5742
Langmuir 1999, 15, 5742-5752
Reactive Sites at the Surface of Crocidolite Asbestos† Gianmario Martra, Elena Chiardola, Salvatore Coluccia, Leonardo Marchese, Maura Tomatis, and Bice Fubini* Dipartimento di Chimica IFM, Universita` degli Studi di Torino, Via P. Giuria 7, I-10125 Torino, Italy Received October 16, 1998. In Final Form: January 7, 1999 The reactive sites at the surface of crocidolite (amphibole asbestos) which may play a role in the toxicity of the fibrous mineral have been investigated by measuring the adsorption of nitric oxide (infrared spectroscopy and adsorption calorimetry) and the release of free radicals in aqueous suspensions. The fibers have been thermally modifiedsprogressively heated in vacuo up to 800 °C, calcined in air at 400 °C, and kept under hydrothermal conditions at 200 °Csin order to mimic modifications likely to occur upon weathering. While the crystal structure, as revealed by XRD, was stable up to 600 °C, IR spectroscopy indicated that from 400 to 600 °C OH groups are eliminated and changes in the lattice vibrations occur. Calcination at 400 °C, which oxidizes part of Fe2+ to Fe3+, as evidenced by diffuse reflectance UV-vis spectroscopy, eliminates OH groups and modifies lattice vibrations. The hydrothermal treatment causes oxidation of Fe2+ only to a limited extent. Upon contact with deuterated water, irreversibly adsorbed H2O is displaced by D2O and exposed OH groups are exchanged into OD. Nitric oxide adsorbs onto poorly coordinated surface cations from which water and hydroxyl groups have been removed, the maximum adsorptive capacity being attained on samples heated at 400 °C. Mononitrosylic species irreversibly held on different types of surface iron ions are formed, and a part of them is converted into dinitrosyls when increasing the NO pressure. The heat of adsorption decreases from 130 kJ mol-1 (strongest mononitrosyls) to 20 kJ mol-1 (addition of NO to a mononitrosyl). NO is also adsorbed on hydrothermally treated crocidolite, but the IR spectra reveal a different site distribution. No nitric oxide is adsorbed on calcined crocidolite. Original crocidolite generates free radicals in aqueous suspensions, revealed by a carboxylate radical originating from the formate ion, but this property is lost both upon calcination and hydrothermal treatment.
1. Introduction Asbestos is the common name given to a group of naturally occurring silicate minerals in the serpentine and amphibole series possessing a fibrous habit.1 Exposure to asbestos fibers is associated with the development of lung cancer and pleural mesothelioma.2 Consequently the International Agency for Research on Cancer has declared all asbestos carcinogenic to humans3 and their industrial use has been strictly regulated or banned from most Western countries. The mechanism of action of the minerals at the molecular level is still unclear despite massive work performed by biologists, biochemists, and toxicologists.4 It is generally agreed that the fiber length and width, on one hand, are important parameters in determining toxicity but also that mineralogical and chemical composition of the fibers plays a crucial role in the ultimate pathogenicity.4 Iron present in the fiber or deposited on it has been reported to be implied in the pathogenic mechanism by several authors as reviewed in Hardy and Aust.5 The difficulties in defining the chemical role played by the fibers in the onset of the pathogenic response largely * To whom correspondence should be sent. Phone: +39-0117607566. Fax: +39-011-6707855. E-mail:
[email protected]. † Presented at the Third International Symposium on Effects of Surface Heterogeneity in Adsorption and Catalysis on Solids, held in Poland, August 9-16, 1998. (1) Veblen, D. R.; Wylie, A. G. In Reviews in Mineralogy; Guthrie, G. D., Mossman, B. T., Eds.; Book Crafters: Washington, USA, 1993; Vol. 28, p 61. (2) Mossman, B. T.; Bignon, J.; Corn, M.; Seaton, A.; Gee, A. J. Science 1990, 247, 294. (3) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Asbestos IARC: Lyon, France, 1977; Vol. 14, p 11. (4) Kane, A. B. In Mechanisms of Fibre Carcinogenesis; Kane, A. B., Boffetta, P., Saracci, R., Wilburn, J. D., Eds.; IARC Scientific Publication No. 140; International Agency for Research on Cancer: Lyon, France, 1996; p 11. (5) Hardy, J. A.; Aust, A. E. Chem. Rev. 1995, 95, 97.
resides on the characteristics of particulate toxicants which act in the body in solid form and not as a singular molecular entity. Particulates act through their surface which are heterogeneous in nature, exhibiting a large variety of surface sites. Furthermore, particulates interact at various stages with living matter and different surface sites may be involved in each stage. A clear picture of the distribution of surface reactive sites is thus of paramount importance for the understanding of the mechanism of toxicity.6 The identification of the surface sites implied in asbestos toxicity is important for at least two main reasons: (i) indicate which action other than costly and dangerous removal could be taken to inactivate asbestos placed in buildings, etc.; (ii) suggest appropriate routes to prepare safe asbestos substitutes. We have therefore planned a systematic investigation on the surface topology and reactivity of various asbestos and on the modifications in surface propertiesshence in biological activityswhich can be attained by aimed thermal or chemical treatments. In the present paper we have considered crocidolite (fibrous riebeckite) which, due to its high toxicity, is one of the most studied amphibole asbestos.2,4,5,7-9 Crocidolite, chemical composition Na2Fe3+2(Fe2+,Mg2+)3Si8O22(OH)2, has the typical amphibole structure reported in Chart 1 with a double chain of linked silica tetrahedra which forms the asbestos axis. These chains are cross linked with bridging cations in octahedral coordination. The ions are (6) Fubini, B.; Aust, A. E.; Bolton, R. E.; Borm, P. J. A.; Bruch, J.; Ciapetti, G.; Donaldson, K.; Elias, Z.; Gold, J.; Jaurand, M. C.; Kane, A. B.; Lison, D.; Muhle, H. ATLA, in press. (7) Lund, L. G.; Aust, A. E. Carcinogenesis 1992, 13, 637. (8) Adachi, S.; Yoshida, S.; Kawamura, K.; Takahashi, M.; Uchida, H.; Odagiri, Y.; Takemoto, K. Carcinogenesis 1994, 15, 753. (9) Werner, A. J.; Hochella, M. F.; Guthrie, G. D.; Hardy, J. A.; Aust, A. E.; Rimstidt, J. D. Am. Miner. 1995, 80, 1093.
