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Effect of MnO2 and α-Fe2O3 on organic binders degradation investigated by Raman spectroscopy
Vibrational Spectroscopy 70 (2014) 70–77
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Effect of MnO2 and ␣-Fe2 O3 on organic binders degradation investigated by Raman spectroscopy Nathália D’Elboux Bernardino, Thiago Sevilhano Puglieri, Dalva L.A. de Faria ∗ Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, Butantã, São Paulo 05508-000, SP, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 9 November 2013 Accepted 12 November 2013 Available online 20 November 2013 Keywords: Methyl linoleate Fat Raman Fe2 O3 MnO2
a b s t r a c t Vibrational spectroscopy and GC–MS were used to investigate the effect of MnO2 and ␣-Fe2 O3 on the degradation of methyl linoleate and vegetal and animal fatty. The metal oxides are among the most employed pigments in rock art paintings, whereas the organic compounds were used to mimic organic binders potentially used in such paintings. Both oxides were very effective in the catalytic oxidation of the organic substrates and light had no significant effect, qualitatively or quantitatively, on the final products. In the case of methyl linoleate without metal oxide, the effect of light (visible) was investigated and it was demonstrated that the samples kept in the dark produced relatively less oxidation products, although the main products were the same (hexanal, methyl 9-oxononanoate and methyl octanoate). In the presence of MnO2 and ␣Fe2 O3 methyl 9-oxononanoate was the main product, followed by hexanal. The spectral patterns of the oxidation products were different for manganese and iron oxide and GC–MS demonstrated that more compounds are formed in the former than with ␣-Fe2 O3 . Vegetal and animal fatty presented the same behavior that methyl linoleate did. The results here reported indicated that the two pigments considered actively contribute to fat degradation and the presence of inorganic pigments is the main factor to take into account when organic binders degradation in rock art paintings are investigated. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Organic binders are reported to be extensively used in the manufacture of archaeological artifacts and also in rock art [1,2]. Their main function is to improve the mechanical properties of the object or, in the case of rock art, to help the application and adhesion of pigments in substrates. The most common examples of binders are proteinaceous compounds such as albumin, casein and fish glue, polysaccharides (starch, gum Arabic and cherry gum, for example), lipids, as in the case of beeswax, linseed and walnut oils and resins such as dammar, amber, mastic, dragon’s blood and gamboge [3]. Since organic compounds are susceptible to degradation through chemical and microbial routes [4], they are rarely found in archaeological artifacts or paintings and some examples are the works of Pallecchi et al. [5] who found egg as organic binder in a Etruscan painting using GC–MS, and Maier et al. [6] who identified animal fat in archeological samples from Patagonia using GC–MS and vibrational spectroscopy. Particularly in the case of rock art, the minerals used as pigments can potentially interfere in the organic binders chemical stability.
∗ Corresponding author. Tel.: +55 1130913853. E-mail address: [email protected] (D.L.A. de Faria). 0924-2031/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vibspec.2013.11.004
While organic binders are easily degraded, metal oxides are stable and known to catalyze several chemical reactions [7]. Iron and manganese oxides are among the most used rock art pigments [8] and, concerning fatty acid degradation in homogeneous media, the catalytic action of Mn4+ [9] and Fe2+ [10] ions is very well established. As far as the authors are aware, there is only one study reporting an inhibitory action of hematite (␣-Fe2 O3 ) in linseed oil degradation [11]. This finding suggests that the same protective effect was observed in investigations carried out by Maier et al. [6] who found animal fat in ␣-Fe2 O3 crayons buried in an archaeological site in the Patagonia Argentina. Manganese oxides are also frequently found as pigment in rock art and the catalytic properties of Mn in lipids oxidation are well established [12], with the metal ion favoring the formation of radicals either in the initial or propagation reaction step. However, it is recognized that metal oxides can act as free radicals scavengers [13], thus interfering in the reaction and eventually this could be the main effect observed with hematite, thus causing the protective effect reported for linseed oil [11]. Vegetal and animal fats and methyl linoleate were chosen as binders simulants. Fats present a complex chemical composition and their degradation can follow several routes [14], thus methyl linoleate was chosen to mimic their behavior, furthermore, linoleic acid is one of the most abundant fatty acid in
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both superior plants and animal fats and, therefore, there is a vast literature on its chemical properties [15] and on the properties of its methyl ester derivative [16]. Clearly, in both cases the main degradation mechanism is autoxidation [17–19] leading to the formation of hydroperoxides that are unstable and decompose forming molecules with lower molecular weight (aldehydes, ketones and acids) and, in the case of products with unsaturation, several C C cis-trans isomerization are present [10]. Other degradation route is methyl linoleate hydrolysis, resulting in linoleic acid formation that, nevertheless, follows the same auto-oxidative degradation mechanism as methyl linoleate does [20]. Thermal degradation [19,21,22] and photosensitized reactions [23,24] were also investigated, but the degradation products are very similar, differing only in number and proportion of isomers formed [24]. Summing up, in this investigation the effect of MnO2 and ␣Fe2 O3 on the degradation of methyl linoleate and fats (vegetal and animal) was addressed as well as the possible synergic effect of light, aiming at a better understanding of the role played by inorganic pigments on organic compounds eventually used as binders in rock art. 2. Experimental 2.1. Materials Linoleic acid (Sigma Aldrich, 99+%), HCl 37% (F. Maia, P.A.) and methanol (Merck 99.9+%) were used to prepare methyl linoleate (ML), while vegetable fat (VF) and animal fat (AF) were edible products commercially available (Mesa and Sadia, respectively). Iron and manganese oxides (respectively ␣-Fe2 O3 , Companhia de aminas and MnO2 , Merck) were used as received. Chloroform (Merck, P.A.) and methanol were employed to extract the organic compounds from the mixtures with oxides and the suspensions were filtered with cellulose Millipore membrane (0.45 m in diameter). N2 (Air products, Brazil) was used when purging was necessary.
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GC–MS analyses were conducted using a Shimadzu 14B/QP5050A equipment with quadrupole analyzer. A BPX5 non polar column (30 m long and 0.25 mm internal diameter) was used and the temperature program started at 60 ◦ C and reached 320 ◦ C with a heating rate of 10 ◦ C/min. Total elution time was 31 min. UV–vis spectra (200–600 nm) were collected from solutions (chloroform/methanol or cyclohexane) with a Shimadzu UV3101PC equipment. FTIR spectra were obtained with a Bomem MB100 equipment and also with a Bruker Alpha equipment, both fitted with a DTGS detector and KBr optics. The spectra were obtained from films between KBr windows and the spectral resolution was 4 cm−1 . Raman spectra were obtained using a Renishaw inVia Reflex fitted with a CCD camera (Renishaw, 600 × 400 pixels) and coupled to a Leica Microscope. The 785 nm laser line from a diode laser (Renishaw) was focused onto the sample by an 50× Leica objective (NA 0.75) and the laser power was kept below 15 mW to avoid sample heating or degradation. The 632.8 nm laser line (He–Ne, Renishaw) was tested, but the spectra (not shown) were hampered by a strong luminescent background. Raman and FTIR spectra were analyzed using the Grams package (Thermo Sci. Inc.). Principal Component Analyses (PCA) was made using the Unscrambler package (CAMO, version 10.1). VF and AF spectra (800–1600 cm−1 ) were pre-processed using baseline correction and mean normalization while ML spectra (800–1600 cm−1 ) were baseline corrected and their first derivative (Savitzky-Golay, 13 points) was obtained. PCA was conducted using Varimax rotation and the NIPALS algorithm; the first four principal components explained ca. 95% of the results. To identify bands that can be used as internal reference, PCA was carried out using a matrix composed by six non-aged ML spectra (2800–3050 cm−1 ) and one aged ML spectrum (8 days in the presence of light and MnO2 ); the matrix was pre-processed with baseline correction and only 2 PCs were considered. Using such procedure the first principal component essentially represents the non-aged ML spectrum whereas the second is representative of the spectral changes associated with the aging process.
