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Functional group analysis during ozonation of sunflower oil methyl esters by FT-IR and NMR
Chemistry and Physics of Lipids 126 (2003) 133–140
Functional group analysis during ozonation of sunflower oil methyl esters by FT-IR and NMR Nestor U. Soriano, Jr a , Veronica P. Migo b , Masatoshi Matsumura c,∗ a
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-0006, Japan b National Institute of Molecular Biology and Biotechnology (BIOTECH), University of Philippines Los Banos, College 4031, Laguna, Philippines c Institute of Applied Biochemistry, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Received 29 May 2003; accepted 25 July 2003
Abstract Ozonation of neat sunflower oil (SFO) methyl esters was monitored by FT-IR and 1 H and 13 C NMR spectroscopy. During the early stage of ozonation, ozone absorption was essentially quantitative. This was accompanied by the formation of 1,2,4-trioxolane. IR and NMR spectra of ozonated samples showed that scission of ozonide to give aldehyde were minimal. 1 H NMR analysis revealed that the amount of ozonide relative to aldehyde was more than 90% regardless of the extent of ozonation. Complete ozonation was attained after supplying around 0.20 g O3 /ml methyl ester after which ozone absorption suddenly dropped to around 25%. At the latter part of ozonation, ozonide and aldehyde reacted with excess ozone to give carboxylic acid. Reaction products were identified according to Criegee mechanism. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ozonation; IR and NMR spectra; Criegee mechanism
1. Introduction Ozonolysis of olefins is well understood as described by the Criegee mechanism. The electrophilic ozone molecule adds to the carbon–carbon double bond to give an unstable cyclic intermediate called initial ozonide. In the absence of any participating solvent, this intermediate leads to the formation of 1,2,4-trioxolane and peroxide oligomer (Scheme 1). Various workers have investigated the ozonolysis of fatty acid methyl esters both in participating and ∗
Corresponding author. E-mail address: [email protected] (M. Matsumura).
non-participating solvents. Spectroscopic analyses of the products used include FT-IR, NMR, LC–MS and GC–MS. Rebrovic (1992) used 1 H NMR spectroscopy to identify the three major peroxidic species and aldehyde formed from the ozonolysis of methyl oleate in carboxylic acid medium. These peroxidic compounds include 1-acyloxyalkyl-1-hydroperoxides, 1,2,4-trioxolane and bis(1-acyloxy-1-alkyl) peroxides. Identification of the reaction products of ozonolysis of methyl oleate in the presence and absence of protic solvents was done by Ledea et al. (1998) using GC–MS and 1 H NMR. In the presence of methanol, ethoxy-hydroperoxide was found to be the major product and only small amount of the ozonides was
0009-3084/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2003.07.001
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of glycerol in the crude methyl ester was removed by centrifugation. The crude methyl ester was further purified by distilling-off the unreacted methanol under normal atmospheric pressure, washing several times with water, centrifugation and drying with anhydrous Na2 SO4 . 2.2. Ozonation
Scheme 1.
detected. The analysis of the reaction product when water was added revealed only the presence of aldehydes (hexanal, nonanal and 9-oxo-methylnonanoate). Diaz et al. (1998) investigated the effects of solvents (water and ethanol) and degree of unsaturation of the sample to the rate of ozonolysis. 1 H NMR spectroscopy at 250 MHz was used to follow up the course of the reaction. Diaz and co-workers found out that there was a higher consumption of the double bonds during the ozonation of methyl oleate with the addition of the solvents especially with the addition of water. The consumption of the double bonds was even faster when methyl linoleate was used. In this paper, we monitored the absorption of ozone in neat SFO methyl esters and the product was analyzed by FT-IR and 1 H and 13 C NMR spectroscopy. These analyses provide information on the functional group changes during ozonation as well as the identification of the products without prior separation techniques.
