Spin Adducts Research Articles

Photocatalytic Performance of Titania Nanosheets Templated by Graphene Oxide

Abstract A facile process was developed to fabricate two dimensional titania nanosheets (t-NS) by using graphene oxide (GO) as template. In-situ growth of anatase TiO 2 on GO suspended in butanol was achieved by combining sol-gel and solvothermal process without further calcination. Stoichiometric amounts of acetic acid were added to introduce esterification with butanol to release sufficient water for the rate-controlled hydrolysis of titanium butoxide. The two-dimensional GO prevented the agglomeration of TiO 2 particles, and simultaneously established the interaction between two phases. For comparison, a following thermal treatment in air was applied to partially remove GO and increase the crystalline size of t-NS. TiO 2 -decorated samples showed better charge transfer efficiency and thus produced more photoinduced radicals and weaker photoluminescence. Electron paramagnetic resonance (EPR) spectra were utilized to in-situ identify the concentrations and types of photo-induced radicals by mixing various nitroxide spin traps with t-NS suspensions under ambient conditions. In aqueous suspension, hydroxyl radicals produced from illuminated t-NS and trapped by 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) immediately within 30 second. The following decay in the hydroxyl adducts of DMPO suggested the presence of strong reductants in the illuminated sample suspension. A sample with a higher production rate of spin adduct (DMPO-OH) or a higher reduction rate of DMPO-OH demonstrated a more efficient decoloration on methylene blue (MB). Spin trapping results indicated that the hydroxyl radicals were produced from both photoinduced holes and electrons. Hence, an optimum rate of hole titration by ethylenediaminetetraacetic acid was essential to efficiently reduce the recombination of charge carriers and consequently to produce more active radicals to decolor MB. The strong interaction between graphitic and titania structures, rather than the crystallite size of anatase, dominated the photoreduction capability of t-NS.

DFT calculation of isotropic hyperfine coupling constants of spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide in benzene and water

Isotropic hyperfine coupling constants (HFCCs) of spin adducts of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) are calculated by DFT method with solvation effect. Geometries of ring conformers and rotational conformers of trapped radicals are optimized and their HFCCs are calculated. Boltzmann weighted average of HFCCs of low energy conformations shows very good agreement with the experimental value in benzene. Two or four water molecules hydrogen bonded to the spin adduct have to be explicitly incorporated in the case of water. The results show time-independent averaging of relevant conformers is a very practical method for calculation of HFCCs of DMPO spin adducts.

Alkylperoxyl spin adducts of pyrroline-N-oxide spin traps: Experimental and theoretical CASSCF study of the unimolecular decomposition in organic solvent, potential applications in water

Alkylperoxyl spin adducts of pyrroline-N-oxide spin traps: Experimental and theoretical CASSCF study of the unimolecular decomposition in organic solvent, potential applications in water

Anti-ulcerogenic Action Mechanism of <i>Maytenus ilicifolia</i> Detected by mRNA RT-PCR and Free Radical Scavenging Activity Evaluated by ESR

Maytenus ilicifolia, known as “espinheira-santa”, is used in the popular medicine for peptic ulcer treatment. This work evaluates the preventive and curative actions of M. ilicifolia in animals, the healing activity by EGF expression detected by RT-PCR as well as the anti-oxidative activity by Electron Spin Resonance spectroscopy (ESR). Oral administration of M. ilicifolia (300 and 500 mg/kg, p.o.) one hour before the ulcerogenic agent application prevented the ulcer formation 42 ± 0.98% and 72 ± 1.2% (p <0.05 and p <0.001, respectively), in comparison with the negative control tween 80, preserving the cytoprotection characteristics of the gastric mucosa and assuring the integrity of gastric glands and gastric fossets. The animals treated with tween, had the epithelium and the mucosa layer damaged and accentuated vascularization. The healing activity of M. ilicifolia (500mg/kg, p.o.) was 71 ± 1.4% (p <0.001) in chronic ulcer experiments induced by acetic acid. EGF expression detected by RT-PCR confirmed the healing activity. Histological analysis showed the recovery of the mucosal layer and the epithelium surface harmed by the acetic acid. Studies in vitro by Electron Spin Resonance spectroscopy assessed the anti-oxidative action of M ilicifolia. Experiments with DPPH (2.2-Diphenyl -1-picrylhydrazyl) demonstrated that the polar fraction of M. ilicifolia presents an IC50 of 0.680.09 mg/ml. The value of 92 mg/ml was found for IC50 in experiments with the radical OH, produced by the Fenton Reaction and detected through spin adduct DMPO-OH. Mass spectrometry analysis of crude extracts and fractions from M. ilicifolia was carried out and compounds of pentacyclic triterpenes class were identified.

