Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR

GRASAS Y ACEITES 65 (3) July-September 2014, e030 ISSN-L: 0017-3495 doi: http://dx.doi.org/10.3989/gya.121913 Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR N. Tena, R. Aparicio-Ruiz and D.L. García-González* Instituto de la Grasa (CSIC), Padre García Tejero 4, E-41012 Sevilla, Spain *Corresponding author: [email protected] Submitted: 2 December 2013; Accepted: 7 February 2014 SUMMARY: Fourier transform infrared (FTIR) spectroscopy has been proposed to study the degradation of virgin olive oils (VOO) in samples undergoing thermoxidation. The polar and nonpolar fractions of oxidized oils have been analyzed by FTIR to provide further information on the minor spectral changes taking place during thermoxidation. This information assists in the interpretation of the spectra of the samples. For this purpose polar and nonpolar fractions of 47 VOO samples thermoxidized (190 °C) in a fryer were analyzed by FTIR. The time-course change of the band area assigned to single cis double bonds was explained by their correlation with the decrease in oleic acid (adjusted-R2=0.93). The bands assigned to the hydroxyl groups and the first overtone of ester groups was better studied in the spectra collected for the polar and nonpolar fractions, respectively. The bands assigned to peroxide, epoxy, tertiary alcohols and fatty acids were clearly observed in the spectra of the polar fraction while they are not noticeable in the spectra of the oils. KEYWORDS: FTIR spectroscopy; Polar and nonpolar fractions; Thermoxidation; Virgin olive oil RESUMEN: Uso de las fracciones polares y no polares como fuente de información adicional al estudio de aceites de oliva vírgenes por FTIR. La espectroscopía de infrarrojos por transformada de Fourier (FTIR) se ha propuesto para estudiar la degradación de los aceites de oliva vírgenes (AOV) sujetas a termoxidación. Las fracciones polares y no polares de aceites oxidados se analizaron mediante FTIR para obtener más información sobre los cambios espectrales menores que tienen lugar durante la termoxidación. Esa información ayuda en la interpretación de los espectros de las muestras puras. Con este objetivo, fracciones polares y no polares de 47AOV termoxidados (190 °C) en una freidora se analizaron mediante FTIR. La banda asignada a dobles enlaces cis se explica por su correlación con la disminución de ácido oleico (R2-ajustado=0,93). Las bandas asignadas a los grupos hidroxilos y del primer sobretono de los grupos éster se estudió mejor en los espectros recogidos para la fracción polar y no polar, respectivamente. Grupos asignados a peróxidos, epoxi, alcoholes terciarios y los ácidos grasos se observan claramente en los espectros de la fracción polar. PALABRAS CLAVE: Aceite de oliva virgen; Espectroscopia FTIR; Fracciones polares y no polares; Termoxidación Citation/Cómo citar este artículo: Tena N, Aparicio-Ruiz R, García-González DL. 2014. Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR. Grasas Aceites 65 (3): e030. doi: http://dx.doi.org/10.3989/gya.121913. Copyright: © 2014 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence. 2 • N. Tena, R. Aparicio-Ruiz and D.L. García-González Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 1. INTRODUCTION The consumption of frying products and fast food has dramatically increased over the last decades. The high concern for the toxic hazard of fried products (Friedman and Levin, 2008; Billek, 2000) and the economical implications of the oil stability during frying (Piper, 2001) have led to a major focus on virgin olive oil (VOO) as a frying oil. In fact, deep- and pan-frying is the second most important application of olive oil, which is a millenarian culinary practice of the Mediterranean countries (García-González et al., 2009). The high stability of olive oil is mainly due to its low content in polyunsaturated fatty acids and its high content in antioxidants (e.g. phenols). Although the official method of percentage of total polar compounds (TPC) is well correlated with the thermal degradation of the oil and oxidized fatty acids formed at frying temperatures (Sanibal and Mancini-Filho, 2004), this methodology is lengthy, expensive and laborious. Some alternative