10.1021/la9814541 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/25/1999
Active Sites on Asbestos
Langmuir, Vol. 15, No. 18, 1999 5743 Chart 1
located in four distinct sites, of which two (indicated as M1 and M3) are coordinated to four oxygen atoms and two hydroxyl groups, one (M2) to six oxygen atoms, and another one (M4) to eight oxygens in lower symmetry. The M1, M2, and M3 sites may be occupied by Mg2+, Fe2+, and Fe3+ ions, while M4 sites are generally filled with Na+. Iron in the fiber promotes free radical generation yielding ultimately reactive oxygen species (ROS) which by damaging DNA cause mutations and possible initiation of a neoplastic process.5,10 Only few of the iron ions actually exposed at the surface appear implied in the free radical reaction:11 assignment of the active ions to a defined M site is however not straightforward. The comparison of long and short amosite fibers and the use of chelators to modify the state of the iron in the fibers12 has led to the conclusion that isolated iron ions at the M1M3M1 positions undergo more readily redox reactions. This investigation however, carried out by IR spectroscopy, only monitored the stretching bands of structural OH in the coordination sphere of cations in the bulk of the fibers, so that results could be obtained neither on bulk M2 or M4 sites, which are only coordinated to oxygen atoms, nor on the cationic centers exposed at the surface of the fibers. To evidence the redox and coordination characteristics of surface iron ions we have studied the adsorption of nitric oxide on crocidolite, as this molecule has been usefully employed to probe the properties of iron centers on the surface of various materials.13-15 Moreover the adsorption of nitric oxide is relevant per se in asbestos toxicity. It has been recently reported that NO is readily adsorbed on various asbestos and that it may play a role in the enhanced toxicity of mineral fibers caused by cigarette smoke.16 The adsorption of NO may be also involved in the pathogenic process for several reasons: (i) NO is one of the chemicals produced by alveolar macrophages attempting to phagocyte the inhaled fibers. Exposure of rats to crocidolite asbestos by inhalation caused an increase in nitric oxide metabolites from alveolar macrophages.17 (ii) NO or products of reactions of NO with other small molecules (e.g. the superoxide anion) can damage a variety of biomolecules leading to toxicity. (iii) NO is produced enzymatically from arginine in cells by constitutive or inducible nitric oxide synthase (iNOS). Adsorption on asbestos may continuously activate these enzymes, with excessive production of it. (10) Kamp, D. W.; Graceffa, P.; Pryor, W. A.; Weitzman, S. A. Free Radical Biol. Med. 1992, 12, 293. (11) Fubini, B.; Mollo, L. Toxicol. Lett. 1995, 951, 82. (12) Graham, A.; Higinbotham, J.; Allan, D.; Donaldson, K.; Turvey, K.; Beswisk, P. H. Ann. Occup. Hyg. 1998, submitted for publication. (13) Busca, G.; Lorenzelli, V. J. Catal. 1981, 72, 303 and references therein. (14) Yuen, S.; Chen, Y.; Dumesic, J. A.; Topsoe, N.; Topsoe, H. J. Phys. Chem. 1982, 86, 3022. (15) Boccuzzi, F.; Guglielminotti, E.; Pinna, F.; Signoretto, M. J. Chem. Soc., Faraday Trans. 1995, 91, 3237. (16) Leanderson, P.; Lagesson, V.; Tagesson, C. Envir. Health Persp. 1997, 105, suppl. 5, 1037.
It has been recently reported that NO participated with iron in the oxidation of DNA in asbestos-treated human lung epithelial cells18 and that asbestos inhalation induced activation of iNOS in exposed rats19 suggesting a key role for NO in the mechanisms of asbestos-induced injury. In this paper we report a systematic study of NO adsorption on crocidolite samplesssubmitted to various treatments aimed to progressively modify their surface featuressin order to identify the surface sites able to interact with NO and investigate their adsorption behavior. Experiments were carried out also on crocidolite fibers oxidized by calcination in air at 400 °C or kept under hydrothermal conditions at 200 °C, as these treatments may mimic the effect of weathering over very long periods of time, thus, the situations of asbestos kept for long periods of time in open air buildings. The potential for free radical release of the heat-modified asbestos has also been evaluated by means of a spin trapping technique previously employed by us and other authors with aqueous suspensions of asbestos fibers.10,11,20,21 2. Experimental Section 2.1. Materials. Crocidolite was from UICC (Union Internationale Contre le Cancer) from the same batch employed in previous research.11,21 The specific surface area of crocidolite (BET method, Quantasorb) is 8 m2 g-1. High-purity O2 and NO (Matheson) were employed for oxidation and adsorption experiments, respectively. D2O from Stohler Isotope Chemicals (98.3% D atoms) was used in isotopic exchange experiments. O2 was admitted onto the samples without further purification except liquid-nitrogen trapping, while NO and D2O were previously purified by several freeze-pump-thaw cycles. The spin trap DMPO (5,5′-dimethyl-1-pyrroline N-oxide), from Fluka, was purified by filtration through activated charcoal according to the method proposed by Buettner and Oberley.22 Sodium formate was from Sigma. Phosphate buffers (monoand disodium phosphate) were from Merck. 2.2. Thermal Treatments. Outgassing treatments at increasing temperature were carried out on samples placed in infrared or calorimetric cells connected to conventional vacuum lines (residual pressure: 1.0 × 10-6 Torr; 1 Torr ) 133.33 Pa) allowing all thermal treatments and adsorption-desorption experiments to be carried out in situ. The samples for the IR experiments were kept at the desired temperature for 45 min, whereas the treatment time was prolonged to 2 h in the case of samples for the calorimetric measurements. In fact, in these cases significantly higher amounts of fibers were treated, and consequently, a longer time to attain equilibrium conditions was needed. Calcination of fibers was performed by heating in air at 400 °C for 2 h. Similar oxidative treatments were carried out on crocidolite in the IR cell. In these cases, the fibers, previously outgassed at room temperature, were heated in situ at 400 °C for 2 h in the presence of 160 Torr O2. Such treated samples were then outgassed at the same temperature for 45 min. Calcination in air slightly reduced the specific surface area to 7 m2 g-1. Hydrothermal conditions were achieved by keeping the fibers for 72 h at 200 °C in a Teflon-lined autoclave. This procedure slightly increased the specific surface area from 8 to 9 m2 g-1. 2.3. Experimental Techniques. X-rays Diffraction. To control the crystallinity all the samples have been examined by (17) Quinlan, T. R.; Berube, K. A.; Hacker M. P.; Taatjes, D. J.; Timblin, C. R.; Goldberg, J.; Kimberley, P.; O’Shaughnessy, P.; Hemenway, D.; Torino, J.; Jimenez, L. A.; Mossman, B. T. Free Radical Biol. Med. 1998, 24, 778. (18) Chao, C. C.; Park, S. H.; Aust, A. E. Arch. Biochem. Biophys. 1996, 326, 152. (19) Tanaka, S.; Choe, N.; Hemenway, D. R.; Zhu, S.; Matalon, S.; Kagan, E. J. Clin. Invest. 1998, 102, 445. (20) Zalma, R.; Bonneau, L.; Jaurand, M. C.; Guignard, J.; Pezerat, H. Can. J. Chem. 1987, 652, 338. (21) Gold, J.; Amandusson, H.; Krozer, A.; Kasemo, B.; Ericsson, T.; Zanetti, G.; Fubini, B. Envir. Health Persp. 1997, 105, suppl. 5, 1021.
