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Investigations of the adulteration of extra virgin olive oils with seed oils using their weak chemiluminescence
Analytica Chimica Acta 464 (2002) 135–140
Investigations of the adulteration of extra virgin olive oils with seed oils using their weak chemiluminescence K. Papadopoulos a,∗ , T. Triantis a , C.H. Tzikis b , A. Nikokavoura a , D. Dimotikali b a
b
Institute of Physical Chemistry, NRCPS “Demokritos”, 15310 Ag. Paraskevi Attikis, Athens, Greece Chemical Engineering Department, NTU Athens, Iroon Polytechniou 9, 15780 Zografou, Athens, Greece Received 17 December 2001; received in revised form 22 April 2002; accepted 17 May 2002
Abstract A weak chemiluminescence (CL) emission was observed in commercial Greek extra virgin olive oils (Knossos, Spitiko, Ananias, Altis, Minerva, Xenia) and in refined seed oils such as sunflower oils (Marata, Sanola, Sun, Mana, Sol, Minerva) as well as in corn oils (Flora, Minerva, Marata Sun and Sol) with potassium superoxide in the aprotic solvent dimethoxyethylene. On measuring the CL of mixtures of extra virgin olive oils with the cheaper refined seed oils, calibrations were produced which can be used for the determination of the adulteration of olive oils with seed oils down to 3%. Furthermore, depending on the kind of oils, “low” authenticity-CL-factors for olive oils (0.8–2.15 mol l−1 gallic acid) and “high” for seed oils (4.5–11.2 mol l−1 gallic acid) were calculated. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Chemiluminescence; Olive oils; Seed oils; Potassium superoxide
1. Introduction The determination of food authenticity and the detection of adulteration are problems of increasing importance in the food industry. Virgin olive oils are frequently adulterated with other vegetable oils of lower commercial value. Adulteration has been known to exist for a long time and various physical and chemical tests have been devised to address the problem [1]. Ultraviolet (UV) spectroscopy is widely used to detect the adulteration of extra virgin oil with refined olive oil [2,3]. By this technique adulteration of virgin olive oil by refined oils can be detected down to 5%. Other analytical procedures including gas chromatography [4–7] and liquid chromatography ∗ Corresponding author. Tel.: +30-16503647; fax: +30-16511766. E-mail address: [email protected] (K. Papadopoulos).
[8–15] have more recently been developed. By all these techniques, certain compounds contained in oils (triglycerides, trilinoleins, tripalmitins, tocotrienols or tocopherols) are detected, analyzed and used for detecting the adulteration of virgin olive oils. However, all these methods are time-consuming and require skilled operators. More recently, Fourier transform infrared (FTIR) [16–19], Raman [20,21] as well as nuclear magnetic resonance (NMR) [22] spectroscopy have been shown to have potential for discriminating between extra virgin olive and seed oils. Finally, the use of neural networks in combination with pyrolysis-mass spectroscopy for the detection of the adulteration of virgin olive oils must be mentioned [23]. To our knowledge, there is no report of the use of chemiluminescence (CL) which can form the base of most sensitive methods for analytical applications— in the analysis of potential adulteration or presentation of authenticity factors of olive oils. In this paper,
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 4 3 6 - 1
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K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
we present “authenticity factors” of extra virgin olive oils and refined seed oils, which make possible the discrimination of the former from seed oils, and some calibrations graphs, which can be used for the detection of the adulteration of extra virgin olive oils by seed oils. We used commercial extra virgin olive oils and refined seed oils such as sunflower and corn oils because these oils make up the most common consumed edible oils in the market and is of interest to know if one can discriminate between seed oils and olive oils using CL authenticity factors, i.e. adulteration of the more expensive extra virgin olive oils by the cheaper seed oils using CL.
