FTIR Spectroscopy and DFT Calculations to Probe the Kinetics of β

May 14, 2019 - Due to its high volatility, β-ionone was not considered in the present analysis. ... (C–C–H), with a large transition dipole momen...
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FTIR Spectroscopy and DFT Calculations to Probe the Kinetics of β‑Carotene Thermal Degradation Daniel Martin,† Ana M. Amado,† Alicia G. Gonzaĺ vez,‡ M. Paula M. Marques,†,§ Luís A. E. Batista de Carvalho,*,† and Á ngel Gonzaĺ ez Ureña‡ †

Unidade de I&D Química-Física Molecular, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal Departamento de Química-Física Aplicada, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain § Department of Life Sciences, University of Coimbra, 3000-456 Coimbra, Portugal Downloaded via KEAN UNIV on July 20, 2019 at 03:29:49 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: The thermal degradation of β-carotene in air was investigated. The sample was heated at different temperatures (90, 100, 115, and 130 °C) for periods of up to 8 h to perform a complete kinetic study, the product analysis having been carried out via infrared spectroscopy in attenuated total reflectance mode coupled to density functional theory (DFT) calculations. The kinetics of this thermal degradation process was found to follow a first-order scheme, with rate coefficients varying from k90 °C = (2.0 ± 0.3) × 10−3 to k130 °C = (11.0 ± 0.7) × 10−3 min−1, the experimental activation energy having been calculated as (52 ± 1) kJ mol−1. This Ea value is close to the DFT energies corresponding to a C15−15′ or a C13−14 cis−trans isomerization, followed by the formation of a carotene−oxygen diradical, which was characterized for the first time. Comparison between the experimental and calculated infrared data confirmed the C15−15′-cis rupture as the predominant reaction pathway and retinal as the major degradation product.

1. INTRODUCTION Carotenoids, especially β-carotene and lutein, are the most predominant pigments in nature1 along with chlorophyll, as well as one of the most abundant natural products2,3 (e.g., bacteria, algae, and plants4). In biological matrixes, these compounds occur predominantly in an all-trans conformation (Figure 1), thermodynamically the most stable one as compared with the cis-form.5−7 These fat-soluble micronutrients8 are prone to undergo oxidation reactions, upon an energy transfer between the pigments and hydroxyl or oxygen free radicals,9 thus behaving as biological antioxidants and being able to effectively quench singlet oxygen. These render carotenoids as very relevant compounds in the food industry since they can be used as antioxidant additives apart from their nutritional interest. In addition, these compounds have been intensively studied as promising chemopreventive agents against oxidative-related human diseases (e.g., neurodegenerative disorders, inflammation, or cancer).9−14 Due to the nutritional and biological interest of carotenoids, it is important to accurately determine their thermal stability. Several studies can be found in the literature on this subject, especially those focused on the kinetics of thermal degradation of β-carotene. Achir et al.15 investigated this process in oil solutions (palm olein and copra) through multiresponse © 2019 American Chemical Society

modeling using high-performance liquid chromatography with diode array detection (HPLC-DAD) and concluded that the oxidation and cleavage mechanisms involve the formation of the 9-cis isomer, which was suggested as a biomarker of the reaction progression. Qiu et al.,16 in turn, analyzed thermal stability of β-carotene in oils by HPLC and UV/vis spectroscopy, having obtained rate constant and activation energy values dependent on the type of oil and the concentration of dissolved oxygen. These studies considered a first-order kinetic approximation, having achieved identification of the majority of the degradation products (e.g., apocarotenals).15−26 Apart from these studies on pure β-carotene, Lemmens et al.17 studied the thermal isomerization of carrot puree samples between 80 and 150 °C, having obtained an all-trans-to-cis activation energy of 11 kJ mol−1. In addition, after thermal treatment, these authors could identify all-trans, 9-cis, 13-cis, and 15-cis-β-carotene species, which were also detected by other researchers as the main products formed under these conditions.27 The 15-cis isomer was found to be the less stable Received: March 12, 2019 Revised: May 6, 2019 Published: May 14, 2019 5266

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Figure 1. Most stable β-carotene calculated structures and schematic representation of its thermal decomposition process. Three routes are shown, according to the most stable isomer formed in the first step (prior to oxygen binding).