2.2. Methods 3. Results and discussion Home built chambers [25] for controlled aging were used to study the degradation process of fats and ML under controlled temperature, illumination (FLC fluorescent lamp, 45 W, 6400 K color temperature, and 2436 lm luminous flux) and relative humidity (RH). The commercially available fats (vegetable and animal) were used as received and ML was prepared by treating linoleic acid with HCl 2% solution in methanol [6]. The effect of iron and manganese oxides was evaluated mixing the pigments (0.033 g) with the respective binders (VF, AF or ML, 0.100 g). Binders and their mixtures with pigments were placed in a teflon cup (15 mm diameter and 5 mm depth) and these samples were submitted to different aging conditions, i.e., illumination (with or without) and exposure time (8 or 22 days). All the experiments were conducted at 50% RH and 35 ◦ C. In the case of 22 days of exposure time, the experiments were duplicated, whereas they were triplicated for the 8 days exposure and quadriplicated in the case of ␣-Fe2 O3 experiments. After the aging experiments, the samples were extracted with 2.00 mL of a methanol/chloroform solution (1:2, v/v) followed by filtration with a cellulose membrane. Solvents were removed by a N2 stream. VF, AF, freshly prepared ML and their degradation products were analyzed by gas chromatography–mass spectrometry (GC–MS), UV–vis and Fourier Transform Infrared (FTIR) absorption spectroscopies and Raman spectroscopy.
3.1. Methyl linoleate ML was prepared as previously described [6] and was characterized by FTIR and Raman Microscopy (main characteristic IR bands at 3010, 2930, 2855, 1742, 1465, 1434, 1198, 1173 and 726 cm−1 and Raman bands at 3013, 2904, 1740, 1656, 1441, 1301, 1263, 970 and 840 cm−1 ). The spectra were in agreement with the literature [16], although a weak band assigned to hydroperoxide (840 cm−1 ) is also observed. Hydroperoxides are formed as primary products of poly-unsaturated fatty acids oxidation [26], probably because the heating temperature used in the esterification reaction (60 ◦ C) is enough to trigger a thermo-oxidation reaction [24]. ML aging experiments were conducted, initially, with 8 days of exposure time and Fig. 1 shows the Raman and FTIR spectra of freshly prepared ML (Fig. 1a) and pure ML aged in absence (Fig. 1b) and presence (Fig. 1c) of light. As can be seen from Fig. 1, the Raman spectra are simpler and, as such, easier to interpret than FTIR ones, due to the low intensity of O H bands as well as overtones and combination bands. In order to discuss the ML spectral changes in the Raman spectra, the band at 2904 cm−1 ((CH3 )) was taken as internal standard, according to the statistical procedure described in Section 2. Such procedure showed that the first PC was mainly representative of the non-aged ML spectrum and the second PC was representative of the
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Fig. 1. Raman and FTIR spectra of (a) freshly prepared ML, (b) ML aged in the dark and (c) ML aged with illumination. The inset details the 1500–1800 cm−1 region of the Raman spectra.
spectral changes. The second PC loadings plotted against wavenumbers showed that negative values were associated with the bands that decreased in intensity during the aging process; the same was observed with the bands whose intensity increased, assuming positive loading values. This lead to the conclusion that the bands which do not appear in the second PC loadings vs. wavenumbers plot correspond to chemical groups that are not affected by the structural changes occurring in the aging process. This is the case of the band at 2904 cm−1 assigned to C H stretching vibration of CH3 groups; indeed, fatty acids and esters degradation mechanisms are not expected to involve such chemical group [27], thus confirming the validity of the adopted PCA procedure. Comparing Fig. 1a and b, a decrease is observed in the intensity of the 3013, 1263 and 970 cm−1 Raman bands, assigned respectively to ML C H stretching and in-phase and out-of-phase C H deformation of unsaturated carbon (non-conjugated) [19]. This behavior could be indicative of cross-linking, as reported in the literature [16], however, no evidence of high molecular weight compounds
was found in the GC–MS data here reported, thus, the intensity decrease of the 3013, 1263 and 970 cm−1 bands was here assigned to the formation of conjugated C C bonds in the degradation process (as shown in Fig. 2) since C H vibrations of conjugated carbons are downshifted when compared to non-conjugated ones. Accordingly, it was also observed a little decrease in the band intensity at 1656 cm−1 (cis C C symmetric stretching) together with new bands showing up at 1670 and 1640 cm−1 , assigned to C C trans and C C stretching in conjugated mixtures, respectively [16]. The conversion of cis to trans isomers and conjugated mixtures is indicative of autoxidation reactions [28]. Furthermore, new bands at 1720 and 1695 cm−1 are observed, indicating the formation of carbonyl compounds. The intense band that shows up at 858 cm−1 is here assigned to O O stretching in hydroperoxides, in agreement with the autoxidation mechanism proposed by Agbenyega et al. [16]. Since the selection rules for Raman and IR absorption are different, the usually weak C O or O H modes in the Raman spectrum, give rise to intense absorption bands in the FTIR one. The latter
Table 1 Band positions and respective relative intensities* and assignment: Raman and FTIR spectra of ML and degraded ML (with MnO2 and under illumination). Assignment was made according to Refs. [3,11,16,19,29]. ML
Degraded ML −1
−1
−1
Assignment −1
Raman/cm
FTIR/cm
Raman/cm
FTIR/cm
– 3013 (m) 2933 (w) 2904 (m) 2855 (s) 1740 (m) – – – 1656 (vs) – – 1441 (s) – 1301 (m) 1263 (s) – 970 (m) – 840 (m) – 722 (w)
– 3010 (m) 2930 (vs) – 2855 (m) 1742 (vs) – – – 1655 (vw) – 1457 (m) – 1434 (m) – – 1173 – – – 726 (m) –
– – 2933 (w) 2904 (m) 2855 (s) 1740 (m) 1720 (w) 1695 (w) 1670 (m) 1656 (m) 1640 – 1441 (s) – 1301 (m) – – – 858 (s) – – –
3446 (m) – 2930 (vs) – 2855 (m) 1742 (vs) 1718 (w) 1698 (w) – 1655 (vw) – 1457 (m) – 1434 (m) – – 1173 – – – 726 (w) –
*
vw = very weak, w = weak, m = medium, s = strong, vs = very strong, oop = out of phase.
(O H) s (=C H) cis as ( CH2 ) s ( CH3 ) s ( CH2 ) (C O) ester of fatty acid (C O) fatty acid (C O) aldehydes (C C) trans unsaturated fatty acid (C C) cis unsaturated fatty acid (C C) in conjugated mixtures ı(CH3 ) (rocking) ı(CH2 ) ı(CH2 ) ı(CH2 ) (twisting) ıcis ( C H) in plane cis (C C) + (C O) (CH CH)oop (O OH) (O OH) + (C C) + ı (CH2 ) (rocking) ı( C H) cis (CH CH) cis
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Fig. 2. Reaction steps in fatty acids and esters autoxidation. Adapted from Ref. [34].
technique is thus more suitable to access the behavior of carbonyl or carboxyl bands in the samples here investigated as shown in Fig. 1. As the relative intensity of the 3010 cm−1 band in the FTIR spectrum, assigned to C H (from C C H groups) [29] decreases, a new broad band shows up at 3400 cm−1 ( O H), indicating the formation of hydroxy compounds, in agreement with the Raman spectroscopy data, and eventually other degradation products with OH groups (see Fig. 2). Furthermore, the band at 1745 cm−1 ( C O) slightly broadens, suggesting that distinct carbonyl or carboxyl compounds are being formed. The spectral behavior can be interpreted considering that, in the dark, autoxidation depends (apart from O2 concentration) only on the temperature which, in the experiment, is 35 ± 1 ◦ C. Such temperature, although not substantially higher than room temperature, is enough to produce hydroperoxides, identified in the Raman spectrum (Fig. 1b) by a broad band centered at 858 cm−1 . In fact, an investigation on the thermal degradation of linoleic acid at 60 ◦ C for six days in the dark, showed that the main reaction products were hydroperoxides, aldehydes and different cis and trans isomers [19] and the reported Raman spectra and band assignment are in full agreement with the data here reported. The fatty acid eventually formed by hydrolysis is subjected to the same degradation mechanisms as ML and, as such, would not affect the conclusions based on the ester degradation. The ML Raman spectrum is very similar without or with light (Fig. 1b and c, respectively), except by the changes in the
1600–1750 cm−1 range, where (C C) and (C O) are observed, indicating that the relative contribution of cis, trans and conjugated mixtures are different when light is present [30]. As it will be shown later in the text, the changes are the same as in the presence of MnO2 , although less pronounced (see Table 1). UV radiation, particularly in the 180–400 nm range, can act directly or indirectly on fatty acids and esters. In the first case, the energy has to be enough to break a chemical bond, (typically C H bond) or to favor electron abstraction, producing radicals that initiate the chain reaction. Table 2 present some typical chemical bond energies both in kJ/mol and nm, showing that UV radiation is able to decompose not only hydroperoxides present in the sample, but also other oxygen-containing compounds, generating the free radicals necessary to the initiation step in the degradation process (Fig. 2). Indirectly, UV radiation is able to excite molecular oxygen to
Table 2 Selected bond energies in kJ/mol and nm. The emission spectrum of the light source used in the experiments starts at 320 nm, but with a very weak intensity. Peroxide linkage is thus the most susceptible bond to illumination. Bond
C C C O
H O C O
Dissociation energy kJ/mol
nm
412 360 348 157
290 332 343 761
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Fig. 3. Raman and FTIR spectra of (a) freshly prepared ML, (b) ML in admixture with MnO2 aged in the dark and (c) ML in admixture with MnO2 aged with illumination. The inset details the 1500–1800 cm−1 region of the Raman spectra.