Amidinoquinoxaline N-oxides: spin trapping of O- and C-centered radicals.

Amidinoquinoxaline N-oxides represent a novel family of heterocyclic spin traps. In this work, their ability to trap O- and C-centered radicals was tested using selected derivatives with different structural modifications. All the studied nitrones were able to trap radicals forming persistent spin adducts, also in the case of OH and OOH radicals which are of wide biological interest as examples of ROS. The stability of the adducts was mainly attributed to the wide delocalization of the unpaired electron over the whole quinoxaline moiety. The nitroxide spectral parameters (hfccs and g-factors) were analyzed and the results were supported by DFT calculations. The N-19 hfccs and g-factors were characteristic of each aminoxyl and could aid in the identification of the trapped radical. The enhanced stability of the OH adducts under the employed reaction conditions could be ascribed to their possible stabilization by IHBs with two different acceptors: the N-O˙ moiety or the amidine functionality. DFT calculations indicate that the preferred IHB is strongly conditioned by the amidine ring size. While five membered homologues show a clear preference for the IHB with the N-O˙ group, in six membered derivatives this stabilizing interaction is preferentially established with the amidine nitrogen as an IHB acceptor.

Spin-Trapping Analysis of Thermal Degradation Reaction of Poly(butylene terephthalate)

Poly(butylene terephthalate) (PBT), and its oligomer, were doped with the spin-trapping reagent, 2-methyl-2-nitrosopropane (MNP), by a solution-casting method in order to detect the short-lived radical intermediates during thermal degradation. In the case of PBT, two types of spin adduct were observed by electron spin resonance (ESR) spectroscopy upon stepwise heating from room temperature to 200 °C and during annealing at 130 °C; the ESR spectrum exhibited an isotropic three-line signal, with a hyperfine coupling constant (hfcc) aN ≈ 0.8 mT, as well as an anisotropic component. These adducts were present on the order of 10–5 mol/L. The isotropic component was assigned to arise uniquely from the benzoyl radical, •CO-ϕ. The anisotropic component was carefully investigated by ESR spectroscopy and high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC-ESI-MS), using low-molecular-weight model compounds such as n-butyl benzoate (BB), di-n-butyl terephthalate (DBT), and deuterat...

Hydroxyl radical generation by dissociation of water molecules during 1.65 MHz frequency ultrasound irradiation under aerobic conditions

The dissociation of water molecules by ultrasound irradiation under aerobic conditions was demonstrated experimentally. To be able to detect the dissociation of water molecules, we performed the ultrasound irradiation of 17O-labelled water (H217O) under aerobic conditions. The hydroxyl and hydrogen radicals generated during the ultrasound irradiation process were trapped with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and electron spin resonance (ESR) spectroscopy was performed on the DMPO spin adducts. In the ESR spectrum, a 15-line signal attributable to the trapping of the hydroxyl radicals containing 17O (17OH radicals) by DMPO together with a 4-line signal attributable to the trapping of the hydroxyl radicals containing 16O (16OH radicals) by DMPO were observed. The generation of 17OH radicals indicated that H217O was dissociated by the sonolysis process under aerobic conditions. On the other hand, the ESR signal attributable to the trapping of hydrogen radicals by DMPO was not observed, suggesting that hydrogen radicals were not generated during the dissociation of water molecules.

α-Phenyl-N-cyclohexyl Nitrones: Preparation and Use as Spin-Traps.

Two bifunctional α-phenyl-N-cyclohexyl nitrones were synthesized with the expectation that the cyclohexyl ring will impart lipophilicity to the molecule, high reactivity to the nitronyl group, and stability to the spin adducts formed. The synthesis of the acid nitrone 4 and its corresponding tert-butyl ester 3 was initiated by a Michael reaction to introduce the cyclohexyl ring. A Zn/AcOH-mediated reduction of the nitro functionality followed by condensation onto benzaldehyde generated the nitronyl function. In agreement with their high lipophilicity values, nitrone 3 was insoluble in water, while nitrone 4 exhibited a poor water solubility. It was determined that the presence of the cyclohexyl ring did not affect either the reduction or oxidation potentials of the nitronyl group in comparison to the classical α-phenyl-N-tert-butylnitrone (PBN). The spin trapping ability of 3 and 4 was investigated by EPR for oxygen- and carbon-centered radicals. In most cases, the nitrones gave rise to a standard six-line EPR spectrum whose values were in agreement with the literature, accompanied by a minor second species. In DMSO, the half-lives of nitrone 3 and 4-OOH adducts were double that of PBN, suggesting that the stabilization comes from the cyclohexyl ring and/or the electronic effect of the carboxylic acid.