methods (determination of free fatty acids, percentage of polymers, peroxide value, Lovibond color, etc) have the general drawback of providing only partial information that is not necessarily correlated to the quality and safety decay of the frying oil (Stier, 2001). For that reason, several rapid spectroscopic methods are being proposed because they provide information on the representative markers of the chemical processes that occur during frying (hydrolysis and oxidation reactions). Fourier transform infrared spectroscopy (FTIR) has already been proven to be an appropriate technique in the quantitative determination of some parameters of edible oils during frying, such as free fatty acids (Lietal., 2008), quantification of trans fatty acids (van de Voort et al., 2008), peroxide values (Yuetal., 2007) and iodine value (Sedman et al., 1998; van de Voort et al., 2008a). In one of our publications (Tena etal., 2009) we described a method based on attenuated total reflectance (ATR)-FTIR to predict the deterioration degree of virgin olive oil during frying. The optimum infrared region to predict the TPC of thermoxidized oil was assigned to trans isomers (978–960 cm−1) and it is highly correlated with the percentage of elaidic acid (trans isomer of oleic acid). This compound rises as the frying process progresses at the same pace as TPC and the FTIR band assigned to single trans double bonds. However, during the thermoxidation process other kinds of degradation reactions occur, most of them being related to hydrolysis, oxidation and other reactions favored by high temperatures. The compounds formed or degraded in these reactions may not follow the same trend as TPC and trans double bonds, and consequently their control needs a further study beyond a simple correlation with single spectral regions. The ability of FTIR to study chemical groups associated with these reactions also makes this technique optimal for studying these chemical processes. Although ATR-FTIR provides information about the entire medium IR spectral region with an easy and rapid output, this technique has less sensitivity for small spectral changes. On the basis of Beer’s law, this limitation means that the use of ATR for sample handling may cause difficulties in tracking low concentrations of chemical species. Alternatively, the use of transmission cells permits studying bands of low intensity that require higher path lengths (~100 μm vs. few μm for ATR). A higher path length allows for maximizing the signal intensity of bands, hence they are recorded at a higher signal-to-noise ratio compared to the spectra collected with ATR. Thus, using transmission cells for sample handling in FTIR analysis provides better results (higher sensitivity and precision) for low intensity bands, although it has the problem of saturation of the detector signal for the most intense bands. Thus, transmission cells of several path lengths (100–350 μm) and window materials (KBr, CaF2, etc.) have been used to study hydroperoxides, the unsaturation degree and fatty acids, among other chemical species, in oxidized edible oils (van de Voort et al., 2008; Moya Moreno et al., 1999). An approach to increasing the intensity of certain spectral bands is to extract the polar and nonpolar fractions of the oil. Separating the oil into two fractions additionally entails simplifying the spectra, avoiding, to some extent, the overlapping bands. Thus, the individual study of fractions obtained from complex matrices with solvents or solid-extraction cartridges has been addressed for a further interpretation of spectroscopic properties (Christophoridou and Dais, 2009; Scano et al., 2008). Most of the fractionation methods are based on polarity or solubility, and extraction of the unsaponifiable matter. Thus, the examination of unsaponifiable matter of olive oil with FTIR and Raman spectroscopies allowed for the development of authentication methods with a better performance compared to the same methods applied to the oils (Baeten et al., 2005). To our knowledge fractionation into polar and nonpolar fractions has not yet been employed in the FTIR studies of edible oils, although it has been successfully applied to other kinds of spectroscopies. Thus, Valavanidis et al., (2004) studied the antioxidant activity of methanol soluble and non soluble phases by ultraviolet-visible (UV-Vis) and by electron paramagnetic resonance (EPR) spectroscopies. Zamora et al., (2002a, 2002b) also studied the polar fractions of several edible oils with 13C-NMR spectroscopy to characterize the oxidative and hydrolytic states of the samples, as well as to assure their genuineness with an improved degree of certainty. Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR • 3 Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 The selective removal of the triacylglycerol base of thermoxidized oils by chromatography leads to the separation of polar and nonpolar fractions. The individual study of these fractions permits a better characterization of the chemical changes catalyzed by high temperatures. The nonpolar fraction contains the unaltered triacylglycerols and consequently it has a composition similar to the original unaltered oils. Most of the newly formed compounds during thermoxidation are expected to be included in the polar fraction so the study of this fraction reveals the most relevant chemical changes taking place. Further chromatographic analysis of the polar fraction has classified the polar compounds into 4 major groups: polymerized triacylglycerols, oxidized triacylglycerols, diacylglycerols and free fatty acids (Dobarganes et al., 2000). The quantitative determination of these polar compounds by high-performance high-exclusion chromatography (HPSEC) (Marmesat et al., 2007) allows for studying in detail the degradation of thermoxidized oils and the progress of oxidative, hydrolytic and thermal reactions. This information can be a useful tool for a proper interpretation of FTIR spectra. The aim of the present study is to examine the major and minor spectral changes occurring in virgin olive oil during thermoxidation by means of FTIR spectroscopic analysis of the samples, and their polar and nonpolar fractions of the samples. Special attention is given to the FTIR spectra of polar fractions and the spectral bands that are not observed in the spectra of the oils, or they are barely noticed. The polar fractions were analyzed by HPSEC and the results served to explain the time-course change in the different bands and the chemical reactions associated with them. Special attention is given to the FTIR spectra of polar fractions and the spectral bands that are not observed in the spectra of the oils, or they are barely noticed. 2. MATERIALS AND METHODS 2.1. Sample Preparation Forty-seven samples of thermoxidized virgin olive oil were obtained from a discontinuous domestic thermoxidation process. Commercial Spanish virgin olive oil, purchased at a local supermarket for frying purposes, was used as a model system. Although the oxidative protection of virgin olive oils greatly depends on their cultivar due to differences in composition (e.g. fatty acids, phenols, etc.), this oil was considered to be representative enough for tracking and assessing oxidation through a qualitative observation of the spectral changes. The thermoxidation process was carried out in a 4 L domestic fryer with an automatic temperature controller (Heidolph EKT 3001, Schwabach, Germany). Three liters of virgin olive oil were heated at 190°C for 94 hours in cycles of 8 hours per day. A sample of 40mL was collected every 2hours until the end of the heating process. Samples were kept in brown glass vials at 4°C until further chemical and spectroscopic analyses. This experiment was repeated with another commercial virgin olive oil to validate the results obtained with the first sequence of samples. In this second experiment a sample was collected every 10hours until obtaining 10 samples of 40mL. These samples were used to validate the regression analysis in those bands that allowed for a linear mathematical modeling and to check the consistency of the trends observed for each spectral change. 