5744 Langmuir, Vol. 15, No. 18, 1999 X-rays diffraction using Philips diffractometer PW1830 using a Co KR radiation. Infrared spectra (4 cm-1 resolution) of the samples were obtained by a Bruker IFS88 spectrometer equipped with an MCT detector. Vibrational studies of the framework structure of the samples were performed on disks containing crocidolite diluted in KBr. The fibers were submitted to the various treatments (heating at increasing temperature under vacuum, calcination, and hydrothermal treatment) and then mixed with KBr powder. The ratio by weight of the two compounds was varied in the 1/100 to 1/200 range, depending on the intensity of the crocidolite bands present in the different spectral regions to analyze. Infrared spectra of adsorbed NO were obtained by using self-supporting pellets of crocidolite (ca. 15 mg cm-2). Because of the extremely poor transparency of the samples in such form in the 4000-2500 cm-1 range, due to light scattering, a system of condenser mirrors, placed between the IR cell and the detector, was employed. In this way, most part of the heavily scattered IR radiation transmitted through the pellets was again focused onto the MCT. Parallel experiments carried out on reference samples with wellknown IR features (Al2O3, TiO2) indicated that neither artifacts nor distortion of the spectral profile was introduced by using this procedure. Diffuse reflectance (DR) UV-vis-NIR spectra of the samples were recorded by a Perkin-Elmer Lambda 19 spectrophotometer equipped with an integrating sphere and using BaSO4 as reference powder, placed in the same type of cell employed for the samples. All spectra were recorded in air. Adsorption Calorimetry. Heats of adsorption have been determined by means of a Tian-Calvet microcalorimeter (Setaram) connected to a volumetric apparatus which allowed simultaneous measurement of adsorbed amount (uptake, na), heat released (Q), and equilibrium pressure (p) for small increments of nitric oxide dosed onto the crocidolite sample. The procedure has been thoroughly described in previous papers for other solids and adsorbates.23 The samples were outgassed in the calorimetric cells for 2 h at 400 °Csthe temperature at which IR spectroscopy reveals a maximum in the NO adsorptive capacitysand subsequently located into the calorimetric vessel without exposure to the atmosphere. The temperature of the calorimeter was maintained at 30 °C throughout the adsorption experiment. A typical adsorption sequence comprised two runs, with the following procedure: (i) dosing successive amounts of NO to the sample up to a pressure of typically 10 Torr (Ads I); (ii) desorption at 30 °C under vacuum; (iii) readsorption of similar doses in order to evaluate the extent of adsorption reversible at room temperature (Ads II). The fraction of adsorbed NO irreversibly held at the surface is evaluated from the difference between Ads I and Ads II. Free Radical Detection by Means of the Spin Trapping Technique. The technique employed for the detection of free radicals generated in aqueous suspensions of particulates has been previously described.11 A water-soluble nitroxide, 5,5′dimethyl-1-pyrroline N-oxide (DMPO), was employed as spin trap. In aqueous medium it can form stable radical adducts, allowing quantitative evaluation of free radical release. The pH in all experiments was kept at 7.4 (phosphate buffer). The intensity of the EPR signal measures the number of radicals trapped, which is proportional to those formed in the solution. The formate ion is traditionally used as a target molecule to measure the radical-generating power of solids. Following homolytic cleavage of the C-H bond of formate, a carboxylate radical is formed, which, with the spin trap, gives rise to the DMPO-COO•- adduct, characterized by a typical EPR spectrum. The test is performed as follows: 0.5 mL of aqueous phosphate buffer (0.5 M HPO42- and 0.5 M in H2PO4-, pH ) 7.4), 1 mL of DMPO (0.05 M) in distilled water (treated with chelex 100 to eliminate traces of metal ions and purified by filtering through activated charcoal), and 1 mL of 2.0 M HCOONa are placed in contact with 45 mg of the solid sample. The suspension is then incubated at 37 °C and shaken in a dark reactor. Aliquots of the suspension are withdrawn at 25 and 55 min intervals and then filtered through porosity filters. The liquid (22) Buettner, G. R.; Oberley, L. W. Biochem. Biophys. Res. Commun. 1978, 83, 69. (23) Fubini, B. Thermochim. Acta 1988, 135, 19.
Martra et al. is introduced into a flat cell, appropriate for aqueous solutions, and the corresponding EPR spectrum recorded at room temperature some 5 min after withdrawal of the aliquots. EPR spectra were obtained by using a Varian E109 EPR spectrometer working in the X band (9-9.5 GHz) with a double resonant cavity. For the evaluation of the number of spins DPPH (purified diphenylpicrylhydrazyl from Sigma containing about 0.95 unpaired spins per molecule) was employed.
3. Results and Band Assignment As reported in the Introduction, this work was aimed to study the surface centers of crocidolite fibers. However, preliminary investigations of the effects of the various treatments on the internal crystal structure of the crocidolite fibers were performed by IR, DR UV-vis-NIR, and XRD spectroscopies. This allowed us to ascertain whether the observed evolution of surface centers caused by the adopted treatments are only related to modifications of the surface and/or first subsurface layers of the fibers or are accompanied by structural changes of the bulk material. 3.1. Study of the Internal Fibrous Structure by Infrared Spectroscopy. Infrared spectroscopy offered many opportunities for identification and structure elucidation of amphiboles.24-26 In particular, the infrared spectra of these materials contain two regions of specific interest:26,27 the 3680-3580 cm-1 range, where absorption bands due to the stretching vibration of structural hydroxyl species are observed, and the 1200-400 cm-1 range, where lattice vibrations absorb. Figure 1A,B displays the spectral features in these two ranges of crocidolite samples heated at increasing temperature in vacuo and then dispersed in KBr. The spectra in the hydroxyl stretching region (Figure 1A) were recorded by diluting the fibers in KBr in a 1/100 ratio by weight, while, due to the presence of some very intense absorptions, a 1/200 ratio by weight was used to observe the lattice vibration bands (Figure 1B). In the high-frequency region of the spectra of the original sample three narrow bands are present at 3649 (weak), 3635, and 3619 cm-1 (Figure 1A,a). Each is assignable to a hydroxyl group interacting with a different ensemble of three cations located in M1M3M1 positions. These three components are associated with an OH species coordinated to two magnesium and one iron ions (band at 3649 cm-1), one magnesium and two iron ions (band at 3635 cm-1), and three iron ions (band at 3619 cm-1).27 The presence of Fe2+ or Fe3+ ions in one position should involve a shift in the frequency of each components of about 5 cm-1.27 However, the narrowness of the bands in Figure 1A, exhibiting full widths at half-maximum in the 4-3 cm-1 range suggests that Fe2+ and Fe3+ are located in the three cases at fixed position and are not interchangeable one with the other. At lower frequency, the lattice spectral features of the original sample are dominated by an intense and complex band with a main component at 991 cm-1 and two weaker and partly resolved absorptions at 1048 and 975 cm-1 (Figure 1B,a). These absorptions are assignable to the asymmetric stretching vibrations of species of the type X-O-Y (X ) Si; Y ) Mg, Fe).28,29 Weaker bands are observed at 1145 and 1106 cm-1, due to the symmetric stretching modes of tetrahedral and octahedral X-O-X (24) Luce, R. W. U.S. Geol. Surv., Prof. Pap. 1971, 750B, 199. (25) Yariv, S.; Helle-Kallai, L. Clays Clay Miner. 1975, 23, 145. (26) Luys, M.-J.; De Roy, G.; Vansant E. F.; Adams, F. J. Chem. Soc., Faraday Trans. 1 1982, 78, 3561. (27) Lewis, I. R.; Chaffin, N. C.; Gunter, M. E.; Griffiths, P. R. Spectrochim. Acta, Part A 1996, 52, 315.
Active Sites on Asbestos
Langmuir, Vol. 15, No. 18, 1999 5745
Figure 1. Effect of the treatment in vacuo at increasing temperature on the vibrational features of the bulk structure of crocidolite. IR spectra in the 3680-3580 cm-1 (section A) and in the 1200-400 cm-1 range (section B) of the following: (a) original sample; crocidolite outgassed for 45 min at (b) 400 °C, (c) 600 °C, and (d) 800 °C. These spectra were obtained by diluting the original or the treated fibers in KBr (see Experimental Section and section 3.1).
species, respectively.28,30 In the 800-600 cm-1 range several weak bands due to symmetric O-X-O stretching and to X-O-X deformation modes are present, while the region at lower frequency exhibits two weak and complex absorptions at 546 and 510 cm-1 and an intense peak at 446 cm-1, assignable to O-X-O deformation modes.28,29 Essentially the same features are present in both ranges for the fibers heated in vacuo at 400 °C (curves a in Figure 1A,B). In contrast, the three bands due to structural hydroxyl groups disappear by treating the sample at 600 °C (Figure 1A,c), and changes in the lattice vibrational absorptions occur (Figure 1B,c). All components appear broader, less resolved, and shifted to lower frequency. Heavier modifications of these bands are observed in the spectrum of the sample heated in vacuo at 800 °C, where essentially two very broad and complex bands, constituted by several heavily overlapped components, are present in the 1200-850 and 600-400 cm-1 ranges (Figure 1C,d). Noticeably, the XRD diffractograms of the fibers heated in vacuo at increasing temperature (not reported) indicate that no modification of the crystalline features occurs up to 600 °C, whereas the treatment at 800 °C produces a collapse of the structure. Similar investigations were carried out on crocidolite samples treated under hydrothermal conditions or calcined in air at 400 °C. The results obtained are shown in Figure 2A,B, where the spectrum of the original sample (curves a) in the hydroxyl and lattice vibrations regions is compared with those of the treated fibers (curves b and c). The vibrational features of the original sample (Figure 2A,B,a) and those of crocidolite which experienced hydrothermal conditions (Figure 2A,2B,b) appear very similar, indicating that this treatment does not affect the bulk structure of the fibers. In the case of the calcined sample, no bands are detected in the 3680-3580 cm-1 range (Figure 2A,c), and absorptions in the lattice modes region appear quite different from those observed for the untreated crocidolite (Figure 2B,c). Calcination, besides (28) Flanigen, E. In Catalysis by Zeolites; Rabo, J. A., Ed.; ACS Monograph; American Chemical Society: Washington, DC, 1976; Vol. 171, p 80. (29) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds; Wiley-Interscience: New York, 1978; Chapter 2.