Accuracy and Validity of AREC (Alkoxy Radical Elimination Capacity) Assay in Evaluating the Antioxidant Abilities of Various Biosubstances

Abstract The AREC (alkoxy radical elimination capacity) assay was developed to observe the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin adducts of free radicals produced by thermal decomposition of AAPH (2,2′-azobis(2,4-amidinopropane) dihydrochloride) using a flow-injection ESR (FI-ESR) system. The γ50 value is defined as [DMPO]0/ID50 = kS/k1, where k1 is the rate constant of spin-trapping of the alkoxy radical by DMPO, kS, that of alkoxy radical-elimination by the substrate, and ID50, 50% inhibition of the alkoxy radical by the substrate, and the AREC value is the ratio of γ50 value of the substrate to that of Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid, TRX). The γ50 and AREC values show fairly wide variations and can be determined for most biosubstances. The highest AREC values were observed for sinapic acid, l-glutathione, caffeic acid, and chlorogenic acid, followed by TRX, syringic acid, trans-ferulic acid, and homogentisic acid. Little correlation is observed between the AREC values and the hydroxy and superoxide radical-elimination abilities. The AREC values of 4-hydroxycinnamic acid derivatives (HCAs) are linearly related to the aryloxy radical-elimination abilities, which indicate that the alkoxy radical-elimination by HCAs is mainly caused by hydrogen-atom transfer. The newly defined AREC value is applicable for various biosubstances, and is far superior and a more reliable indicator than the oxygen radical absorption capacity (ORAC) value determined by the ORAC-fluorescein assay. Thus, the AREC value is an excellent indicator to characterize the antioxidant activities of a wide range of biologically important antioxidants present in fruits, vegetables, and beverages.

Pathways of the photocatalytic reaction of acetate in H2O and D2O: A combined EPR and ATR-FTIR study

The adsorption and photocatalytic degradation of acetate on TiO2 surfaces were investigated in H2O and D2O by ATR-FTIR and EPR Spectroscopy respectively. These studies were carried out in the dark and under UV(A) illumination to gain additional insights into the adsorption behaviour with the identification of paramagnetic species formed during the oxidation of acetate. Isotopic exchange during the adsorption of D2O on the TiO2 surface led to different interactions between the adsorbate and OD groups. At different pH levels, several surface complexes of acetate can be formed such as monodentates or bidentates. Under UV(A) irradiation of TiO2 aqueous suspensions, the formation of hydroxyl and methoxy radicals evidenced as the corresponding spin-adducts, was found to dominate in alkaline and acidic suspensions respectively. Two possible pathways for the oxidation of acetate have been suggested at different pH levels in solution in terms of the source of the spin adduct formed. These proposed pathways were found to be in good agreement with ATR-FTIR and EPR results.

Reactions of nitroxide radicals in aqueous solutions exposed to non-thermal plasma: limitations of spin trapping of the plasma induced species

Low temperature (‘cold’) atmospheric pressure plasmas have gained much attention in recent years due to their biomedical effects achieved through the interactions of plasma-induced species with the biological substrate. Monitoring of the radical species in an aqueous biological milieu is usually performed via electron paramagnetic resonance (EPR) spectroscopy using various nitrone spin traps, which form persistent radical adducts with the short-lived radicals. However, the stability of these nitroxide radical adducts in the plasma-specific environment is not well known. In this work, chemical transformations of nitroxide radicals in aqueous solutions using a model nitroxide 4-oxo-TEMPO were studied using EPR and LC-MS. The kinetics of the nitroxide decay when the solution was exposed to plasma were assessed, and the reactive pathways proposed. The use of different scavengers enabled identification of the types of reactive species which cause the decay, indicating the predominant nitroxide group reduction in oxygen-free plasmas. The 2H adduct of the PBN spin trap (PBN-D) was shown to decay similarly to the model molecule 4-oxo-TEMPO. The decay of the spin adducts in plasma-treated solutions must be considered to avoid rendering the spin trapping results unreliable. In particular, the selectivity of the decay indicated the limitations of the PTIO/PTI nitroxide system in the detection of nitric oxide.