2. Experimental 2.1. Equipment CL measurements were performed on a 1250 Bio-Orbit luminometer with the timer circuitry disconnected. The luminometer (output range 1.0 mV–10 V) is provided with a Hamamatsu photomultiplier tube (HAM 105–21) with side window and spectral range from 300 to 620 nm and connected to a potentiometric chart recorder (GOW-MAC Instrument CO., Model 70–150) or personal computer equipped with a home made software program which allows the continuous monitoring and analysis of the output signal. UV spectra and fluorescence spectra are recorded on a JASCO Spectrophotometer V-560 and a JASCO spectrofluorimeter FP-777 (scanning speed: 200 nm min−1 ; photomultiplier sensitivity: low; excitation and emission bandwidth: 5 nm). 2.2. Reagents All commercial edible oils (olive, sunflower and corn oils) were purchased from Greek supermarkets and used without any further elaboration. According to the manufacturers’ procedures, the extra virgin olive oils are extracted by first cold pressing of hand picked olives. The oxidizing reagent, potassium superoxide, was purchased from Aldrich and used as a saturated solution in dry dimethylsulfoxide (DMSO). The organic solvents 1,2-dimethoxyethylene (DME) (HPLC grade) and DMSO were purchased from Aldrich, Germany. Working solutions were freshly
made. At this point, it must be said that DME is the only solvent in which all oils are quite soluble and give homogeneous and clear solutions, necessary for the CL measurements. 2.3. Preparations of oil–DME-solutions (a) For the evaluation of authenticity factors the oil solutions were prepared as follows: to 10 ml of oil, 20 ml of DME was added and stirred for few minutes until the solution was clear. (b) For the evaluation of the adulteration curves, the oil solutions were prepared as follows: to mixtures of total volume 10 ml, seed oil/extra virgin olive oil, of different proportions (0/10; 0.5/9.5; 1/9; 2/8; 4/6; 6/4; 8/2; 10/0) and 20 ml of DME were added and stirred until the solutions were clear. From these solutions 1.0 ml was taken and used for CL measurements. 2.4. Chemiluminescence measurements The light reactions were started by adding saturated potassium superoxide solution in DMSO (250 l) into Table 1 CL intensities of commercial extra virgin olive oils and seed oils expressed in mV and in authenticity factors No. Olive 1 2 3 4 5 6 7 8
Commercial brandname oils Xenia Ananias Spitiko Knossos Kalamata Altis Marata Minerva
CL intensities (mV)
Authenticity factor (mol l−1 gallic acid)
260 340 540 600 860 920 940 970
0.81 0.94 1.21 1.32 1.92 2.09 2.15 2.24
Sunflower oils 1 Marata 2 Sanola 3 Sun 4 Mana 5 Sol 6 Minerva
1460 1500 1750 1640 2090 3080
4.52 4.78 6.92 5.85 11.22 45.8
Corn oils 1 Flora 2 Lydia 3 Mana 4 Marata 5 Sol
1800 1850 1900 2100 1500
7.35 7.90 8.48 11.29 4.78
K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
the solutions of diluted oils in DME (1:2, 1 ml) with a Hamilton syringe through a septum. At this point, it should be mentioned that pure olive oils or seed oils could not be measured undiluted due to their high viscosity. Oils diluted by DME, in proportion 1:2, could be taken easily with Hamilton syringes and gave the highest CL signals (Table 1). More dilute solutions gave lower CL signals. Saturated potassium superoxide solutions in DMSO gave the highest CL signals and volumes of 250 l of these DMSO solutions are necessary to avoid inhomogeneous solutions (formation of two phases). The relative light intensities (mean value of five measurements) and the calculated authenticity factors, expressed in mol l−1 gallic acid, are shown in Table 1. The CL signals of all measured oils occurred as strong intense bursts. The duration of light emission was not longer than a few seconds.