Figure 2. Effect of the calcination and hydrothermal treatments on the vibrational features of the bulk structure of crocidolite. IR spectra in the 3680-3580 cm-1 (section A) and in the 1200-400 cm-1 range (section B) of the following: (a) original sample; (b) crocidolite hydrothermally treated in Teflonlined autoclave at 200 °C for 72 h; (c) crocidolite calcined in air at 400 °C for 2. These spectra were obtained by diluting the original or the treated fibers in KBr (see Experimental Section and section 3.1).
the transformation of most part of Fe2+ ions into Fe3+ species (vide infra), results in the disappearance of structural hydroxyl groups and causes therefore some rearrangement of the internal structure of the fibers. These changes, however, affect only slightly the crystal features of crocidolite, as the XRD diffractogram of the calcined sample resulted very close to the original one (not reported). 3.2. Diffuse Reflectance UV-Vis-NIR Characterization. As reported in the Introduction, both Fe2+ and Fe3+ ions are present in the crocidolite fibers. The various treatments carried out on the samples may affect the relative amounts of these two species: a diffuse reflectance UV-vis-NIR study of the differently treated fibers thus was performed (spectra not reported). The DR UV-vis-NIR spectrum of the original crocidolite, gray-blue in color, exhibit ligand to metal charge transfer (LMCT) and d-d internal transitions of both Fe2+ and Fe3+ ions in octahedral coordination, and essentially the same features are present in the case of the sample outgassed at 400 °C. After outgassing of the sample at
5746 Langmuir, Vol. 15, No. 18, 1999
Figure 3. IR spectra of crocidolite (self-supporting pellet) outgassed at increasing temperature: (a) crocidolite in air; (b) after outgassing at room temperature for 45 min; after outgassing at (c) 150 °C; (d) 300 °C, and (e) 400 °C for 45 min. Inset: zoom of the 3780-3680 cm-1 range (lettering as in the main frame).
600 °C, crocidolite fibers turn to light gray, and the spectral components related to Fe2+ and Fe3+ ions appear superimposed on a continuos absorption ranging from the UV to the NIR region. By subsequent outgassing of the sample at 800 °C, the color turns to black, and only an intense continuous absorption is observed in the whole spectral range. This behavior results either from the formation of nonstoichiometric defects involving iron ions or to intervalence effects between adjacent ions. IR data presented above indicate that structural hydroxyl groups disappear after outgassing at 600 °C (Figure 1A,c). These OH- species act as counteranions for Fe2+ and Fe3+ centers, and then their removal is likely to produce some defect and/or redox reactions affecting the electronic properties of the iron ions. DR UV-vis-NIR measurements were carried out also on the calcined and hydrothermally treated samples. After calcination, bands due to electronic transitions related to Fe3+ species exhibit an intensity significantly higher with respect to the original sample, owing to the expected conversion of a large fraction of ferrous ions into ferric ones. The overall features of the electronic spectrum of the sample treated in hydrothermal conditions appear quite similar to those of the original one, except for a slightly higher intensity of the absorptions related to Fe3+ ions, which monitors the oxidation of a few Fe2+ to Fe3+ species. 3.3. Infrared Studies of the Surface Hydration Species. 3.3.1. Infrared Spectra of Crocidolite Outgassed at Increasing Temperature. The original fibers retain at the surface hydroxyl groups and adsorbed molecular water. These species play a quite important role, being the actual interfacial layer between the fibers and the biological medium. To investigate their features, an infrared analysis of the crocidolite sample in the form of self-supporting pellets was carried out. Owing to the rather low specific surface of the samples, sufficient amounts of surface species are only present in a nondiluted pellet. On the other hand, in this case, the bands due to vibrations of the lattice become so intense to completely absorb below 1200 cm-1. Figure 3 reports the IR spectra in the 3800-1500 cm-1 range of crocidolite in air (Figure 3a) and then progressively outgassed at increasing temperature (Figure 3bd). The spectral profile of the sample in air (Figure 3a) is
Martra et al.
dominated by three very intense absorptions in the 37003600 cm-1 range (maxima out of scale in the figure) due to the structural hydroxyl groups (see Figure 1A). On the low-frequency side of this peak a broad absorption is present in the 3600-2700 cm-1 range, resulting from the overlapping of the asymmetric and symmetric stretching bands of molecular water adsorbed on the surface and of the stretching modes of surface hydroxyl groups hydrogen bonded to neighbor surface OH species and/or molecular water.31 A series of weaker bands is present at lower frequency. Among these components, the one at 1630 cm-1 is ascribed to the bending mode of adsorbed molecular water,31 while the others are overtones and combination bands of the fundamental lattice vibrations observed in the 1200-400 cm-1 range (Figure 1B). By outgassing at room temperature, the 3600-2700 cm-1 absorption and the band at 1630 cm-1 decrease in intensity (Figure 3b), indicating that a part of the adsorbed molecular water has been removed. This fraction corresponds to multilayers of “liquidlike” water, usually present at the surface of any oxide exposed to ambient moisture, in weak interaction with the surface. By increase of the outgassing temperature to 150 °C a further decrease of the 3600-2700 and 1630 cm-1 absorptions occurs (Figure 3c) together with a parallel increase in the intensity of the 3740 cm-1 component (Figure 3c, inset). This indicates that further water molecules are desorbed from the fibers, and higher amount of free silanols are left at their surface. Treatment at 300 °C leads to a further decrease in intensity of the 3600-2700 cm-1 absorption (Figure 3d), which disappears after outgassing at 400 °C (Figure 3e). In contrast, the 1630 cm-1 band appears only slightly affected by these treatments. As water molecules are expected to be completely desorbed by outgassing at such high temperatures, the residue of the 1630 cm-1 component observed in the spectra after outgassing at 300 and 400 °C is ascribed to an overtone or combination band of lattice vibrational modes. In conclusion, the spectral behavior indicates that most part of the molecular water is desorbed from the surface by outgassing at 150 °C. The decrease of the 36002700 cm-1 absorption occurring above 150 °C is due to the removal from the surface of hydroxyl groups interacting one another through hydrogen bond. At the same time, increasing amounts of free silanols are left on the surface of the fibers, as indicated by the further increase in intensity of the 3740 cm-1 band (Figure 3d,e, inset). 3.3.2. Isotopic Exchange Experiment with D2O. The above results concern surface silanols and adsorbed molecular water but yield no information on the presence of hydroxyl groups bonded to magnesium and iron ions in octahedral layers exposed at the surface, as the 37003600 cm-1 range is dominated by the very intense absorptions due to these species present in overwhelming amounts in the bulk. To overcome this difficulty, isotopic exchange experiments of the surface hydration species with D2O were performed. In this way, besides molecular water and silanol groups, also hydroxyl species bonded to surface magnesium and iron ions, if present, are deuterated, whereas the analogous groups in the bulk, not accessible to D2O, must keep their OH nature unchanged. In the first part of the experiment, D2O was admitted onto the crocidolite fibers outgassed at room temperature (rt), kept in contact with the sample for 30 min, and then again outgassed at rt. The complete cycle was repeated until the bands due to D2O molecules irreversibly adsorbed (30) Whittaker, E. J. Acta Crystallogr. 1960, 13, 291. (31) Little, L. H. Infrared Spectra of Adsorbed Species, 1st ed.; Academic Press: London, 1966; Chapter 10.