An identification of the C–C bonding spin adduct in the spin trapping of N-methyl benzohydroxamic acid radical by 5,5-dimethyl-1-pyrroline N-oxide

A detailed knowledge of the spin trapping of the radicals by spin traps is crucial for the elucidation of the reaction mechanisms involving radicals and the rational design of the novel efficient spin traps experimentally. In this study, the spin trapping of N-methyl benzohydroxamic acid radical (·N-MeBHA) produced in the reaction of 2,5-dichloro-1,4-benzoquinone (DCBQ) with N-methyl benzohydroxamic acid has been systematically investigated employing 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap theoretically. The tautomerization behavior of ·N-MeBHA radical has been firstly investigated, and four tautomers including C- and N-centered forms have been located. After that, the nature of the formed spin adducts has been explored as well as the thermodynamic and kinetic parameters associated with the spin-trapping process. Moreover, the reaction of the ·N-MeBHA radical with the C-centered quinone ketoxy radical has been studied. Besides the available C–N bonding product identified experimentally, more stable C–C bonding products have also been observed. Additionally, significant catalytic role of explicit water molecules should be highlighted in the tautomerization reaction of the ·N-MeBHA radical and the keto–enol tautomerization reaction of the final products. This study demonstrates for the first time the possibility of the existences of the C-centered ·N-MeBHA radical and the C–C bonding product in the reaction of DCBQ and N-MeBHA, providing new insights into the reaction mechanisms between polyhalogenated quinones and hydroxamic acids.

Metal-Diazo Radicals of α-Carbonyl Diazomethanes.

Metal-diazo radicals of α-carbonyl diazomethanes are new members of the radical family and are precursors to metal-carbene radicals. Herein, using electron paramagnetic resonance spectroscopy with spin-trapping, we detect diazo radicals of α-carbonyl diazomethanes, induced by [RhICl(cod)]2, [CoII(por)] and PdCl2, at room temperature. The unique quintet signal of the Rh-diazo radical was observed in measurements of α-carbonyl diazomethane adducts of [RhICl(cod)]2 in the presence of 5,5-dimethyl-pyrroline-1-N-oxide (DMPO). DFT calculations indicated that 97.2% of spin density is localized on the diazo moiety. Co- and Pd-diazo radicals are EPR silent but were captured by DMPO to form spin adducts of DMPO-N∙ (triplet-of-sextets signal). The spin-trapping also provides a powerful tool for detection of metal-carbene radicals, as evidenced by the DMPO-trapped carbene radicals (DMPO-C∙, sextet signal) and 2-methyl-2-nitrosopropane-carbene adducts (MNP-C∙, doublet-of-triplets signal). The transformation of α-carbonyl diazomethanes to metal-carbene radicals was confirmed to be a two-step process via metal-diazo radicals.

Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase

The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that 1O2 generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte.

Theoretical studies on the spin trapping of the 2-chloro-5-hydroxy-1,4-benzoquinone radical by 5,5-dimethyl-1-pyrroline N-oxide (DMPO): the identification of the C–O bonding spin adduct

The C–O bonding spin adduct has been identified in the spin trapping of 2-chloro-5-hydroxy-1,4-benzoquinone radical by DMPO.

Implications of Xanthohumol Enrichment on the Oxidative Stability of Pale and Dark Beers

Xanthohumol, a hop-derived flavonoid, has been linked to a number of positive health benefits, which has spurred an interest in the production of xanthohumol-enriched beers. The aim of the present study was to investigate the implications of xanthohumol enrichment on the oxidative stability of wort and beer. The levels of radicals in a model beer (5.8% ethanol, v/v) spiked with xanthohumol were evaluated by electron spin resonance. Xanthohumol exhibited radical scavenging properties at lower concentrations, reducing the formation of spin adducts by around 10% at 0.1 mg L−1. However, a trend toward radical formation was observed for concentrations higher than 5 mg L−1, indicative of a pro-oxidant effect. The influence of xanthohumol on the oxidative stability of the model beer was further determined by measuring the consumption of O2 in the presence of EBC worts made from different malts (pilsner, melano, and black). The addition of 10 mg L−1 of xanthohumol was also responsible for an increase of O2 uptake in the pilsner wort, whereas no effect was observed for black wort because of a possible interaction of xanthohumol with high-molecular-weight compounds generated during roasting. It has thus been demonstrated that xanthohumol-enriched pilsner beers could be prone to oxidation and, therefore, be less stable than xanthohumol-enriched beers brewed from dark malts.