3. Results and discussion All edible oils, extra virgin olive oils as well as sunflower or seed oils, tested in this work were chemiluminescent with the seed oils being 2–3-fold stronger than olive oils. This is very interesting and can be used, as we will show later, in the production of authenticity factors and possibly for the measurement of adulteration of extra virgin olive oils with seed oils. A representative example of CL for an olive oil with potassium superoxide is shown in Fig. 1. For the evaluation of
137
authenticity factors, gallic acid was used as reference because of its intense CL signals and its broad linear range from 1×10−6 to 1×10−2 mol l−1 . Fig. 2 shows the increase of CL intensity versus concentration of gallic acid which is well described by the linear equation: y = a + b log x, where y is the CL intensity and x the concentration of gallic acid (a = 10080 ± 72.7 and b = 1613 ± 17.6, R = 0.9958, S.D. 68.6, N = 9). This diagram can eventually also be used for the quantitation of gallic acid down to 1 × 10−6 mol l−1 . The CL intensities and the authenticity factors of all oils expressed in gallic acid values (mol l−1 ) are given in Table 1. All tested seed oils show stronger CL than olive oils and have authenticity factors, except Minerva, between 4.52 and 11.29, while the extra virgin olive oils give factors between 0.81 and 2.24 mol l−1 gallic acid. The impressive differences in CL intensities between seed and extra virgin olive oils encouraged us to test this novel and simple method for the determination of the adulteration of commercial virgin olive oils with the cheaper seed oils. The method was applied in four different extra virgin olive oil–seed oil pairs. As shown in Fig. 3, the adulteration (by ourselves) of the expensive extra virgin olive oils, Ananias, Xenia and spitiko with the cheaper sunflower oils, Sol, Sun and Minerva can be determined down to 3%. This low percentage value was determined as the mean value of five measurements of the CL intensity of pure extra virgin olive oil used plus its three-fold standard
Fig. 1. Typical example of CL intensity vs. time diagram of a virgin olive oil.
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K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
Fig. 2. Relative CL intensities vs. the logarithm of concentration of gallic acid.
deviation. The adulteration is well described by the linear equations log I = d + mA, where I is the CL intensity and A the adulteration in percentage of seed oil. The analytical characteristics of each linear equation are given in Table 2. Note that the CL intensities of olive oils produced in our laboratory, from fresh green olives after pressing, drying over magnesium sulfate, filtering and dilution as mentioned earlier in DME (1 ml of oil in 2 ml of DME) gave CL signals of the same range of measured
values as by the extra virgin olive oils bought in supermarkets (400–600 mV). Regarding the mechanism of the weak CL of oils, it is assumed that the light is produced by the oxidation of polyunsaturated fatty acid esters contained in oils (linoleic or linolenic) with superoxide either through excited ketones (direct CL) or after energy transfer to fluorescent compounds (sensitized CL) contained in oils. The oxidation products of polyunsaturated fatty acids or their esters, especially of linoleic
Fig. 3. Typical plots of relative CL intensities vs. the percentage of seed oils (Sol, Sun, Minerva) contained in mixtures of extra virgin olive oils (Ananias, Xenia, spitiko).
K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
139
Table 2 Analytical characteristics of the linear regression equations log I = d +mA, where I is the chemiluminescence intensity and A the percentage of seed oil added to extra virgin olive oil Olive oil–seed oil
d
m
S.D.a
rb
Adulteration (%)
Xenia–Sol Ananias–Sol Ananias–Sun Spitiko–Minerva
2.42 2.54 2.50 2.70
0.0075 0.0079 0.0071 0.0076
0.0157 0.0120 0.0135 0.0219
0.9986 0.9993 0.9989 0.9975
5.1 3.2 5.8 7.7
a b
S.D. of slope (n = 8). Correlation coefficient (n = 8).
acid are known to be weak CL reactions, which can be enhanced (sensitized) in the presence of fluorescent molecules [24–28]. Recently, Kyriakidis and Skarkalis [29] published the fluorescence spectra of some common vegetable oils, including olive oil, corn oil, soybean oil, sunflower oil and cotton oil and showed that each oil exhibits a strong fluorescence band at 430–450 nm. We have also measured the fluorescence of all oils used in this work and found that they show fluorescence in three different regions with maxima at ca. 490 (λexc , 317 nm), 448 (λexc , 380 nm) and at 472, 503 nm (λexc , 420 nm). All seed oils gave much stronger fluorescence bands than the extra virgin olive oils, one fact that must be considered in the interpretation of the observed stronger CL signals of seed oils. With this in mind and the bibliographic information that: (a) the linoleic acid concentration in seed oils is five times more (ca. 50%) than in virgin olive oils (ca. 10%) [30]; (b) it is also possible that energy transfer from the excited CL product back to the reactant linoleic ester could increase the fluorescence intensity, although, the fluorescence of the spent reaction mixture was not much different from that of the seed oil itself explain the higher CL intensities of seed oils. Another important factor that explains the lower CL intensities in virgin olive oils is the higher total phenolic content in these oils [31] which are known to act as antioxidants [30,32] and consequently also reduce the CL intensities. As mentioned earlier, we were unable to obtain a meaningful CL spectrum due to the relatively low intensities, which did not allow the recording of a continuous spectrum which could give some information about the nature of the light emitting species. Attempts to obtain intensity-time diagrams at different emission wavelengths and plot the peaks versus wavelength did not give a meaningful emission spectrum.
