Rapid Evaluation of the Debromination Mechanism of Eosin in Oil

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Rapid Evaluation of the Debromination Mechanism of Eosin in Oil Paint by Direct Analysis in Real Time and Direct Infusion-Electrospray Ionization Mass Spectrometry Alba Alvarez-Martin, Timothy P. Cleland, Gwénaëlle Kavich, Koen Janssens, and G. Asher Newsome Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02568 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

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Analytical Chemistry

Rapid Evaluation of the Debromination Mechanism of Eosin in Oil Paint by Direct Analysis in Real Time and Direct InfusionElectrospray Ionization Mass Spectrometry Alba Alvarez-Martin†,‡*, Timothy P. Cleland†, Gwénaëlle M. Kavich†, Koen Janssens‡, G. Asher Newsome† †Smithsonian ‡AXES,

Institution Museum Conservation Institute, 4210 Silver Hill Road, Suitland MD, United States of America

Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium

ABSTRACT: Eosin is a synthetic organic colorant prone to fading under the influence of light. Based on the growing interest in the understanding of the discoloration mechanism of eosin-based lakes, this study compares the ability of two ultrafast and ultrasensitive mass spectrometry techniques to detect eosin derivatives in complex matrices such as oil media without the use of conventional separation columns or additional sample preparation protocols. Direct analysis in real time mass spectrometry (DART-MS) and direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) were used to characterize the degradation pathway of eosin in oil media. The analysis protocols developed in this study are applied to discern the degradation mechanism of the lake pigment eosin (comprising the molecule per se complexed to an inorganic substrate) dispersed in linseed oil to create an oil paint. The analysis of oil paints by high resolution MS without an extraction methodology that modifies the system chemistry allowed us to identify the degradation forms without causing any additional fragmentation. Both techniques revealed the primary photo-degradation pathway of eosin in linseed oil, and DI-ESI-MS provided additional information on the native conformation of the lake.

Eosin, a tetra-brominated xanthene red dye (CI 45380, Acid Red 87), was first synthesized in 1873 by BASF1. From 1880 onwards, eosin was commercialized as a dye and was widely applied in a variety of fields as a cosmetic colorant and as a colorant in red inks. Eosin has also become a standard histological stain, because metal complexes of eosin enhances the visibility of specific components of cells and tissues2. Eosin has been used in artworks as a lake, formed by the precipitation of an organic dye onto a metallic salt. In the late 19th century, the beginning of Modern Art and a period of tremendous change for both oil painters and watercolorists, eosin became available in oil paint tubes as a lake pigment. Vincent Van Gogh (18531890), the most well-known Dutch post-impressionist artist, immediately introduced this new color in his palette, giving a vibrant appearance to his oeuvre. However, it has been demonstrated that the original colors used by Van Gogh are losing their brightness, particularly when eosin lakes are present3. This may be because eosin is a photosensitizer4,5 and its degradation mechanism can be strongly affected by the surrounding media6, leading to the discoloration of masterworks within an artist’s lifetime7. In this context, it is also very relevant to have a full understanding of the mechanism of breakdown and discoloration of the original molecules, as such knowledge can be used to design optimal conditions of storage and display of the art works. Many analytical techniques have been utilized for the identification of eosin in paintings even after extensive fading. The detection of bromine, an element rarely found in pigments, on paintings by macroscopic X-Ray Fluorescence (MA-XRF) and/or Scanning Electron Microscopy-Energy Dispersive X-ray Spectrometry (SEM–EDS) analysis has been hypothesized to indicate the presence of eosin8-10. The use of Surface-Enhanced