Oxidative stress and DNA damages induced by 1-nitropyrene in human lung fibroblasts: New insights into the mechanisms of genotoxicity and EPR-spin trapping direct monitoring of free radicals at subcellular levels

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs are ubiquitous contaminants released in the atmosphere from the incomplete combustion of organic matter and pesticide intermediates nitration. Many studies have established that 1-nitropyrene (1-NP), one the most abundant nitro-PAH pollutant in diesel exhaust, is associated with increased risk of cancer and inflammatory diseases via possible metabolic pathways, including ring oxidation, nitroreduction or conjugation reactions. In addition, genotoxicity tests (e.g. comet assay and Ames test), demonstrated that 1-NP is a potent mutagen and genotoxic agent. However, the mechanisms underlying the promotion of DNA damages remain to be fully elucidated in relation with the progression of oxidative stress due to the stimulation of cellular free radical sources. Methods: On human lung fibroblasts exposed to environmentally relevant doses of 1-NP (1-10 microM) for 24–48 h, we performed: (i) cytokinesis-blocked micronucleus (CBMN) assay in association with immunofluorescence staining of centromere protein A in micronuclei to distinguish between induction of structural or numerical chromosome changes; (ii) EPR experiments using a set of spin trapping agents (i.e. the phosphorylated nitrone DEPMPO (Culcasi et al., 2012), the new stereoselective superoxide probe Me4CyDEPMPO (Gosset et al., 2011)) and mitochondrial-targeted nitrones; and (iii) the evaluation of oxidative stress from protein carbonyls, lipid peroxidation and antioxidant depletion. Major results: One advantage of the CBMN assay is its ability to elucidate the genotoxic mechanism of agents with clastogenic or aneugenic modes of action. The data provide the first evidence for a superoxide-related aneugenic mode of action at low 1-NP doses and a dominant clastogenic mechanism as the 1-NP doses and exposure time increased. This two-steps mechanism differs from that induced by CeO2-nanoparticles, an oxidant present in diesel exhausts (Benameur et al., 2014). From the EPR detection of superoxide and hydroxyl radical spin adducts, two free radical cellular sources activated by 1-NP exposure were hypothetized, i.e. NADPH oxidase and mitochondria, this latter being likely involved in oxidative stress damages development. References Culcasi, et al., 2012. Chem. Biol. Interact.. Gosset, et al., 2011. Bioorg. Med. Chem.. Benameur, et al., 2014. Nanotoxicology.

Generation Mechanism of Deferoxamine Radical by Tyrosine-Tyrosinase Reaction

Nitroxide radical formations of deferoxamine mesylate (DFX) that is used clinically to treat iron-overload patients was examined by a tyrosine-tyrosinase reaction system as models of the H-atom transfer or proton-coupled electron transfer. When DFX was exposed to the tyrosine-tyrosinase reaction, nine-line ESR spectrum (g = 2.0063, hfcc; aN = 0.78 mT, aH(2) = 0.63 mT) was detected, indicating that the oxidation of DFX leads to a nitroxide radical. The signal intensity of the DFX radical increased dependently on the concentrations of tyrosine and tyrosinase. The amounts of DMPO-OH spin adducts via the tyrosine-tyrosinase reaction declined with DFX. Furthermore, mass spectra of an extra removed from the tyrosine-tyrosinase reaction mixture showed that the enzyme reactions might not be degradations of DFX. Therefore, there might be two types of DFX reaction passways, which could be through an internal electron transfer from tyrosine and hydrogen absorptions by ·OH directly.

Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study.

We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity.

Atmospheric nonequilibrium mini-plasma jet created by a 3D printer

In this study, a small-sized plasma jet source with a 3.7 mm head diameter was created via a 3D printer. The jet’s emission properties and OH radical concentrations (generated by argon, helium, and nitrogen plasmas) were investigated using optical emission spectrometry (OES) and electron spin resonance (ESR). As such, for OES, each individual gas plasma propagates emission lines that derive from gases and ambient air inserted into the measurement system. For the case of ESR, a spin adduct of the OH radical is typically observed for all gas plasma treatment scenarios with a 10 s treatment by helium plasma generating the largest amount of OH radicals at 110 μM. Therefore, it was confirmed that a plasma jet source made by a 3D printer can generate stable plasmas using each of the aforementioned three gases.