2. Materials and methods 2.1. Transesterification The reaction was carried out by mixing 1000.0 g sunflower oil, 218.6 g methanol and 5.00 g NaOCH3 in a 2 l jar fermentor. The temperature was maintained at 60–70 ◦ C for 2 h under reflux with stirring at 500 rpm. The reaction mixture was allowed to stand overnight and the methyl ester layer was separated from the glycerol layer using separatory funnel. Residual amount
Ozone/oxygen mixture containing 30–40 g/m3 ozone was bubbled into a 2 l jar fermentor containing 500 ml SFO methyl esters at a rate of 2.0 l/min for 35 h while stirring at 250 rpm. The amount of ozone absorbed by the carbon–carbon double bond in SFO methyl esters was monitored every hour. The concentration of ozone (g/m3 ) coming in and going out from the reactor was measured using TOA OZ-30 ozone meter. 2.3. Spectroscopic analysis IR spectra were recorded on thin films cast onto KBr plates using Jasco FT-IR 300 Spectrometer. All spectra were the result of 20 scans with a 4 cm−1 resolution. For 1 H and 13 C NMR spectroscopy, about 30 mg of the sample was dissolved in CDCl3 containing TMS as standard. Both spectra were obtained using Bruker Avance 600 Spectrometer. Trace amount of CHCl3 in the solvent used exhibits signal at 7.26 and 77.0 ppm in 1 H and 13 C NMR, respectively. 3. Results and discussions 3.1. FT-IR analysis FT-IR analyses were done to investigate the changes in the functional groups found in SFO methyl esters during ozonation. Fig. 1 shows the IR spectra of neat and ozonated SFO methyl esters. Summary of characteristic bands found in SFO methyl esters is shown in Table 1. Several major changes were observed in IR spectra of ozonated samples. As expected, intensities of all bands corresponding to the presence of carbon–carbon double bonds decreased with increasing duration of ozonation. These include the relatively weak band due to C=C stretch at 1650 cm−1 and the =C–H stretch and bend at 3008 and 723 cm−1 , respectively.
N.U. Soriano Jr et al. / Chemistry and Physics of Lipids 126 (2003) 133–140 Fig. 1. IR spectra of neat and ozonated SFO methyl esters (A: neat, B: 15 h, C: 25 h and D: 35 h ozonated). Amount of ozone consumed for samples B, C and D were 0.119, 0.196 and 0.222 g O3 /ml methyl ester.
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Table 1 IR interpretation of SFO methyl esters Functional group C=C stretch =C–H stretch =C–H bend C=O stretch C–O stretch O–C stretch CH3 bend
Table 2 1 H and 13 C NMR assignments of SFO methyl esters Wavenumber (cm−1 ) 1650 3008 723 1743 1170 1195 1361
The band due to carbonyl stretch of SFO methyl esters at 1743 cm−1 is relatively sharp. With increasing duration of ozonation, this band became broader suggesting the formation of new carbonyl compounds during ozonation. In fact after 30 h of ozonation, a shoulder appeared at around 1700 cm−1 . These could only be aldehyde and/or carboxylic acid. Aldehyde may be formed from the scission of either initial or final ozonide. Accompanying the former reaction is the formation of oligomer while the latter, could have resulted from the further reaction of ozonide with ozone to give carboxylic acid. However, presence of aldehydic C–H stretch at around 2900–2700 cm−1 was absent, 1 H NMR though confirmed their presence in ozonated samples. The appearance of relatively broad but strong band at 1105 cm−1 with increasing duration of ozonation was assigned to C–O stretch of the ozonide consistent with the reported value in the literature (Wu et al., 1992). This suggests that scission of the final ozonide was minimal. Early studies of ozonide by FT-IR revealed that its trans isomer absorbed at around 1300 cm−1 while its cis isomer at around 800 cm−1 (Bailey, 1978; Murray et al., 1967). During ozonation of SFO methyl esters, a broad peak appeared at around 1365 cm−1 that became stronger with increasing dosage of ozone absorbed, suggesting that the major ozonide product was the trans isomer. 3.2. NMR analysis Fig. 2 shows the NMR spectra of neat SFO methyl esters and assignments to all pertinent peaks were summarized in Table 2 (Diaz et al., 1998; Miyake et al., 1998; Lie Ken Jie and Mustafa, 1997). NMR analyses further confirmed the structural changes undergone by SFO methyl esters during