The fluorescence spectrum, however, of the CL spent reaction mixture is usually a good approximation to the CL spectrum and this is similar to that of the fluorescence of oils before the CL reaction. This could be an indication that the light is partly produced by energy transfer from oxidized unsaturated hydrocarbons back to the initial linoleic or linolenic esters, or that the oxidized product is not much different from the reactant. Finally, it must be mentioned that the triglyceride ester of linoleic acid, synthesized in our laboratory, gave weak and brief CL signals, similar to those of the oils.
4. Conclusions In this work, we have shown that the weak CL of extra virgin olive oils as well as of seed oils (sunflowers or corn oils) gave significant differences in CL intensities which can be used to discriminate extra virgin olive oils from seed oils (authenticity factors) and possibly for the determination of the adulteration of extra virgin olive oils with cheaper seed oils down to 3%. We tentatively believe that the light is produced by the oxidation of polyunsaturated fatty acid esters, such as linoleic or linolenic acid, and possibly energy transfer to fluorescent species contained in edible oils. References [1] J. Gracian, The chemistry and analysis of olive oil, in: H.A. Boekenoogen (Ed.), Analysis and Characterization of Oils and Fat Products, Vol. 2, Wiley/Interscience, New York, 1968, pp. 315–606. [2] S. Passaloglou-Emmanouilidou, Z. Lebensm, Unters. Forsch. 191 (1990) 132. [3] V.M. Kapoulias, N.K. Andrikopoulos, Food Chem. 23 (1987) 183.
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[4] G. Morchio, A. DiBello, C. Mariani, E. Fedeli, Riv. Ital. Sostanze Grasse 66 (1989) 251. [5] C. Mariani, S. Venturini, E. Fedeli, Riv. Ital. Sostanze Grasse 68 (1991) 283. [6] V.M. Kapoulas, S. Passaloglou-Emmanouilidou, J. Am. Oil Chem. Soc. 58 (1981) 694. [7] N.K. Andrikopoulos, I. Giannakis, V. Tsamtzis, J. Chrom. Sci. 39 (2001) 137. [8] E. Casadei, Riv. Ital. Sostanze Grasse 64 (1987) 373. [9] A. Serani, G. Staiano, Riv. Ital. Sostanze Grasse 66 (1989) 327. [10] J. Sanchis Rodrigues, J. Rodrigues Serano, Alimentaria 28 (1991) 27. [11] E. Salivaras, A.R. McCurdi, J. Am. Oil Chem. Soc. 69 (1992) 935. [12] A.H. El-Hamdy, N.K. El-Fizga, J. Chromatogr. A 708 (1995) 351. [13] R. Apricio, R. Aparicio-Ruiz, J. Chromatogr. A 881 (2000) 93. [14] M. Tsimidoy, R. Macrae, I. Wilson, Food Chem. 25 (1987) 251. [15] F. Dionisi, J. Prodolliet, E. Tagliaferri, J. Am. Oil Chem. Soc. 72 (1995) 1505. [16] Y.W. Lai, E.K. Kemsley, R.H. Wilson, J. Agric. Food Chem. 42 (1994) 1154. [17] Y.W. Lai, E.K. Kemsley, R.H. Wilson, Food Chem. 53 (1995) 95. [18] L. Kuepper, H.M. Heise, P. Lampen, A.N. Davies, P. McIntyre, Appl. Spectrosc. 55 (2001) 563.
