Spin Trapping Organic Radicals - American Chemical Society

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Spin Trapping Organic Radicals

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ree radicals play an important role in a number of toxicologically sig nificant gas-phase environments, including smoke resulting from combus tion, gases formed by pyrolytic processes, and polluted air. The reactivity associated with many radicals has led to considerable speculation about their association with the toxic effects of smoke or polluted air. Thus the development of methods for studying these species has been an impor tant goal in many laboratories. Because reactive radicals in smoke or polluted air typically occur at low concen trations in complex matrices, techniques that are sensitive and selective are needed for their unambiguous detection, identifi cation, and accurate quantitation. The re quirement that they be radical-specific places fairly stringent limitations on the methods available for studying radicals in the gas phase. IR, UV-vis, and fluores cence spectroscopies have all been used to study gas-phase radicals (i) ; however, it becomes increasingly difficult to apply these techniques as the composition of the gas-phase mixture becomes more complex. Electron paramagnetic resonance (EPR) spectroscopy appears to be well suited for the determination of low con centrations of free radicals in complex gaseous media. EPR spectroscopy is highly selective; it detects only species that are paramagnetic (radicals, diradi-

Daniel F. Church Louisiana State University 0003-2700/94/0366-419A/$04.50/0 © 1994 American Chemical Society

netic species (2). For larger radicals in Spin trapping the gas phase, the strong coupling of the rotational angular momentum to the elec with EPR tronic spin and orbital angular momenta in a large number of transitions; spectroscopy is a results the distribution of the signal between so lines effectively causes extreme sig promising technique many nal broadening and a corresponding loss of sensitivity. for studying Laser magnetic resonance (LMR) spectroscopy is another radical-specific gas-phase free radicalstechnique applicable to gas-phase studies. Its principal advantage over EPR spectros in smoke copy is sensitivity; its detection limit is at

cals, etc.). Furthermore, the spectroscopic characteristics of many organic free radi cals are not drastically affected by the na ture of the matrix. As a result, even the most rudimentary separation steps are often unnecessary. Also, the hyperfine interaction between the unpaired electron and nearby nuclear spins can sometimes provide sufficient information to allow unambiguous determination of the struc ture of the radical. In practice, however, there are severe limitations to applying EPR spectroscopy to gas-phase studies. Although EPR spec troscopy is reasonably sensitive (under optimal conditions, it can detect radicals at levels of ΙΟ"8 Μ in a 1-mL sample vol ume) , the concentrations of many toxico logically important free radicals are below the detection limit. Moreover, direct EPR studies of radicals in the gas phase gener ally are limited to systems containing atomic, diatomic, and triatomic paramag

least 4-5 orders of magnitude lower than the limit for the EPR technique. Structural data and identification, however—espe cially of unknown organic radicals—are less readily accessible from LMR spectra. Both EPR and LMR techniques have additional disadvantages that limit their use for directly detecting and studying gas-phase radicals, especially in environ mental work. The complex and bulky in strumentation generally is more suited for permanent installations than for transport to field sites. Moreover, neither technique by itself can be used to unambiguously assign structure, especially in the case of larger radicals. A related problem is that neither technique offers the capability to easily resolve complex mixtures of similar radicals. Many such problems can be overcome by using the spin-trapping tech nique. This method provides the en hanced sensitivity traditionally associated with trap-sampling techniques and retains the selectivity for paramagnetic species inherent in EPR spectroscopy. In addition,

Analytical Chemistry, Vol. 66, No. 7, April 1, 1994 4 1 9 A

REPORT recent advances hold promise for improved structural determination capabilities. The focus of this Report is the application of the spin-trapping method to study gas-phase free radicals in smoke resulting from combustion and/or pyrolysis processes and smoke in polluted air. Detecting reactive radicals

Spin trapping is by far the most widely applied method for the detection and identification of reactive radicals (3-5). Although solution-phase studies constitute the vast majority of spin-trapping applications, numerous gas-phase studies have been published. The essential feature of this method— regardless of the medium in which it is used—is that an unstable radical is allowed to react with a molecule (the spin trap), resulting in the formation of a new, more stable radical (the spin adduct) that can be studied conveniently by EPR spectroscopy. The most common spin traps are nitrone and nitroso compounds, both of which react with radicals to form stable nitroxide (aminoxyl) radicals, as shown in Equations 1 (nitrone spin trap) and 2 (nitroso spin trap) below.

The radical adds directly to the nascent nitroxide nitrogen of the nitroso spin traps, whereas radicals add to the carbon adjacent to the nitrogen in ni420 A