Chemical shift (ppm)
Functional group
1H
13 C
0.89 1.30 1.62 2.04 2.30 2.77 3.67 5.34 –
14.1 29.1–31.9 24.9 27.2 34.1 24.9 51.5 128.0–130.0 174.3
CH2 CH3 (CH2 )n CH2 CH2 COOCH3 CH2 CH=CHCH2 CH2 COOCH3 CH=CHCH2 CH=CH COOCH3 CH2 CH=CHCH2 COOCH3
ozonation (Fig. 3). New signal at 5.17 ppm found in 1 H NMR spectra of ozonated samples were assigned to the ring proton of 1,2,4-trioxolane (Diaz et al., 1998; Ewing et al., 1989; Ledea et al., 1998; Nishikawa et al., 1995; Rebrovic, 1992; Wu et al., 1992). This was supported by the appearance of signal in 13 C NMR spectra at 104.5 ppm assigned by Wu et al. (1992) as the ring carbon in the same structure. The protons of the methylene group ␣ to the sp2 -hybridized carbons, which resonated at 2.04 and 2.77 ppm shifted to 1.35 and 1.75 ppm upon formation of 1,2,4-trioxolane (Table 3) (Anachkov et al., 2000). Ozonated samples exhibited peaks at 9.75 and 2.50 ppm in 1 H NMR corresponding to the aldehydic proton and the methylene protons ␣ to the carbonyl carbon, respectively. In 13 C NMR, the carbonyl carbon and the carbon ␣ to it, resonated at 202.8 and 43.9 ppm, respectively. The time course of ozone absorption during the ozonation of neat SFO methyl esters is shown in Table 3 1 H and 13 C NMR assignments of new signals found in ozonated SFO methyl esters Chemical shift (ppm)
Functional group
1H
13 C
5.17 1.35, 1.71 9.75 2.50
104.5 202.8 43.9 178.5 35.6
1,2,4-Trioxolane ␣-Methylene group Aldehyde ␣-Methylene group Carboxylic acid ␣-Methylene group
N.U. Soriano Jr et al. / Chemistry and Physics of Lipids 126 (2003) 133–140
Fig. 2. 1 H and
13 C
137
NMR spectra of neat SFO methyl esters.
Fig. 4. The ozone absorption was essentially quantitative during the early stage of ozonation and suddenly dropped after 25 h. The sudden drop in absorption was explained by the complete consumption of carbon–carbon double bond in SFO methyl esters. Fig. 5 shows the NMR spectra of completely ozonated sample. After 25 h of ozonation amounting to around 0.20 g O3 /ml methyl ester, the peak at 2.04 and 5.34 ppm in 1 H NMR spectra and 128.0–130.0 ppm in 13 C NMR spectra completely disappeared. To understand more the trend in the changes in functional groups in SFO methyl esters during ozonation, the ratio between the integrated signals due to various functionalities and the average area per proton
were plotted against the amount of ozone absorbed. The average area per proton was calculated from the integrated intensity of the signals due to terminal methyl group of the fatty acid moiety (0.89 ppm), the methylene group ␣ to the carbonyl carbon (2.30 ppm) and the free methyl group of the ester moiety (3.67 ppm). These functionalities were chosen because they were not affected by ozonation. The consumption of carbon–carbon bond was evaluated considering the integrated intensity of the signal due to methylene protons ␣ to sp2 -hybridized carbon (2.04 ppm). The formation of ozonide and aldehyde were monitored from the integrated intensity of the signals at 1.35, 1.71 and 9.75 ppm, respectively.
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Fig. 3. 1 H NMR spectra of neat and partially ozonated SFO methyl esters (A: neat; B: ozonated for 15 h amounting to 0.119 g O3 /ml methyl ester).
Fig. 6 shows the rapid consumption of ozone by the carbon–carbon double bonds present in SFO methyl esters. This is accompanied by the formation 1,2,4-trioxolane. Linear regression analysis on series of data corresponding to the methylene groups
␣ to the sp2 -hybridized carbon and 1,2,4-trioxolane suggests that the rate of disappearance of unsaturation and the formation of ozonide was almost equal (Table 4). This also suggests that the major product of the reaction is ozonide and that scission of ozonide was very minimal. Table 4 Linear regression on data series on the disappearance of unsaturation and formation of ozonide during ozonationa 1H
NMR shift (ppm)
2.04 1.31 1.71 Fig. 4. Time course of ozone absorption.