2.2. Analysis of Total Polar Compounds The percentage of total polar compounds (TPC) was determined gravimetrically according to the IUPAC Standard Method N° 2.507 (IUPAC, 1992). Nonpolar and polar fractions were separated from 1g of oil by silica gel column chromatography (20g silica adjusted to a water content of 5%, w/w) hexane/diethyl ether (90:10, v/v) and 150 mL diethyl ether as elution systems, respectively. The nonpolar fraction was eluted with 150mL of n-hexane/ diethyl ether (90:10, v/v), while the polar fraction was eluted with 150mL of diethyl ether. Efficiency of the separation was confirmed by TLC using n-hexane/diethyl ether/acetic acid (80:20:1, v/v/v) and visualized with iodine vapor. The percentage of polar fraction was calculated by weighing both fractions after the evaporation of the solvents. 2.3. Distribution of Polar Compounds by High- Performance Size-Exclusion Chromatography (HPSEC) This method determines the contents of polymerized triacylglycerols, oxidized triacylglycerols, and diacylglycerols in oils. It was applied to the polar fraction of heated virgin olive oil. A solution of sample polar fraction in hexane (15mg/mL) was analyzed by high performance liquid chromatography (Agilent Technologies1200) on two columns Hewlett Packard PL gel, 30cm×0,75cm i.d., packed with high-performance spherical gel made of styrene-divinylbenzene co-polymer, particle size 5μm, pore size 100Å and 500Å, respectively (Agilent Technol., Madrid, España), connected in a series in the order 500Å and 100Å, eluting with tetrahydrofuran at a flow rate of 1mL/min. The injection volume was 10μL. A refractive index detector (Agilent Technologies 1200) was used. Thequantification was done according to Dobarganes et al., 2000. The calculation is achieved assuming that all compounds of the sample areeluted. The contents 4 • N. Tena, R. Aparicio-Ruiz and D.L. García-González Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 of the different groups of compounds were expressed as percentages in the oil. 2.4. Fatty Acid Analysis Total fatty acids were transmethylated according to ISO 5509. About 0.2g of commercial virgin olive oil were dissolved in 4mL of hexane and 0.4mL of a solution of 2N de KOH in methanol. The mixture was vigorously shaken for 10–15seconds. The sample (0.2 μl) was injected into a gas chromatograph (Varian 3900) equipped with a split-splitless injector and a flame ionization detector. An SP-2380, 60×0.25mm ID and 0.25μm column (Supelco) was used. The injector and detector temperatures were set at 225°C and 250°C respectively. The oven temperature was held at 170°C for 10min, then programmed from 170°C to 210°C at 1.5°C·min−1 and finally held at the last temperature for 8min. Hydrogen was used as carrier gas at a flow rate of 1.0mL·min−1. Each sample was analyzed in duplicate. The results were expressed as g fatty acid·100g−1 total fatty acids (%). The results were normalized according to Dobarganes and Pérez-Camino (1988). 2.5. Analysis of Phenols A standard solution (0.5mL) made of p-hydroxy- phenylacetic (0.12mg·mL−1) and o-coumaric acids (0.01mg·mL−1) in methanol was added to a sample of filtered virgin olive oil (2.5g). A rotary evaporator at 40°C under vacuum was used to evaporate the solvent and the oily residue was dissolved in 6mL of hexane. The diol-bonded phase cartridge was conditioned according to Mateos et al., (2001). After the sample loading, the final residue was extracted with 500μL of methanol-water (1:1v/v) at 40°C and a filtrated aliquot (20μL) of the final colorless solution was injected into the HPLC system (LaChrom Elite Tokio, Japan), equipped with a diode array detector. The column was a Lichrospher 100RP-18column (4.0 mm i.d.×250 mm; 5 μm, particle size) (Darmstadt, Germany) maintained at 30 °C. The gradient elution, at a flow rate of 1.0mL·min−1, was created using the following mobile phases: A mixture of water/phosphoric acid (95.5:0.5v/v) (solvent A) and methanol-acetonitrile (50:50 v/v) (solvent B). The change in solvent gradient was programmed as follows: from 95% (A)−5% (B) to 70% (A)−30% (B) in 25 min; 62% (A)−38% (B) in 10 min; 62% (A)−38% (B) in 5min; 55% (A)−45% (B) in 5min; 47,5% (A)−52,5% (B) in 5 min and 100% (B) in 5min, followed by 5min of maintenance. The chromatographic signals were obtained at 235, 280, and 335nm. The response factors and recoveries were based on the procedure developed by Mateos et al., (2001). 