trones. This positions nuclei having spin (e.g., protons) in the trapped radical closer to the nitroxide radical center and results in larger, more readily measured hyperfine interactions. Thus the experimentalist can often obtain more detailed structural information using nitroso spin traps than is possible using nitrones. Moreover, because the rates of radical trapping by nitroso spin traps generally are faster than the rates for spin trapping by nitrones, substantially better initial trapping efficiencies are often obtained (6). However, nitroso compounds typically do not form stable adducts with oxygencentered radicals such as alkoxyls or peroxyls. In addition, the nitrosoalkanes are somewhat photochemically and thermally labile and therefore can be awkward to handle, especially over the longer sampling periods required for trapping species in environmental samples. Largely because of the limitations of the nitroso spin traps, nitrones are more commonly used. In this article, use of nitrone spin traps and, in particular, a-phenyl-Nteri-butylnitrone (PBN), can be assumed whenever the trap is not explicitly identified. The identities of the trapped radicals occasionally can be determined unambiguously from the EPR spectra of the spin adducts; more typically, only the general type(s) of radicals present (alkoxyl, peroxyl, alkyl, etc.) can be determined by the EPR spectrum alone. Figure 1 shows a typical EPR spin adduct spectrum. This spectrum was obtained by photolytically decomposing tetraethyltin vapor in a stream of nitrogen and then passing the gas mixture over silica gel coated with PBN (5% w/w). To obtain maximum spectral quality, the spin adducts were washed off the silica gel with benzene and the solution was deoxygenated by nitrogen bubbling before acquisition of the spectrum. Figure 1 also indicates the measurement of the hyperfine splitting constants (HFSCs) that arise from the couplings between the unpaired electron and the nitroxide nitrogen (aN) and the hydrogen on the carbon adjacent to the nitroxide (%); these HFSCs are the principal spectroscopic parameters used to characterize the spin adduct. The measured HFSCs from this spectrum are aN = 1.46 mT and

Analytical Chemistry, Vol. 66, No. 7, April 1, 1994

eH = 0.34 mT, which are consistent with literature values for the ethyl radical spin adduct of PBN (3). In experiments such as the one in the preceding paragraph, in which just a single adduct is formed, accurate measurement of the HFSC is a trivial matter. The HFSC parameters for the PBN spin adducts of several radicals in benzene are presented in Table 1. These parameters are very similar, especially for PBN spin adducts of similar radicals. For example, both the aN and aH HFSCs for the methyl and ethyl spin adducts are virtually identical. Given the peak width of EPR lines, it would not be possible to distinguish between or to individually quantitate these two spin adducts if they were present in the same mixture. Even when the HFSCs of two spin adducts are different (e.g., those for the methyl and methoxyl radicals), it is often difficult to obtain accurate resolution of overlapping spectra. Two approaches for overcoming such problems are the use of higher derivatives of the spin adduct spectra and spectral simulation (7,8). However, even when these techniques are effective, the best one can typically expect from HFSC data alone is the correct classification of a spintrapped radical (i.e., as carbon- or oxygencentered); more detailed structural elucidation requires other techniques. The preceding discussion reveals the two principal weaknesses of spin trapping with detection by conventional EPR spectroscopy: It does not by itself allow unam-

Figure 1 . EPR spin adduct spectrum of tetraethyltin.

Table 1 . HFSC parameters for spin adducts resulting from the reaction of α-phenyl-N-tert-butylnitrone with various radicals Radical

«N

«H

CH3

1.424

0.345

3

CH2CH3 CH2CBH5 C6H5 0(CH2)3CH3 OCH3 OOC(CH3)3

1.433 1.445 1.441 1.360 1.359 1.334

0.322 0.254 0.221 0.200 0.184 0.125

17 47 48 49 47 48

biguous structural identification and can not adequately deal with even relatively simple mixtures of radicals. In recent years, several research groups have tried to address these limitations by integrating EPR spectroscopy with HPLC, LC/MS, GC/MS, and MS (9-14). Although efforts have been somewhat successful with model systems, these hybrid techniques have not yet been routinely applied to practical spin-trapping problems. One problem is associated with the lability of most spin adducts, especially at the elevated temperatures required for separation by GC. Furthermore, analysis of spin-trapping mixtures by MS suffers from at least two significant problems. The first is the lack of specificity for free radicals. The radical chemistry of many practical systems is complex, as is the chemistry of spin trapping; therefore, the number of products is large. Unambigu ous identification of products that arise from the reaction of radicals with the spin trap is not easily accomplished by MS alone. Use of the hybrid technique of LC/ EPR/MS has been reported (12) but does not yet appear to have sufficient sensitiv ity. For example, using known concentra tions of stable nitroxides, Iwahashi et al. found a detection limit for LC/EPR/electrospray MS of at best 100 μΜ (12); in many practical applications of spin trap ping, particularly in biological or environ mental applications, the concentration of individual spin adducts is often 10-100 times lower. Spin trapping is integrative and, in principle, its sensitivity should be virtually unlimited. Even if the radical steady-state concentrations are substantially below the detection limits of EPR spectroscopy, it should only be necessary to expose the spin trap long enough to accumulate a

Reference

desirable concentration of spin adducts. In practice, nitroxide spin adducts are re active species that are converted to other nitroxides or to nonradical species by nu merous processes (11,15). The observed spin adduct intensities may, in fact, reflect the steady-state levels of the spin adduct rather than actual concentrations of the radical species of interest in the gas-phase system under investigation. Another potential problem with the application of spin trapping is that for any given trap, different radicals react with that trap with very different rate constants (6,11,15). For example, alkylperoxyl radicals typically are several orders of magnitude less reactive to PBN than are alkyl or alkoxyl radicals (11). Thus, one must apply a certain degree of caution when estimating gas-phase radical con centrations based on the relative intensi ties of spin adducts in a mixture. Simple gas-phase systems Spin trapping has been applied to a variety of relatively "simple" gas-phase chemical systems (16-21). Many such systems also have been studied extensively with other methods. The results obtained from spin trapping can be used to determine the method's utility and limitations for the study of gas-phase radicals in more com plex environmental or toxicological sys tems. Janzen's group has used the photolytic decomposition of volatile metalalkyls, azoalkanes, and ketones to generate gasphase free radicals that were subse quently trapped either by PBN or by 2-methyl-2-nitrosopropane (MNP) (16,17, 19). In these experiments, gases were photolyzed in nitrogen atmospheres at pressures of 1 atm or lower in quartz reac tors and then immediately passed over the