Slope (ratio/ozone dosage)
R2
−17.7 17.9 17.6
0.998 0.992 0.992
a Linear regression analysis was done only from time 0 to time 25 h (0.196 g O3 /ml methyl ester).
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Table 5 Ozonide to aldehyde ratio Duration of Ozone dosage ozonation (h) (g O3 /ml methyl ester)
5 10 15 20 25 30 35
0.041 0.080 0.119 0.158 0.196 0.211 0.222
Ozonide:aldehyde ratio 1.35 ppm: 1.71 ppm: 9.76 ppm 9.76 ppm 94.1:5.9 92.9:7.1 92.0:8.0 95.1:4.9 95.6:4.4 97.1:2.9 97.2:2.8
93.8:6.2 93.0:7.0 92.4:7.6 94.7:5.3 95.3:4.7 97.1:2.9 97.0:3.0
with only one mole of ozone. The formation of allylic 1,2,4-trioxolane shifted the signal of these protons from 5.34 ppm in the original structure to 5.58 ppm.
Fig. 5. 1 H and ester.
13 C
NMR of completely ozonated SFO methyl
The signals at 1.35 ppm and 1.71 ppm do not discriminate the oleate and linoleate methyl ester ozonide. However, new signal at 5.58 ppm appeared in early stage of ozonation, gradually increased until 15 h, and finally disappeared after 25 h of ozonation. This was assigned to the protons bonded to sp2 hybridized carbon of linoleate methyl ester ozonide resulted from the reaction of linoleate methyl ester
In the latter part of ozonation when all the carbon–carbon double had been consumed, the amount of ozone absorbed was only about 25%. In the absence of unsaturation, ozonide formed in the early stage of ozonation was cleaved to give carboxylic acid. The signal due to carboxyl carbon was observed at 178 ppm in 13 C NMR. Table 5 shows the ozonide to aldehyde ratio obtained from various stages of ozonation. The amount of ozonide regardless of the extent of ozonation was
Fig. 6. Ratios of 1 H NMR peaks intensity relative to area per proton against amount of ozone absorbed by SFO methyl esters.
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always above 90%. The relatively small amount of aldehyde suggests that oligomerization was also minimal. The decreasing amount of aldehyde relative to ozonide with increasing duration of ozonation could be attributed to volatilization and/or reaction with excess ozone to give carboxylic acid. Acknowledgements This research was conducted under the support from NEDO International Joint Research Grant. References Anachkov, M.P., Rakovski, S.K., Stefanova, R.V., 2000. Ozonolysis of 1,4-cis-polyisoprene and 1,4-trans-polyisoprene in solution. Polym. Deg. Stab. 67, 355–363. Bailey, P.S., 1978. Ozonation in Organic Chemistry, vol. 1. Academic Press, New York, pp. 25–37. Diaz, M., Hernandez, F., Alvarez, I., Velez, H., Ledea, O., Molerio, J., 1998. The 1 H NMR spectroscopy in following ozone reaction with unsaturated fatty acids. Revista CENIC Ciencias Quimicas 29, 89–93.