Raman Spectroscopy (SERS) has also been investigated with the aim to identify eosin in dyestuff mixtures11,12. However, the correlation between the mechanisms of discoloration and eosin breakdown remains one the main challenges to be overcome in the context of the conservation of historical paintings in which this fugitive lake pigment has been used. Traditionally, identification of organic pigments in works of art involves sampling followed by chromatography analysis (e.g. high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD-MS) and pyrolysis gas chromatography mass spectrometry (Py-GCMS)); this usually involves time-consuming preparation steps, long analysis times from column separation, and poor sensitivity due to the use of small sample sizes9,13-15. Moreover, traditional extraction protocols may cause chemical modification in the original conformation of the pigment by hydrolysis, esterification, complexation or other forms of modification of the conformation/structure of the chromophore13,16,17. A breakthrough technology relatively unexplored in the field of conservation science is the new generation of ambient ionization techniques that can be coupled to mass spectrometry detectors for chemical analysis. Direct analysis in real time mass spectrometry (DART-MS) is an ambient ionization technique that samples material at atmospheric pressure in the open laboratory environment18. Reflection mode DART enables the direct examination of a surface without physically sectioning a sample or other forms of mechanical actions19,20. The sample surface is heated up to several hundred degrees Celsius by a gas plume directed at this surface, causing thermal desorption of some of the material. The latter is ionized and transferred into the MS. DART-MS has been used as a

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screening method to detect traces of explosives or illicit drugs21, pesticide monitoring22 and for food quality and authenticity assessment23,24, among many other applications. In only a few reports, the application of DART-MS to the study of historical artifacts25-27 has been addressed. In what follows, we described for the first time the use of DART-MS for monitoring and elucidating the degradation pathways of organic pigments in oil paint. In order to obtain insights into the possible source fragmentation resulting from the DART process, a comparative analysis of the studied samples was performed using a second, possibly competitive method. Electrospray ionization mass spectrometry (ESI-MS) results in little fragmentation of the target analyte for conformation analysis in a complex mixture such as oil paint. ESI-MS can provide information beyond the molecular mass of the analyte, having the ability to preserve noncovalent interactions and to distinguish between various conformations and geometries in molecular complexes28,29. ESI-MS has been applied as an effective tool for the analysis of complex samples that include proteins30, metal complexes31 and biodegradable copolymers32. The conformation of metallic lakes in an oil paint medium has not yet been studied by ESIMS. Another key benefit of this technique is the possibility of performing direct infusion electrospray ionization (DI-ESIMS), where the analytes are solubilized, ionized and transferred to a high mass accuracy spectrometer without the need of column separation, significantly reducing the analysis time. Thus, both DART-MS and DI-ESI-MS can be considered as methods with which the analysis of organic components in artwork might be rendered less destructive, more rapid and more robust in comparison to the traditional approaches based on HPLC-DAD-MS and Py-GC-MS. In what follows, we report the results of analysis by both ionization techniques coupled to a high-resolution mass spectrometer to determine the degradation pathways of an aluminum-based eosin lake and obtain detailed information about its breakdown products. To identify the degradation products formed during the discoloration process, oil paint samples were analyzed at multiple time points during accelerated photo-aging using reflection mode DART-MS and DI-ESI-MS. In addition, to evaluate the effect of inorganic pigments present in the paint as possible catalysts of the discoloration rate of eosin, a set of samples was prepared by mixing eosin-based lakes in different ratios to lead white, the most important white pigment used in Europe until the 19th century. In parallel to the MS techniques two spectroscopic methods attenuated total reflectance – Fourier transform infrared (ATR-FTIR) spectroscopy and fiber optic reflectance spectroscopy (FORS), were employed to monitor changes during the photo-aging in the studied samples. This also allowed the demonstration of the improved performance by MS analysis in this type of degradation study.

MATERIALS AND METHODS Chemicals. Eosin-Y (99% dye content) was purchased from Sigma-Aldrich. Aluminum chloride hexahydrate (AlCl3·6H2O) was purchased from Fluka. Lead white was synthetized “in house” on a small scale following the Dutch stack process33. The bleached linseed oil (Talens, NL) employed was diluted with turpentine. Paint models preparation and artificial photo-aging. Eosin-based lake (Eo) was synthesized by precipitating the organic dye eosin-Y with the aluminum salt (AlCl3·6H2O). The