one and therefore the harder to detect. Furthermore, the reverse reaction could be also monitored.22 Moreover, β-carotene has been investigated as to its structural preferences over the last decades, using computational methods to get insights into its most stable conformations and radical species.28,29 Mordi et al.30 proposed a mechanism through which the compound undergoes a cis− trans isomerization followed by diradical generation, a C−O bond being subsequently created between the carotenoid and molecular oxygen. The next step was found to be the generation of a peroxide intramolecular cycle that triggered the formation of apocarotenoids upon its rupture. Mohamed et al.31 investigated the different cis−trans carotene stabilities as well as the peroxide cycle stabilities. However, the diradical formed by the carotenoid species displaying only one C−O bond was not considered, and consequently, there is still a lack of information on the molecule stabilization via the peroxide cycle pathway. Using a molecular mechanics approach, these authors concluded that the 13-cis and 9-cis carotenoids were more stable than the 15-cis species. In addition, they proposed that the peroxide cycle created in the central position of the molecule (C15−15′) was less probable than those formed either in C9−10 or C13−14. Schlücker et al.29 carried out the first complete investigation on β-carotene, including the IR and Raman spectral prediction by density functional theory (DFT) at the BPW91/6-31G(d) level, shedding light on the stability of the molecule in different conformations along with their associated relative energies. Despite several reports on thermal degradation of βcarotene, no quantitative analysis of this process is still available: neither on the reaction intermediate relative energies nor on the relative stabilization when the cyclic peroxide is formed, which constitute valuable information for unraveling the corresponding mechanism at a molecular level. This study

combines experimental Fourier transform infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) with theoretical calculations at the DFT level, with a view to analyze both reactant consumption and product formation during degradation of β-carotene in the presence of oxygen, a relevant but still ill-understood process. Indeed, it is well known that βcarotene prevents the formation of 1O2 (1Δg), which otherwise would be produced by the chlorophyll triplet state, but it can also quench this excited molecular oxygen and even react with the ground-state triplet oxygen molecule 3O2 (3∑g). It should be emphasized that this investigation was performed for the isolated pure compound (all-trans-β-carotene), as opposed to most of the reported works that were carried out for extracts or samples where the carotenoid was embedded in either a biological or oil matrix. This kind of thermal decomposition is of major relevance in the food industry, where solid β-carotene is usually milled and heated during the manufacture of numerous food products (namely when β-carotene is added as a color additive).32 A full vibrational assignment was currently attained for the most relevant experimental bands of thermal degradation products of β-carotene, assisted by the corresponding DFTcalculated frequencies. In addition, the activation energy of the process was measured and compared with theoretical calculations for different intermediates, to try and clarify the detailed reaction mechanism and therefore identify some of the main products. Hence, when β-carotene is used as a food additive, the results currently obtained are of significant practical interest.

2. MATERIALS AND METHODS 2.1. Sample Preparation and Thermal Treatment. Commercial all-trans-β-carotene (purity ≥ 97%) was purchased from Sigma-Aldrich Chemical S.A. (Sintra, Portugal). 5267