Ewing, J.C., Cosgrove, J.P., Giamalva, D.H., Church, D.F., Pryor, W.A., 1989. Autooxidation of methyl linoleate initiated by the ozonide of allylbenzene. Lipids 24, 609–615. Ledea, O., Molerio, J., Diaz, M., Jardines, D., Rosado, A., Correa, T., 1998. Analysis of ozonides and peroxidic compounds from methyl oleate ozonation. Revista CENIC Ciencias Quimicas 29, 75–78. Lie Ken Jie, M.S.F., Mustafa, J., 1997. High-resolution nuclear magnetic resonance spectroscopy—application to fatty acids and triglycerols. Lipids 32, 1019–1034. Miyake, Y., Yokomizo, K., Matsuzaki, N., 1998. Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy. J. Am. Oil Chem. Soc. 75, 1091–1094. Murray, R.W., Youssefyeh, R.D., Story, P.R., 1967. Ozonolysis. Steric and stereochemical effects in the olefin. J. Am. Oil Chem. Soc. 89, 2429–2434. Nishikawa, N., Yamada, K., Matsutani, S., Higo, M., Kigawa, H., Inagaki, T., 1995. Structures of ozonolysis products of methyl oleate obtained in a carboxylic acid medium. J. Am. Oil Chem. Soc. 72, 735–740. Rebrovic, L., 1992. The peroxidic species generated by ozonolysis of oleic acid or methyl oleate in a carboxylic acid medium. J. Am. Oil Chem. Soc. 69, 159–165. Wu, M., Church, D.F., Mahier, T.J., Barker, S.A., Pryor, W.A., 1992. Separation and spectral data of the six isomeric ozonides from methyl oleate. Lipids 27, 129–135.
Functional group analysis during ozonation of sunflower oil methyl esters by FT-IR and NMR Nestor U. Soriano, Jr a , Veronica P. Migo b , Masatoshi Matsumura c,∗ a
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-0006, Japan b National Institute of Molecular Biology and Biotechnology (BIOTECH), University of Philippines Los Banos, College 4031, Laguna, Philippines c Institute of Applied Biochemistry, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Received 29 May 2003; accepted 25 July 2003
Abstract Ozonation of neat sunflower oil (SFO) methyl esters was monitored by FT-IR and 1 H and 13 C NMR spectroscopy. During the early stage of ozonation, ozone absorption was essentially quantitative. This was accompanied by the formation of 1,2,4-trioxolane. IR and NMR spectra of ozonated samples showed that scission of ozonide to give aldehyde were minimal. 1 H NMR analysis revealed that the amount of ozonide relative to aldehyde was more than 90% regardless of the extent of ozonation. Complete ozonation was attained after supplying around 0.20 g O3 /ml methyl ester after which ozone absorption suddenly dropped to around 25%. At the latter part of ozonation, ozonide and aldehyde reacted with excess ozone to give carboxylic acid. Reaction products were identified according to Criegee mechanism. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ozonation; IR and NMR spectra; Criegee mechanism
1. Introduction Ozonolysis of olefins is well understood as described by the Criegee mechanism. The electrophilic ozone molecule adds to the carbon–carbon double bond to give an unstable cyclic intermediate called initial ozonide. In the absence of any participating solvent, this intermediate leads to the formation of 1,2,4-trioxolane and peroxide oligomer (Scheme 1). Various workers have investigated the ozonolysis of fatty acid methyl esters both in participating and ∗
Corresponding author. E-mail address: [email protected] (M. Matsumura).
non-participating solvents. Spectroscopic analyses of the products used include FT-IR, NMR, LC–MS and GC–MS. Rebrovic (1992) used 1 H NMR spectroscopy to identify the three major peroxidic species and aldehyde formed from the ozonolysis of methyl oleate in carboxylic acid medium. These peroxidic compounds include 1-acyloxyalkyl-1-hydroperoxides, 1,2,4-trioxolane and bis(1-acyloxy-1-alkyl) peroxides. Identification of the reaction products of ozonolysis of methyl oleate in the presence and absence of protic solvents was done by Ledea et al. (1998) using GC–MS and 1 H NMR. In the presence of methanol, ethoxy-hydroperoxide was found to be the major product and only small amount of the ozonides was
0009-3084/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2003.07.001
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of glycerol in the crude methyl ester was removed by centrifugation. The crude methyl ester was further purified by distilling-off the unreacted methanol under normal atmospheric pressure, washing several times with water, centrifugation and drying with anhydrous Na2 SO4 . 2.2. Ozonation
Scheme 1.
detected. The analysis of the reaction product when water was added revealed only the presence of aldehydes (hexanal, nonanal and 9-oxo-methylnonanoate). Diaz et al. (1998) investigated the effects of solvents (water and ethanol) and degree of unsaturation of the sample to the rate of ozonolysis. 1 H NMR spectroscopy at 250 MHz was used to follow up the course of the reaction. Diaz and co-workers found out that there was a higher consumption of the double bonds during the ozonation of methyl oleate with the addition of the solvents especially with the addition of water. The consumption of the double bonds was even faster when methyl linoleate was used. In this paper, we monitored the absorption of ozone in neat SFO methyl esters and the product was analyzed by FT-IR and 1 H and 13 C NMR spectroscopy. These analyses provide information on the functional group changes during ozonation as well as the identification of the products without prior separation techniques.