[19] N.A. Marigheto, E.K. Kemsley, M. Defernez, R.H. Wilson, J. Am. Oil Chem. Soc. 75 (1998) 987. [20] A.N. Davies, P. McIntyre, E. Morgan, Appl. Spectrosc. 54 (2000) 1664. [21] V. Baeten, M. Meurens, J. Morales, R. Aparicio, J. Agric. Food Chem. 44 (1996) 2225. [22] T. Mavromoustakos, Z. Zervou, G. Bonas, A. Coulouris, P. Petrakis, J. Am. Oil Chem. Soc. 77 (2000) 405. [23] R. Goodacre, D. Kell, G. Bianchi, Analysis Europa, J. Sci. Agric. 63 (1993) 297. [24] T. Miyazawa, H. Kunika, K. Fujimoto, Y. Endo, T. Kaneda, Lipids 30 (1995) 1001. [25] I. Neeman, D. Joseph, W.H. Biggley, H.H. Seliger, Lipids 20 (1985) 729. [26] T. Metsa-Ketela, A. Montfoort, T. Kunnas, Basic Life Sci. 49 (1988) 233. [27] G.A. Velding, G.J. Garssen, S. Slappendel, J.F. Bolding, Biochem. Biophys. Res. Commun. 78 (1977) 424. [28] T. Watanabe, N. Shirai, H. Okada, Y. Honda, M. Kuwahara, Eur. J. Biochem. 268 (2001) 6114. [29] N. Kyriakidis, P. Skarkalis, J. AOAC Intern. 83 (2000) 1435. [30] R.W. Owen, A. Giacosa, W.E. Hull, R. Haubner, B. Spiegelhalder, H. Bartsch, Eur. J. Cancer 36 (2000) 1235. [31] D. Rayan, K. Robards, S. Lavee, J. Chromatogr. A 832 (1999) 87. [32] R.W. Owen, W. Mier, A. Giacosa, W.E. Hull, B. Spiegelhalder, H. Bartsch, Food Chem. Toxicol. 38 (2000) 647.
Investigations of the adulteration of extra virgin olive oils with seed oils using their weak chemiluminescence K. Papadopoulos a,∗ , T. Triantis a , C.H. Tzikis b , A. Nikokavoura a , D. Dimotikali b a
b
Institute of Physical Chemistry, NRCPS “Demokritos”, 15310 Ag. Paraskevi Attikis, Athens, Greece Chemical Engineering Department, NTU Athens, Iroon Polytechniou 9, 15780 Zografou, Athens, Greece Received 17 December 2001; received in revised form 22 April 2002; accepted 17 May 2002
Abstract A weak chemiluminescence (CL) emission was observed in commercial Greek extra virgin olive oils (Knossos, Spitiko, Ananias, Altis, Minerva, Xenia) and in refined seed oils such as sunflower oils (Marata, Sanola, Sun, Mana, Sol, Minerva) as well as in corn oils (Flora, Minerva, Marata Sun and Sol) with potassium superoxide in the aprotic solvent dimethoxyethylene. On measuring the CL of mixtures of extra virgin olive oils with the cheaper refined seed oils, calibrations were produced which can be used for the determination of the adulteration of olive oils with seed oils down to 3%. Furthermore, depending on the kind of oils, “low” authenticity-CL-factors for olive oils (0.8–2.15 mol l−1 gallic acid) and “high” for seed oils (4.5–11.2 mol l−1 gallic acid) were calculated. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Chemiluminescence; Olive oils; Seed oils; Potassium superoxide
1. Introduction The determination of food authenticity and the detection of adulteration are problems of increasing importance in the food industry. Virgin olive oils are frequently adulterated with other vegetable oils of lower commercial value. Adulteration has been known to exist for a long time and various physical and chemical tests have been devised to address the problem [1]. Ultraviolet (UV) spectroscopy is widely used to detect the adulteration of extra virgin oil with refined olive oil [2,3]. By this technique adulteration of virgin olive oil by refined oils can be detected down to 5%. Other analytical procedures including gas chromatography [4–7] and liquid chromatography ∗ Corresponding author. Tel.: +30-16503647; fax: +30-16511766. E-mail address: [email protected] (K. Papadopoulos).