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The Journal of Physical Chemistry A The samples were kept at −18 °C until their use, avoiding any decomposition before heating. A known amount of β-carotene was weighed and placed on a glass surface, and heated for different time periods (from 1 up to 8 h) at distinct temperatures (90, 100, 115, and 130 °C), in an aerobic environment and avoiding light exposure. The FTIR measurements were performed immediately after the samples were removed from the oven, without further treatment. 2.2. Infrared Spectroscopy. FTIR-ATR spectra were recorded using a Bruker Optics Vertex 70 FTIR spectrometer purged by CO2-free dry air, equipped with a Bruker Platinum ATR single reflection diamond accessory, using a Ge on KBr substrate beamsplitter with a liquid-nitrogen-cooled wide-band mercury cadmium telluride detector for the mid-IR interval (400−4000 cm−1). Each spectrum was the sum of 128 scans, at 2 cm −1 resolution, and the 3-term Blackman−Harris apodization function was applied. Under these conditions, the wavenumber accuracy was better than 1 cm−1. The spectra were corrected for the frequency dependence of the penetration depth of the electric field in ATR (considering a mean refractive index of 1 in consonance with the transmission acquisitions) using the Opus 7.5 spectroscopy software (“extended ATR correction” option). The spectra were normalized according to the 1673 cm−1 band, assigned to the (CC) stretching mode and always present in conjugated alkenes.33,34 As it will be shown during the experiments, this signal hardly suffers any variation during the heating process, a feature that justified its choice for spectral normalization in this kinetic study. Nevertheless, in the spectral comparison with the DFT-calculated data, the band used for normalization was the one ascribed to ν(CO) due to its very high intensity. 2.3. DFT Calculations. The DFT calculations were performed with the Gaussian program (G03w and G16w releases). 35 The mPW1PW91 functional was applied, combined with the widely used 6-31G(d) basis set, as well as with the more extensive 6-31+G(d,p) (comprising one additional polarization function on hydrogen atoms and diffusion functions on nonhydrogen atoms), to compare the resulting activation energies. The choice of mPW1PW91, a one-parameter hybrid functional, is supported by previous works36,37 on the validation of this theory level as an appropriate method for this kind of studies. Additionally, the more recent wB97XD functional (which includes corrections for long-range interactions and dispersion effects) was tested, yielding slightly worse results.33 All geometries were fully optimized within the Berny algorithm, using redundant internal coordinates and considering the Gaussian default convergence criteria. Vibrational frequency calculations were carried out for all optimized geometries, at the same theory level, with a view to verify convergence to a real minimum within the potential energy surface (no negative eigenvalues) and to assist in the vibrational mode description. Atomic displacements characterizing each vibrational mode were visualized using the GaussView program.38 For an accurate comparison between the calculated and experimental vibrational frequencies, the former were corrected for anharmonicity and incomplete electron correlation treatment, using the scaling factor proposed by Merrick and co-workers39 for the theory level presently used (0.9828 and 0.9499 for predicted frequencies below and above 500 cm−1, respectively).

3. RESULTS AND DISCUSSION 3.1. Product Analysis. It is well known that apocarotenals, apocarotenones, and several apocarotenols (apocarotenoids, i.e., carotenoid derivatives with less than 40 carbon atoms) are the main products of carotenoid thermal degradation.23,24 Among these, some of the most relevant are retinal (C20H28O),21,22,26 β-apo-13-carotenone (C18H26O),25,26 βapo-14′-carotenal (C22H30O),21,22,26 β-apo-10′-carotenal (C27H36O),21,22 and β-ionone (C13H20O)26,40 (Figure 1). Due to its high volatility, β-ionone was not considered in the present analysis. According to Mordi et al.,30 the first step in thermal decomposition of β-carotene is the cis−trans isomerization that may take place between the 9−10 or 13−14 carbons and even in the central part of the molecule, i.e., between the 15− 15′ carbons (Figure 1). Obviously, depending on the site where this conformational rearrangement occurs, the products can be quite different. After isomerization, diradical formation takes place followed by the creation of a 4-unit intramolecular ring. Figure 2 shows a comparison between the experimental IR spectrum of the most degraded product upon thermal

Figure 2. (A) Experimental FTIR-ATR spectrum of the product of degradation of β-carotene upon heating at 130 °C for 3 h, and calculated spectra (black) for (B) isolated retinal, (C) β-apo-14′carotenal, (D) β-apo-13-carotenone, and (E) β-apo-10′-carotenal. (For the sake of clarity, the spectra were vertically displaced).

decomposition, measured after 3 h of exposure to 130 °C, and the calculated spectra for each of the corresponding products, namely, retinal, β-apo-14′-carotenal, β-apo-13carotenone, and β-apo-10′-carotenal. Two main calculated signals in the 1580−1820 cm−1 range, assigned to the in-phase CC and CO stretching modes, were found to overlap into a broad experimental band centered at 1715 cm−1 that is ascribed to νCO (in agreement with reported data41 that places this signal between 1645 and 1765 cm−1). For this analysis, the experimental spectra were normalized relative to the most intense CO stretching band. A closer look at the spectral profiles reveals a remarkable similarity for most of the products, especially for retinal (Figure 2B). In the high-wavenumber region, two different features were clearly resolved in the theoretical spectra: a sharp peak at ca. 2900 cm−1 assigned to the C−H stretching of the aldehyde 5268