the highly reactive (electrophilic) singlet state that attacks the double bonds producing hydroperoxides [30]. Photosensitizers, such as chlorophyll and methylene blue [23,31], act as antennas, enhancing the effect of visible light. The presence of chromophoric groups other than hydroperoxides was discarded using UV–vis absorption spectroscopy (spectra not shown). In the experiments here reported the light source was in the visible range of the spectrum, however, emission although weak, starts at 320 nm (spectral profile not shown) and is thus enough to produce hydroperoxide decomposition, as described above (Fig. 2). The radicals so generated are active in the propagation step of the chain reaction, favoring the formation of C C isomers and, effectively, a significant change in the 1600–1680 cm−1 range of both Raman and IR spectra is observed, when compared with the spectra obtained from samples kept in the dark. Raman spectroscopy is thus able to differentiate the degradation routes and, therefore, can be used in the understanding of the effect of inorganic pigments on organic binders degradation, particularly, to identify synergism between environment parameters (in this case, illumination) and inorganic pigments (MnO2 and ␣-Fe2 O3 ). The effect of the metal oxide on ML in the presence of light and in the dark, at constant temperature and RH was investigated. Fig. 3 shows the spectra (Raman and FTIR) of ML in admixture with MnO2 aged during 8 days in darkness (Fig. 3b) and under constant illumination (Fig. 3c); one spectrum of freshly prepared ML (Fig. 3a) was included for comparison purposes. Comparing the Raman and FTIR spectra shown in Figs. 1 and 3, it is clear that the MnO2 has a significant effect on ML degradation. Although the degradation products seem to be the same, the spectral changes are much more accentuated, clearly indicating that the metal oxide acts as a catalyzer in ML degradation. This conclusion is not new, but demonstrates that vibrational spectroscopy can be used as a fast tool in the investigation of degradation mechanisms and that Raman spectroscopy can be employed as a non-destructive technique even in the quantification of the degradation products as recently shown [19]. Comparing the Raman spectra in Fig. 3b and c, which are similar in terms of both band positions and intensities, it is evident that light does not affect ML degradation when in the presence of MnO2 . The same is observed with FTIR spectroscopy. This result clearly indicates that no synergism is observed between visible light and MnO2 .
Since several products with close chemical structures can be formed in the degradation process and that the spectral differences can be very subtle, the spectra obtained from the aged samples were statistically processed by Principal Component Analysis (PCA). PCA allows not only the identification of tendencies but also the grouping of compounds with similar behavior [32]. PCA was conducted in the 700–1800 cm−1 range of the above discussed samples, that is where the most significant changes appear, and 4 principal components we considered. A PC-1 vs. PC-2 plot (Fig. 4) shows 4 different sample groups: (i) fresh (non aged) ML; (ii) pure ML aged in the dark; (iii) pure ML under illumination and (iv) ML in admixture with MnO2 in the dark and with illumination. Such behavior confirms the discussions of the experimental data made below and the fact that, under the experimental conditions here employed, there is no synergism between MnO2 and light, since it was not possible to separate the results from samples aged in the darkness and under illumination. MnO2 is known to act as oxidizing agent on several organic substrates [33] subtracting a hydrogen atom from allylic carbons in the initiation step [12]. The resulting radical is delocalized over the carbon chain but, by far, the most stable configuration present the unpaired electron on carbons 9 and 13. A fast reaction with O2
Fig. 4. PCA analysis of aged ML: PC-1 vs. PC-2 scores plot. Fresh ML (+), ML aged in darkness (|), ML aged under illumination (), ML aged in admixture with MnO2 and absence of light () and ML aged in admixture with MnO2 and under illumination (•).
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Fig. 5. Raman spectra of ML in admixture with ␣-Fe2 O3 aged in the dark in three different experiments: the spectral pattern (relative intensities) in the 1600–1800 cm−1 region is not reproducible.
(triplet) and further hydrogen abstraction from the organic substrate leads to hydroperoxides at the 9 and 13 positions. Important to the following discussions is the formation of methyl octanoate and methyl 9-oxononanoate from the latter and hexanal from the former [34]. As it will be discussed below, GC–MS proved that these three compounds are the most abundant ML degradation products, however, many other compounds were also detected although in minor amounts. Fig. 5 shows the Raman spectra of ML samples in admixture with ␣-Fe2 O3 in 3 different experiments; the spectra were obtained in the same conditions as described for MnO2 . Contrarily to MnO2 , the results were not reproducible, with the main differences in the 1600–1800 cm−1 range. The effect of hematite on organic binders such as linseed oil and egg yolk, was investigated by IR absorption spectroscopy and the reported conclusion was that the iron oxide had a protective effect on linseed oil degradation [11]. There is no information, however, on how reproducible such result was. In the 1600–1800 cm−1 spectral window bands assigned to C O and C C stretching vibrations are observed and for such reason this region is sensitive to C C isomers formation as described in Fig. 2. The spectra shown in Fig. 5 indicate that although hydroperoxides are present, isomerization involving the unsaturated carbons is not as evident as in the case of MnO2 . Furthermore, the vibrational spectra are not reproducible, indicating that other factors, which were not controlled, were affecting the reaction. A search in the literature revealed that ␣-Fe2 O3 chemical behavior is strongly affected by particle size and morphology [35]. These parameters can potentially be important in the reactions here reported. At this point, GC–MS was used to help in the identification of the degradation products and in the understanding of ␣-Fe2 O3
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behavior. Table 3 reports the relative amounts of main degradation products; only compounds with percentages higher than 5% were considered. From Table 3 it is clear that methyl 9-oxononanoate is the most favored product of ML degradation when hematite is present (ca. 50%) followed by hexanal. The latter results from the 9hydroperoxide decomposition whereas the former is originated by the decomposition of the 13-hydroperoxide (Fig. 2). It is also evident from Table 3 that light had a minor role in ML degradation in the presence of ␣-Fe2 O3 . When MnO2 was used a larger number of compounds were separated in the chromatogram but with a much smaller contribution than methyl 9-oxononanoate and hexanal. GC–MS data thus indicates that although the main products are the same when manganese and iron oxides are used, hematite seems to be more efficient in decomposing the hydroperoxides present, not allowing further radical reactions. It is thus likely that a heterogeneous Fenton-type reaction [36] takes place with ␣Fe2 O3 , that both accelerates ML degradation by generating highly oxidizing agents (HO• , O2 −• and H2 O2 ) and by decomposing the hydroperoxides formed in the autoxidation step. The irreproducibility observed in the 1600–1800 cm−1 range of the Raman and FTIR spectra would then reflect the heterogeneous phase condition and the fact that ␣-Fe2 O3 is acting in initiation, propagation and termination steps, contrarily to MnO2 whose main role seems to be in the initiation step of the chain reaction [12]. 3.2. Animal fat (AF) When animal fat was used to mimic organic binders of archeological interest, the exposure time used for ML (8 days) was not enough to produce significant spectral changes and it was necessary to extend it to 22 days. When ML was left for 22 days in the controlled environment chamber, the vibrational spectra showed the same trend as with 8 days, except by a larger degradation, evidenced by a luminescent background in the spectra. It is thus reasonable to compare the spectral behavior of AF aged for 22 days with the results obtained for ML aged for 8 days. AF Raman and FTIR spectra are shown in Fig. 6. The spectra of fresh AF (Fig. 6a) and AF aged for 22 days in admixture with MnO2 in the dark (Fig. 6b) and under illumination (Fig. 6c) are presented; without MnO2 the AF vibrational spectra of aged (dark and with illumination) and non-aged samples are similar and were not included in the figure. AF aging in the presence of MnO2 lead to the same changes in the vibrational spectra as in the case of ML, with a decrease in the relative intensities of the bands at 3009 cm−1 and 1263 cm−1 , assigned to the formation of conjugated C C bonds [16], and a decrease in the relative intensity of the band at 1656 cm−1 (cis isomer), with two new bands concomitantly showing up at 1670 cm−1 and 1638 cm−1 , assigned to trans and conjugated mixture of isomers [19]. Relative intensities were obtained using the 2904 cm−1 band as internal standard, following the same procedure used in the case of ML. The effect of aging is much less significant in AF when compared to ML, as demonstrated by the changes in its vibrational spectra.