[8–15] have more recently been developed. By all these techniques, certain compounds contained in oils (triglycerides, trilinoleins, tripalmitins, tocotrienols or tocopherols) are detected, analyzed and used for detecting the adulteration of virgin olive oils. However, all these methods are time-consuming and require skilled operators. More recently, Fourier transform infrared (FTIR) [16–19], Raman [20,21] as well as nuclear magnetic resonance (NMR) [22] spectroscopy have been shown to have potential for discriminating between extra virgin olive and seed oils. Finally, the use of neural networks in combination with pyrolysis-mass spectroscopy for the detection of the adulteration of virgin olive oils must be mentioned [23]. To our knowledge, there is no report of the use of chemiluminescence (CL) which can form the base of most sensitive methods for analytical applications— in the analysis of potential adulteration or presentation of authenticity factors of olive oils. In this paper,
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 4 3 6 - 1
136
K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
we present “authenticity factors” of extra virgin olive oils and refined seed oils, which make possible the discrimination of the former from seed oils, and some calibrations graphs, which can be used for the detection of the adulteration of extra virgin olive oils by seed oils. We used commercial extra virgin olive oils and refined seed oils such as sunflower and corn oils because these oils make up the most common consumed edible oils in the market and is of interest to know if one can discriminate between seed oils and olive oils using CL authenticity factors, i.e. adulteration of the more expensive extra virgin olive oils by the cheaper seed oils using CL.
2. Experimental 2.1. Equipment CL measurements were performed on a 1250 Bio-Orbit luminometer with the timer circuitry disconnected. The luminometer (output range 1.0 mV–10 V) is provided with a Hamamatsu photomultiplier tube (HAM 105–21) with side window and spectral range from 300 to 620 nm and connected to a potentiometric chart recorder (GOW-MAC Instrument CO., Model 70–150) or personal computer equipped with a home made software program which allows the continuous monitoring and analysis of the output signal. UV spectra and fluorescence spectra are recorded on a JASCO Spectrophotometer V-560 and a JASCO spectrofluorimeter FP-777 (scanning speed: 200 nm min−1 ; photomultiplier sensitivity: low; excitation and emission bandwidth: 5 nm). 2.2. Reagents All commercial edible oils (olive, sunflower and corn oils) were purchased from Greek supermarkets and used without any further elaboration. According to the manufacturers’ procedures, the extra virgin olive oils are extracted by first cold pressing of hand picked olives. The oxidizing reagent, potassium superoxide, was purchased from Aldrich and used as a saturated solution in dry dimethylsulfoxide (DMSO). The organic solvents 1,2-dimethoxyethylene (DME) (HPLC grade) and DMSO were purchased from Aldrich, Germany. Working solutions were freshly
made. At this point, it must be said that DME is the only solvent in which all oils are quite soluble and give homogeneous and clear solutions, necessary for the CL measurements. 2.3. Preparations of oil–DME-solutions (a) For the evaluation of authenticity factors the oil solutions were prepared as follows: to 10 ml of oil, 20 ml of DME was added and stirred for few minutes until the solution was clear. (b) For the evaluation of the adulteration curves, the oil solutions were prepared as follows: to mixtures of total volume 10 ml, seed oil/extra virgin olive oil, of different proportions (0/10; 0.5/9.5; 1/9; 2/8; 4/6; 6/4; 8/2; 10/0) and 20 ml of DME were added and stirred until the solutions were clear. From these solutions 1.0 ml was taken and used for CL measurements. 2.4. Chemiluminescence measurements The light reactions were started by adding saturated potassium superoxide solution in DMSO (250 l) into Table 1 CL intensities of commercial extra virgin olive oils and seed oils expressed in mV and in authenticity factors No. Olive 1 2 3 4 5 6 7 8
Commercial brandname oils Xenia Ananias Spitiko Knossos Kalamata Altis Marata Minerva
CL intensities (mV)
Authenticity factor (mol l−1 gallic acid)
260 340 540 600 860 920 940 970
0.81 0.94 1.21 1.32 1.92 2.09 2.15 2.24
Sunflower oils 1 Marata 2 Sanola 3 Sun 4 Mana 5 Sol 6 Minerva
1460 1500 1750 1640 2090 3080
4.52 4.78 6.92 5.85 11.22 45.8
Corn oils 1 Flora 2 Lydia 3 Mana 4 Marata 5 Sol
1800 1850 1900 2100 1500
7.35 7.90 8.48 11.29 4.78
K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
the solutions of diluted oils in DME (1:2, 1 ml) with a Hamilton syringe through a septum. At this point, it should be mentioned that pure olive oils or seed oils could not be measured undiluted due to their high viscosity. Oils diluted by DME, in proportion 1:2, could be taken easily with Hamilton syringes and gave the highest CL signals (Table 1). More dilute solutions gave lower CL signals. Saturated potassium superoxide solutions in DMSO gave the highest CL signals and volumes of 250 l of these DMSO solutions are necessary to avoid inhomogeneous solutions (formation of two phases). The relative light intensities (mean value of five measurements) and the calculated authenticity factors, expressed in mol l−1 gallic acid, are shown in Table 1. The CL signals of all measured oils occurred as strong intense bursts. The duration of light emission was not longer than a few seconds.