solid spin trap in a nitrogen stream. Al though much of the subsequent work has been carried out by bubbling the radicalcontaining gas phase through a solution of the spin trap, these early papers clearly demonstrate that gas-phase radicals also can be successfully trapped by a solidphase spin trap. In fact, as discussed be low, the solvent is a potential source of artifactual signals because it might react with gas-phase free radicals and lead to the formation of secondary radicals that are then spin-trapped. Although EPR spectra of the spin ad ducts can be obtained by direct EPR ex amination of the solid spin trap, the spec tra obtained in this manner are highly anisotropic. Better spectra are obtained when the spin adducts are first dissolved in a suitable low-dielectric solvent and deoxygenated by bubbling with either nitrogen or argon. The experiments discussed above illustrate an important feature of radicals in gas-phase reaction systems. Even very reactive radicals have significant lifetimes at the low concentrations encountered in the gas phase. Thus, they can be formed at one location and still migrate some dis tance in a flowing gas stream before react ing, either with other species in the gas phase or with the spin trap. This is a criti cal issue in terms of which sampling pro tocols are used. This sort of reactivity re gime has allowed traditional gas-phase bubblers to be used fairly effectively for sample collection in gas-phase spin-trap ping experiments. Another study in this series by Jan zen's group clearly illustrates the problem that often there is no "ideal" spin trap (19). When acetone is photolyzed in the gas phase, it undergoes homolytic decom position to both methyl and acetyl radicals (Equation 3).

Analytical Chemistry, Vol. 66, No. 7, April 1, 1994 421 A

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However, when the gas phase resulting from the photolysis of acetone was passed over solid PBN, only the methyl radical spin adduct was observed. This result can be explained by assuming that the unimolecular decomposition of acetyl radicals to carbon monoxide and methyl radicals (Equation 4) is too fast for the acetyl radicals to be trapped by PBN (19).

This conclusion is consistent with later estimates of rate constants for radical trapping by PBN (6). The photochemical decomposition of acetone was repeated and everything was kept the same except that MNP was used as the spin trap. The spin adduct in this latter experiment was attributed to the acetyl radical; the methyl adduct was not observed. This result is consistent with the observation that nitroso traps react more rapidly with carbon-centered radicals (both alkyl and acyl) than do nitrones (6). It is not clear, however, why the methyl radical is not also trapped by MNP; Maeda and Ingold have shown that both acyl and alkyl radicals react with MNP with similar rate constants (6). One likely possibility is that the methyl spin adduct of MNP is much less stable than the spin adduct derived from the acetyl radical. It is well known that the nitroxides with an alpha hydrogen atom are prone to undergo a bimolecular disproportionation to nonradical products, namely a new nitrone and a hydroxylamine (Equation 5).

422 A

The preceding results make it clear that neither of these two spin traps alone can provide a meaningful measure of even the relative gas-phase concentrations of the two radical species present in this simple gas-phase system. Combustion and pyrolytic processes

Most combustion and many pyrolytic processes involving organic materials occur via radical-mediated mechanisms (22). Indeed, the spontaneity of most combustions arises from the fact that they are branched radical chain oxidations. Moreover, the high temperatures to which organic materials are exposed during either spontaneous combustion or pyrolyses at temperatures higher than - 400 °C are sufficient to cause the homolysis of many of the bonds in organic molecules. In recent years researchers have questioned whether organic radicals might in some way contribute to the toxicity of the smoke or fumes produced by combustion and/or pyrolytic processes (22,23). The key premise is that organic free radicals are highly reactive and, if inhaled, would be able to react with critical target biomolecules in the lung, a process that would lead to their oxidative destruction. The toxicological interest in combustionderived gas-phase free radicals relates primarily to the exposure of individuals to the gases produced during structural fires; there are also industrial hygiene concerns for workers exposed to fumes produced by the high-temperature processing of various materials. A key observation is that some of the pathology resulting from smoke inhalation resembles the pathology associated with acute respiratory distress syndrome (24), a condition known to involve radicalmediated damage to lung tissue (25,26). In this context, a number of studies using EPR spin trapping have been designed to identify and quantitate gas-phase radicals formed by combustion or pyrolytic processes in order to confirm their presence, characterize them chemically, and thus gain a better understanding of the mechanism (s) for their toxicity. One of the first applications of spin trapping to an environmental gas-phase problem was studied by Westerberg, Pfaffli, and Sundholm (27), who detected air-


borne free radicals in air samples from a plant during the processing of polyethylene and polystyrene plastics. Air samples were collected into a spin trap solution. Gas-phase organic free radicals were formed in air samples collected during injection molding, extruding, seam welding, and wire-cutting of the plastics. In all of these processes the materials are subjected to high temperatures and mechanical stress. There are undoubtedly many other high-temperature industrial processes that also produce high gas-phase radical concentrations; however, there are no other reports of comparable studies. The results of this simple experiment raise a fundamental question about the nature of radicals in the more complex environmentally related systems. The radicals detected by spin trapping typically are characterized as highly reactive small organic species that would not be expected to have lifetimes of more than a few seconds under ambient conditions (28). Consistent with this expectation, results from early gas-phase spin-trapping experiments using simple chemical systems demonstrated that a short time period between radical formation and trapping was needed to obtain maximal spin adduct concentrations (16). One could predict that as gas-phase mixtures become increasingly complex, there will be additional pathways for the destruction of reactive radicals, and effective radical lifetimes will become even shorter. However, gas-phase radicals in combustion systems can often be detected for many seconds or even minutes after the combustion event in which they were formed. These observations constitute a paradox that can best be resolved if it is assumed that the reactive radicals actually being spin trapped are in fact being continuously formed in the gas phase and are present in a steady state. These radical steady states may be formed through a variety of mechanisms; the particular mechanism is obviously a function of the material burned (or pyrolyzed) and the nature of the combustion process itself. EPR spin trapping has been used to probe for the presence of gas-phase radicals in the smoke produced by the combustion of various natural and synthetic materials in an effort to understand the toxicity of the smoke produced when