2. Materials and methods 2.1. Transesterification The reaction was carried out by mixing 1000.0 g sunflower oil, 218.6 g methanol and 5.00 g NaOCH3 in a 2 l jar fermentor. The temperature was maintained at 60–70 ◦ C for 2 h under reflux with stirring at 500 rpm. The reaction mixture was allowed to stand overnight and the methyl ester layer was separated from the glycerol layer using separatory funnel. Residual amount
Ozone/oxygen mixture containing 30–40 g/m3 ozone was bubbled into a 2 l jar fermentor containing 500 ml SFO methyl esters at a rate of 2.0 l/min for 35 h while stirring at 250 rpm. The amount of ozone absorbed by the carbon–carbon double bond in SFO methyl esters was monitored every hour. The concentration of ozone (g/m3 ) coming in and going out from the reactor was measured using TOA OZ-30 ozone meter. 2.3. Spectroscopic analysis IR spectra were recorded on thin films cast onto KBr plates using Jasco FT-IR 300 Spectrometer. All spectra were the result of 20 scans with a 4 cm−1 resolution. For 1 H and 13 C NMR spectroscopy, about 30 mg of the sample was dissolved in CDCl3 containing TMS as standard. Both spectra were obtained using Bruker Avance 600 Spectrometer. Trace amount of CHCl3 in the solvent used exhibits signal at 7.26 and 77.0 ppm in 1 H and 13 C NMR, respectively. 3. Results and discussions 3.1. FT-IR analysis FT-IR analyses were done to investigate the changes in the functional groups found in SFO methyl esters during ozonation. Fig. 1 shows the IR spectra of neat and ozonated SFO methyl esters. Summary of characteristic bands found in SFO methyl esters is shown in Table 1. Several major changes were observed in IR spectra of ozonated samples. As expected, intensities of all bands corresponding to the presence of carbon–carbon double bonds decreased with increasing duration of ozonation. These include the relatively weak band due to C=C stretch at 1650 cm−1 and the =C–H stretch and bend at 3008 and 723 cm−1 , respectively.
N.U. Soriano Jr et al. / Chemistry and Physics of Lipids 126 (2003) 133–140 Fig. 1. IR spectra of neat and ozonated SFO methyl esters (A: neat, B: 15 h, C: 25 h and D: 35 h ozonated). Amount of ozone consumed for samples B, C and D were 0.119, 0.196 and 0.222 g O3 /ml methyl ester.