3. Results and discussion All edible oils, extra virgin olive oils as well as sunflower or seed oils, tested in this work were chemiluminescent with the seed oils being 2–3-fold stronger than olive oils. This is very interesting and can be used, as we will show later, in the production of authenticity factors and possibly for the measurement of adulteration of extra virgin olive oils with seed oils. A representative example of CL for an olive oil with potassium superoxide is shown in Fig. 1. For the evaluation of
137
authenticity factors, gallic acid was used as reference because of its intense CL signals and its broad linear range from 1×10−6 to 1×10−2 mol l−1 . Fig. 2 shows the increase of CL intensity versus concentration of gallic acid which is well described by the linear equation: y = a + b log x, where y is the CL intensity and x the concentration of gallic acid (a = 10080 ± 72.7 and b = 1613 ± 17.6, R = 0.9958, S.D. 68.6, N = 9). This diagram can eventually also be used for the quantitation of gallic acid down to 1 × 10−6 mol l−1 . The CL intensities and the authenticity factors of all oils expressed in gallic acid values (mol l−1 ) are given in Table 1. All tested seed oils show stronger CL than olive oils and have authenticity factors, except Minerva, between 4.52 and 11.29, while the extra virgin olive oils give factors between 0.81 and 2.24 mol l−1 gallic acid. The impressive differences in CL intensities between seed and extra virgin olive oils encouraged us to test this novel and simple method for the determination of the adulteration of commercial virgin olive oils with the cheaper seed oils. The method was applied in four different extra virgin olive oil–seed oil pairs. As shown in Fig. 3, the adulteration (by ourselves) of the expensive extra virgin olive oils, Ananias, Xenia and spitiko with the cheaper sunflower oils, Sol, Sun and Minerva can be determined down to 3%. This low percentage value was determined as the mean value of five measurements of the CL intensity of pure extra virgin olive oil used plus its three-fold standard
Fig. 1. Typical example of CL intensity vs. time diagram of a virgin olive oil.
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K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
Fig. 2. Relative CL intensities vs. the logarithm of concentration of gallic acid.
deviation. The adulteration is well described by the linear equations log I = d + mA, where I is the CL intensity and A the adulteration in percentage of seed oil. The analytical characteristics of each linear equation are given in Table 2. Note that the CL intensities of olive oils produced in our laboratory, from fresh green olives after pressing, drying over magnesium sulfate, filtering and dilution as mentioned earlier in DME (1 ml of oil in 2 ml of DME) gave CL signals of the same range of measured
values as by the extra virgin olive oils bought in supermarkets (400–600 mV). Regarding the mechanism of the weak CL of oils, it is assumed that the light is produced by the oxidation of polyunsaturated fatty acid esters contained in oils (linoleic or linolenic) with superoxide either through excited ketones (direct CL) or after energy transfer to fluorescent compounds (sensitized CL) contained in oils. The oxidation products of polyunsaturated fatty acids or their esters, especially of linoleic
Fig. 3. Typical plots of relative CL intensities vs. the percentage of seed oils (Sol, Sun, Minerva) contained in mixtures of extra virgin olive oils (Ananias, Xenia, spitiko).