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The Journal of Physical Chemistry A functional group and a mixture of signals associated with ν(C− H) from methyl and CC−H groups. Although these two features were not well resolved in the experimental spectrum, a rounded feature due to ν(C−H), as well as a smaller signal, at a lower frequency, probably associated with ν(C−H)aldehyde, can be detected. Regarding the spectral profiles of the other possible thermal degradation products, shown in Figure 2C−E, it is evident that the overall agreement is not as satisfactory as for retinal. However, it is still acceptable and therefore one cannot rule out their presence although in lower concentrations than that of retinal (see further discussion on this issue). 3.2. Kinetic Study. To obtain valuable kinetic information on thermal degradation process of β-carotene, FTIR-ATR spectra were recorded for samples heated in air for distinct heating temperatures, as a function of heating time. As an example, Figure 3 comprises data for β-carotene at room

opposite to that of the hydrogen atoms from the main carbon chain. Additionally, the intensity of the signal centered at 1715 cm−1 [ν(CO)] was found to increase with heat exposure (dominating the spectrum for longer heating times), which is justified by an increase in the products containing carbonyl groups as β-carotene oxidation develops. Aiming at a better understanding of the β-carotene degradation reaction, the time variation of the 966 cm−1 band as a function of temperature was analyzed (for distinct heating periods), using a first-order kinetics model, according to which the time-dependent β-carotene concentration [β-carotene(t)] should follow the kinetic equation given by [β ‐carotene(t )] = [β ‐carotene(0)]·e−kt

(1)

in which [β-carotene(0)] represents the initial β-carotene concentration, whereas k and t are the rate coefficient and the heating time, respectively. Since this degradation process takes place in open air, the oxygen concentration is in excess compared with that of β-carotene, and the first-order scheme is, in fact, pseudo-first-order kinetics. Consequently, one should rather represent the k value as k = k′·[O2 ] = constant ·k′

(2) −1

Figure 4 depicts a semilogarithmic plot of the 966 cm band area (considered as directly proportional to β-carotene

Figure 3. FTIR-ATR spectra of a β-carotene sample at room temperature (t = 0 h) and after thermal treatment at 100 °C for 4, 6, and 8 h. (For the sake of clarity, spectra in the upper part of the figure were vertically displaced). Figure 4. Semilogarithmic representation of the IR peak area obtained at 966 cm−1 vs the heating time, for each temperature tested. (Experimental error < 10%, represented in the last point for each temperature. For the sake of clarity, the experimental points for 90 °C were displaced by 0.1 units with respect to the values at 100, 115, and 130 °C).

temperature (t = 0 h) and after exposure to 100 °C for several time periods (4−8 h). The spectra were normalized relative to the 1673 cm−1 signal [assigned to ν(CC−C), which in the case of polyenes appears in the 1660−1580 cm−1 region with medium or weak intensities].33,34 The red spectrum in Figure 3, from pure β-carotene with no thermal treatment, displays a prominent band centered at 966 cm−1 due to the out-of-plane deformation mode of the conjugated carbon chain, with negligible contributions from the rings associated with βionone. This vibrational mode comprises in-phase displacements involving methyl groups (C−C−CH3) and hydrogen atoms (C−C−H), with a large transition dipole moment perpendicular to the molecular plane.29 For increasing heating times, this band displays a gradually lower intensity due to βcarotene decomposition. This is a consequence of the strong dependence of this signal on the extent of chain conjugation that diminishes as the molecular degradation progresses. In addition, the DFT calculations currently performed revealed that in retinal the aldehydic hydrogen vibrates in the direction

concentration) as a function of the heating time, for different temperatures. The observed linear dependence supports the assumed first-order kinetics. Table 1 lists the rate coefficients and half-life values of the degradation process at 90, 100, 115, and 130 °C, as well as a comparison with values reported in the literature. The agreement of the presently obtained results with those from Chen and Huang18 is noteworthy. Analyzing this data, it should be noted that when the temperature increases from 50 to 100 °C the rate coefficient also rises, up to 7-fold for the higher temperature. Regarding the temperature range 100−150 °C, the rate is 3.5 times higher. In our case, for a temperature increase of 40 °C, the rate coefficient increased 5.5 times, 5269