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synthesis of the lakes was carried out according to the protocol proposed by Claro et al.34. Oil paint models were prepared by mixing eosin lake (Eo) and lead white (LW) in different ratios and painting them out on glass slides; paint films of approximately 50-100 μm thickness were obtained in this manner. Table S1 lists the different ratios used to prepare the paints. Samples were subjected to accelerated light aging up to 192 h in a controlled climate chamber (Model UV 200RB/20DU, Weiss Technik®) equipped with a UVA (350400 nm) lighting system (Actinic BL TL, Philips). The temperature (21 ºC) and relativity humidity (30 %) were kept constant during the photo-aging. A set of samples was removed from the climate chamber after 0, 24, 48, 96, 144 and 192 h of light exposure for analysis (Figure S1). DART-MS. A DART-100 probe using helium discharge gas and operated by a small volume prober (SVP) controller (IonSense, Saugus, MA) was custom-mounted to interface to a linear trap quadrupole (LTQ) Orbitrap Velos mass spectrometer (ThermoFisher Scientific, Waltham, MA). An IonSense Vapur flange differentially pumped by a MZ2NT diaphragm pump (Vacuubrand, Essex, CT) was installed over the mass spectrometer heated ion inlet. The DART was mounted in reflection mode at 45° to the sample surface. A 30 cm long, ¼” outer diameter uncoated stainless steel transfer tube at room temperature was connected to the Vapur flange, terminating 45° to the sample surface, at 2.5 mm from the DART ceramic cap. Paint models were placed 7.5 mm below the DART and transfer tube. An electronically-controlled shutter20 was positioned between the sample and DART configuration. The experimental set up is shown in Figure 1A. The helium plasma temperature setting and exposure time were optimized in the range from 50 °C to 500 °C and from 0.25 s to 10 s, respectively. The optimal plasma temperature setting was identified to be 400 °C while the sample exposure time was limited to 3 s. Mass spectra were acquired in positive mode from m/z 50 to 900 at a resolving power of 30,000.

Figure 1. (A) Custom DART configuration (with sample lowered for the photograph) used to produce (B) extracted ion chromatogram at m/z 648.7117, [C20H9O5Br4]+, the most abundant isotope from tetra-brominated eosin.

DI-ESI-MS. Before DART-MS analysis, ca 1 mg of sample was removed from each paint with a scalpel and transferred to a 2 mL extraction tube with 2.8 mm ceramic beads (Omni International, Kennesaw, GA). To find the optimal method to recover eosin from the paint samples four extraction solvents were tested: i) (2:1 v/v) MeOH:chloroform; ii) (2:1 v/v) EtOH:chloroform; iii) (1:1 v/v) acetonitrile:MeOH and; iv) ammonium acetate. The organic based extractions (i) to (iii) were found to be ineffective at solubilizing the lake. Ammonium acetate was chosen as an extraction solvent as all of the tested samples were completely solubilized in this solvent and no additional products resulting from the interaction between the sample and the solvent were found. Samples were


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prepared as follows: i) addition of 500 µL of ammonium acetate (50 mM); ii) homogenization in a BeadRuptor Elite (Omni International, Kennesaw, GA) (4 m/s, 30s, 2 cycles); iii) dilution of the extract in ammonium acetate (1:10 v/v); iv) centrifugation (18500 rcf, 2min). A color change in the solution was observed, indicating the extraction of the red dye into the solvent supernatant. Filtration was not required because no precipitate was observed. The homogenate was infused at 3-5 µL/min and electrosprayed into an LTQ Orbitrap Velos mass spectrometer. High resolution MS1 and MS2 were acquired in positive mode at multiple mass range windows (m/z 150-2000 and 1000-1500) at 30,000 resolving power. For tandem mass analysis, each selected analyte was isolated with a 4 m/z window for 0-45% collision energy (CE) for collision induced dissociation (CID) and 0-45% normalized collision energy (NCE) for higher-energy collisional dissociation (HCD) to obtain information on fragmentation pathways of selected analytes.