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Table 1 IR interpretation of SFO methyl esters Functional group C=C stretch =C–H stretch =C–H bend C=O stretch C–O stretch O–C stretch CH3 bend
Table 2 1 H and 13 C NMR assignments of SFO methyl esters Wavenumber (cm−1 ) 1650 3008 723 1743 1170 1195 1361
The band due to carbonyl stretch of SFO methyl esters at 1743 cm−1 is relatively sharp. With increasing duration of ozonation, this band became broader suggesting the formation of new carbonyl compounds during ozonation. In fact after 30 h of ozonation, a shoulder appeared at around 1700 cm−1 . These could only be aldehyde and/or carboxylic acid. Aldehyde may be formed from the scission of either initial or final ozonide. Accompanying the former reaction is the formation of oligomer while the latter, could have resulted from the further reaction of ozonide with ozone to give carboxylic acid. However, presence of aldehydic C–H stretch at around 2900–2700 cm−1 was absent, 1 H NMR though confirmed their presence in ozonated samples. The appearance of relatively broad but strong band at 1105 cm−1 with increasing duration of ozonation was assigned to C–O stretch of the ozonide consistent with the reported value in the literature (Wu et al., 1992). This suggests that scission of the final ozonide was minimal. Early studies of ozonide by FT-IR revealed that its trans isomer absorbed at around 1300 cm−1 while its cis isomer at around 800 cm−1 (Bailey, 1978; Murray et al., 1967). During ozonation of SFO methyl esters, a broad peak appeared at around 1365 cm−1 that became stronger with increasing dosage of ozone absorbed, suggesting that the major ozonide product was the trans isomer. 3.2. NMR analysis Fig. 2 shows the NMR spectra of neat SFO methyl esters and assignments to all pertinent peaks were summarized in Table 2 (Diaz et al., 1998; Miyake et al., 1998; Lie Ken Jie and Mustafa, 1997). NMR analyses further confirmed the structural changes undergone by SFO methyl esters during
Chemical shift (ppm)
Functional group
1H
13 C
0.89 1.30 1.62 2.04 2.30 2.77 3.67 5.34 –
14.1 29.1–31.9 24.9 27.2 34.1 24.9 51.5 128.0–130.0 174.3
CH2 CH3 (CH2 )n CH2 CH2 COOCH3 CH2 CH=CHCH2 CH2 COOCH3 CH=CHCH2 CH=CH COOCH3 CH2 CH=CHCH2 COOCH3
ozonation (Fig. 3). New signal at 5.17 ppm found in 1 H NMR spectra of ozonated samples were assigned to the ring proton of 1,2,4-trioxolane (Diaz et al., 1998; Ewing et al., 1989; Ledea et al., 1998; Nishikawa et al., 1995; Rebrovic, 1992; Wu et al., 1992). This was supported by the appearance of signal in 13 C NMR spectra at 104.5 ppm assigned by Wu et al. (1992) as the ring carbon in the same structure. The protons of the methylene group ␣ to the sp2 -hybridized carbons, which resonated at 2.04 and 2.77 ppm shifted to 1.35 and 1.75 ppm upon formation of 1,2,4-trioxolane (Table 3) (Anachkov et al., 2000). Ozonated samples exhibited peaks at 9.75 and 2.50 ppm in 1 H NMR corresponding to the aldehydic proton and the methylene protons ␣ to the carbonyl carbon, respectively. In 13 C NMR, the carbonyl carbon and the carbon ␣ to it, resonated at 202.8 and 43.9 ppm, respectively. The time course of ozone absorption during the ozonation of neat SFO methyl esters is shown in Table 3 1 H and 13 C NMR assignments of new signals found in ozonated SFO methyl esters Chemical shift (ppm)
Functional group
1H
13 C
5.17 1.35, 1.71 9.75 2.50
104.5 202.8 43.9 178.5 35.6
1,2,4-Trioxolane ␣-Methylene group Aldehyde ␣-Methylene group Carboxylic acid ␣-Methylene group
N.U. Soriano Jr et al. / Chemistry and Physics of Lipids 126 (2003) 133–140
Fig. 2. 1 H and
13 C
137
NMR spectra of neat SFO methyl esters.
Fig. 4. The ozone absorption was essentially quantitative during the early stage of ozonation and suddenly dropped after 25 h. The sudden drop in absorption was explained by the complete consumption of carbon–carbon double bond in SFO methyl esters. Fig. 5 shows the NMR spectra of completely ozonated sample. After 25 h of ozonation amounting to around 0.20 g O3 /ml methyl ester, the peak at 2.04 and 5.34 ppm in 1 H NMR spectra and 128.0–130.0 ppm in 13 C NMR spectra completely disappeared. To understand more the trend in the changes in functional groups in SFO methyl esters during ozonation, the ratio between the integrated signals due to various functionalities and the average area per proton
were plotted against the amount of ozone absorbed. The average area per proton was calculated from the integrated intensity of the signals due to terminal methyl group of the fatty acid moiety (0.89 ppm), the methylene group ␣ to the carbonyl carbon (2.30 ppm) and the free methyl group of the ester moiety (3.67 ppm). These functionalities were chosen because they were not affected by ozonation. The consumption of carbon–carbon bond was evaluated considering the integrated intensity of the signal due to methylene protons ␣ to sp2 -hybridized carbon (2.04 ppm). The formation of ozonide and aldehyde were monitored from the integrated intensity of the signals at 1.35, 1.71 and 9.75 ppm, respectively.