2.6. FTIR Spectroscopy Spectral data were collected using a Bruker 55 Equinox S FTIR spectrometer equipped with a DGTS detector (Bruker Optics, Ettlingen, Germany). The sampling station was equipped with a support of transmission cell. The cell used to collect the spectra was a removable cell model 20510 (Specac, Orpington, UK), with windows of KBr and an optical path length of 150μm. Each spectrum was recorded at room temperature in the region of 4000–400cm−1 from an average of 22 scans at a resolution of 4cm−1. Each sample (20μL) was injected into the interior of the cell using a mic ropipette and analyzed in duplicate. Between samples, the cell was thoroughly cleaned by using a membrane vacuum pump (Vacuubrand MD 4C NT, Wer theim, Germany). The sample was removed from the cell by aspiration and by passing hexane through the cell. The cell was later dried with nitrogen gas and a spectrum was scanned to test the cleanliness of the windows and the total absence of sample. The spectra were manipulated with OPUS version 4.0 (Bruker Optics, Ettlingen, Germany). Peak areas were computed on the raw spectra and the results were exported as ASCII data for further statistical analysis. The spectra of oleic, linoleic and linolenic acids and trioleine were acquired from pure standards (Sigma-Aldrich, St. Louis, MO). 2.7. Statistical Analysis Univariate and multivariate algorithms have been applied by means of Statistica version 6.0 (Statsoft, Tulsa OK). Stepwise multiple linear regression analysis (SMLRA) was the statistical procedure for the selection of IR bands that better correlate with the polar compound percentage and the percentage of the different groups of compounds identified in the polar fraction. The objective of this statistical procedure was to select the wavenumbers that could predict which kind of degradation reaction is happening in the frying oil during the process, using the TPC and the percentage of the different groups of compounds in the polar fraction as reference for the alteration degrees of the samples. The F-to-enter and F-to-remove values specified a priori determined the wavenumbers included or excluded from the model (Tabachnick and Fidell, 1983). In order to assure the significance of the results and avoid a good result by chance the F-to-Enter/Remove values were selected under the strictest conditions (Fisher-distribution table at p=0.05). The concentration of some compounds did not show a linear trend during the thermoxidation process, and for this reason no linear regression model was used in those cases. Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR • 5 Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 3. RESULTS AND DISCUSSION 3.1. FTIR spectra of neat thermoxidized virgin oliveoils The FTIR spectra of the virgin olive oils (VOOs) thermoxidized from 0 to 94 hours show remarkable changes in the bands assigned to (i) hydroxyl groups (-OH) indicative of the oxidation reactions (3584– 3508cm−1), (ii) first overtone of the stretching vibration of the ester group of triacylglycerols (C=O) (3472cm−1), (iii) single cis double bonds (3005cm−1), (iv) changes in the band assigned to single trans double bonds, the band at 967cm−1 (976–963cm−1). All these spectral regions provide information about the chemical structures that are involved in the complex degradation reactions occurring in the oil during the frying process. The band that better correlated with the percentage of total polar compounds (TPC) was the band assigned to single trans double bonds, the band centered at 967 cm−1 (976–963cm−1), which was already observed in a previous work carried out with ATR (Tena et al., 2009). The equation resulting from regressing TPC against the area of the single trans double bonds has a regression coefficient adjusted-R2 of 0.99 and a standard deviation in the prediction of RSD=1.44%: TPC (%)=−3.78+124.06×[FTIR trans band area] The regression line points out a linear relationship when TPC is lower than 30% (Figure 1A), and the goodness of the regression model allowed for a successful validation with the other 10 thermoxidized olive oil samples collected every 10 hours from a domestic fryer for 90 hours. However, the prediction value is overestimated from 30% onwards (Figure1B). This overestimation is due to the heterocedastic behavior of the regression, where the lower values of band