RESULTS AND DISCUSSION In a first explorative experiment, ATR-FTIR analysis was carried out to confirm the formation of the lake pigment after the synthesis6,35, Supporting Information (Figure S2). During the artificial photo-aging, FORS spectroscopy analysis (Supporting Information, Figure S3) was used to follow the color changes occurring during the discoloration process observed on the paint models. FORS measurements support the hypothesis that the rate of the discoloration may be influenced by the ratio of the pigments eosin and lead white present in the paint, as described in a previous paper6. However, spectroscopic analysis could not determine the structure of the degradation intermediates and thus could not answer questions about whether eosin was brominated or un-brominated with or without the xanthene moiety, or about the fragmentation pathway of the xanthene moiety. The spectroscopic methods employed were also unable to detect eosin below the degraded outermost surface6. To identify the degradation products formed during the discoloration process, oil paint samples were examined at multiple time points during accelerated aging in positive mode by DART-MS and DI-ESI-MS. DART-MS The most intense peak associated with DART-MS of eosin was observed at m/z 648.710 (Figure 1B); thus the spectra were subsequently acquired from m/z 50 to 900. Ionization of eosin forms resulted in the formation of protonated species [M+H]+ listed in Table 1. DART analysis of all samples produced characteristic isotope patterns from tetra-, tri- and di-brominated eosin species. The most intense peaks derived from the tetra-brominated eosin form were accompanied by the peaks associated with the triand di-brominated forms (Figure 2A). However, during aging the abundance of the characteristic species changes as shown in Figures 2B-D, and their abundance was also affected by the presence of lead white in the paint, as described below. When eosin was the only pigment in the oil sample, the abundance of tetra-brominated species was equivalent before and after aging (Figure 2B, sample Eo(1)). However, in paint models also containing the semiconductor pigment lead white (LW), the eosin-to-lead white ratio modified the degradation rate of the eosin and this effect become more pronounced with increasing LW abundance (see Figure 2B, samples Eo-LW(2) and EoLW(5)).

The trend vs aging time presented by the tri-brominated forms (Figure 2C), showing only a very modest decrease within the time window investigated, may indicate the simultaneous occurrence of formation and degradation processes of this/these species. We suggest that the presence of tri-brominate species in the non-aged oil paint sample is related to (a) the early generation of these forms, either during the preparation of the lake complex or during the drying process that oil samples require (between 3-4 weeks) and (b) to the general low stability of eosin. In our previous study, where a fresh aqueous solution of pure eosin-Y was analysed by HPLC-MS, no tri- and dibrominated forms were detected36. In a recent study focused on the detection of eosin in solution by LC-MS37, the tribrominated form was detected in an un-aged sample. In this case, the presence of the tri-brominated eosin was tentatively attributed to either (a) the influence of the pH in the solution38,39, (b) the interaction with the solvent during the extraction protocol40, and/or (c) the natural aging of the sample because it was part of a historical reference collection.

Figure 2. (A) DART-MS spectra of Eo-LW(5) (1:5 ratio) after 96 h aging. (B-E) Peak area of brominated products identified at different aging time for three different Eo:LW ratios: Eo(1): 2:0; Eo-LW(2): 2:2; Eo-LW(5): 10:2. (B) Tetra-brominated eosin peak area normalized to peak area sum of all eosin species, (C) tribrominated eosin peak area over tetra-brominated eosin peak area; (D) di-brominated eosin peak area over tetra-brominated eosin peak area, (E) di-brominated eosin peak area over tri-brominated eosin peak area. For clarification, polynomial fitting has been performed to easily follow the trend of each eosin specie in the plot.

In the present study, the abundance of the di-brominate species generally increased with the photo-aging process (Figure 2D). The di-brominate species appears to be most