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Fig. 3. 1 H NMR spectra of neat and partially ozonated SFO methyl esters (A: neat; B: ozonated for 15 h amounting to 0.119 g O3 /ml methyl ester).
Fig. 6 shows the rapid consumption of ozone by the carbon–carbon double bonds present in SFO methyl esters. This is accompanied by the formation 1,2,4-trioxolane. Linear regression analysis on series of data corresponding to the methylene groups
␣ to the sp2 -hybridized carbon and 1,2,4-trioxolane suggests that the rate of disappearance of unsaturation and the formation of ozonide was almost equal (Table 4). This also suggests that the major product of the reaction is ozonide and that scission of ozonide was very minimal. Table 4 Linear regression on data series on the disappearance of unsaturation and formation of ozonide during ozonationa 1H
NMR shift (ppm)
2.04 1.31 1.71 Fig. 4. Time course of ozone absorption.
Slope (ratio/ozone dosage)
R2
−17.7 17.9 17.6
0.998 0.992 0.992
a Linear regression analysis was done only from time 0 to time 25 h (0.196 g O3 /ml methyl ester).
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Table 5 Ozonide to aldehyde ratio Duration of Ozone dosage ozonation (h) (g O3 /ml methyl ester)
5 10 15 20 25 30 35
0.041 0.080 0.119 0.158 0.196 0.211 0.222
Ozonide:aldehyde ratio 1.35 ppm: 1.71 ppm: 9.76 ppm 9.76 ppm 94.1:5.9 92.9:7.1 92.0:8.0 95.1:4.9 95.6:4.4 97.1:2.9 97.2:2.8
93.8:6.2 93.0:7.0 92.4:7.6 94.7:5.3 95.3:4.7 97.1:2.9 97.0:3.0
with only one mole of ozone. The formation of allylic 1,2,4-trioxolane shifted the signal of these protons from 5.34 ppm in the original structure to 5.58 ppm.
Fig. 5. 1 H and ester.
13 C
NMR of completely ozonated SFO methyl
The signals at 1.35 ppm and 1.71 ppm do not discriminate the oleate and linoleate methyl ester ozonide. However, new signal at 5.58 ppm appeared in early stage of ozonation, gradually increased until 15 h, and finally disappeared after 25 h of ozonation. This was assigned to the protons bonded to sp2 hybridized carbon of linoleate methyl ester ozonide resulted from the reaction of linoleate methyl ester
In the latter part of ozonation when all the carbon–carbon double had been consumed, the amount of ozone absorbed was only about 25%. In the absence of unsaturation, ozonide formed in the early stage of ozonation was cleaved to give carboxylic acid. The signal due to carboxyl carbon was observed at 178 ppm in 13 C NMR. Table 5 shows the ozonide to aldehyde ratio obtained from various stages of ozonation. The amount of ozonide regardless of the extent of ozonation was
Fig. 6. Ratios of 1 H NMR peaks intensity relative to area per proton against amount of ozone absorbed by SFO methyl esters.
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always above 90%. The relatively small amount of aldehyde suggests that oligomerization was also minimal. The decreasing amount of aldehyde relative to ozonide with increasing duration of ozonation could be attributed to volatilization and/or reaction with excess ozone to give carboxylic acid. Acknowledgements This research was conducted under the support from NEDO International Joint Research Grant. References Anachkov, M.P., Rakovski, S.K., Stefanova, R.V., 2000. Ozonolysis of 1,4-cis-polyisoprene and 1,4-trans-polyisoprene in solution. Polym. Deg. Stab. 67, 355–363. Bailey, P.S., 1978. Ozonation in Organic Chemistry, vol. 1. Academic Press, New York, pp. 25–37. Diaz, M., Hernandez, F., Alvarez, I., Velez, H., Ledea, O., Molerio, J., 1998. The 1 H NMR spectroscopy in following ozone reaction with unsaturated fatty acids. Revista CENIC Ciencias Quimicas 29, 89–93.
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