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which can be attributed to the distinct experimental protocols used in both studies. Indeed, while Chen and collaborators dissolved the original carotene sample in an organic solvent that was evaporated before heating, in this work, β-carotene was not solubilized prior to heat exposure. These conditions might have influenced the particle size of the sample and consequently the activation energy of the degradation process. In fact, not solubilizing the sample before heating ensures the presence of solid matrixes not as reactive as the fine particle type of sample in solution, which is much more prone to oxygen attack. This comment, however, should be taken as a mere hypothesis for future work. Koca and co-workers42 analyzed the decomposition of βcarotene in samples of dehydrated carrots blanched in boiled water, which were then subjected to different temperatures from 27 to 57 °C. The activation energy thus obtained was (66.2 ± 3.4) kJ mol−1, which compares quite well with the one currently measured (52 ± 1 kJ mol−1). 3.4. On the Reaction Mechanism. According to the earlier cited Mordi mechanism,30 the β-carotene cis−trans isomerization may take place between the 9 and 10 or 13 and 14 chain carbons or in the central part of the molecule, at C15−15′. Naturally, the degradation products are expected to be different for each case. In this section, we discuss the prering intermediates comprising only one oxygen atom bound to one of the carbons belonging to the cis conformation (Figure 1) and compare the results with those reported by Mordi et al.30 An estimation of the energy associated with the formation of these three isomers, as well as the energy barrier of the reaction yielding the corresponding activated complexes, was obtained by DFT calculations. Figure 6 comprises the energy differences between distinct molecular configurations for several isomerization pathways. The starting point in this energy diagram corresponds to the most stable β-carotene conformation and the most stable species for triplet oxygen (see the Supporting Information for geometrical details). Regarding the first reaction step, i.e., the cis−trans isomerization, the calculated energy differences between the all-trans β-carotene and the cis species were 10.02 kJ mol−1 for C15−15′-cis, 4.29 kJ mol−1 for C13−14-cis, and 3.60 kJ mol−1 for C9−10-cis. These results are comparable with those of Mohamed et al.,31 who reported a 0.5 kJ mol−1 energy difference between the 9-cis and 13-cis species, compatible with the present value of 0.69 kJ mol−1. Likewise, these authors found a difference of 7.78 kJ mol−1 between the 9-cis and 15-cis isomers, which is in fair agreement with our result of 6.52 kJ mol−1. As to the energy required for a trans-to-cis isomerization, the highest value was found for the C15−15′ case. Again, our result of 10.02 kJ mol−1 compares satisfactorily with 8.63 kJ mol−1 reported by Xiao et al.43 for the total of cis isomers (since this study did not discriminate between the distinct cis species). The next step in the mechanism proposed by Mordi30 is the rupture of the cis double bond and the creation of a diradical with an oxygen bound to just one carbon atom. In this study, the criterion for carbon selection is based on the steric effects of the methyl group that presumably blocks the position for a first attack of the O2 molecule. The calculated energies required to overcome the barrier from the cis isomer to the formation of the diradical species depend on the starting cis conformation: 60.14 kJ mol−1 for C15−C15′−O, 69.25 kJ mol−1 for C13−C14−O, and 86.68 kJ mol−1 for C9−C10−O. These values should be added to those obtained for the cis−trans

Table 1. Rate Coefficient (k) and Half-Lifetime (τ1/2) Values for the β-Carotene Thermal Degradation in Air at Different Temperatures (Present Work vs Published Values) T/°C