efficiently generated during the aging of the samples when the ratio of LW increases. The photocatalytic potential of semiconductor nanoparticles during the degradation of eosin has been widely evaluated41,42, showing the enhancement of the degradation when semiconductor particles where present. In the present study, the catalytic effect of LW is evidenced by comparing all the intensities of all eosin forms before and after the photo-aging. In previous studies, the analysis of an aqueous solution of eosin after photo-aging by HPLC allowed the identification of two different di-brominated products that only differ in the position of the remaining Br atoms36,37 showing evidence for the formation of isomers during the degradation. However, since DART-MS operates without involving any separation between isomeric species it is not possible to assign a structure of these intermediate products. The trend observed in Figure 2E shows the different degradation/formation rates between the tri- and di-brominated forms. Generally similar to Figure 2D, it nevertheless highlights that the formation of the di-brominated form is faster than the formation of the tri-brominated form. This observation is consistent with the assumption that the limited variation in tribrominated species observed in Figure 2C is indicative of the fact that only tri-brominated (and no di-brominated) species present at the initial stage of the aging while all newly formed tri-brominated forms, mainly formed during the light-aging and in the presence of LW, are rapidly converted to di-brominated species. Neither the mono-brominated nor the non-brominated form of eosin (fluorescein) were detected under the conditions studied here. These forms were also not detected in a previous study when an aqueous solution of eosin was irradiated with different laser wavelengths under oxic or anoxic conditions36. The detection of fluorescein at early stages of the aging experiment or in historical real paint samples could indicate a low purity of the sample because fluorescein can be an impurity during the synthesis, and/or an advanced state of degradation of the pigment37,43-45. The decrease observed in the abundance of tetra-brominated species (Figure 2B) is accompanied by a change in the red hue observed during photo-aging (Supplementary information, Figure S1). In addition, the slow variation observed in red hue suggests that during the initial stages of the fading process, one or more colored compounds are generated by the dissociation of the tetra-brominated eosin form, such as the di-brominated eosin forms (Figure 2D). The moderate decrease of the absorption maximum of the red hue in the early stages of the discoloration process has been previously ascribed to the decrease of the degree of bromination in organic pigments46 and it will be addressed in the following section. Previous studies of the photochemistry behavior of eosin in solution have suggested a cascade mechanism giving rise the progressive formation of debrominated forms45,47. Here we report for first time the clear correlation between the general discoloration process of eosin and the degree of (de)bromination of eosin lakes in an oil-based medium. The reduced abundance of the tetra-brominated form and the increased abundance of the di-brominated form during the photo-aging experiment suggest that at least one of the degradation pathways under the applied photo-aging conditions48 gives rise to these species. Here, it is of special importance to note that the influence of the light used during the

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artificial aging because the type of light applied can lead to different degradation mechanisms49. In the study presented here, UVA light is used to induce the excitation of the eosin (in the form of a triplet state) and these wavelengths do not match the absorption maximum of the chromophore group responsible for the color of the tetra-, tri- and di-brominated species. In this manner rapid structural breakdown is avoided and the degradation mechanism is expected to be slowed but not necessarily altered in its fundamental nature37,50. The lighting conditions used here are considered to be more suitable to retain intermediate species formed during the artificial aging for detection by MS. It is relevant to note that the same debromination pattern was observed in our previous study when light with a wavelength generally matching the absorption peak of the differently brominated Eosin species was used to induce degradation in solution36. This suggests that the type of irradiation affects the discoloration speed but not (necessarily) the mechanism of degradation.


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Analytical Chemistry ageing. Tandem mass spectra, acquired in positive mode 45% CE CID of all eosin forms observed: (B) EoBr4 = [C20H9O5Br4]+, m/z 648.7138, (C) EoBr3 = [C20H10O5Br3]+, m/z 568.7964, (D) EoBr2 = [C20H11O5Br2]+, m/z 490.8956.

Figure 3. (A) DI-ESI spectra of Eo-LW(5) after 96h of UVA light

Table 1. Summary of compounds identified by DART-MS and DI-ESI-MS. Assigned code

Assigned formula

EoBr2 EoBr3

[C20H11O5Br2]+ [C20H10O5Br3]+

EoBr4

[C20H9O5Br4]+

EoBr3-C3H6O

[C23H14O6Br3]+

EoBr4-C3H6O

[C23H15O6Br4]+

Observed m/z *most intense peak 488.8981, 490.8959*, 492.8936 566.8085, 568.8064*, 570.8042, 572.8015 644.7188, 646.7161, 648.7117*, 650.7118, 652.7102 624.8612, 626.8592, 628.8572*, 630.8555 702.7714, 704.7694, 706.7673*, 708.7653, 710.7636