areas have more error than the higher values, probably due to a problem of lack of sensitivity. As a consequence of the formation of trans double bonds during thermoxidation (Tena et al., 2009), the band assigned to cis double bonds (3013– 2991 cm−1) decreases as the thermal degradation progresses. The area of this band in the initial non thermoxidized sample is higher than any value of band area of the single trans double bonds quantified in the rest of the samples, and it decreases up to 50% of the initial value at the end of the experiment. This band area is correlated with TPC by linear regression (adjusted-R2=0.98; RSD=2.48%). However, the behavior of the band area assigned to the single cis double bonds is not completely linear, unlike the band area assigned to trans double bonds. The lack of linearity explains that the former band was not selected as a control parameter of the thermoxidation process in a previous study carried out with ATR-FTIR (Tena et al., 2009). Thus, the band area of cis double bonds (3013–2991cm−1) explains TPC with an adjusted-R2 of 0.99 (RSD=0.96%) by means of the following non linear regression model: TPC (%)=56.22 + 42.78×[FTIR cis band area] - 25.45×[FTIR cis band area]2 The content of cis double bonds follows two approximately linear behaviors with intersection at around 45% of the total polar compounds (60h of thermoxidation). The two lines intersect forming a five degree angle between them. The composition of fatty acids in the samples was determined by gas chromatography to provide a chemical explanation for the observed changes in the band assigned to single cis double bonds. Oleic acid accounts for 75.93% of the total fatty acids present in the unaltered oil (non thermoxidized sample), while linoleic and linolenic acids account for 4.55% FIGURE 1. Linear regression between the area of the single trans double bonds band (976–963cm−1) and the percentage of total polar compounds for percentages lower than 30% (A), andfor the entire experiment (B). Validation samples arelabelled with the letter “v” and a number corresponding tothe hours of thermoxidation . 6 • N. Tena, R. Aparicio-Ruiz and D.L. García-González Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 and 0.67%, respectively (Table 1). The relatively high percentage of oleic acid suggests that a significant amount of single cis double bonds, quantified by the FTIR band area (3013–2991cm−1), is due to oleic acid. This hypothesis was verified by regressing the percentage of oleic acid using the band area assigned to single cis double bonds (3013–3991cm−1). The linear model that explains the percentage of oleic acid has an adjusted-R2 of 0.93 (RSD=1.84%): Oleic acid (%)=19.95+22.32×[FTIR cis band area] In addition to cis/trans transformation during thermoxidation, the use of FTIR spectroscopy is also particularly useful for monitoring hydroperoxides and hydroxyl derivatives that are produced during oxidation (van de Voort et al., 2001). Thus, the spectral region of 3600–3410cm−1 was examined to extract information about the oxidation process involved in a thermal degradation (Figure2). This region includes the band assigned to hydroperoxide moieties due to the –OO-H stretching vibration (~3425cm−1) and the band associated to alcohol groups (~3544cm−1) (van de Voort et al., 2001; Russin et al., 2003; van de Voort et al., 1994). The locations of these bands strongly depend on sample polarity and they gradually shift as the oxidation progresses (Russin et al., 2003). The first overtone of stretching vibration of ester group C=O of triacylglycerols, whose fundamental band absorbs at ~1746cm−1, is also located in this region (~3472cm−1) (van de Voort et al., 2001; Russin et al., 2003; van de Voort et al., 1994). Although the concentration of hydroperoxides in oils undergoing thermoxidation is expected to be low due to the breakdown of these compounds at high temperatures (Velasco and Dobarganes, 2002), it is important to note that hydroperoxides may be formed shortly after taking each sample and they are cooled at room temperature before being frozen. Furthermore, the thermoxidation process was carried out emulating a common discontinued frying process, where the oil is kept at room temperature between