the spin density. Protons in free radicals can be classified as alpha- or betaprotons. Alpha-protons are those directly attached to the carbon (or other nuclei) which has the unpaired electron while beta-protons are attached to nuclei next to the radical site. The difference between alpha- and beta-protons is shown in the following examples: CH3CH ch3chchch3 8 cx 7 & (x & For planar radicals with tt systems such as the benzene anion, the magnitude of the alpha-proton coupling («) is linearly related to the unpaired spin density (px) in an adjacent carbon s pz orbital, that is, Qpc'r with Q «3 G. However, a beta-proton is related not only to the pi-spin density but also to its orientation to the p orbital on the radical center, a ; 50p,r cos0. Consequently, the beta-proton splitting changes orientation from 0 G in the x, y plane of the Figure 3. (A) Side view of RCHC< radical fragment showing the pz orbital at the radical center (B). The same radical viewed along the C-C bond axis from the right. The angel 0 is the dihedral angle between the C-H bond and the z axis of the pz orbital. The /3-proton splitting varies with this angle. It is large ca. 50G when C-H bond is aligned with the z axis and small, near zero, when at p orbital (6 90 ) to up to 50 G when it rotates and is aligned with the p orbital, 0 0 {see Fig. 3). Applications to Radiation Chemistry In Situ Steady State Techniques Free radical generation can be accomplished by in situ techniques or by sample irradiation and transfer. The in situ techniques are those in which radiation of the sample takes place within the EPR cavity. This very successful technique often employs the steady state method in which the ESR spectrum is taken while a sample is continuously irradiated Volume 58 Number February

3 - 8 ~ ** by light or another radiation source such as an electron beam from a Van de Graf generator (6'-9). Under these conditions the steady state spectra of the longer lived radicals are observed. These may result from primary mechanisms such as ionization, bond cleavage or from secondary reactions, such as abstraction. The signal intensity is a function of the rate of production, the rate of loss, the line width and the number of magnetic nuclei. A larger number of magnetic nuclei lowers the signal intensity because each nucleus further splits the spectra. Among the first investigations employing this now classical technique was the study of electron beam irradiated hydrocarbons by Fessenden and Schuler (6). Virtually all the simple hydrocarbons were investigated. In 1968, this technique was successfully employed with an aqueous solution (7). Since this time, the reactions of e~, OH-, and H- have been successfully investigated with many compounds. For example, the reaction of electrons with peptides found that primary deamination results (.9): e" + K H -,+C 111 (CON H V H K 0<V NH3 + CHRCOHNCHRC0- Or, e_ and OH- attack on DNA bases has been shown to produce the anion, and OH- adduct, respectively. The disadvantage of the electron beam technique and other steady state techniques is that the radiation chemical sequence that produces the observed radical is most often not observed. To understand the mechanisms on a shorter time scale pulse, ESR techniques have been developed (10-11). However, they lack the sensitivity of the steady state methods and are complicated by nonequilibrium spin populations. One of the chief advantages of this technique is that very high resolution spectra are obtained which make radical identification straightforward; thus, subtle changes in environment or structure can be detected by changes in hyperfine splittings. For example, the state of protonation of a radical may be determined by the detection of both protonated and unprotonated forms or the effect of the protonation on the other hfc constants. In the latter case a plot of a. versus ph will often yield a change in a hfc at the pka. Irradiation of Neat Solids In order to observe the details of the radiation chemical mechanism by ESR, investigators have often irradiated pure samples with high energy radiation at low temperatures and then observed the ESR spectra as a function of temperature (5). In this manner, the initial radical species formed by the radiation can be observed since they are stabilized by the low temperature. These radicals, which are often the primary ionic species, can then be observed to decay to more stable radical species as the temperature is raised. In order to use this technique to its full advantage, single crystals of the compound of interest have often been employed. In polycrystalline matrices the anisotropic hyperfine splittings cause large line-broadening effects which make spectrum analysis difficult. For example, a typical ESR spectrum containing a single a-proton isotropic splittings of 0 G will show anisotropic splittings of approximately 10 G. Thus, depending on the orientation of the radical to the external magnetic field, splittings from (0 10) 10 G to (0 + 10) 30 G are observed. Since in a polycrystalline sample all orientations are present in one spectrum, anisotropic splittings cause broadening and severe loss of resolution. However, in a single crystal the magnetic field can be oriented along a single molecular axis (Fig. 4). Thus, by rotation of the