Active Sites on Asbestos
Figure 4. Isotopic exchange experiment of surface hydration species of crocidolite with D2O. IR spectra (self-supporting pellet) of the following: (a) original crocidolited outgassed at room temperature (rt) for 45 min; (b) after admission of 18 Torr D2O; (c) after outgassing D2O at rt for 45 min; (d) after two further D2O adsorption/desorption cycles and subsequent outgassing at rt for 45 min. Bands labeled with the asterisk are due to adsorbed hydrocarbon impurities.
on the surface at rt reached their maximum intensity. The spectra of the sample during the first cycle and in final conditions (after three cycles) in the 3800-2000 cm-1 range are compared in Figure 4. The intense peaks due to bulk hydroxyl species and the broad absorption at 36002700 cm-1 related to hydrogen bonded OH groups and molecular water adsorbed on the surface are not affected by the admission of D2O vapor which causes instead the appearance of a broad and complex band at 2750-2000 cm-1 (Figure 4b), assigned to D2O molecules adsorbed on the fibers. As expected, this adsorption is partly decreased by the subsequent outgassing at rt, but also the 36002700 cm-1 band, mainly due to adsorbed H2O adsorbed molecule, slightly decreases in intensity (Figure 4c). As outgassing at room temperature of the original sample prior to the admission of D2O was long enough to remove all H2O molecules reversibly adsorbed on the surface at rt, it can be concluded that a fraction of H2O molecules initially irreversibly adsorbed on the surface become reversibly adsorbed after the contact with D2O. Similar features were observed for each stage of the exchange procedure, the band due to irreversibly (at rt) adsorbed D2O progressively increasing in intensity and that of the irreversibly adsorbed H2O species appearing progressively eroded. After three cycles these two absorptions reached their maximum and minimum intensity respectively (Figure 4d), indicating that equilibrium conditions for the H2O/D2O displacement at rt was attained. In this conditions the OD stretching (νOD) region is dominated by a broad band centered at 2620 cm-1, partly superimposed to a component at 2500 cm-1 due to an overtone or combination mode of lattice vibrations (see Figure 3). The 2620 cm-1 absorption should result from the overlapping of the stretching bands of D2O molecules adsorbed onto the surface and the νOD band of surface deuterioxyl, if any, interacting via hydrogen bond with D2O molecules. Furthermore, a weak absorption is observed at 2765 cm-1, due to the stretching vibration of free deuterated silanol groups. In a second part of the experiment the exchanged crocidolite was progressively outgassed at increasing temperatures, to desorb D2O molecules and leave unperturbed OD species at the surface. The results are shown in Figure 5, which also reports, for comparison, the spectrum in the 2800-2000 cm-1 range of the D2O exchanged crocidolite simply outgassed at rt (Figure 5a). By increase of the outgassing temperature the band at 2620 cm-1 undergoes a progres-
Langmuir, Vol. 15, No. 18, 1999 5747
Figure 5. IR spectra (self-supporting pellet) of the following: (a) crocidolite exchanged with D2O and then outgassed at room temperature for 45 min (the same as curve d in Figure 4); after outgassing at (b) 150 °C; (c) 300 °C and (d) 400 °C for 45 min. Inset: zoom of the 2700-2650 cm-1 range; curves c′ and d′ are as curves c and d, respectively, in the main frame.
sive erosion (Figure 5b-d), disappearing almost completely after the treatment at 400 °C (Figure 5d). Noticeably, after outgassing at 300 °C three well-resolved bands appear at 2692 (very weak), 2681, and 2669 cm-1 (Figure 5c and curve c′ in the inset) and slightly increase in intensity by subsequent outgassing at 400 °C (Figure 5d and curve d′ in the inset). These absorptions are located at the frequencies expected for the deuterated form of hydroxyl groups coordinated to magnesium and iron ions in the octahedral sheets on the basis of the ratio of the reduced mass of the OH and OD oscillators. As these bands appear after the exchange with D2O, which cannot penetrate inside the bulk of the structure, these deuterioxyl species must have surface character. This clearly indicates that octahedral layers of the crocidolite structure are exposed at the surface of the fibers. It must be noticed that the three νOD bands at 2692, 2681, and 2669 cm-1 exhibit a very low intensity with respect to the corresponding absorption in the 3680-3580 cm-1 due to bulk OH species, as a consequence of the low specific surface area of the fibers. 3.4. Characterization of Surface Centers by NO Adsorption. Besides hydroxyl groups, octahedral sheets at the surface of crocidolite fibers are expected to expose also magnesium and iron ions. To investigate the properties of these surface centers infrared and calorimetric experiments were carried out by using NO as probe molecule. 3.4.1. Infrared Study of Adsorbed NO. Figure 6 reports the IR spectra of NO adsorbed on crocidolite outgassed at increasing temperature. No nitric oxide is adsorbed on crocidolite simply evacuated at rt. This is somehow expected because under these circumstances the surface is still covered by a layer of molecular water (see Figure 3), hindering the interaction between probe molecules and cationic surface centers. By outgassing of the sample at increasing temperature, water molecules are progressively removed from the surface, and bands due to NO adsorbed on cations appear in the 2000-1500 cm-1 range. The admission of NO onto the sample outgassed at 150 °C produces a very weak and complex band at 1800 cm-1, with two evident shoulders at 1842 and 1750 cm-1 (Figure 6a). More intense and better resolved features are obtained by adsorbing NO on crocidolite outgassed at 300 °C, where
5748 Langmuir, Vol. 15, No. 18, 1999
Figure 6. IR spectra of NO (100 Torr) adsorbed on crocidolite (self-supporting pellet) outgassed at (a) 150 °C, (b) 300 °C, (c) 400 °C, and (d) 600 °C for 45 min each.
a peak at 1809 cm-1, with a poorly resolved shoulder at 1790 cm-1, and two weak bands at 1900 and 1740 cm-1 are observed (Figure 6b). With the sample outgassed at 400 °C the overall intensity of the bands of adsorbed NO appears slightly higher, and the spectrum is dominated by a main peak at 1800 cm-1, slightly asymmetric on the high-frequency side, accompanied by a band at 1730 cm-1 and a weak component at 1897 cm-1 (Figure 6c). Finally, only one weak band at 1807 cm-1 is present in the spectrum of NO adsorbed on crocidolite outgassed at 600 °C (Figure 6d), whereas no bands due to adsorbed NO were observed in the case of the sample outgassed at 800 °C. The overall intensity of the bands observed in all cases is very low, even for the sample pre-outgassed at 400 °C (Figure 6d), because of the low specific surface of crocidolite fibers. Due to this reason, the original spectra recorded in the presence of a high NO pressure had a large contribution from the rotovibrational spectrum of gaseous NO, which has been subtracted by means of a reference spectrum of gaseous nitric oxide. On the basis of the literature,14,15 the main component at ca. 1800 cm-1 and the weak absorption at ca. 1900 cm-1 present in the spectra of NO adsorbed on crocidolite outgassed at 300 and 400 °C (Figure 6,c,d) are assigned to a dinitrosylic species stabilized on highly coordinatively unsatured Fe2+ ions. The same assignment is proposed for the band at 1800 cm-1 in the spectrum of NO adsorbed on the sample outgassed at 150 °C (Figure 6a). In this case the overall spectrum is weaker, and the partner expected at 1900 cm-1 is probably too weak to be observed. Spectra of NO adsorbed on crocidolite outgassed at up to 400 °C also exhibit a component in the 1750-1730 cm-1 range, where mononotrosylic adducts on highly coordinatively unsatured Fe2+ centers were observed to absorb.14,15 The shoulder at 1842 cm-1 present in the spectrum of NO adsorbed on the sample outgassed at 150 °C (Figure 7a) is assigned to a mononitrosylic species on Fe3+ centers.15,32 Finally, the weak band observed by admitting NO onto the crocidolite outgassed at 600 °C (Figure 6d) is ascribed to a mononitrosylic species on a different type of Fe2+ ions (vide infra). In the case of the samples outgassed at 300 and 400 °C, the higher intensity of the bands allowed us to obtain a reasonable signal-to-noise ratio also for the spectra recorded when decreasing the NO pressure. The behavior (32) Harrison, P. G.; Thornton, E. W. J. Catal. 1978, 74, 2703.