frying cycles and hydroperoxides are accumulated before the next frying session. Although the FTIR absorption of hydroperoxides is observable in this region, the band assigned to the first overtone of the ester groups C=O (~3472cm−1) clearly interferes in the surrounding regions and makes any study of this area difficult to carry out (Figure 2). Thus, this overlapping negatively affects the quantification of the hydroperoxide band (~3425cm−1) and the overtone of the ester group, which provides information on the hydrolysis of triacylglycerols during thermoxidation. Furthermore, the small concentrations of hydroperoxydes –~12meq of O2·kg−1 of oil (Moya Moreno et al., 1999) – prevent them from being quantified accurately (van de Voort et al., 1994). The extraction of the polar and nonpolar fractions of the samples and their subsequent study by FTIR can resolve the overlapping problem of the hydroxyl region by simplifying the whole spectrum and would provide the added advantage of supplying more details about the chemical changes that take place. 3.2. FTIR spectra of polar and non polar fractions of thermo-oxidized virgin olive oils Figure 3 shows the variance spectra computed from the whole set of spectra collected for the neat oil, polar and nonpolar fractions. These spectra highlight the most significant changes during the whole thermoxidation process. Comparing the variance spectra of polar and nonpolar fractions, it is noticeable that most of the spectral changes are observed in the former (Figure3). The near absence of relevant spectral changes in the nonpolar fraction spectrum is explained by the fact FIGURE 2. Time-course change in the bands assigned to hydroxyl groups (3544cm−1), hydroperoxides (3425cm−1) and the first overtone of esters groups (3472cm−1) during the thermoxidation process in virgin olive oil. 3420344034603480350035203540356035803600 wavenumber (cm–1) 92h 67.95% TPC 30h 20.70% TPC 14h 9.07% TPC 0h 3.45% TPC 0.120 0.124 0.128 0.132 0.136 0.140 0.144 0.148 Absorbance unit Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR • 7 Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 that most of the new products generated in thermal processes are polar (Marmesat et al., 2007). Thus, the compounds containing hydroxyl groups are exclusively present in the polar fraction, and the high intensity of the band assigned to hydroperoxides allows for their quantification. Furthermore, this band is free of the interference of the overtone band associated to the ester linkage (Figure 2). Figure 4 shows the spectral region between 3600–3200cm−1 collected from the polar fraction and nonpolar fraction of a thermoxidized virgin olive oil sample. The spectrum of the nonpolar fraction only shows a FIGURE 3. Variance spectra computed from the whole set of spectra of 47 thermoxidation samples, polar and nonpolar fractions. FIGURE 4. Polar and nonpolar fraction spectra in the region (3600–3200cm−1) of a thermoxidized virgin olive oil (VOO) samples. 0.26 0.27 0.28 0.29 0.30 0.31 0.32 0.33 0.34 0.35 0.36 0.37 0.38 0.39 0.40 Polar fraction Hydroxyl groups Alcohols (-OH) Hydroxyl groups Hydroperoxide (O-OH) Non-polar fraction 1 st Overtone Ester groups (C=O) 1 st Overtone Ester groups (C=O) 32503300335034003450350035503600 Wavenumber (cm –1) Absorbance unit 8 • N. Tena, R. Aparicio-Ruiz and D.L. García-González Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 single band centered within 3510–3430cm−1, which is due to the first overtone of the stretching vibration of the ester group in triacylglycerols (van de Voortet al., 2001). By contrast, in the polar fraction, the spectrum has a higher absorption, which can be divided into three bands: (i) a weak band located within 3510–3430 cm−1, assigned to the first overtone of the ester group of triacylglycerols; (ii) a shoulder at higher wavenumbers assigned to hydroxyl groups of alcohols (~3544cm−1) (van de Voort et al., 2001; Russin et al., 2003; van de Voort etal., 1994); and (iii) a shoulder at lower wavenumbers assigned to the hydroxyl groups of hydroperoxides (3425cm−1) (van de Voort et al., 2001). The hydroperoxides, already present in the non thermoxidized sample, are decomposed into secondary oxidation products during the whole