Table 3 ML degradation products and respective relative amounts from GC–MS (estimated from integrated peak areas in the chromatogram). System ML dark ML illumination ML + MnO2 dark ML + MnO2 illumination ML + ␣-Fe2 O3 dark ML + ␣-Fe2 O3 illumination a
Not measured.
ML 16% 5% 11% 8% a a
Hexanal a
13% 7% 7% 14% 12%
Methyl 9-oxononanoate 16% 25% 37% 35% 51% 50%
Methyl octanoate 7% 8% – – – –
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Fig. 6. Raman and FTIR spectra of (a) fresh AF, (b) AF in admixture with MnO2 aged in the dark and (c) AF in admixture with MnO2 aged with illumination.
Fig. 7. Raman and FTIR spectra of (a) fresh VF, (b) VF in admixture with MnO2 aged in the dark and (c) VF in admixture with MnO2 aged with illumination.
The higher stability can be probably explained either by the chemical composition of the animal fat (myristic, palmitic, stearic, oleic and linoleic fatty acids) [27,37], which contains proportionally less unsaturation, or by the presence of industrial stabilizers. Principal Component Analysis using 7 PCs explains 90% of the experimental data that in a PC4 vs. PC1 plot are arranged in 3 groups, one containing non-aged and AF aged in the dark, another one with AF aged with illumination and a third group with AF aged in the presence of MnO2 . It is thus clear that the presence of the metal oxide is the most important factor in AF degradation. Results from experiments carried out with AF in admixture with ␣-Fe2 O3 were comparable to the ML ones; the measurements were conducted in duplicate and, contrarily to ML, were fully reproducible, probably reflecting the fact that the unsaturated fatty acids content is smaller in AF than in ML, thus reducing the number of possible reaction paths. Compared to MnO2 the spectral changes are the same except by the 1600–1750 cm−1 region, as observed for ML, although in a smaller scale.
intensity of the band at 1670 cm−1 assigned to the C C stretching vibration of the trans isomer (not shown). The observed spectral changes (shown in Fig. 7) were, however, the same as reported for ML and AF. The spectra of fresh VF (Fig. 7a) and VF aged for 22 days in admixture with MnO2 in the dark (Fig. 7b) and under illumination (Fig. 7c) are presented in Fig. 7; without MnO2 the VF vibrational spectra of aged (illuminated and in the dark) and non-aged samples are similar and were not included in the figure. PCA showed a similar although poorer grouping (not shown) when compared to ML, as in the case of AF, and the hematite had the same effect on the spectra. This means that VF autoxidation is accelerated in the presence of both MnO2 and ␣-Fe2 O3 and the vibrational spectra indicate that the degradation mechanism is the same.
4. Conclusions 3.3. Vegetable fat (VF) Vegetal fats are richer in trans isomers when compared to animal fats [38,39] and in the Raman spectrum this is evidenced by a higher
The combined use of vibrational spectroscopy and GC–MS allowed the understanding of the effect of MnO2 and ␣-Fe2 O3 on methyl linoleate degradation, with both oxides catalyzing autoxidation reactions; synergic effect of light was also investigated.
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In the presence of the metal oxides, the major products formed were hexanal, methyl octanoate and methyl 9-oxononanoate. The main difference is that hematite generates a much narrower degradation products distribution than MnO2 suggesting that hydroperoxides are decomposed quickly by Fenton-type reactions. The poorer reproducibility observed with ␣-Fe2 O3 reinforces this idea, considering that in this case hydroperoxide decomposition depends on the Fe3+ and Fe2+ availability close to the oxide surface. Curiously, the degradation inhibitory action of hematite was not observed, contrarily to a previous report using linseed oil [11]. Visible light had no significant effect on the degradation process in the presence of metal oxides but, without them, illumination had a clear effect in the degradation rate as clearly observed in the Raman and FTIR spectra. The major reaction products were, however, the same as demonstrated by GC–MS. Metal oxides are among the most employed pigments used in rock art paintings and it was here demonstrated that they have an important role in organic binders degradation. Acknowledgements The authors wish to express their gratitude to CNPq (132258/2010-2 and 309288/2009-6) and Fapesp (2006/587487, 2008/56127-0, 2011/13760-8 and 2012/05643-4) for financial support and Kelliton José Mendonc¸a Francisco for the chambers maintenance. References [1] A. Duran, M.C. Jimenez De Haro, J.L. Perez-Rodriguez, M.L. Franquelo, L.K. Herrera, A. Justo, Archaeometry 52 (2010) 286–307. [2] A. Nevin, I. Osticioli, D. Anglos, A. Burnstock, S. Cather, E. Castellucci, Analytical Chemistry 79 (2007) 6143–6151. [3] P. Vandenabeele, B. Wehling, L. Moens, H. Edwards, M. De Reu, G. Van Hooydonk, Analytica Chimica Acta 407 (2000) 261–274. [4] S. Adachi, T. Ishiguro, R. Matsuno, Journal of the American Oil Chemists Society 72 (1995) 547–551. [5] P. Pallecchi, G. Giachi, M.P. Colombini, F. Modugno, E. Ribechini, Journal of Archaeological Science 36 (2009) 2635–2642. [6] M.S. Maier, D.L.A. Faria, M.T. Boschín, S.D. Parera, M.F. del Castillo Bernal, Vibrational Spectroscopy 44 (2007) 182–186. [7] K.M. Schaich, in: F. Shahidi (Ed.), Edible Oil and Fat Products: Chemistry, Properties, and Health Effects, vol. 1, 6th ed., John Wiley & Sons, Inc., Hoboken, 2005, pp. 269–356. [8] A. Prous, Arqueologia Brasileira, 1992.