Martra et al.
Figure 7. IR spectra of NO adsorbed on crocidolite (selfsupporting pellet) outgassed at 400 °C for 45 min, recorded in the presence of the following: (a) 100, (b) 50, and (c) 10 Torr NO and after outgassing at (d) room temperature; (e) 100 °C and (f) 200 °C for 45 min.
of the nitrosyl species as a function of the NO coverage is described in Figure 7, as it concerns crocidolite outgassed at 400 °C, this case being where the maximum adsorptive capacity toward NO was developed. Curve a in Figure 7 is the spectrum of crocidolite outgassed at 400 °C in the presence of a high NO coverage. By decrease of the NO pressure, a progressive erosion of the weak band at 1897 cm-1 and of the main peak at 1800 cm-1 occurs, while the 1735 cm-1 absorption increases in intensity (Figure 7b-d). A similar behavior was observed in the case of NO adsorbed on ferrous ions dispersed on silica14, 15 and attributed to the conversion of a dinitrosylic species stabilized on highly coordinatively unsaturated Fe2+ ions into a mononitrosylic one. Moreover, it must be noticed that a component at 1735 cm-1 is already present, with a weaker intensity, in the spectrum recorded under a high NO pressure (Figure 7a). This suggests that, after outgassing at 400 °C, highly uncoordinated Fe2+ ions able to absorb only one NO molecule are present. The reduced availability to coordinate ligands could reflect some particular structure of their environment, hindering the adsorption of a second NO molecule. During outgassing, a band at 1812 cm-1 is progressively revealed (Figure 7bd). A similar absorption was found in the spectra of NO dosed on Fe2+/SiO2 systems14,15 and assigned to a mononitrosylic adduct adsorbed on more coordinated Fe2+ ions. These species should be the same formed by admitting NO onto the sample outgassed at 600 °C (band at 1807 cm-1, Figure 6d). Finally, upon outgassing of the sample at 100 °C, the 1735 cm-1 band is depleted, and the 1812 cm-1 absorption is strongly reduced in intensity (Figure 7e). By subsequent outgassing of the sample at 200 °C also this last component disappears (Figure 7f). Similar NO adsorption experiments were carried out on crocidolite fibers exchanged with D2O and then outgassed at 400 °C (see Figure 5d). Admission of nitric oxide onto this sample produced the bands due to adsorbed NO molecules on iron ions previously described but did not produce any perturbation of the bands related to the deuterioxyl species, which also are coordinated to iron surface centers. This behavior suggests that NO molecules are not stabilized on the M1 and M3 iron cations carrying the deuterioxyl groups but probably are adsorbed on coordinatively unsaturated iron centers located in M2 position or at M1 or M3 surface sites lacking the OH in
Active Sites on Asbestos
Langmuir, Vol. 15, No. 18, 1999 5749
Figure 8. IR spectra of NO adsorbed on crocidolite treated in hydrothermal conditions (self-supporting pellet) and then outgassed at 400 °C for 45 min, recorded in the presence of (a) 100, (b) 50, and (c) 10 Torr NO and (d) after outgassing at room temperature for 45 min.
their coordination sphere. Furthermore, the presence of iron ions in surface defect positions (steps, kinks, corner, etc.) is to be considered.5,6,33 Nitric oxide adsorption was also carried out on crocidolite fibers heated in oxygen or treated in hydrothermal conditions and then outgassed at 400 °C. No bands due to adsorbed NO were observed in the case of the oxidized sample. Admission of nitric oxide on the hydrothermally treated crocidolite produced a spectral pattern with an overall intensity and shape similar to that obtained for the original sample (Figure 8). At the highest NO coverage, however, the main peak at 1800 cm-1 and the weak component at 1897 cm-1 are less intense with respect to the band at 1735 cm-1 (Figure 8a) than in the case of the crocidolite simply outgassed at 400 °C (Figure 7a). The hydrothermal treatment seems thus to reduce slightly the number of highly coordinately unsatured Fe2+ sites which can adsorb two NO molecules with respect to the analogous centers able to stabilize only one NO ligand. Furthermore, the absorption progressively revealed by decreasing the nitric oxide pressure and assigned to NO stabilized on less coordinately unsaturated Fe2+ centers appears located at 1820 cm-1 (Figure 8e), whereas it was observed at 1812 cm-1 for the original sample (Figure 7d). As in the adsorption on surface centers with partially occupied d orbitals, NO acts as a σ donor and a π acceptor;34 this behavior probably reflects a decreased availability of electron charge in the d orbitals of the surface center, with a consequent shift of the NO stretching vibrations toward the value observed for nitric oxide in the gas phase (1880 cm-1). This suggest that the hydrothermal treatment results in an increase of the oxidation state of these less uncoordinated iron surface centers. However, this oxidation should not correspond to the transformation of such ferrous ions into ferric ones, as the band due to mononitrosyl adsorbed on Fe3+ was observed at ca. 1830-1840 cm-1.15,32 A partial oxidation of these Fe2+ centers could occur through some rearrangement in the coordination sphere of oxygen atoms, leading to a partial charge transfer from Fe2+ ions to these surface ligands. Furthermore, DR UV-vis-NIR spectra have evidenced a slight increase in (33) Fubini, B.; Mollo, L.; Giamello, E. Free Radical Res. 1995, 23, 593. (34) Sheppard, N. In Vibration Properties of Adsorbates; Willes, E. F., Ed.; Cambridge Proceedings; Springer-Verlag: Berlin, 1980; p 165.
Figure 9. Adsorption of nitric oxide on crocidolite outgassed at 400 °C: full symbols, total adsorption (Ads I); empty symbols, reversible adsorption (Ads II): A, isotherms; B, isotherms calorimetric; C, differential heat vs uptake.
the amount of Fe3+ species after the hydrothermal treatment. It could be then supposed that the partial increase in oxidation state of these ferrous sites is due to the conversion from Fe2+ to Fe3+ of an iron cations in neighbor position. This newly formed ferric centers could then partly deplete the electron density of the neighbor ferrous sites. 3.4.2. Heat of Adsorption of NO on Crocidolite. Heats of adsorption of NO on crocidolite have been measured on a sample outgassed at 400 °C for 2 h in a vacuum in the calorimetric cell. The relevant data for the first (Ads I) and second (Ads II) runs are reported in Figure 9A (adsorption isotherms), Figure 9B (calorimetric isotherms), and Figure 9C (differential heat vs uptake). The two isotherms exhibit a different shape. Ads I is typical of the superposition of various phenomena, while Ads II, except for the first points, exhibits a nearly linear increase with pressure. At the same equilibrium pressure the adsorption values measured on Ads I are more than twice what adsorbed in Ads II, in agreement with the large amount of irreversibly adsorbed NO indicated by the spectra in Figure 7. The differences between Ads I and Ads II are even more pronounced between the two calorimetric isotherms, as the irreversible process is characterized by a much higher energy of adsorption than the reversible one (Ads II). When measured at the equilibrium pressure of 8 Torr, the average heat of irreversible adsorption, obtained by the differences in adsorbed amounts and released heat between Ads I and Ads II, is 120 kJ mol-1. The plot of differential heat vs adsorbed amounts (Figure 9C) reveals a substantial heterogeneity in adsorption sites as expected, the mononitrosyls and dinitrosyls being formed in parallel. The initial value extrapolated to zero coverage, 130 kJ mol-1, has to be assigned to the strongest NO coordinating
5750 Langmuir, Vol. 15, No. 18, 1999
Martra et al.