thermoxidation process, reducing their concentration until 20h of thermoxidation (14% of TPC). At this moment, the intensity of this band varies slightly with time and tends to reach a plateau. One of the most relevant bands in the variance spectrum of the polar fraction is located around ~1167 cm−1 (Figure 3). This band is assigned to tertiary alcohols (C-O stretching) (Coates, 2000), which they can be formed as secondary oxidation products. This band rises during thermoxidation following a trend opposite to that of the hydroperoxide band (Figure 5). Thus, this band reaches a maximum intensity at 20 hours of thermoxidation (14% of TPC), probably due to the end of the hydroperoxide breakdown. This band may also be assigned to phenols (~1200 cm−1, C-O stretching) (Coates, 2000), although their contribution to this band may be irrelevant since its intensity rises during the process while the concentration of phenols decreases (Table 1). The moment in which the hydroperoxide and the tertiary alcohols reach a plateau matches with a trend change observed for the different polar chemical groups analyzed by HPSEC. Table 1 shows the percentages of TPC, polymerized triacylglycerols, oxidized triacylglycerols, and diacylglycerols throughout the experiment. Polar polymers and oxidized triacylglycerols are the main compounds produced by oxidation at high temperatures (Dobarganes and Marquez-Ruiz, 1998; Caldwell et al., 2011), and they accounted for 45.61% and 21.91% in most of the thermoxidized oil. The concentration of these compounds rises more rapidly after 20h of the process. This inflection at 20 hours is not observed for other polar compounds resulting from hydrolysis (diacylglycerols and free fatty acids), whose formation is not directly related to hydroperoxide breakdown. The observed inflection at 20h also coincides with the moment in which the concentration of o-diphenols abruptly decreases to 8.71mg·kg−1, which means 6.5% of the initial concentration (Table 1). After that moment, the oil is hardly protected against oxidation by means of antioxidants, and the oil undergoes oxidation at a higher rate, which depends on its composition of fatty acids (Barrera-Arellano et al., 2002). Similar inflections in the increasing trend of polar polymers and oxidized triacylglycerols are observed after 20h (Table 1). In addition to the O-H vibration observed for hydroperoxides at ~3425 cm−1, the C-O-O stretching of peroxides and epoxy produce a weak absorption FIGURE 5. Time-course trend in the the band assigned to hydroperoxides (~3400cm−1) and tertiary alcohols (~1167cm−1) in the spectra of polar fractions during the thermoxidation process. Use of polar and nonpolar fractions as additional information sources for studying thermoxidized virgin olive oils by FTIR • 9 Grasas Aceites 65 (3), July–September 2014, e030. ISSN-L: 0017–3495 doi: http://dx.doi.org/10.3989/gya.121913 TABLE 1. Percentages of total polar compounds, polar polymers, oxidized triacylglycerols, diacylglycerols, oleic, linoleic and linolenic acids, and the content of o-diphenols (mg/kg) in the virgin olive oil samples during the thermoxidation experiment. Note:TAG, Triacylglycerols; DAG, Diacylglycerols; tr, trace levels. Thermoxidation time(hours) Total polar compounds Polar polymers Oxidized TAG DAG C18:1 C18:2 C18:3 o-diphenols 0 3.5 0.2 1.4 1.3 75.9 4.6 0.7 134.1 6 5.1 1.5 1.7 1.4 72.9 4.0 0.6 70.0 10 7.1 2.7 2.5 1.5 72.4 3.9 0.5 29.8 16 9.4 3.9 3.5 1.6 72.2 3.4 0.5 11.6 20 13.0 5.9 4.9 1.8 71.4 3.4 0.5 8.7 26 18.6 8.9 7.2 2.0 70.8 3.1 0.5 7.4 30 20.8 10.9 7.9 1.6 69.5 3.0 0.5 7.2 36 31.0 16.3 12.7 2.2 69.3 2.7 0.4 tr 40 36.1 18.9 15.9 2.0 67.3 2.3 0.2 tr 46 37.9 21.6 14.5 2.6 65.2 2.0 0.2 tr 50 45.7 25.9 18.3 2.6 64.0 1.8 0.2 – 56 50.6 30.5 18.8 3.0 63.0 1.6 0.2 – 60 53.9 32.8 18.8 4.0 62.2 1.6 0.2 – 66 60.5 37.2 21.7 4.0 60.8 1.4 0.2 – 70 61.9 39.7 21.6 3.8 58.4 1.2 0.2 – 76 63.8 40.3 21.9 4.9 58.0 1.2 0.1 tr 80 64.8 42.1 22.1 4.6 55.8 1.0 0.1 – 86 66.0 42.8 22.3 5.1 53.8 0.8 0.1 – 90 67.7 44.7 22.2 6.0 51.3 0.7 tr – 94 68.1 45.6 21.9 5.5 50.5 0.7 tr – FIGURE 6. Time-course change in the peak area and the absorption in the region (890–800cm−1) assigned to peroxide and epoxy groups for the polar fraction during the thermoxidation process.