k·103/min−1

τ1/2/min

ref

50 90 100 100 115 125 130 150

1 2.0 ± 0.3 2.6 ± 0.2 7 5.5 ± 0.6 18 11.0 ± 0.7 26

693 346 ± 2 267 ± 3 99 126 ± 1 38.5 60 ± 3 26.7

ref 18 present present ref 18 present ref 18 present ref 18

work work work work

which is an intermediate value between those obtained by Chen and Huang. Henry and co-workers20 investigated the thermal degradation of β-carotene in safflower oil, by heating the samples in this matrix within the 75−95 °C interval. The degradation products were extracted with hexane and further analyzed by high-performance liquid chromatography. The measured rate coefficient (k85 °C) was (2.0 ± 0.1) × 10−3 min−1, which is very similar to the value currently reported, k90 °C = (2.0 ± 0.2) × 10−3 min−1. Actually, one would have expected a greater difference between the two values since while in this study pure β-carotene was exposed to air, in the work reported by Henry et al.,20 the sample was inside an oleaginous matrix. However, this similarity between both values may be justified by the fact that in the latter an air flux was used to enrich the reaction media in oxygen. Interestingly, another team19 investigated β-carotene degradation (between 60 and 120 °C) in olive oil, in the absence of oxygen, having obtained a significantly lower rate coefficient than the one found in this study, k100 °C = (0.17 ± 0.2) × 10−3 min−1 versus (2.6 ± 0.2) × 10−3 min−1, respectively. This difference is not surprising, as one should expect a lower degradation rate when carotene is maintained in an oleaginous matrix (that exerts a protective effect toward the heating process). 3.3. Activation Energies. The temperature dependence of the rate coefficients presently obtained for thermal degradation of β-carotene is plotted in Figure 5, in an Arrhenius-like representation. An activation energy (Ea) equal to (52 ± 1) kJ mol−1 was obtained, higher than the one previously reported by Chen and Huang18 for the same process (39 kJ mol−1),

Figure 5. Semilogarithmic representation of the kinetic rate (k) vs 1000/T. (Due to the small experimental errors, the error bars are almost overruled by the points). 5270

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Figure 6. Representation of the energy variation for different stages of the β-carotene thermal degradation process. (The three possible routes, reactions 1−3, were considered.) The most relevant portion of the molecule, for each key step, is highlighted in the bottom panel.

First, the theoretical values are generally higher than the experimental ones, this difference being explained by the fact that the DFT calculations were implemented in an isolated molecule scenario, i.e., one molecule of β-carotene and one molecule of (ground state) oxygen. However, our experiment employed a solid matrix of β-carotene in which an extra surface reaction cannot be ruled out. As a result, it is not surprising that the activation energy of the measured degradation reaction is higher for the isolated molecule (calculated) relative to the solid matrix (experimental). On the other hand, the similar activation energies for routes 1 and 2 (lower than that obtained for reaction 3) led us to conclude that both routes (1 and 2) are equally probable for the thermal degradation of carotene. Indeed, when the 15−15′-cis isomer is formed, the reactants evolve to the final product through the generation of two retinal molecules, followed by 13−14-cis rupture yielding βapo-14′-carotenal and β-apo-13-carotenone. Figure 2 compares the experimental infrared spectra of the final degradation products (mixture) with the calculated spectra of each possible product (retinal, β-apo-14′-carotenal, β-apo-13-carotenone, and β-apo-10′-carotenal). In turn, Figure 7 matches the same experimental data with the calculated spectra of all reaction products for two different routes (see

isomerization process to get the total energy required to originate the most reactive intermediates (diradicals, Figure 1), such that they may be compared with the experimental value of the activation energy. Table 2 lists these results [obtained with Table 2. Experimental and Calculated Activation Energies (Ea) for the Different Step Reactions of Thermal Degradation Process of β-Carotene cis-isomer precursor

Ea (exp.) (kJ mol−1)

Ea (calc.) (kJ mol−1)

route 1 (15−15′-cis) route 2 (13−14-cis) route 3 (9−10-cis)

52 ± 1 52 ± 1 52 ± 1

70.16 (68.64)a 73.54 (71.97)a 90.28 (88.75)a

a

Calculated values at the 6-31+G(d,p) level.

both the 6-31G(d) and 6-31+G(d,p) basis sets] for an easier comparison of these three possible reactions, corresponding to increasing energies (C15−C15′−O, C13−C14−O, and C9−C10− O). It should be emphasized that the activation energies delivered by the two basis sets were very similar, displaying differences of up to 2% in absolute terms and identical relative values (Table 2). Analysis of these Ea values deserves some comments. 5271