these materials burn. In particular, many deaths attributed to smoke inhalation occur several hours after exposure and cannot be explained by high levels of identifiable toxins such as carbon monoxide or hydrogen cyanide. Lowry et al. were among the first to propose that free radicals might play a role in this sort of "inexplicable" smoke toxicity (29). These workers conducted a full-scale test burn of a furnished room and sampled the combustion gases produced in this controlled fire into a solution of the spin trap PBN. They showed that reactive free radicals were present in substantial concentrations. Furthermore, they proposed that the radical concentrations reached levels that were sufficiently high to account for otherwise inexplicable toxicity associated with the smoke from fires of this type. The radicals produced in this experimental fire were not identified conclusively; on the basis of the published spectra of the spin adducts, they appear to result from the trapping of alkoxyl radicals. There is, however, a serious question about the interpretation of the spin adducts detected in this study. Toluene was used as the solvent to dissolve the PBN for sampling. This solvent is itself highly oxidizable, and the observed spin adducts might actually have been derived from oxidation of this solvent. Although nearly all materials burn or oxidatively degrade with heat to form gasphase radicals that can be detected by spin trapping (8,30), radical production, as measured by spin trapping, varies widely. For example, various materials were heated to a fixed temperature in a quartz combustion tube in a constant velocity air stream that carried the combustion products into a solution of PBN (8). Polyethylene and butyl rubber gave the highest concentrations of spin adducts observed under these conditions; cellulosic materials such as wood gave two to three times lower spin adduct concentrations. Other materials, such as nylon, gave nearly a 50-fold decrease in spin adducts compared with polyethylene. Some materials, such as perfluoropolymers (PFPs), did not give any detectable spin adducts. The striking variations in these spin adduct yields can be attributed to several factors. First, different materials may burn

to produce gas-phase radicals with significantly different stabilities. Second, if different radicals are produced, there may be significant differences in their trapping rates. Third, gas-phase radicals are produced by different mechanisms during the combustion and/or pyrolysis of the different materials. Wood smoke has been thoroughly studied with respect to gas-phase radical lifetimes. The smoke from burning wood or any other cellulosic material can be aged for many minutes, either in the gas phase or after bubbling the smoke through and allowing it to dissolve in a suitable solvent. The aged gas phase can then be bubbled through a solution of spin trap, or spin trap can be added to the aged solution of wood smoke. Substantial levels of spin adducts are observed when wood smoke is aged by either method (30). Possibly the combustion of wood produces a gas phase that contains sub-

Wood smoke has been thoroughly studied with respect to gas-phase radical lifetimes

stantial concentrations of exceptionally stable free radicals. However, it is unlikely that such stable free radicals would react with spin traps. It is more likely that the radicals givingriseto spin adducts in aged wood smoke are derived from the continous decomposition of metastable nonradical species (30). Another smoke of particular toxicological interest is formed when PFPs are oxidatively pyrolyzed. Under certain conditions, these PFP pyrolysis gases are more toxic than the gases produced by the pyrolysis or combustion of other common

construction materials (31). This toxicity is typified by massive lung damage that is consistent with what would be expected to result from oxidative damage (31). On the basis of this characterization, it has been hypothesized that the pyrolysis of PFPs results in the formation of gas-phase free radicals and that these radicals might contribute to the toxicity of PFP pyrolysis products (32). As discussed above, initial experiments in which the fumes from oxidatively pyrolyzed PFPs were spin-trapped did not result in the formation of observable spin adducts. However, subsequent work demonstrated that with sufficiently high pyrolysis temperatures, longer sampling times, and minimum gas-phase residence time (aging), the spin adducts of many radical species—including those of an organic oxy radical (alkoxyl or peroxyl) and the fluorine atom—could be observed when unfiltered smoke was bubbled through a solution of spin trap (8). In addition, the spin trap (PBN) was oxidized to benzoyl-tert-butylnitroxide (PBNOJ. Of special note in these experiments was the observation and unambiguous characterization of a spin adduct attributable to the trapping of chlorine atoms. The source of the chlorine is not certain, although it is most likely related to the presence of chlorine-containing impurities in the PFPs, even though chlorine was not detectable by traditional methods for determining the elemental composition of PFP. The fact that comparable concentrations of the spin adducts of the fluorine and chlorine atoms were observed as their PBN spin adducts can be attributed to one or more of the following factors. First, the somewhat less reactive chlorine atoms may have substantially longer gasphase lifetimes than do fluorine atoms. In addition, chlorine atoms may be trapped more efficiently than fluorine atoms. The chlorine atom spin adducts may also be much more stable than fluorine atom spin adducts. Finally, the chlorine atoms may be produced during the pyrolysis by a more efficient mechanism than fluorine atoms, which may lead to higher steadystate concentrations. This observation of chlorine atom spin adducts clearly demonstrates the caution necessary when using spin adduct yields to assess steady-state radical concentrations.