(water and/or contaminants) on the original sample but once exposed at the surface following outgassing remain available for radical generation. If the heating is carried out in air (Figure 10e) or samples are heated at 600 °C and above (Figure 10c,d), at the opposite no radical yield was detectable; i.e., all radical generating sites are destroyed. Similarly also the hydrothermal treatment inactivates nearly completely the fibers for this reaction, as just vestiges of the EPR spectrum are detectable (Figure 10f). 4. Discussion
Figure 10. Free radical release from aqueous suspensions of crocidolite fibers. EPR spectra of the [DMPO-CO2•-] adduct produced by contacting the DMPO aqueous solution (see Experimental Sections) with the following: (a) original sample; samples outgassed at (b) 400 °C, (c) 600 °C, and (d) 800 °C; (e) sample calcined in air at 400 °C for 2 h; (f) sample kept in hydrothermal conditions at 200 °C for 72 h.
sites. Upon increasing coverage, differential heat decreases down to 60 kJ mol-1 at about 10 Torr equilibrium pressure, where both mononitrosyls and dinitrosyls are formed (see Figure 7ab). The Ads II curve also decreases from an initial value of about 100 kJ mol-1 down to 20 kJ mol-1. 3.5. Free Radical Release from Aqueous Suspensions of Crocidolite. The variously treated crocidolite fibers were tested for free radical release in the presence of the formate ion. This test, widely described in previous papers21,33 evidences any moiety capable of hydrogen abstraction from the formate ion via a homolytic rupture of the carbon hydrogen bond. The reactions taking place may be summarized as follows in the absence of other radical intermediates:
Alternatively short-lived radicals may extract the hydrogen atom from formate before being trapped by DMPO, the latter just acting as a secondary trap. In any case the DMPO-CO2•- adduct is formed whose EPR spectrum intensity reveals the extent of the radicals generated. The EPR spectra recorded with the following samples are reported in Figure 10: original sample (a); heated in vacuo at 400 °C (b); 600 °C (c); 800 °C (d); calcined at 400 °C (e); treated at 200 °C in hydrothermal conditions (f). As previously reported,33 the original sample (Figure 10a) is active, indicating that in aqueous suspensions the radical generating sites are accessible to the molecules in solution. The intensity of the spectrum (Figure 10b), i.e., the radical yield, is greater when the samples have been previously outgassed at 400 °C, cooled, and briefly exposed to the atmosphere before the test in the aqueous suspensions. This indicates that a consistent fraction of the radical generating sites are hindered by adsorbed molecules
4.1. Structural Features. The results obtained on the characteristics of the bulk of the crocidolite specimen employed are in agreement with old26,35 and recent papers.27,36 Both lattice vibrations and the stretching modes of hydroxyls coordinated to cations fall at the same frequencies reported by Lewis et al.27 The three OH bands reveal that the association of three iron ions (lower frequency band at 3619 cm-1) is much more abundant than that of two iron ions and one magnesium (3635 cm-1), while the associations of two magnesium and one iron ions (3649 cm-1) are very few. Surface exposed hydroxyls, evaluated by the presence of the OD stretching bands following contact with D2O and removal of adsorbed water (Figure 5), reflect more or less the same abundance of the bulk OH species. 4.2. Stability of the Crystal and of the Surface Structure. As expected, the crystal structure of crocidolite is fairly stable up to 600 °C.35 At 800 °C both XRD patterns and IR lattice vibrations indicate a substantial modification in the bulk. Calcination at 400 °C and the hydrothermal treatment do not involve detectable lattice modification. Some modifications however occur below 600 °C without affecting much the lattice structure. The hydroxyl groups disappear (Figure 1A,c), and the lattice vibrations start to be modified at this temperature (Figure 1B,c). The elimination of the hydroxyls in vacuo is likely accompanied by release of hydrogen and oxidation to ferric of the ferrous ions coordinating the hydroxyl.35 Note that the 600 °C sample heated in vacuo behaves similarly to that oxidized at 400 °C and that a remarkable loss in reactivitysboth toward nitric oxide and in free radical generationstakes place when heating from 400 to 600 °C. The octahedral layer is thus deeply modified in respect of the redox and coordination state of the metal ions. Calcination involves oxidation of ferrous ions to ferric, clearly visible in the modifications of the DR UV-visNIR spectrum both in the charge transfer and d-d transitions regions. Only slight changes are visible following the hydrothermal treatment. 4.3. Surface Active Sites: Availability and Redox State. The surface of crocidolite is largely heterogeneous. When the silica frame layer is exposed, both siloxane bridges (Si-O-Si) and silanols (SiOH) are present. The IR band at 3740 cm-1 characteristic of isolated silanols is in fact visible in Figure 3b-e when the adsorbed molecular water is removed by thermal treatments. The octahedral layer is also exposed. This is proven by the experiment with deuterated water which indicates that some of the hydroxyl coordinated to the metal ions, associated with the three bands at 3619, 3635, and 3649 cm-1, become deuterated, following isotopic exchange. The intensity of (35) Rouxhet, P. G.; Gillard, J. L.; Fripiat, J. J. Miner. Magn. 1972, 105, 1037. (36) Rendall, R E. G. Mineral Fibers and Health; CRC Press: Boca Raton, FL, 1991; Chapter 2.