Theoretical monoisotopic m/z 488.8978 566.8078

Detected by DART ESI    

644.7183





624.8496

nd



702.7602

nd




EoBr4-Al-EoBr3

[C40H13Br7O10Al]+

EoBr4-Al-EoBr3-O

[C40H13Br7O11Al]+

EoBr4-EoBr4

[C40H17Br8O10]+

EoBr4-Al-EoBr4

[C40H12Br8O10Al]+

EoBr4-Al-EoBr4-O

[C40H12Br8O11Al]+

1232.5084, 1234.5036, 1236.5016, 1238.5002*, 1240.4978, 1242.4961, 1244.4955, 1246.4958 1248.4729, 1250.4788, 1252.4737, 1254.4713, 1256.4681*, 1258.4681, 1260.4686, 1262.4750 1288.4331, 1290.4319, 1292.4307, 1294.4292, 1296.4267*, 1298.4256, 1300.4240, 1302.4222, 1304.4197 1310.4119, 1312.4116, 1314.4109, 1316.4094, 1318.4087*, 1320.4071, 1322.4058, 1324.4042, 1326.3990 1326.3990, 1328.3875, 1330.3849, 1332.3826, 1334.3796*, 1336.3788, 1338.3777, 1340.3753, 1342.3771

A factor not yet discussed is the possible presence of reactive species in the medium in which eosin is present. This may trigger different degradation pathways, as will be discussed in the following section. DI-ESI-MS analysis of eosin monomeric species To control for chemical changes from thermally desorbing samples, 0.5 mg of each paint sample, in all aging stages, were sampled before DART-MS. Samples were extracted and infused for positive-ion electrospray ionization (ESI). DI-ESI-MS revealed the presence of the monomeric eosin forms detected by DART-MS. DI-ESI-MS data confirmed the primary degradation pattern of monomeric species of eosin, by a sequential loss of bromine atoms (m/z = 648.7138, 568.7964 and 490.8956, see Figure 3A and Table 1). The loss of the first bromine atom leads to the formation of four (tri-brominated) isomers because the bromine atoms present in the initial form have different electron densities. The consecutive losses of the remaining bromine atoms lead to six (di-brominated) and four (mono-brominated) different isomer forms. However, to confirm the structural conformations of these species further NMR analysis is required. In addition to the MS data, the presence of intermediary products appearing during the photo-aging were supported by the changes observed in the spectroscopic measurements of the aqueous extracts of the studied samples (see Supporting Information, Figure S4). The photochemical process can be detected by monitoring the changes in the region between 300-400 nm in the absorption spectra assigned to π/π* transitions in the aromatic rings. As described in detail in previous studies45,47,51, the debromination takes place in photo-excited semi-reduced eosin forms. These forms are revealed by changes at low wavelengths in the absorption spectra due to a stepwise procedure by electron transfer between neutral-brominated and semireducedbrominated species. Gas phase fragmentation of the tetra-, tri- and di-brominated forms with varying collision energies helps elucidate the breakdown pathway of eosin previously proposed in solution36,37. Similarities in fragmentation were observed between all of the monomeric eosin species (Figure 3B-D). A plausible fragmentation scheme has been proposed in Figure 4 where the de-bromination of eosin takes place in a step-by-step fashion followed by other fragmentation steps. CID generates fragments at the weakest bonds52, that may reflect the types of breakdown possibilities of eosin monomeric species. The detection of other fragmentation products

Page 6 of 10 1232.4607

nd



1248.4556

nd



1288.4288

nd



1310.3712

nd



1326.3661

nd



exclusively by tandem MS analysis may indicate that such species either do not form or are rapidly degraded in oil based medium. Fragmentation products ascribed to the loss of the carboxylic group may be generated by two different, parallel paths (Figure 4, m/z 604.7251, 524.7986, 444.8997). The first scenario is a two-step process with the loss of the carboxylic occurring first, followed by the loss of the bromine. In the other proposed path, the loss of the carboxylic group and bromine occurs simultaneously. Products formed from the replacement of the carboxylic group by a hydroxyl group accompanied by a de-bromination step were also observed (Fig. 4, m/z 541.8013, 461.8925, 383.9821). The formation of these three forms may be explained by the oxygenation mechanism, proposed by Pirok et al.37, and further opening of the epoxy group. However, the species formed by the opening of the epoxy group detected in this study have not been reported in previous literature. Additionally, a minor species (Figure 3B, m/z 415.8875) and its methylated form (Figure 3D, m/z 430.8898), formed from the chromophore moiety opening and further fragmenting, has been identified. However, the formation of this fragmentation product, identified in previous studies as 2-(3,5-dibromo-4hydroxybenzoyl)-benzoic acid41,53, may only occur during the extraction process in solution and not during the photo-aging experiment. Furthermore, eosin forms produced by the selective reactions through the carboxylate group, keeping intact the xanthenic moiety, were identified only by DI-ESI-MS.