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Another complexity is associated with the smoke resulting from the pyrolysis of PFPs. Upon pyrolytic degradation, these materials formed extremely fine particu lates and the animals were protected against the toxic effects of PFP smoke by small-pore filters (31,33,34). Further more, in our experiments, the spin adduct intensities were greatly reduced by the in sertion of a 0.2-μπι glass-fiber filter in the gas stream between the pyrolysis chamber and the spin-trapping solution (35). Examination of the particulate col lected on the filter by EPR spectroscopy revealed a relatively stable population of end-chain peroxyl radicals (33,36,37). Although they are stable for many hours under ambient conditions, these particulate-bound perfluoroalkyl peroxyl species are nevertheless quite reactive if they are suspended in a solution containing a suit able substrate. For example, the particles from pyrolyzed PFP initiated lipid peroxi dation in liposomes formed from soy phosphatidylcholine (37). It also was found that when the particles were sus pended in a solution of PBN, the same spin adducts were observed as when the gaseous pyrolysis products were passed into a solution of the same spin trap. Taken together, these results suggest that the spin-trapping protocol primarily de tects radicals associated with the particu late fraction of PFP smoke, although it is also possibile that some of the observed spin adducts arise from the trapped gasphase radicals. Cigarette smoke Tobacco smoke is formed in a unique combustion process, and the relationship between its free-radical chemistry and its toxicity has received considerable atten tion. What distinguishes tobacco smoke from the gases produced by the combus tion and/or pyrolysis of most synthetic and many natural materials is the com plexity of the smoke. Unburned tobacco itself has a complex chemistry; the burn ing process not only pyrolytically decom poses the tobacco, it also results in the volatilization of many of the compounds in the tobacco. Many thousands of com pounds have been identified in smoke, and an even larger number remain un known. Although the nature of tobacco smoke is complex, spin-trapping studies 424 A

have given us a remarkably clear under standing of its radical chemistry. Tobacco smoke contains two very di verse radical populations (38). The tar phase (material that does not pass through a submicron glass-fiber Cam bridge filter) contains several stable radi cal species; the principal tar radical spe cies has been characterized as a mixture of quinone, semiquinone, and hydroquinone functionalities held in a poly meric matrix. In marked contrast to the extremely stable tar-phase radicals, the gas phase of tobacco smoke (material that passes through the Cambridge filter) con tains radicals that are too reactive to be detected directly; however, researchers have used spin trapping to study them

(38-42). Most spin-trapping studies of gasphase cigarette smoke have tried to mimic the way in which people smoke cigarettes (38). A "puff" (35-mL volume) is drawn through the burning cigarette into a glass syringe, and the resulting gas phase is subsequently bubbled through a solution of the spin trap. This protocol was used by Bluhm, Weinstein, and Sousa to detect free radicals in the smoke produced by

Figure 2. EPR spin adduct spectra from cigarette smoke. Spectra are observed when smoke from research cigarettes passes through (a) a solution of PBN in benzene or (b) PBN adsorbed onto silica gel followed by elution of the spin adducts with benzene.


burning cigarettes, cigars, or pipe tobacco (43). All three types of smoke showed a spin adduct spectrum that had an HFSC consistent with the trapping of an alkoxyl radical. Although quantitative measure ments were not reported in this prelimi nary communication, cursory inspection of the data reveals that the intensities of the spin adduct signals from cigars or pipe tobacco are substantially stronger than is the case for cigarettes based on similar sampling volumes for the respective smokes. Menzel, Vincent, and Wasson used a similar approach to trap radicals in the gas phase of the smoke from research ciga rettes (44). These workers observed sub stantial changes in the spin adduct spectra when the samples were aged; their data suggested the presence of at least two long-lived spin adducts. This example illustrates the difficulty associated with using spin trapping for quantitation; different spin adducts may have different stabilities, and the relative intensitites of spin adducts may change over a long period of time. This can pose practical limitations on the use of very long sampling times in studies of environ mental radicals that occur at very low con centrations. Menzel and co-workers also bubbled their smoke through several solutions of spin traps connected in series and deter mined the radical trapping efficiency to be - 47%. Finally, they used measurements of the dipolar broadening of the reso nance lines in their spin adduct spectra to estimate the concentration of the gasphase free radicals in cigarette smoke at - 1 χ 1018 free radicals per puff. A num ber of subsequent studies of gas-phase cigarette smoke have confirmed and ex tended these results (38-40,42,45). When the smoke from research ciga rettes is bubbled through a solution of PBN in benzene, the spectrum shown in Figure 2a is observed. This spectrum con sists of three principal types of spin ad ducts: alkoxyl radicals (aN = 1.36 mT, aH = 0.19 mT), PBNO, (% = 0.080 mT), and a carbon-centered species (aN = 1.44 mT, aH = 0.20 mT). On the basis of these ob served HFSCs, researchers proposed that this carbon-centered radical was attribut able to a cyclohexadienyl radical formed by the addition of radicals in the cigarette

smoke to the aromatic solvent, as shown in Equation 6 (40).