K. Papadopoulos et al. / Analytica Chimica Acta 464 (2002) 135–140
139
Table 2 Analytical characteristics of the linear regression equations log I = d +mA, where I is the chemiluminescence intensity and A the percentage of seed oil added to extra virgin olive oil Olive oil–seed oil
d
m
S.D.a
rb
Adulteration (%)
Xenia–Sol Ananias–Sol Ananias–Sun Spitiko–Minerva
2.42 2.54 2.50 2.70
0.0075 0.0079 0.0071 0.0076
0.0157 0.0120 0.0135 0.0219
0.9986 0.9993 0.9989 0.9975
5.1 3.2 5.8 7.7
a b
S.D. of slope (n = 8). Correlation coefficient (n = 8).
acid are known to be weak CL reactions, which can be enhanced (sensitized) in the presence of fluorescent molecules [24–28]. Recently, Kyriakidis and Skarkalis [29] published the fluorescence spectra of some common vegetable oils, including olive oil, corn oil, soybean oil, sunflower oil and cotton oil and showed that each oil exhibits a strong fluorescence band at 430–450 nm. We have also measured the fluorescence of all oils used in this work and found that they show fluorescence in three different regions with maxima at ca. 490 (λexc , 317 nm), 448 (λexc , 380 nm) and at 472, 503 nm (λexc , 420 nm). All seed oils gave much stronger fluorescence bands than the extra virgin olive oils, one fact that must be considered in the interpretation of the observed stronger CL signals of seed oils. With this in mind and the bibliographic information that: (a) the linoleic acid concentration in seed oils is five times more (ca. 50%) than in virgin olive oils (ca. 10%) [30]; (b) it is also possible that energy transfer from the excited CL product back to the reactant linoleic ester could increase the fluorescence intensity, although, the fluorescence of the spent reaction mixture was not much different from that of the seed oil itself explain the higher CL intensities of seed oils. Another important factor that explains the lower CL intensities in virgin olive oils is the higher total phenolic content in these oils [31] which are known to act as antioxidants [30,32] and consequently also reduce the CL intensities. As mentioned earlier, we were unable to obtain a meaningful CL spectrum due to the relatively low intensities, which did not allow the recording of a continuous spectrum which could give some information about the nature of the light emitting species. Attempts to obtain intensity-time diagrams at different emission wavelengths and plot the peaks versus wavelength did not give a meaningful emission spectrum.
The fluorescence spectrum, however, of the CL spent reaction mixture is usually a good approximation to the CL spectrum and this is similar to that of the fluorescence of oils before the CL reaction. This could be an indication that the light is partly produced by energy transfer from oxidized unsaturated hydrocarbons back to the initial linoleic or linolenic esters, or that the oxidized product is not much different from the reactant. Finally, it must be mentioned that the triglyceride ester of linoleic acid, synthesized in our laboratory, gave weak and brief CL signals, similar to those of the oils.
4. Conclusions In this work, we have shown that the weak CL of extra virgin olive oils as well as of seed oils (sunflowers or corn oils) gave significant differences in CL intensities which can be used to discriminate extra virgin olive oils from seed oils (authenticity factors) and possibly for the determination of the adulteration of extra virgin olive oils with cheaper seed oils down to 3%. We tentatively believe that the light is produced by the oxidation of polyunsaturated fatty acid esters, such as linoleic or linolenic acid, and possibly energy transfer to fluorescent species contained in edible oils. References [1] J. Gracian, The chemistry and analysis of olive oil, in: H.A. Boekenoogen (Ed.), Analysis and Characterization of Oils and Fat Products, Vol. 2, Wiley/Interscience, New York, 1968, pp. 315–606. [2] S. Passaloglou-Emmanouilidou, Z. Lebensm, Unters. Forsch. 191 (1990) 132. [3] V.M. Kapoulias, N.K. Andrikopoulos, Food Chem. 23 (1987) 183.
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