crystal and analysis of the now well resolved ESR spectrum at each angle, the isotropic and anisotropic hyperfine splittings can be observed. The anisotropic hyperfine splittings provide new information about the radical. For example, an alpha-proton has the minimum in its total splitting (10 G) when the magnetic field is along the C-H bond and its maximum (30 G), 90 away, still in the plane of the molecule. Beta-protons only have a slight degree of anisotropy. Thus, the single crystal analysis can provide information about the type of proton and its location on the molecule. As an example of this type of analysis, splittings from a radical produced in the irradiation of a single crystal of uracil are given below (1): xx yy zz G G 1 G The hyperfine splittings show coupling to two 0-protons and one a-proton. The position of the alpha-coupling on the molecule can be assigned by a knowledge of the crystallographic structure of uracil assuming the position of the radical in the crystal is the same as the original uracil molecule. The 0-proton splittings show very little anisotropy. This fact along with their magnitude identifies them as due to 0-protons. Free radical mechanisms can be elucidated by use of irradiated single crystals. The method normally employed is to irradiate at low temperatures and observe radical formation and decay upon warming by use of ESR (5,13). A generalized reaction mechanism involving a crystalline amino acid is given below (5): (1) Ionization NH3+CHRC0 -* NH3+CHRC0- + e~ () Anion Formation NH3+CHRC0~ + NHa+CHRCO-j- (3) Decay of Cation NH3+CHRC0- - NH3+CHR + C0 (4) Decay of Anion NH3+CHRC0 NH3 + -( HUGO? A B SOLUTION C D SINGLE CRYSTAL HIIX HIIY H IfZ POLYCRYSTALLINE Figure 4. ESR spectra from the a-proton coupling in the >C-H radical fragment diagramed in A, (B) In solution showing the isotropic coupling of 0G. The anisotropic couplings average to zero for a rapidly rotating radical. (C) In a single crystal showing the change in splitting with orientation to the external magnetic field. The splitting is 10G when the magnetic field is along the C-H bond axis <H [X). It is 30G 90 away in the xy plane, and it is 0G when the field is aligned with the pz orbital on the carbon. (D) In a polycrystalline matrix where all possible orientations to the external magnetic field are present. Notice that although the spectrum is broadened there is structure. This structure can often be analyzed for both the isotropic and anisotropic couplings. 108 Journal of Chemical Education

4 - LiCI, 5JW KCO;j, m NaC104,5MMgCl), acids (8M HS04, 5M H3PO4) or bases (8Af NaOH, 8M KOH) when cooled to 77 K will form glasses. Radiolysis of these glasses will produce trapped electrons, OH-, H-, as well as radicals from the inorganic compounds used to make the glass. Electron and H- reactions with solutes can he investigated at low temperatures since they can be made mobile with visible light (the electron) or warming slightly (H-). As an alternative to radiolysis each of the radiolytic intermediates, e~, *H, and OH-, can be produced individually by photolysis in aqueous glasses. For example electrons can be produced by the photolysis of 10-3 M Fe(CN)6-4 in most of the above glasses (14). This photolysis produces trapped electrons and FefCNle-3. Fe(CN)6- l ^-e- + Fe(CN)<r3 Figure 5. First derivative ESR spectra in 10 M LiCI aqueous (D0) glasses. (A) The trapped electron at 100K generated by photoionization of ferrocyanide ion. (B) The spectrum of the acetic acid anion at 160K produced by electron attachment to acetic acid. The markers in the center of each spectrum are separated by 13.1G, The general steps outlined above (ionization, anion formation, etc.) are repeated in many molecular systems and are often followed by hydrogen abstraction reactions leading to a more stable radical species. This species ultimately decays by recombination and disproportionation reactions. The advantages of the use of irradiated single crystals are (1) easy radical production, () good resolution of ESR spectra in the solid state, (3) identification of proton type (a or /3) and possible location, and (4) the determination of radical mechanisms. The greatest disadvantage of this technique is that interpretation of spectra is time consuming and requires a great deal of care and expertise. In addition, there is less control over the radicals environment than in solutions where the concentration, ph, and ionic strength can be altered. Aqueous Glasses ESR studies of radical reaction mechanisms are limited in a crystalline matrix since it allows for little radical mobility albeit with long radical lifetimes. An aqueous solution allows for high radical mobility; however, radical lifetimes are very short. In an aqueous glass the radical mobility can be controlled because the viscosity of the glass is a function of temperature. As a consequence, radical mobility is combined with long radical lifetimes (14-16). Since the environment (concentrations, ph, etc.) can be varied also, the aqueous glass provides a useful medium for investigation of radical mechanisms (15). The major disadvantage of the glassy matrix is that the anisotropic hyperfine splitting causes broadening of spectra and thus creates difficulty in radical identification (Fig. 4D). This can be overcome to some