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Information). Additionally, the carotene−oxygen diradical intermediate was characterized for the first time and the energy changes along the three different possible routes of the overall process could be evaluated. A first-order kinetics was found for the global process, and the rate coefficients, [k90 °C = (2.0 ± 0.3) × 10−3 min−1], [k100 °C = (2.6 ± 0.2) × 10−3 min−1], [k115 °C = (5.5 ± 0.6) × 10−3 min−1], and [k130 °C = (11 ± 0.7) × 10−3 min−1], as well as the activation energy(52 ± 1) kJ mol−1, were calculated. The geometrical parameters, relative energies, and infrared profile of reagents, products, and intermediates were calculated at the mPW1PW91/6-31G(d) level. This approach allowed to obtain information not only regarding the initial stabilities of reagents and products (mainly apocarotenals) but also on how much energy is required to attain each intermediate species, specifically those comprising an oxygen bound to one carbon atom (diradical) and those displaying an intramolecular peroxide cycle (stable molecule). The calculated energies for the creation of a diradical at the C15−15′-cis or the C13−14-cis positions were found to be quite similar in comparison with the experimental activation energy, with a minor contribution from the C9−10 species. Thus, the kinetic profile and the key steps of thermal degradation process of β-carotene (in the presence of oxygen), as well as the conformational features of the main reaction products and intermediates, were definitely identified.

Figure 7. Comparison between the experimental FTIR-ATR spectrum of the product of degradation of β-carotene upon heating at 130 °C for 3 h (red), and the average calculated spectra (black) for (A) isolated retinal, β-apo-14′-carotenal, and β-apo-13-carotenone (reactions 1 and 2), and (B) isolated retinal, β-apo-14′-carotenal, βapo-13-carotenone, and β-apo-10′-carotenal (reactions 1−3, except for the volatile ionone). (The main differences are highlighted by the dashed lines).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.9b02327.

Figure 1), reactions 1 and 2 (Figure 7A) or reactions 1−3 (Figure 7B). In these cases, an averaging of the calculated infrared profiles for several isolated compounds was performed (with equal weighting), with a view to better compare with the mixture of degradation products experimentally obtained and analyzed by FTIR. A closer look reveals small differences, which is good evidence that route 3 constitutes a minor channel in degradation of β-carotene (under the conditions of our study) and provides additional support for considering retinal production (reaction 1) as the most relevant step in the global process. Finally, a short comment should be added on the spin change of the thermal degradation reaction. Since the global electron spin of the reactants is a triplet state and that of the products is a singlet, an intersystem crossing (ISC) must take place along the reaction coordinate, which may have some influence on the reaction yield. Unfortunately, the lack of detailed information on the potential energy surfaces in the vicinity of such ISC region does not allow a proper assessment of the underlying reaction dynamics whose knowledge would require extensive theoretical calculations, a task outside the scope of this study.

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Main vibrational assignments for β-carotene and retinal; calculated geometrical parameters related to the key intermediates with only one oxygen atom bonded (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

M. Paula M. Marques: 0000-0002-8391-0055 Luís A. E. Batista de Carvalho: 0000-0002-8059-8537 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support is acknowledged from the Portuguese Foundation for Science and Technology (FCT)(UID/ MULTI/00070/2019) and the European Regional Development Fund (FEDER), through Portugal 2020Programa Operacional Competitividade e Internacionalizaçaõ (POCI-010145-FEDER-029305), and from Project ReNATURE Valorization of the Natural Endogenous Resources of the Centro Region (Centro 2020, Centro-01-0145-FEDER000007).

4. CONCLUSIONS The thermal stability of pure β-carotene samples in air was evaluated by infrared spectroscopy (in ATR mode), for solid samples subjected to different temperatures for distinct heating times. A complete vibrational assignment was performed (assisted by DFT calculations), and the major degradation product was identified, i.e., retinal (see the Supporting

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REFERENCES

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DOI: 10.1021/acs.jpca.9b02327 J. Phys. Chem. A 2019, 123, 5266−5273

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DOI: 10.1021/acs.jpca.9b02327 J. Phys. Chem. A 2019, 123, 5266−5273