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[9] G. Maayan, G. Christou, Inorganic Chemistry 50 (2011) 7015–7021. [10] H. Kaneda, Y. Kano, M. Kamimura, T. Osawa, S. Kawakishi, Journal of Agricultural and Food Chemistry 38 (1990) 1363–1367. [11] R.J. Meilunas, J.G. Bentsen, A. Steinberg, Studies in Conservation 35 (1990) 33–51. [12] K.M. Schaich, Lipids 27 (1992) 209–218. [13] E.R. Rie, Studies in Conservation 33 (1988) 9–22. [14] K. Sato, Chemical Engineering Science 56 (2001) 2255–2265. [15] A.K.Y. Lee, C.K. Chan, The Journal of Physical Chemistry A 111 (2007) 6285–6295. [16] J.K. Agbenyega, M. Claybourn, G. Ellis, Spectrochimica Acta Part A: Molecular Spectroscopy 47 (1991) 1375–1388. [17] H.W. Gardner, D. Weisleder, Lipids 7 (1972) 191–193. [18] D.A. Pratt, J.H. Mills, N.A. Porter, Journal of the American Chemical Society 125 (2003) 5801–5810. [19] N.F.L. Machado, L.A.E.B. de Carvalho, J.C. Otero, M.P.M. Marques, Journal of Raman Spectroscopy 43 (2012) 1991–2000. [20] R.P. Evershed, S.N. Dudd, M.S. Copley, R. Berstan, A.W. Stott, H. Mottram, S.A. Buckley, Z. Crossman, Accounts of Chemical Research 35 (2002) 660–668. [21] F. Destaillats, P. Angers, European Journal of Lipid Science and Technology 107 (2005) 167–172. [22] T. Toschi, A. Costa, G. Lercker, Journal of the American Oil Chemists Society 74 (1997) 387–391. [23] E.N. Frankel, W.E. Neff, E. Selke, D. Weisleder, Lipids 17 (1982) 11–18. [24] J.N. Chacón, P. Gaggini, R.S. Sinclair, F.J. Smith, Chemistry and Physics of Lipids 107 (2000) 107–120. [25] R.I. Paiva, J.R.C. Rocha, A. Cavicchioli, F.S. Lopes, D.L.A. Faria, Química Nova 33 (2010). [26] K. Rekdal, T.B. Mel, Chemistry and Physics of Lipids 75 (1995) 127–136. [27] G. Solomons, C. Fryhle, Organic Chemistry, 8th ed., Wiley, Hoboken, NJ, 2004. [28] M. Colditz, E. Kunkel, K.-H. Bohne, Ullmann’s Encyclopedia of Industrial Chemistry, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2000. [29] N. Vlachos, Y. Skopelitis, M. Psaroudaki, V. Konstantinidou, A. Chatzilazarou, E. Tegou, Analytica Chimica Acta 573–574 (2006) 459–465. [30] M.C. Cabús Llauradó, Catalytic Non-oxidative Dehydrogenation and Reactivity of Biobased Fatty Acid Derivatives, Universitat Rovira i Virgili, Tarragona, Spain, 2007. [31] H.R. Rawls, P.J. Santen, Journal of the American Oil Chemists Society 47 (1970) 121–125. [32] S. Wold, K. Esbensen, P. Geladi, Chemometrics and Intelligent Laboratory Systems 2 (1987) 37–52. [33] R.J.K. Taylor, M. Reid, J. Foot, S.A. Raw, Accounts of Chemical Research 38 (2005) 851–869. [34] E. Choe, D.B. Min, Comprehensive Reviews in Food Science and Food Safety 5 (2006) 169–186. [35] I.V. Chernyshova, S. Ponnurangam, P. Somasundaran, Physical Chemistry Chemical Physics 12 (2010) 14045–14056. [36] F. Herrera, A. Lopez, G. Mascolo, P. Albers, J. Kiwi, Water Research 35 (2001) 750–760. [37] G. Solomons, C. Fryhle, Organic Chemistry, 8th ed., Wiley, Hoboken, NJ, 2004. [38] R. Garcés, E. Martínez-Force, J.J. Salas, M. Venegas-Calerón, Lipid Technology 21 (2009) 79–82. ˝ [39] Z. Kemény, K. Recseg, G. Hénon, K. Kovári, F. Zwobada, Journal of the American Oil Chemists Society 78 (2001) 973–979.
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Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec
Effect of MnO2 and ␣-Fe2 O3 on organic binders degradation investigated by Raman spectroscopy Nathália D’Elboux Bernardino, Thiago Sevilhano Puglieri, Dalva L.A. de Faria ∗ Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, Butantã, São Paulo 05508-000, SP, Brazil
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 9 November 2013 Accepted 12 November 2013 Available online 20 November 2013 Keywords: Methyl linoleate Fat Raman Fe2 O3 MnO2
a b s t r a c t Vibrational spectroscopy and GC–MS were used to investigate the effect of MnO2 and ␣-Fe2 O3 on the degradation of methyl linoleate and vegetal and animal fatty. The metal oxides are among the most employed pigments in rock art paintings, whereas the organic compounds were used to mimic organic binders potentially used in such paintings. Both oxides were very effective in the catalytic oxidation of the organic substrates and light had no significant effect, qualitatively or quantitatively, on the final products. In the case of methyl linoleate without metal oxide, the effect of light (visible) was investigated and it was demonstrated that the samples kept in the dark produced relatively less oxidation products, although the main products were the same (hexanal, methyl 9-oxononanoate and methyl octanoate). In the presence of MnO2 and ␣Fe2 O3 methyl 9-oxononanoate was the main product, followed by hexanal. The spectral patterns of the oxidation products were different for manganese and iron oxide and GC–MS demonstrated that more compounds are formed in the former than with ␣-Fe2 O3 . Vegetal and animal fatty presented the same behavior that methyl linoleate did. The results here reported indicated that the two pigments considered actively contribute to fat degradation and the presence of inorganic pigments is the main factor to take into account when organic binders degradation in rock art paintings are investigated. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Organic binders are reported to be extensively used in the manufacture of archaeological artifacts and also in rock art [1,2]. Their main function is to improve the mechanical properties of the object or, in the case of rock art, to help the application and adhesion of pigments in substrates. The most common examples of binders are proteinaceous compounds such as albumin, casein and fish glue, polysaccharides (starch, gum Arabic and cherry gum, for example), lipids, as in the case of beeswax, linseed and walnut oils and resins such as dammar, amber, mastic, dragon’s blood and gamboge [3]. Since organic compounds are susceptible to degradation through chemical and microbial routes [4], they are rarely found in archaeological artifacts or paintings and some examples are the works of Pallecchi et al. [5] who found egg as organic binder in a Etruscan painting using GC–MS, and Maier et al. [6] who identified animal fat in archeological samples from Patagonia using GC–MS and vibrational spectroscopy. Particularly in the case of rock art, the minerals used as pigments can potentially interfere in the organic binders chemical stability.
∗ Corresponding author. Tel.: +55 1130913853. E-mail address: [email protected] (D.L.A. de Faria). 0924-2031/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vibspec.2013.11.004
While organic binders are easily degraded, metal oxides are stable and known to catalyze several chemical reactions [7]. Iron and manganese oxides are among the most used rock art pigments [8] and, concerning fatty acid degradation in homogeneous media, the catalytic action of Mn4+ [9] and Fe2+ [10] ions is very well established. As far as the authors are aware, there is only one study reporting an inhibitory action of hematite (␣-Fe2 O3 ) in linseed oil degradation [11]. This finding suggests that the same protective effect was observed in investigations carried out by Maier et al. [6] who found animal fat in ␣-Fe2 O3 crayons buried in an archaeological site in the Patagonia Argentina. Manganese oxides are also frequently found as pigment in rock art and the catalytic properties of Mn in lipids oxidation are well established [12], with the metal ion favoring the formation of radicals either in the initial or propagation reaction step. However, it is recognized that metal oxides can act as free radicals scavengers [13], thus interfering in the reaction and eventually this could be the main effect observed with hematite, thus causing the protective effect reported for linseed oil [11]. Vegetal and animal fats and methyl linoleate were chosen as binders simulants. Fats present a complex chemical composition and their degradation can follow several routes [14], thus methyl linoleate was chosen to mimic their behavior, furthermore, linoleic acid is one of the most abundant fatty acid in
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both superior plants and animal fats and, therefore, there is a vast literature on its chemical properties [15] and on the properties of its methyl ester derivative [16]. Clearly, in both cases the main degradation mechanism is autoxidation [17–19] leading to the formation of hydroperoxides that are unstable and decompose forming molecules with lower molecular weight (aldehydes, ketones and acids) and, in the case of products with unsaturation, several C C cis-trans isomerization are present [10]. Other degradation route is methyl linoleate hydrolysis, resulting in linoleic acid formation that, nevertheless, follows the same auto-oxidative degradation mechanism as methyl linoleate does [20]. Thermal degradation [19,21,22] and photosensitized reactions [23,24] were also investigated, but the degradation products are very similar, differing only in number and proportion of isomers formed [24]. Summing up, in this investigation the effect of MnO2 and ␣Fe2 O3 on the degradation of methyl linoleate and fats (vegetal and animal) was addressed as well as the possible synergic effect of light, aiming at a better understanding of the role played by inorganic pigments on organic compounds eventually used as binders in rock art. 2. Experimental 2.1. Materials Linoleic acid (Sigma Aldrich, 99+%), HCl 37% (F. Maia, P.A.) and methanol (Merck 99.9+%) were used to prepare methyl linoleate (ML), while vegetable fat (VF) and animal fat (AF) were edible products commercially available (Mesa and Sadia, respectively). Iron and manganese oxides (respectively ␣-Fe2 O3 , Companhia de aminas and MnO2 , Merck) were used as received. Chloroform (Merck, P.A.) and methanol were employed to extract the organic compounds from the mixtures with oxides and the suspensions were filtered with cellulose Millipore membrane (0.45 m in diameter). N2 (Air products, Brazil) was used when purging was necessary.