Active Sites on Asbestos
these OD bands, located at 2669, 2681, and 2692 cm-1 in Figure 6, roughly measures the fraction of metal ions in the M1 and M3 position which are actually at or very near to the surface. In the present case the sharpness of the OD bands and the absence of shoulders (Figure 5c,d) suggests fixed positions on the M1 and M3 sites for ferrous and ferric ions. When the octahedral layer is exposed at the surface, the metal ions will be coordinatively insaturated. The extent of unsaturation as well as the oxidation state and the nature of the nearby cations determines their reactivity. The exposed iron ions active in NO adsorption and in free radical generation are fully inactivated by calcination at 400 °C. They are mostly Fe2+ ions which are oxidized to Fe3+ in this process. The few Fe3+ ions coordinating NO in the vacuum heated samples also appear inactivated. This behavior is probably to be ascribed to a full coordination into subsurface layers of the iron centers previously exposed at the surface, promoted by the calcination treatment. Both NO adsorption and free radical release attain their maximum activity following a vacuum treatment at 400 °C. At this stage water molecules and hydroxyl groups have been completely removed from surface iron ions (but the onset of the lattice modifications has not yet been attained), and then a maximum amount of nitric oxide may be adsorbed. Adsorbed water molecules on the original sample fully inhibit NO adsorption but not free radical generation in aqueous suspensions, because of the higher mobility of water molecules in aqueous medium than at the solid/vacuum interface. This can be explained by the different situations of the solid surface at the gas/vacuum or aqueous interface. Molecular water is strongly held at the surface exposed to vacuum or air, because of the intrinsic hydrophobicity of the air phase. In other terms the enthalpy associated with bonding to the surface overwhelms the entropy gain associated to the detachment of water. In fact, as usually, a high temperature is required to remove such water (or other contaminant molecules). This is not the case at the solid/ water interface where any water molecule leaving the surface will interact via hydrogen bonding with water molecules in the liquid phase. A dynamic equilibrium is thus established at the surface immersed in water, so that the active sites which are hindered by water at the gas interface are available to other molecules in aqueous solutions. Note that the experiment with deuterated water also showed that H2O irreversibly held at the surface becomes reversible in the presence of multilayers of D2O. The same iron sites which require outgassing to become available for NO adsorption are thus available for the reaction generating free radicals in solution. The IR spectra of NO adsorbed on the hydrothermally treated sample outgassed at 400 °C indicate that only a very small fraction of highly coordinatively unsaturated Fe2+ ions is modified by this treatment (Figure 8). Surprisingly free radical yield is almost suppressed on this sample. This strongly suggests that only this very small amount of Fe2+ ions is active in the generation of free radical, in agreement with previous findings.11,33 The mechanism of this radical reaction is still not clear as the carboxylate ion may result as a consequence of a direct reaction of either the formate ion with the surface or with a short-lived radical, e.g. the hydroxyl radical. This latter may be generated following the interaction of oxygen with
Langmuir, Vol. 15, No. 18, 1999 5751
the surface and formation of an active oxygen form such as the superoxide ion and hence the hydroxo radical.11,37 It is not possible to assign to a specific M sites the Fe2+ ions involved in both nitric oxide coordination and free radical release. M2 and M4 sites have been reported to be occupied only by iron ions in crocidolite,26 while the triplet of cations associated to hydroxyls may be occupied also by magnesium. The OD stretching frequency was not perturbed by NO adsorption which could suggest M2 and M4 as candidates for the NO coordinating sites. However, NO could be also coordinated to M1M3M1 sites exposed to the surface which are missing one external OH in their coordination sphere, provided that coordination of NO would not perturb the other underlying OH groups. Both these assignments would be in agreement with previous findings on the peculiar reactivity of Fe2+-Fe3+ associated ions in radical release and in the initiation of lipid peroxidation.33,38 4.4. Characteristics of the Adsorption of Nitric Oxide on Crocidolite. Nitric oxide is adsorbed on coordinately unsaturated iron ions (Fe2+) exposed at the surface, similarly with what found on iron deposited on silica.14,15 The process of adsorption is very fast as revealed by both spectra and thermokinetics. The stretching frequency of the adsorbed NO is lower than that of gaseous NO because of substantial back-donation from the Fe2+ ion. The coordinating sites are covered by water in samples kept in air, which is progressively desorbed when the sample is heated in vacuo. This process (water removal) takes place progressively up to 400 °C, at which the maximum number of coordinating sites are exposed. The stretching frequencies of the various bands assigned to surface-coordinated NO shift toward lower frequency values with higher outgassing temperature. This suggests that surface sites which retain more strongly the water molecules (thus only released at high temperature) also hold more tightly nitric oxide, with consequent lowering of the stretching frequency. At least three different sites are involved in the process. One originates as the dinitrosyl whose stretching frequencies fall at 1897 and 1800 cm-1. The other two coordinate a single NO molecule even under high NO pressure, yielding two mononitrosyls absorbing at 1812 and 1735 cm-1. Dinitrosyl species are reversible by NO outgassing at rt and give origin to mononytrosyls absorbing at 1735 cm-1. Upon outgassing of the samples at 100 °C, one of the two mononitrosyl species (1735 cm-1) is fully desorbed while a fraction of the other one (1812 cm-1) is still held at the surface and requires a 200 °C heating to be desorbed completely. We would expect the lower frequency mononitrosyl to be more strongly bound at least for two reasons: First, a larger shift in frequency from the gas values usually corresponds to a higher bonding energy, and second, parts of the ions originating the lower frequency band are able to coordinate two NO molecules. Being more uncoordinated to the solid should also result in a stronger coordinating site. Possibly on this latter site decomposition of NO occurs upon heating, as previously found on other systems, which would explain why the nitrosyl species more stable to heating is the highfrequency one. The heat value extrapolated at zero coverage, about 130 kJ mol-1, may be assigned to the strongest sites coordinating NO in the mononitrosyl form. This species (37) Fubini, B. In Mechanisms of Fibre Carcinogenesis; Kane, A. B., Boffetta, P., Saracci, R., Wilbourn, J. D., Eds.; IARC Scientific Publication No. 140; International Agency for Research on Cancer: Lyon, France, 1996; p 35. (38) Minotti, G.; Aust, S. D. J. Biol. Chem. 1987, 262, 1098.
5752 Langmuir, Vol. 15, No. 18, 1999
should be related to the IR band at 1812 cm-1 in the spectra in Figure 8, which is the less reversible component, disappearing only after outgassing at 200 °C. The heat value of 120 kJ mol-1 for the irreversible adsorption evaluated from the volumetric and calorimetric isotherms (Ads I and Ads II) measures the heat released by the two mononitrosyls not removable by room-temperature evacuation. This is consistent with a strong coordinative bond between NO and the iron ion, strengthened by a backdonation from iron. This should be the average value of formation of the two mononitrosyls, one related to the 1812 cm-1 band and the other, irreversible at room temperature, producing the IR absorption at 1735 cm-1 (Figure 8). Upon increase of the pressure, NO gives rise simultaneously to mono- and dinitrosyls, and the heat progressively decreases with coverage as expected for an intrinsically heterogeneous adsorption. The reversible adsorption decreases from 100 kJ mol-1, corresponding to the formation of mononitrosyls reversible at room temperature, to 20 kJ mol-1, which can be confidently considered the value corresponding to the weak adsorption of a second NO onto a mononitrosyl to form a dinitrosyl species. Both IR and calorimetric data indicate that NO is strongly coordinated at the ferrous sites exposed at the surface. The site which yields the dinitrosyl form at high pressure is likely more coordinatevely unsaturated than the other one. The heterogeneity in bonding energy found among both species may be ascribed either to differences in the cations surrounding the coordinating one or to differences in the depth from the surface of the cation itself, which would require a sort of “extraction” to react.
Martra et al.
turned out to be an excellent probe for coordinately unsaturated Fe2+ ions at the surface. Three different kinds of sites able to bind NO have been evidenced, within each of which a heterogeneity in binding energy was detected. Some of these sites are also active in free radical release in aqueous suspensions, which is a reaction relevant to DNA damage, mutagenicity, and hence carcinogenicity.37 The surface sites which coordinate NO are covered by irreversibly held molecular water when exposed to air, which hinders access to the exposed iron ions. When in aqueous solutions, however, water is loosely bound and the active sites are accessible to other molecules. In vivo asbestos may thus irreversibly bind endogenous generated NO. Asbestos is phagocitized by alveolar macrophages, which consequently release NO among other factors.10 Crocidolite was reported to activate NO production also on human lung epithelial cells18 and in exposed rats.19 In these circumstances NO adsorption may yield various potentially adverse effects. The fiber may act as a carrier of NO and/or may be the site where catalytic reactions involving NO and generating dangerous radical species take place (e.g. •OH radical from peroxinitrite). Moreover the adsorption of NO, thus subtracted from the aqueous medium within the cell, may in turn activate the inducible NO synthase enzyme (iNOS) with prolonged production of other NO. Oxidizing thermal treatments deeply modify the state of the crocidolite structure, well below the temperature at which the crystal structure collapses. Once the surface site directly involved in the initiation of the pathogenic process will be identified, a physicochemical procedure to inactivate asbestos, based on appropriate termic and oxidizing treatments, could be envisaged.
5. Conclusions The present results constitute a first approach to the description of the sites exposed at the surface of asbestos which is required in order to formulate a sound hypothesis on the molecular basis of their toxicity. Nitric oxide has
Acknowledgment. The research has been carried out with financial support by MURST (Ministero della Universita` e della Ricerca Scientifica e Tecnologica). LA9814541