By slightly changing the trapping tech nique, it was possible to demonstrate the likelihood of this mechanism. Figure 2b shows the EPR spectrum observed when the cigarette smoke was passed through PBN adsorbed onto silica gel, followed by elution of the PBN/silica gel with benzene; the EPR spectrum shown was obtained from the resulting benzene solution. Only two types of radicals were observed in this second experiment: alkoxyl radicals ( e N = 1.37 mT and aH = 0.20 mT) and carboncentered radicals (aN = 1.43 mT and aH = 0.32 mT). With this latter protocol, the car bon-centered radicals have HFSCs that are consistent with those of simple alkyls rather than cyclohexadienyl radicals, as in the first protocol. There are two noteworthy aspects of these cigarette smoke spin-trapping re sults. First, even though the two protocols are significantly different from one an other, the ratio of the alkoxyl to carboncentered (cyclohexadienyl vs. alkyl) spin adducts in the two experiments is the same within experimental error. This ob servation lends credence to the hypothe sis that when cigarette smoke is trapped with PBN in benzene, alkyl radicals in the smoke react with the aromatic solvent and lead to the formation of the observed cy clohexadienyl spin adducts. This is an other clear example of the need to be careful about misinterpreting spin-trap ping results. The second interesting aspect is that the quality of the spectrum obtained using PBN adsorbed onto silica gel is much higher than that obtained by trapping with PBN in benzene. Substantial line broaden

ing occurs in the spectrum obtained by bubbling through PBN in benzene. This broadening is almost certainly due to the interaction of some of the many thou sands of compounds in smoke with the nitroxide radicals. With the other protocol, the silica gel not only serves as a support for the spin trap, it also helps to trap and retain many of the more polar compounds in the smoke when the trap is eluted with ben zene prior to obtaining the spectra. The PBN/silica gel technique also makes for a much more convenient trapping system, especially in field studies, because neither fragile bubblers nor toxic solvents are required.

Time profiles for relative nitrogen di oxide and PBN spin adduct levels as a function of the age of the cigarette smoke are shown in Figure 3. Nitrogen dioxide is not present at significant levels in fresh smoke; instead, the principal oxide of ni trogen in fresh smoke is nitric oxide, which is generally unreactive with most organic molecules. In air, however, nitric oxide undergoes a relatively slow oxida tion to the more reactive nitrogen dioxide. The results depicted in Figure 3 strongly suggest that the radicals spin trapped from cigarette smoke are not derived di rectly from the combustion process; in stead, they are formed by reactions of ni trogen dioxide with one or more reactive components of smoke. (Nitrogen dioxide itself does not react with PBN to give spin adducts [40] ; the only product of that re action is PBNO,.) One reactive component of cigarette smoke mat occurs at high concentrations is isoprene, and this reactive diene is a likely candidate for the species in cigarette smoke that is responsible for the formation of the observed spin-trapped radicals. To confirm this hypothesis, it is possible to prepare a mixture of isoprene and nitric oxide in air that closely models the spin adduct concentration time profiles ob served for cigarette smoke (38,40).

Although the nature of tobacco smoke is complex, spin-trapping studies have given us a remarkably clear understanding of its radical Given the numerous examples of the ap plication of spin trapping to the analysis of chemistry Polluted air

Although we have not obtained unam biguous identification of specific gasphase radical species in tobacco smoke as their spin adducts, the spin-trapping re sults have nevertheless provided impor tant clues about the likelihood of a spe cific mechanism for radical formation. The key observation is that the intensity of the spin adducts derived from cigarette smoke actually increases as the smoke is aged over the first several minutes follow ing combustion (38,40), after which the intensity slowly decreases. This is exactly the same concentration time profile exhib ited by the nitrogen dioxide in cigarette smoke (38).

Figure 3. Time profiles for nitrogen dioxide and PBN spin adducts in cigarette smoke as a function of smoke age. Curve A, obtained by extrapolation, represents measured spin adduct intensities. Curve Β represents the relative concentration of nitrogen dioxide.


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gas-phase free radicals, one might think that this technique would have been widely applied to air sampling in pollution studies. Aside from the report of air sampling in a plastics-fabricating factory reported above (27), however, there is just a single example of such an application. Watanabe et al. coated glass-fiber filters with PBN and then exposed these coated filters to the upper atmosphere (14). The filters were then washed with benzene for analysis by EPR spectroscopy, which showed the presence of the spin adduct of the hydroxyl radical. This identification was confirmed by reducing the spin adduct to the hydroxylamine, preparing the trimethylsilyl derivative, and detection with GC/MS. This experiment suggests that spin trapping may well be useful for studying radicals in the atmosphere. However, the results raise a notable question. Why is the hydroxyl radical the only radical observed? This highly reactive species typically occurs at concentrations that are

Figure 4. EPR study of the gas-phase reaction of ozone and propylene. (a) PBN spin adduct spectra, (b) Time profiles for the total spin adducts (alkoxyl plus alkyl), PBNOx, and ozone. The initial concentrations of ozone and propylene were 40 and 2100 ppm, respectively. 426 A