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GC–MS analyses were conducted using a Shimadzu 14B/QP5050A equipment with quadrupole analyzer. A BPX5 non polar column (30 m long and 0.25 mm internal diameter) was used and the temperature program started at 60 ◦ C and reached 320 ◦ C with a heating rate of 10 ◦ C/min. Total elution time was 31 min. UV–vis spectra (200–600 nm) were collected from solutions (chloroform/methanol or cyclohexane) with a Shimadzu UV3101PC equipment. FTIR spectra were obtained with a Bomem MB100 equipment and also with a Bruker Alpha equipment, both fitted with a DTGS detector and KBr optics. The spectra were obtained from films between KBr windows and the spectral resolution was 4 cm−1 . Raman spectra were obtained using a Renishaw inVia Reflex fitted with a CCD camera (Renishaw, 600 × 400 pixels) and coupled to a Leica Microscope. The 785 nm laser line from a diode laser (Renishaw) was focused onto the sample by an 50× Leica objective (NA 0.75) and the laser power was kept below 15 mW to avoid sample heating or degradation. The 632.8 nm laser line (He–Ne, Renishaw) was tested, but the spectra (not shown) were hampered by a strong luminescent background. Raman and FTIR spectra were analyzed using the Grams package (Thermo Sci. Inc.). Principal Component Analyses (PCA) was made using the Unscrambler package (CAMO, version 10.1). VF and AF spectra (800–1600 cm−1 ) were pre-processed using baseline correction and mean normalization while ML spectra (800–1600 cm−1 ) were baseline corrected and their first derivative (Savitzky-Golay, 13 points) was obtained. PCA was conducted using Varimax rotation and the NIPALS algorithm; the first four principal components explained ca. 95% of the results. To identify bands that can be used as internal reference, PCA was carried out using a matrix composed by six non-aged ML spectra (2800–3050 cm−1 ) and one aged ML spectrum (8 days in the presence of light and MnO2 ); the matrix was pre-processed with baseline correction and only 2 PCs were considered. Using such procedure the first principal component essentially represents the non-aged ML spectrum whereas the second is representative of the spectral changes associated with the aging process.
2.2. Methods 3. Results and discussion Home built chambers [25] for controlled aging were used to study the degradation process of fats and ML under controlled temperature, illumination (FLC fluorescent lamp, 45 W, 6400 K color temperature, and 2436 lm luminous flux) and relative humidity (RH). The commercially available fats (vegetable and animal) were used as received and ML was prepared by treating linoleic acid with HCl 2% solution in methanol [6]. The effect of iron and manganese oxides was evaluated mixing the pigments (0.033 g) with the respective binders (VF, AF or ML, 0.100 g). Binders and their mixtures with pigments were placed in a teflon cup (15 mm diameter and 5 mm depth) and these samples were submitted to different aging conditions, i.e., illumination (with or without) and exposure time (8 or 22 days). All the experiments were conducted at 50% RH and 35 ◦ C. In the case of 22 days of exposure time, the experiments were duplicated, whereas they were triplicated for the 8 days exposure and quadriplicated in the case of ␣-Fe2 O3 experiments. After the aging experiments, the samples were extracted with 2.00 mL of a methanol/chloroform solution (1:2, v/v) followed by filtration with a cellulose membrane. Solvents were removed by a N2 stream. VF, AF, freshly prepared ML and their degradation products were analyzed by gas chromatography–mass spectrometry (GC–MS), UV–vis and Fourier Transform Infrared (FTIR) absorption spectroscopies and Raman spectroscopy.
3.1. Methyl linoleate ML was prepared as previously described [6] and was characterized by FTIR and Raman Microscopy (main characteristic IR bands at 3010, 2930, 2855, 1742, 1465, 1434, 1198, 1173 and 726 cm−1 and Raman bands at 3013, 2904, 1740, 1656, 1441, 1301, 1263, 970 and 840 cm−1 ). The spectra were in agreement with the literature [16], although a weak band assigned to hydroperoxide (840 cm−1 ) is also observed. Hydroperoxides are formed as primary products of poly-unsaturated fatty acids oxidation [26], probably because the heating temperature used in the esterification reaction (60 ◦ C) is enough to trigger a thermo-oxidation reaction [24]. ML aging experiments were conducted, initially, with 8 days of exposure time and Fig. 1 shows the Raman and FTIR spectra of freshly prepared ML (Fig. 1a) and pure ML aged in absence (Fig. 1b) and presence (Fig. 1c) of light. As can be seen from Fig. 1, the Raman spectra are simpler and, as such, easier to interpret than FTIR ones, due to the low intensity of O H bands as well as overtones and combination bands. In order to discuss the ML spectral changes in the Raman spectra, the band at 2904 cm−1 ((CH3 )) was taken as internal standard, according to the statistical procedure described in Section 2. Such procedure showed that the first PC was mainly representative of the non-aged ML spectrum and the second PC was representative of the
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Fig. 1. Raman and FTIR spectra of (a) freshly prepared ML, (b) ML aged in the dark and (c) ML aged with illumination. The inset details the 1500–1800 cm−1 region of the Raman spectra.
spectral changes. The second PC loadings plotted against wavenumbers showed that negative values were associated with the bands that decreased in intensity during the aging process; the same was observed with the bands whose intensity increased, assuming positive loading values. This lead to the conclusion that the bands which do not appear in the second PC loadings vs. wavenumbers plot correspond to chemical groups that are not affected by the structural changes occurring in the aging process. This is the case of the band at 2904 cm−1 assigned to C H stretching vibration of CH3 groups; indeed, fatty acids and esters degradation mechanisms are not expected to involve such chemical group [27], thus confirming the validity of the adopted PCA procedure. Comparing Fig. 1a and b, a decrease is observed in the intensity of the 3013, 1263 and 970 cm−1 Raman bands, assigned respectively to ML C H stretching and in-phase and out-of-phase C H deformation of unsaturated carbon (non-conjugated) [19]. This behavior could be indicative of cross-linking, as reported in the literature [16], however, no evidence of high molecular weight compounds
was found in the GC–MS data here reported, thus, the intensity decrease of the 3013, 1263 and 970 cm−1 bands was here assigned to the formation of conjugated C C bonds in the degradation process (as shown in Fig. 2) since C H vibrations of conjugated carbons are downshifted when compared to non-conjugated ones. Accordingly, it was also observed a little decrease in the band intensity at 1656 cm−1 (cis C C symmetric stretching) together with new bands showing up at 1670 and 1640 cm−1 , assigned to C C trans and C C stretching in conjugated mixtures, respectively [16]. The conversion of cis to trans isomers and conjugated mixtures is indicative of autoxidation reactions [28]. Furthermore, new bands at 1720 and 1695 cm−1 are observed, indicating the formation of carbonyl compounds. The intense band that shows up at 858 cm−1 is here assigned to O O stretching in hydroperoxides, in agreement with the autoxidation mechanism proposed by Agbenyega et al. [16]. Since the selection rules for Raman and IR absorption are different, the usually weak C O or O H modes in the Raman spectrum, give rise to intense absorption bands in the FTIR one. The latter
Table 1 Band positions and respective relative intensities* and assignment: Raman and FTIR spectra of ML and degraded ML (with MnO2 and under illumination). Assignment was made according to Refs. [3,11,16,19,29]. ML
Degraded ML −1
−1
−1
Assignment −1
Raman/cm
FTIR/cm
Raman/cm
FTIR/cm
– 3013 (m) 2933 (w) 2904 (m) 2855 (s) 1740 (m) – – – 1656 (vs) – – 1441 (s) – 1301 (m) 1263 (s) – 970 (m) – 840 (m) – 722 (w)
– 3010 (m) 2930 (vs) – 2855 (m) 1742 (vs) – – – 1655 (vw) – 1457 (m) – 1434 (m) – – 1173 – – – 726 (m) –
– – 2933 (w) 2904 (m) 2855 (s) 1740 (m) 1720 (w) 1695 (w) 1670 (m) 1656 (m) 1640 – 1441 (s) – 1301 (m) – – – 858 (s) – – –
3446 (m) – 2930 (vs) – 2855 (m) 1742 (vs) 1718 (w) 1698 (w) – 1655 (vw) – 1457 (m) – 1434 (m) – – 1173 – – – 726 (w) –
*
vw = very weak, w = weak, m = medium, s = strong, vs = very strong, oop = out of phase.