Figure 4. Fragmentation pathway of eosin lake obtained in positive mode, 45% CE CID. The assigned mass (m/z) corresponds to the most intense peak observed in Figure 3.


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The formation of the derivative forms of the tri- and tetrabrominated monomeric eosin, m/z 626.8606 and 706.7683 respectively, were detected in all samples as is shown in Figure 3A. The MS/MS spectrum of the ester derivative of the tetrabrominated form (see Supporting Information, Figure S5A-B) showed a loss of the ester group, resulting in the native eosin form. The mass difference of 58.0536 Da can correspond to the addition of C3H6O (theoretical 58.0418 Da). Two selective reactions that can give rise to the formation of these species are viable in the studied conditions: i) the interaction with the linseed oil during the drying process and further aging steps and ii) the interaction with the solvent used during the extraction protocol. However, the relative abundance of derivatives of the monomeric form was tracked throughout the accelerated aging and no variation of the intensity was observed. This suggests the formation of these species as a results of the interaction between the solvent and the eosin37,40,54 or during the drying process of the oil paint, but not during the artificial aging. The photochemical behavior of eosin in different media has been deeply investigated44,48,51,55, showing that its complex protolytic system may easily react with the media. As a result, the formation of intermediates observed in the present study is explained by the higher acidity of the carboxylic group compared to the phenolic group, due to the presence of the bromine groups. The negative charge of the basic form better stabilized in the carboxylate than in the phenolate, keeping intact the xanthenic moiety40. Intermediates of eosin have been also identified by LC after an extraction protocol carried out with DMSO37,54,56 showing the possible interaction with the oil media and/or the solvent. The same samples analyzed by DART-MS did not show the presence of ester-eosin forms due to: i) the absence of solvent during the analysis or ii) the higher ionization energy of the technique, fragmenting the ester bond during the ionization process. DI-ESI-MS/MS analysis of eosin metallic lake species Unlike DART-MS, DI-ESI-MS allowed for retention and detection of the metallic lake complex in addition to the uncomplexed forms (Figure 5). DI-ESI-MS allows for the identification of the native eosin lake (EoBr4-Al-EoBr4) at m/z 1318.4087, previously characterized by MALDI-MS35, and the presence of its doubly-charged tetramer form (2[EoBr4-AlEoBr4]) that has not been detected previously (Figure S6). In addition, one de-brominated form (EoBr4-Al-EoBr3) at m/z 1238.5002 was also detected.

Figure 5. DI-ESI spectra of (A) all eosin-lake forms observed, and tandem mass spectra, acquired in positive mode 10% NCE HCD, of (B) fragmentation of EoBr4-Al-EoBr4 and (C) EoBr4-Al-EoBr3.

The oxidized forms of the tri- and tetra- eosin-based lakes (EoBr4-Al-EoBr3-O and EoBr4-Al-EoBr4-O) were also detected at m/z 1256.4681 and m/z 1334.3796 respectively (Figure 5A). HCD fragmentation of EoBr4-Al-EoBr4 results in symmetrical fragmentation with neutral loss of EoBr4 and a single fragment peak of EoBr4-Al (m/z 670.6974 Figure 5B). Fragmentation of EoBr4-Al-EoBr3 shows asymmetrical fragmentation with both EoBr4-Al (m/z 670.6973) and EoBr3-Al (m/z 592.7864) peaks and neutral loss of either EoBr3 or EoBr4, respectively (Figure 5C). Tandem MS analysis indicates that during the applied fragmentation conditions the loss of an eosin molecule is more likely than the breakdown of the chromophore, keeping the color intact during the first steps of degradation. Although monomeric eosin forms can be tracked during the photo-aging after normalization for comparison (see Supporting Information, Figure S7) showing a trend similar to that the observed by DART-MS analysis, limited intensity of all eosinlake species prevented accurate tracking of these patterns in the studied conditions. This may indicate the different fragmentation stability between the monomeric forms and the eosin-based lakes under light exposure and the different solubility of the lake, harder to extract than the monomer. In general, this makes it much harder to extract information on relevant time-dependent trends from the DI-ESI-MS data than from the DART-MS data.