orders of magnitude lower than those generally reported for organic species— especially relatively stable ones such as alkyl peroxyl radicals. This could reflect the reactivity difference between hydroxyl and peroxyl radicals toward PBN. Alternatively, the spin adducts of organic radicals may be substantially less stable than those of the hydroxyl radical. Finally, nitrones are known to be hydrolyzed to cc-hydroxy-hydroxylamines that are airoxidized to the same nitroxide formed by spin trapping the hydroxyl radical. These experiments were not repeated, nor were adequate control experiments performed to ensure that the observed nitroxide radical was not an artifact. Unpublished model experiments demonstrate that other atmospheric processes form radicals that are detectable by spin trapping. It is known that ozone reacts with alkenes in solution to produce spin adducts that are detectable by spin trapping (46). When gaseous mixtures of ozone and propylene are allowed to react for various periods of time before the gas mixture passes into a solution of PBN, strong spin adduct spectra are observed. The HFSCs determined for the spin adducts are consistent with trapping primarily alkoxyl radicals (aN = 1.38 mT and o H = 0.20 mT), along with a lower concentration of alkyl radical adducts (aN = 1.48 mT and aH = 0.31 mT), as well as the ubiquitous PBNC\. Figure 4a shows the PBN spin adduct spectra obtained after allowing the ozone/ propylene gas mixture to age for various lengths of time. Figure 4b plots the spintrapping data and measured ozone levels on the same time axis. Note that spin adduct production clearly builds to a maximum, substantially lagging the ozone concentration. In fact, at the point where the ozone has just become undetectable, the total spin adduct yield is - 70% of the apparent maximum value. In addition, this plot demonstrates that the levels of the PBN oxidation product PBNL\ parallel the levels of ozone, a result suggesting that PBNOj may be derived primarily from direct oxidation of the PBN by ozone. Conclusions

There appear to be three principal reasons that spin trapping has not been more


widely applied to environmental problems. First, the currently available spin-trapping compounds are not entirely satisfactory. Important classes of radicals such as peroxyls do not readily react with nitrones. Furthermore, the spin adducts formed by currently available traps are often quite labile. More robust traps that react readily with a wide range of radical types and that form more stable spin adducts are needed. Second, there is a lack of specific structural information provided by the EPR spectrum of the spin adduct. The marriage of HPLC to both EPR spectroscopy and MS has already begun to address this shortcoming and is likely to be used increasingly in coming years. However, currently available MS technology does not readily lend itself to the detection of nitroxide radicals at the levels of spin adducts formed in many biological or environmental systems of interest. Finally, application of spin trapping is subject to many artifacts. In spite of these limitations, spin trapping has advantages that can be exploited in studies of radicals in gas-phase systems. EPR spin trapping can be used effectively to study radical intermediates in complex gas-phase systems. EPR spectroscopy is highly selective, detects only paramagnetic species, and is relatively insensitive to the matrix in which the radicals occur. Spin trapping appears to be suitable for studies of radical intermediates in the atmosphere, especially when remote sampling techniques are employed. Some of the gas-phase work is the result of collaborative efforts with William A. Pryor; the author is grateful for his support and advice over the years. Some of the EPR spectra were acquired on a MicroNow EPR spectrometer purchased with funds provided by the Louisiana Education Quality Support Fund, grant number LEQSF (1991-92)-ENH-15.

References (1) Hack, W. Int. Rev. Phys. Chem. 1985,4, 165. (2) Wertz, J. E.; Bolton, J. R Electron Spin Resonance: Elementary Theory and Practical Applications; Chapman and Hall: New York, 1986; p. 345. (3) Janzen, E. G.; Blackburn, B. }.]. Am. Chem. Soc. 1969,91,4481. (4) Janzen, E. G.Acc. Chem. Res. 1971, 4, 31.

(5) Rosen, G. M.; Cohen, M. S.; Britigan, B. E.; Pou, S. Free Radical Res. Commun. 1990, 9, 187. (6) Maeda, Y.; Ingold, K. /. Am. Chem. Soc. 1979,101,4975. (7) Triolet, J.; Raffi, J.; Agnel, J. P.; Battesti, C; Thiery, C; Vincent, P. Magn. Reson. Chem. 1992, 30.1051. (8) Lachocki, T. M.; Nuggehalli, S. K; Scherer, K. V.; Church, D. F.; Pryor, W. A. Chem. Res. Toxicol. 1989,2,174. (9) Albro, P. W.; Knecht, K. T.; Schroeder, J. L.; Corbett, J. T.; Marbury, D.; Collins, Β. J.; Charles, J. Chem. Biol. Interact. 1992, 82, 73. (10) Kieber, D. J.; Johnson, C. G.; Blough, N. V'. Free Radical Res. Commun. 1992, 16, 35. (11) Janzen, E. G.; Krygsman, P. H.; Lindsay, D. Α.; Haire, D. L./. Am. Chem. Soc. 1990,112,8279. (12) Iwahashi, H.; Parker, C. E.; Mason, R. P.; Tomer, Κ. Β. Anal. Chem. 1992, 64, 2244. (13) Parker, C. E.; Iwahashi, H.; Tomer, Κ. Β. /. Am. Soc. Mass Spectrom. 1991,2,413. (14) Watanabe, T.; Yoshida, M.; Fujiwara, S.; Abe, K.; Onoe, Α.; Hirota, M.; Igarashi, S. Anal. Chem. 1982, 54,2470. (15) Pou, S.; Hassett, D. J.; Britigan, Β. Ε.; Co hen, M. S.; Rosen, G. M. Anal. Biochem. 1989,177,1. (16) Janzen, E. G.; Gerlock, J. L. Nature 1969, 222,867. (17) Janzen, E. G.; Lopp, I. G./ Phys. Chem. 1972, 76,2056. (18) Janzen, E. G.; Kasai, T.; Kuwata, K. Bull. Chem. Soc.Jpn. 1973,46, 2061. (19) Janzen, E. G.; Lopp, I. G.; Morgan, T. V. / Phys. Chem. 1973, 77,139. (20) Chandra, H.; Davidson, I.M.T.; Symons, M.C.R/ Chem. Soc. Faraday Trans. 1 1983, 79,2705. (21) Migita, C. T.; Chaki, S.; Nakayama, M.; Ogura, K./. Chem. Soc. Perkin Trans. 2 1990,1965. (22) Cullis, C. F.; Hirschler, M. M. The Com bustion of Organic Polymers; Oxford Uni versity Press: New York, 1981. (23) Birky, M.; Halpin, B. M.; Caplan, Y. H.; Fisher, R. S.; McAllister, J. M.; Dixon, A. M. Fire Mater. 1979,3,211. (24) Crapo, R. O.; Nellis, N. Management of Smoke Inhalation Injuries; Intermountain Thoracic Society: Salt Lake City, UT, 1980. (25) Tate, R. M.; Repine, J. E. In Free Radicals in Biology; Pryor, W. A, Ed.; Academic Press: New York, 1984; Vol. 6; p. 199. (26) Morganroth, M. L.; Till, G. O.; Kunkel, R G.; Ward, P. A Lab. Invest. 1986, 54, 507. (27) Westerberg, L M.; Pfaffli, P.; Sundholm, F. Am. Ind. Hyg. Assoc. J. 1982,43, 544. (28) Batt, L; Milne, R. T. Int. J. Chem. Kinet. 1976,8,59. (29) Lowry, W. R; Peterson, J.; Petty, C. S.; Badgett, J. L.J. Forensic Set. 1985,30, 73. (30) Lachocki, T. M.; Church, D. F.; Pryor, W. A Free Radical Biol. Med. 1989, 7, 17. (31) Lee, K. P.; Zapp, J. A; Sarver, J. W. Lab. Invest. 1976,35,152. (32) Williams, S. J.; Clarke, F. B. Fire Mater. 1983, 7,96.