(O H) s (=C H) cis as ( CH2 ) s ( CH3 ) s ( CH2 ) (C O) ester of fatty acid (C O) fatty acid (C O) aldehydes (C C) trans unsaturated fatty acid (C C) cis unsaturated fatty acid (C C) in conjugated mixtures ı(CH3 ) (rocking) ı(CH2 ) ı(CH2 ) ı(CH2 ) (twisting) ıcis ( C H) in plane cis (C C) + (C O) (CH CH)oop (O OH) (O OH) + (C C) + ı (CH2 ) (rocking) ı( C H) cis (CH CH) cis
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Fig. 2. Reaction steps in fatty acids and esters autoxidation. Adapted from Ref. [34].
technique is thus more suitable to access the behavior of carbonyl or carboxyl bands in the samples here investigated as shown in Fig. 1. As the relative intensity of the 3010 cm−1 band in the FTIR spectrum, assigned to C H (from C C H groups) [29] decreases, a new broad band shows up at 3400 cm−1 ( O H), indicating the formation of hydroxy compounds, in agreement with the Raman spectroscopy data, and eventually other degradation products with OH groups (see Fig. 2). Furthermore, the band at 1745 cm−1 ( C O) slightly broadens, suggesting that distinct carbonyl or carboxyl compounds are being formed. The spectral behavior can be interpreted considering that, in the dark, autoxidation depends (apart from O2 concentration) only on the temperature which, in the experiment, is 35 ± 1 ◦ C. Such temperature, although not substantially higher than room temperature, is enough to produce hydroperoxides, identified in the Raman spectrum (Fig. 1b) by a broad band centered at 858 cm−1 . In fact, an investigation on the thermal degradation of linoleic acid at 60 ◦ C for six days in the dark, showed that the main reaction products were hydroperoxides, aldehydes and different cis and trans isomers [19] and the reported Raman spectra and band assignment are in full agreement with the data here reported. The fatty acid eventually formed by hydrolysis is subjected to the same degradation mechanisms as ML and, as such, would not affect the conclusions based on the ester degradation. The ML Raman spectrum is very similar without or with light (Fig. 1b and c, respectively), except by the changes in the
1600–1750 cm−1 range, where (C C) and (C O) are observed, indicating that the relative contribution of cis, trans and conjugated mixtures are different when light is present [30]. As it will be shown later in the text, the changes are the same as in the presence of MnO2 , although less pronounced (see Table 1). UV radiation, particularly in the 180–400 nm range, can act directly or indirectly on fatty acids and esters. In the first case, the energy has to be enough to break a chemical bond, (typically C H bond) or to favor electron abstraction, producing radicals that initiate the chain reaction. Table 2 present some typical chemical bond energies both in kJ/mol and nm, showing that UV radiation is able to decompose not only hydroperoxides present in the sample, but also other oxygen-containing compounds, generating the free radicals necessary to the initiation step in the degradation process (Fig. 2). Indirectly, UV radiation is able to excite molecular oxygen to
Table 2 Selected bond energies in kJ/mol and nm. The emission spectrum of the light source used in the experiments starts at 320 nm, but with a very weak intensity. Peroxide linkage is thus the most susceptible bond to illumination. Bond
C C C O
H O C O
Dissociation energy kJ/mol
nm
412 360 348 157
290 332 343 761
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Fig. 3. Raman and FTIR spectra of (a) freshly prepared ML, (b) ML in admixture with MnO2 aged in the dark and (c) ML in admixture with MnO2 aged with illumination. The inset details the 1500–1800 cm−1 region of the Raman spectra.
the highly reactive (electrophilic) singlet state that attacks the double bonds producing hydroperoxides [30]. Photosensitizers, such as chlorophyll and methylene blue [23,31], act as antennas, enhancing the effect of visible light. The presence of chromophoric groups other than hydroperoxides was discarded using UV–vis absorption spectroscopy (spectra not shown). In the experiments here reported the light source was in the visible range of the spectrum, however, emission although weak, starts at 320 nm (spectral profile not shown) and is thus enough to produce hydroperoxide decomposition, as described above (Fig. 2). The radicals so generated are active in the propagation step of the chain reaction, favoring the formation of C C isomers and, effectively, a significant change in the 1600–1680 cm−1 range of both Raman and IR spectra is observed, when compared with the spectra obtained from samples kept in the dark. Raman spectroscopy is thus able to differentiate the degradation routes and, therefore, can be used in the understanding of the effect of inorganic pigments on organic binders degradation, particularly, to identify synergism between environment parameters (in this case, illumination) and inorganic pigments (MnO2 and ␣-Fe2 O3 ). The effect of the metal oxide on ML in the presence of light and in the dark, at constant temperature and RH was investigated. Fig. 3 shows the spectra (Raman and FTIR) of ML in admixture with MnO2 aged during 8 days in darkness (Fig. 3b) and under constant illumination (Fig. 3c); one spectrum of freshly prepared ML (Fig. 3a) was included for comparison purposes. Comparing the Raman and FTIR spectra shown in Figs. 1 and 3, it is clear that the MnO2 has a significant effect on ML degradation. Although the degradation products seem to be the same, the spectral changes are much more accentuated, clearly indicating that the metal oxide acts as a catalyzer in ML degradation. This conclusion is not new, but demonstrates that vibrational spectroscopy can be used as a fast tool in the investigation of degradation mechanisms and that Raman spectroscopy can be employed as a non-destructive technique even in the quantification of the degradation products as recently shown [19]. Comparing the Raman spectra in Fig. 3b and c, which are similar in terms of both band positions and intensities, it is evident that light does not affect ML degradation when in the presence of MnO2 . The same is observed with FTIR spectroscopy. This result clearly indicates that no synergism is observed between visible light and MnO2 .
Since several products with close chemical structures can be formed in the degradation process and that the spectral differences can be very subtle, the spectra obtained from the aged samples were statistically processed by Principal Component Analysis (PCA). PCA allows not only the identification of tendencies but also the grouping of compounds with similar behavior [32]. PCA was conducted in the 700–1800 cm−1 range of the above discussed samples, that is where the most significant changes appear, and 4 principal components we considered. A PC-1 vs. PC-2 plot (Fig. 4) shows 4 different sample groups: (i) fresh (non aged) ML; (ii) pure ML aged in the dark; (iii) pure ML under illumination and (iv) ML in admixture with MnO2 in the dark and with illumination. Such behavior confirms the discussions of the experimental data made below and the fact that, under the experimental conditions here employed, there is no synergism between MnO2 and light, since it was not possible to separate the results from samples aged in the darkness and under illumination. MnO2 is known to act as oxidizing agent on several organic substrates [33] subtracting a hydrogen atom from allylic carbons in the initiation step [12]. The resulting radical is delocalized over the carbon chain but, by far, the most stable configuration present the unpaired electron on carbons 9 and 13. A fast reaction with O2
Fig. 4. PCA analysis of aged ML: PC-1 vs. PC-2 scores plot. Fresh ML (+), ML aged in darkness (|), ML aged under illumination (), ML aged in admixture with MnO2 and absence of light () and ML aged in admixture with MnO2 and under illumination (•).
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Fig. 5. Raman spectra of ML in admixture with ␣-Fe2 O3 aged in the dark in three different experiments: the spectral pattern (relative intensities) in the 1600–1800 cm−1 region is not reproducible.