DI-ESI-MS data does reveal the presence of an eosin dimer, two eosin molecules connected by an intermolecular H bonding without metal anchoring57,58 at m/z 1296.4267 (EoBr4-EoBr4, Figure 5A). The detection of this species can be related to the increase of monomeric reactive species in the solution generated during the synthesis of the lake or during the extraction process. The aggregation behavior of eosin is affected by the solvent polarity; the process of aggregation is enhanced by the increase in polarity59. In addition, the –OH group present in protic solvents may promote the aggregation, as hydrogen bonding plays an important role in the formation of dimers36,58. As a consequence, the dimers are likely formed when the extraction process involves an aqueous solvent such as ammonium acetate. It should be noted that no ester derivative of the dimeric form was detected. This can be explained by the pattern of the hydrogen bonding between the two eosin molecules, through one OH group of the xanthene and the carboxylic group58, preventing any further esterification reaction of the carboxylic group.

CONCLUSIONS Two fast, minimally invasive techniques to analyze the degradation pattern of organic dyes in oil paint have been developed and compared for the specific case of the red eosinbased lake. The studied techniques operate in-situ (DART-MS) or use a single preparation step (DI-ESI-MS) analysis, reducing experiment time and complexity. DART-MS permitted to easily track the light-induced degradation mechanism of eosin in oil paint without any sample preparation. The advantages of this technique are in the simplicity of the analysis with no physical sample sectioning, and without organic solvents such as DMSO and HF commonly used during the extraction of organic dyes13,14,60,61 that can dissociate the organic lake. Monitoring eosin discoloration by spectroscopic techniques does not allow determination of the molecular composition of bottom layers without sampling, but DART-MS was able to detect brominated ions from the remaining eosin in a lower layer. Analysis by DI-ESI-MS allows the detection and characterization of the composition of the original lake without significant fragmentation, in which two dye molecules are complexed to the metal (Eo-Al-Eo). However, DART-MS could not detect the metallic lake complex formed during lake synthesis. One of the hypothesized eosin degradation pathway involving gradual loss of bromine due to light is well-supported by our results. Although the debromination phenomenon has been previously described in solution, de-bromination has not been described before in a more relevant oil-based medium. The debromination pathway, however, is expected not to be the only degradation mechanism, as it cannot readily explain the significant color loss that is observed for eosin under the influence of light. Indeed, all partially debrominated eosin species, still exhibit a relatively high absorption maximum in the 520 nm range, giving rise to an intense red color. Rather, we consider that the debromination and its products must be seen as being the result of the relaxation of the excited triplet state that is created when light is absorbed by brominated eosin species. Yet to realize the significant loss of color, another pathway must be present that gives rise to the destruction of the chromophoroic moiety at the center of the molecule. At this point, no indications of the nature of the products of such a degradation could be identified by us

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and also not from which intermediate form or excited state this additional pathway would start. The photocatalytic effect of lead white in the paint under the discoloration rate of eosin has been tracked throughout the aging experiment, detecting an increase of the di-brominate eosin form directly related to the ratio of LW in the mixture. These data represent the first report of the detection of an organic pigment by two ultrafast and minimally invasive techniques. The implementation of these methods allows us to discern the native structure of the aluminum-based eosin lake and to measure the trend of its discoloration mechanism in oil based medium.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental section, optical photographs, ATR-FTIR, FORS, UV-Vis, characterization of ester derivatives and eosin tetramer by DI-ESI-MS (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Alba Alvarez-Martin: 0000-0002-6756-165X Timothy P. Cleland: 0000-0001-9198-2828 Gwénaëlle M. Kavich: 0000-0002-7706-1173 Koen Janssens: 0000-0002-6752-6408 Asher G. Newsome: 0000-0003-1683-2197

ACKNOWLEDGMENT The authors would like to acknowledge the SolarPaint project (GOA program, Antwerp University Research Council) and Smithsonian Institution for financial support.

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Rapid Evaluation of the Debromination Mechanism of Eosin in Oil

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