(33) Seidel, W. C; Scherer, K. V.; Cline, D.; Olson, A. H.; Bonesteel, J. K.; Church, D. F.; Nuggehalli, S.; Pryor, W. A. Chem. Res. Toxicol. 1991,4,229. (34) Baker, B. B.; Kaiser, M. A. Anal. Chem. 1991, 63, 79. (35) Lachocki, T. M. Ph.D. Dissertation, Louisiana State University, 1988. (36) Metcalfe, E.; Harman, A. R. Fire Mater. 1991,15, 53. (37) Pryor, W. A; Nuggehalli, S. K.; Scherer, K. V.; Church, D. F. Chem. Res. Toxicol.

Say Goodbye to Either/Or Electrochemistry

1990,5,2. (38) Church, D. F.; Pryor, W. A Environ. Health Perspect. 1985, 64, 111. (39) Zhou, B.; Yan, L; Hou, J.; Xin, W. Chin. Med.]. (Beijing, Engl. Ed.) 1991,104, 591. (40) Pryor, W. A; Tamura, M.; Church, D. F. /. Am. Chem. Soc. 1984,106, 5073. (41) Ohkubo, Y.; Kadosima, C; Kaneko, T.; Chuchiya, J.; Akutsu, Y.; Tamura, M.; Yoshida,T././M Soc. Air Pollut. 1 9 9 1 , 26,171. (42) Halpern, Α.; Knieper, J. NBS Spec. Publ. (U.S.) 1986,726,306. (43) Bluhm, A L.; Weinstein, J.; Sousa, J. A. Nature 1971,229, 500. (44) Menzel, E. R; Vincent, W. R.; Wasson, J. R.J. Magn. Reson. 1976,21,321. (45) Pryor, W. A; Church, D. F.; Evans, M. D.; Rice, W. Y; Hayes, J. R. Free Radical Biol. Med. 1990,8,275. (46) Church, D. F.; McAdams, M; Pryor, W. A. In Oxidative Damage and Repair: Chemical, Biological and Medical Aspects; Davies, K.J.A., Ed.; Pergamon Press: New York, 1991; p. 517. (47) Haire, D. L.; Oehler, U. M.; Krygsman, P. H.; Janzen, E. G.J. Org. Chem. 1988, 53,4535. (48) Ohto, N.; Niki, E.; Kamiya, Y.J. Chem. Soc. Perkin Trans. II1977,1770. (49) Rehorek, D.; Winkler, W.; Wagener, R; Hennig, H. Inorg. Chim. Acta 1981, 64, L7.

M

ost electrochemical software is designed to d o either sophisti cated research or routine measure ments. In the past, if your lab did both, y o u were stuck. Either y o u had to buy t w o separate packages, which meant satisfying the compatibility requirements for both and learning two very different environments. Or y o u had to buy one package and make do. Well say g o o d b y e to eitherjor electro chemistry. The Model 270 Electro chemical Analysis software from EG&G Princeton Applied Research is unmatched o n all counts—power, versatility, and ease of use. Consider just this small selection of Model 270 advantages: • Both time-tested hardware (Model 273 Potentiostat) and state-of-theart computer environment (IBM platform, pull-down menus) • Automatic control of both the PARC Model 303A SMDE and a se lection of microelectrodes • Traditional voltammetry/polarography and fast Square Wave • Easy-to-learn Standard Mode for routine use and feature-rich Expert Mode for finer experimental control

Daniel F. Church earned his Ph.D. in chemistry from Oregon State University in 1975. He was a postdoctoral research asso ciate at Louisiana State University, where he worked under the direction of William Pryor. He is currently an associate professor of chemistry at LSU (Baton Rouge, LA 70803), where his research focuses on the application ofEPR spectroscopy and spintrapping techniques to study free-radical mediated processes in chemistry, environ mental toxicology, and biology.

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Spin Trapping Organic Radicals - American Chemical Society

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