(triplet) and further hydrogen abstraction from the organic substrate leads to hydroperoxides at the 9 and 13 positions. Important to the following discussions is the formation of methyl octanoate and methyl 9-oxononanoate from the latter and hexanal from the former [34]. As it will be discussed below, GC–MS proved that these three compounds are the most abundant ML degradation products, however, many other compounds were also detected although in minor amounts. Fig. 5 shows the Raman spectra of ML samples in admixture with ␣-Fe2 O3 in 3 different experiments; the spectra were obtained in the same conditions as described for MnO2 . Contrarily to MnO2 , the results were not reproducible, with the main differences in the 1600–1800 cm−1 range. The effect of hematite on organic binders such as linseed oil and egg yolk, was investigated by IR absorption spectroscopy and the reported conclusion was that the iron oxide had a protective effect on linseed oil degradation [11]. There is no information, however, on how reproducible such result was. In the 1600–1800 cm−1 spectral window bands assigned to C O and C C stretching vibrations are observed and for such reason this region is sensitive to C C isomers formation as described in Fig. 2. The spectra shown in Fig. 5 indicate that although hydroperoxides are present, isomerization involving the unsaturated carbons is not as evident as in the case of MnO2 . Furthermore, the vibrational spectra are not reproducible, indicating that other factors, which were not controlled, were affecting the reaction. A search in the literature revealed that ␣-Fe2 O3 chemical behavior is strongly affected by particle size and morphology [35]. These parameters can potentially be important in the reactions here reported. At this point, GC–MS was used to help in the identification of the degradation products and in the understanding of ␣-Fe2 O3
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behavior. Table 3 reports the relative amounts of main degradation products; only compounds with percentages higher than 5% were considered. From Table 3 it is clear that methyl 9-oxononanoate is the most favored product of ML degradation when hematite is present (ca. 50%) followed by hexanal. The latter results from the 9hydroperoxide decomposition whereas the former is originated by the decomposition of the 13-hydroperoxide (Fig. 2). It is also evident from Table 3 that light had a minor role in ML degradation in the presence of ␣-Fe2 O3 . When MnO2 was used a larger number of compounds were separated in the chromatogram but with a much smaller contribution than methyl 9-oxononanoate and hexanal. GC–MS data thus indicates that although the main products are the same when manganese and iron oxides are used, hematite seems to be more efficient in decomposing the hydroperoxides present, not allowing further radical reactions. It is thus likely that a heterogeneous Fenton-type reaction [36] takes place with ␣Fe2 O3 , that both accelerates ML degradation by generating highly oxidizing agents (HO• , O2 −• and H2 O2 ) and by decomposing the hydroperoxides formed in the autoxidation step. The irreproducibility observed in the 1600–1800 cm−1 range of the Raman and FTIR spectra would then reflect the heterogeneous phase condition and the fact that ␣-Fe2 O3 is acting in initiation, propagation and termination steps, contrarily to MnO2 whose main role seems to be in the initiation step of the chain reaction [12]. 3.2. Animal fat (AF) When animal fat was used to mimic organic binders of archeological interest, the exposure time used for ML (8 days) was not enough to produce significant spectral changes and it was necessary to extend it to 22 days. When ML was left for 22 days in the controlled environment chamber, the vibrational spectra showed the same trend as with 8 days, except by a larger degradation, evidenced by a luminescent background in the spectra. It is thus reasonable to compare the spectral behavior of AF aged for 22 days with the results obtained for ML aged for 8 days. AF Raman and FTIR spectra are shown in Fig. 6. The spectra of fresh AF (Fig. 6a) and AF aged for 22 days in admixture with MnO2 in the dark (Fig. 6b) and under illumination (Fig. 6c) are presented; without MnO2 the AF vibrational spectra of aged (dark and with illumination) and non-aged samples are similar and were not included in the figure. AF aging in the presence of MnO2 lead to the same changes in the vibrational spectra as in the case of ML, with a decrease in the relative intensities of the bands at 3009 cm−1 and 1263 cm−1 , assigned to the formation of conjugated C C bonds [16], and a decrease in the relative intensity of the band at 1656 cm−1 (cis isomer), with two new bands concomitantly showing up at 1670 cm−1 and 1638 cm−1 , assigned to trans and conjugated mixture of isomers [19]. Relative intensities were obtained using the 2904 cm−1 band as internal standard, following the same procedure used in the case of ML. The effect of aging is much less significant in AF when compared to ML, as demonstrated by the changes in its vibrational spectra.
Table 3 ML degradation products and respective relative amounts from GC–MS (estimated from integrated peak areas in the chromatogram). System ML dark ML illumination ML + MnO2 dark ML + MnO2 illumination ML + ␣-Fe2 O3 dark ML + ␣-Fe2 O3 illumination a
Not measured.
ML 16% 5% 11% 8% a a
Hexanal a
13% 7% 7% 14% 12%
Methyl 9-oxononanoate 16% 25% 37% 35% 51% 50%
Methyl octanoate 7% 8% – – – –
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Fig. 6. Raman and FTIR spectra of (a) fresh AF, (b) AF in admixture with MnO2 aged in the dark and (c) AF in admixture with MnO2 aged with illumination.
Fig. 7. Raman and FTIR spectra of (a) fresh VF, (b) VF in admixture with MnO2 aged in the dark and (c) VF in admixture with MnO2 aged with illumination.
The higher stability can be probably explained either by the chemical composition of the animal fat (myristic, palmitic, stearic, oleic and linoleic fatty acids) [27,37], which contains proportionally less unsaturation, or by the presence of industrial stabilizers. Principal Component Analysis using 7 PCs explains 90% of the experimental data that in a PC4 vs. PC1 plot are arranged in 3 groups, one containing non-aged and AF aged in the dark, another one with AF aged with illumination and a third group with AF aged in the presence of MnO2 . It is thus clear that the presence of the metal oxide is the most important factor in AF degradation. Results from experiments carried out with AF in admixture with ␣-Fe2 O3 were comparable to the ML ones; the measurements were conducted in duplicate and, contrarily to ML, were fully reproducible, probably reflecting the fact that the unsaturated fatty acids content is smaller in AF than in ML, thus reducing the number of possible reaction paths. Compared to MnO2 the spectral changes are the same except by the 1600–1750 cm−1 region, as observed for ML, although in a smaller scale.
intensity of the band at 1670 cm−1 assigned to the C C stretching vibration of the trans isomer (not shown). The observed spectral changes (shown in Fig. 7) were, however, the same as reported for ML and AF. The spectra of fresh VF (Fig. 7a) and VF aged for 22 days in admixture with MnO2 in the dark (Fig. 7b) and under illumination (Fig. 7c) are presented in Fig. 7; without MnO2 the VF vibrational spectra of aged (illuminated and in the dark) and non-aged samples are similar and were not included in the figure. PCA showed a similar although poorer grouping (not shown) when compared to ML, as in the case of AF, and the hematite had the same effect on the spectra. This means that VF autoxidation is accelerated in the presence of both MnO2 and ␣-Fe2 O3 and the vibrational spectra indicate that the degradation mechanism is the same.
4. Conclusions 3.3. Vegetable fat (VF) Vegetal fats are richer in trans isomers when compared to animal fats [38,39] and in the Raman spectrum this is evidenced by a higher
The combined use of vibrational spectroscopy and GC–MS allowed the understanding of the effect of MnO2 and ␣-Fe2 O3 on methyl linoleate degradation, with both oxides catalyzing autoxidation reactions; synergic effect of light was also investigated.
N.D. Bernardino et al. / Vibrational Spectroscopy 70 (2014) 70–77
In the presence of the metal oxides, the major products formed were hexanal, methyl octanoate and methyl 9-oxononanoate. The main difference is that hematite generates a much narrower degradation products distribution than MnO2 suggesting that hydroperoxides are decomposed quickly by Fenton-type reactions. The poorer reproducibility observed with ␣-Fe2 O3 reinforces this idea, considering that in this case hydroperoxide decomposition depends on the Fe3+ and Fe2+ availability close to the oxide surface. Curiously, the degradation inhibitory action of hematite was not observed, contrarily to a previous report using linseed oil [11]. Visible light had no significant effect on the degradation process in the presence of metal oxides but, without them, illumination had a clear effect in the degradation rate as clearly observed in the Raman and FTIR spectra. The major reaction products were, however, the same as demonstrated by GC–MS. Metal oxides are among the most employed pigments used in rock art paintings and it was here demonstrated that they have an important role in organic binders degradation. Acknowledgements The authors wish to express their gratitude to CNPq (132258/2010-2 and 309288/2009-6) and Fapesp (2006/587487, 2008/56127-0, 2011/13760-8 and 2012/05643-4) for financial support and Kelliton José Mendonc¸a Francisco for the chambers maintenance. References [1] A. Duran, M.C. Jimenez De Haro, J.L. Perez-Rodriguez, M.L. Franquelo, L.K. Herrera, A. Justo, Archaeometry 52 (2010) 286–307. [2] A. Nevin, I. Osticioli, D. Anglos, A. Burnstock, S. Cather, E. Castellucci, Analytical Chemistry 79 (2007) 6143–6151. [3] P. Vandenabeele, B. Wehling, L. Moens, H. Edwards, M. De Reu, G. Van Hooydonk, Analytica Chimica Acta 407 (2000) 261–274. [4] S. Adachi, T. Ishiguro, R. Matsuno, Journal of the American Oil Chemists Society 72 (1995) 547–551. [5] P. Pallecchi, G. Giachi, M.P. Colombini, F. Modugno, E. Ribechini, Journal of Archaeological Science 36 (2009) 2635–2642. [6] M.S. Maier, D.L.A. Faria, M.T. Boschín, S.D. Parera, M.F. del Castillo Bernal, Vibrational Spectroscopy 44 (2007) 182–186. [7] K.M. Schaich, in: F. Shahidi (Ed.), Edible Oil and Fat Products: Chemistry, Properties, and Health Effects, vol. 1, 6th ed., John Wiley & Sons, Inc., Hoboken, 2005, pp. 269–356. [8] A. Prous, Arqueologia Brasileira, 1992.
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