extent by: (1) replacing a-protons with methyl groups or /3-protons which show very little anisotropy. () the use of deuterated aqueous glasses which reduce matrix-induced linewidths and often allow the resolution of the anisotropic components of the a-proton splittings and (3) the use of spectrum simulation programs which allow for analysis of the glassy spectra in terms of both isotropic and anisotropic splittings and g values. Concentrated solutions of a number of inorganic salts (10M Ferricyanide does not produce an ESR signal in the g region. Thus, electron reactions can be investigated separately from the other free radical intermediates, H-, OH-. As an example of the spectra found in glassy systems we show, in Figure 5A, the spectrum of the electron after photolysis in 10 M LiCI (D0). In glasses containing a solute, the photoejected electron can react with the dissolved solute to produce the anion radical. In Figure 5B the spectrum of the acetic acid anion is shown after its production by electron attachment to acetic acid. 0 CH^COOH + e" CH3COH This technique has been employed by several investigators to elucidate the reactions of electrons with amino acids, peptides, DNA bases, and other biological compounds (14-19). Spin Trapping Often radicals are produced in radiation processes in aqueous solutions which have lifetimes too short to be detected by ESR spectroscopy. Spin Trapping, a technique for conversion of these unstable radicals into long-lived free radicals has been developed recently (0,1). The compound most often used as a spin trap is -methyl--nitrosopropane, (CH;t)CN0. A number of other nitroso compounds are also used as spin traps (0); however, -methyl--nitrosopropane results in radicals which show more resolved hyperfine structure than most other traps. Spin traps react with free radical intermediates to form unreactive nitroxide radicals. As an example, the spin trapping of the methyl radical is shown below. (CH3)3CN0 + CHy -* O' (CH3)3C N CH3 Spin Trap Methyl Radical Long-lived Nitroxide (short-lived) Radical In the case illustrated above the hyperfine coupling of the nitrogen and the methyl group are observed in the ESR spectrum. The protons from the t-butyl group give no resolved structure. The coupling to the methyl group provides excellent evidence for the methyl radical as an intermediate in the system under investigation. The stability of the nitroxide radicals produced by spin trapping are such that they can be separated via column chromatography. This makes the identification of radicals easier by simplifying the ESR spectra from a number of overlapping signals to their individual spectra. Spin trapping as a technique has a number of advantages. It is quite straightforward and can be done with a minimum of equipment. It produces long-lived radicals from short-lived radicals. Experiments do riot take long to perform. Thus, varying conditions and running a number of experiments is possible. The major disadvantage of this technique is that the nitroxide radicals produced do not usually allow for positive identification of the radical intermediate. This occurs because I Volume 58 Number February

5 the unpaired spin on the nitrogen usually interacts only with protons near it (/3 or y-protons). However, often with a little chemical intuition and knowledge of the system under investigation. the identity of the intermediates can be surmised. Literature Cited (1) Wertz, J. E. and Bolton, J. R., Electron Spin Resonance Elementary Theory and Practical Applications, McGraw Hill, New York, 197. () Atherton, N, W,, Electron Spin Resonance: Theory and Applications," John Wiley and Sons, New York, (3) Symons, M. C. R., Chemical and Biochemical Aspects of Electron Spin Resonance Spectroscopy, John Wiley and Sons, New York, (4) Symons, M. C. R. and Atkins, P. W., The Structure of Inorganic Radicals," Elsevier Publishing Co., New York, (5) Box, H. C., Radiation Effects; ESR and ENDOR Analysis," Academic Press, New York, (6) Fessenden, R. W. and Schuler, R. H., J. Chem. Phys., 39, 147, (1963). (7) Eiben, K. and Fessenden, R. W.,J. Phys. Chem., 7, 3387, (1968). (8) Zeldes, H. and Livingston, R., J. Magnetic Resonance, 34, 543, (1979). (9) Neta, P., and Fessenden, R. W., J. Phys. Chemistry, 74, 63, (1970). (10) Nucifora, G., Smaller, B., Remko, R.,and Avery, E. G., Radial. Res., 49, 96 (197). (11) Verma, N. C. and Fessenden, R. W..J, Chem. Phys., 58, 501, (1973). (1) Zehner, H., Flossman, W., Westhof, E. and Muller, A., Mol. Phys., 3,869 (1976). (13) Herak, J. N., Adamic, K. J., "Magnetic Resonance in Chemistry and Biology." Dekker. New York, 1975, pp (14) Ayscough, P. B., Collins, R. G. and Dainton F, S., Nature, 05, 965 (1965), (15) Sevilla, M. D. and Brooks, V. L., J. Phys. Chem., 77, 954 (1973). (16) Kevan, L., in The Chemical and Biological Action of Radiations, M, Haissinsky, (Editor), Masson, Paris, (17) Holroyd, R. A. and Glass, J. W., Int. J. Radial. Biol.. 14,445 (1968). (18) Sevilla, M. D,, Failor, R., Clark.., and Holroyd. R. A., and Pettei, M.. J. Phys. Chem., 80, 353 (1976). (19) Sevilla, M. D., D Arcy, J. B., and Suryanarayana, I)., J. Phys. Chem., 8, 589 (1978). (0) Janzen, E. G., Ace. Chem. Res., 4, 31 (1971). (1) Rustgi, S. and Riesz P., Int. J. Radiat. Biot., 33, ). 110 Journal of Chemical Education