Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 402–407
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Quantitative analysis of cyclic dimer fatty acid content in the dimerization product by proton NMR spectroscopy Kyun Joo Park a, Minyoung Kim a, Seunghwan Seok a, Young-Wun Kim b, Do Hyun Kim a,⇑ a b
Department of Chemical & Biomolecular Engineering, KAIST, Daejeon, Republic of Korea Green Chemistry Research Division, Surfactant & Lubricant Research Team, KRICT, Daejon, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The cyclic dimer acid (CDA) in a
complex mixture was quantified using proton NMR. Quantification of CDA was achieved by using structurally similar compound. Content of CDA is estimated by established standard curve and reported equation. Proposed NMR analysis provides a quantification of CDA as low as micro-gram scale.
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 27 April 2015 Accepted 28 April 2015 Available online 6 May 2015 Keywords: Cyclic dimer acid Dimer acid Quantitative analysis Proton NMR spectroscopy
a b s t r a c t In this work, 1H NMR is utilized for the quantitative analysis of a specific cyclic dimer fatty acid in a dimer acid mixture using the pseudo-standard material of mesitylene on the basis of its structural similarity. Mesitylene and cyclic dimer acid levels were determined using the signal of the proton on the cyclic ring (d = 6.8) referenced to the signal of maleic acid (d = 6.2). The content of the cyclic dimer fatty acid was successfully determined through the standard curve of mesitylene and the reported equation. Using the linearity of the mesitylene curve, the cyclic dimer fatty acid in the oil mixture was quantified. The results suggest that the proposed method can be used to quantify cyclic compounds in mixtures to optimize the dimerization process. Ó 2015 Elsevier B.V. All rights reserved.
Introduction Vegetable oil has won attention as a renewable resource upon the background of environmental concerns and fossil fuel depletion [1–6]. Dimer acids, produced by the dimerization of unsaturated fatty acids from vegetable oil, are highly value-added products and are widely used as adhesives [7–9], plastic additives [10,11], and lubricants [12]. Several synthesis methods have been investigated to date to obtain dimer acids. However, the reported methods inevitably produce a mixture of several isomers of dimer ⇑ Corresponding author. Tel.: +82 42 350 3929. E-mail address: [email protected] (D.H. Kim). http://dx.doi.org/10.1016/j.saa.2015.04.099 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
acids including acyclic and cyclic dimer acids (CDAs) owing to the use of different raw materials. Due to several possible couplings, synthesized CDAs may have several chemically different side arms on the cyclic ring. Moreover, both the detection and separation of specific dimer acids from the product mixture are difficult due to the similar in molecular weights of the acids. The development of analysis techniques for such complex compounds is therefore required. To analyze the dimer acid, several methods including high-performance liquid chromatography (HPLC) [13–16], gas chromatography [17], and capillary electrophoresis [18] have been employed for the quantitative determination of dimer acids. However, in the case of complex mixtures, these methods require
K.J. Park et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 402–407
a complicated and time-consuming pre-treatment to extract and concentrate each target compound from the mixture to prepare high purity analytical grade target materials [15,16]. To determine the contents of different substances in complex mixture, quantitative 1H NMR spectroscopy can also be used. NMR spectroscopy provides high selectivity and absolute quantification of specific compound in a mixture by using an internal standard (IS) reference material [19–21]. The magnitude of the NMR peak is directly proportional to the number of nuclei and the molar concentration of the sample. Based on this intensity relationship, the concentration of the analyte can be measured by quantitative 1H NMR using the ratio between the integral value of the sample’s specific chemical shift and that of the IS [19–21]. Previous studies have demonstrated the advantages of quantitative 1 H NMR such as reduced measurement time, simple sample preparation, and a small volume requirement compared to conventional analysis techniques [19–24]. Additionally, NMR analysis has further advantages of containing chemical structure information and providing nondestructive analytic test [19–24]. Owing to these benefits, quantitative 1H NMR has been adopted to quantify the specific component of crude samples, such as urine, processed foods, and tablets [19–21,23,25]. Based on these strengths, we applied the NMR method to quantify the absolute content of cyclic component in a mixture. We developed a 1H NMR spectroscopic method to quantify CDAs by employing IS and a pseudo-standard material. Owing to the difficulty of obtaining the CDA in pure form and the resemblance of chemical structure and chemical shift with CDA, mesitylene was adopted as a pseudo-standard material. The intensity area ratio of sample to IS was constructed based on NMR spectrums. The relationship between the sample concentration and the peak intensity of 1H NMR showed a high linearity. With this linearity and the standard curve of mesitylene, the cyclic dimer fatty acid was quantified in a mixture containing side products such as acyclic, bicyclic dimer acid, unreacted monomer, overreacted trimer, and oligomers. For the verification of the proposed technique, a known amount of mesitylene was quantified by the curve and compared with the value from the reported equation.
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For the clay catalyzed synthesis, soybean based vegetable oil was used as a source for dimerization. It is composed of palmitic acid (C16, 15.9%), stearic acid (C18, 3.1%), oleic acid (C18, 44.9%), linoleic acid (C18, 31.5%), and some residues (4.6%) [10,12]. In this study, the fatty acids mixture consisted of 19.1% saturated fatty acid (C16) and 80.9% unsaturated fatty acids (C18) which are composed of oleic acid, linoleic acid, and their isomers [10]. The overall synthesis process is illustrated in Fig. 1. For the dimerization, the fatty acids mixture (93.7 wt%), catalyst (3.8 wt%), co-catalysts (0.2 wt% of lithium carbonate, 0.3 wt% of calcium carbonate and 0.1 wt% of hypophosphorous acid), and deionized (DI) water (1.9 wt%) were mixed in a 40 mL high-pressure stainless-steel gastight reactor and sealed tightly. The batch-reactor consists of separable upper and lower parts. The upper part has two bolts to introduce gas for pressurizing. The large chamber in the lower part is designed for the reaction. Once all components were assembled, these two parts were tightly joined and prepared as a gastight reactor to prevent gas or chemical leakage. To pressurize up to 10 bar, nitrogen gas was purged into the reactor 3 times and the reactor was filled until the desired pressure was obtained. After these preparation steps, the reactor was placed into a 250 °C furnace for 4 h. Cooling, filtration, and distillation steps were followed to obtain the dimer acids among the synthesized product. The reaction was terminated after 4 h by lowering the temperature to room temperature. Subsequently, product and catalysts were pulled out from the reactor to remove the solid catalyst. Through filtration, the clay catalyst was removed and the liquid product was prepared for collecting the dimer acids. Finally, the liquid product was distilled at 270 °C to separate the dimer acids. The formation of dimer fatty acids was confirmed by liquid chromatography mass spectroscopy (LC–MS) and 1H NMR. NMR analysis 1
H NMR spectra of the fatty acids mixture, dimer acids mixture, mesitylene, and maleic acid were obtained using an Agilent
Experimental Materials Vegetable oil for synthesizing the dimer acid was provided by the Korea Research Institute of Chemical Technology (KRICT). The catalyst, montmorillonite, was purchased from Dong Hae Chemical Co., Ltd. Lithium carbonate, calcium carbonate, and hypophosphorous acid as co-catalysts, chloroform-d, 1,3,5-trimethylbenzene (mesitylene), and maleic acid for NMR analysis were purchased from Sigma–Aldrich. All chemicals were used as provided without purification. Synthesis of dimer acid The synthesis of the dimer acid using the fatty acid in vegetable oil followed previous reports [10,12,26,27]. The clay catalyzed method is well known as a better strategy to synthesize the dimer acids than radical coupling method, which inevitably leads to greater amounts of undesirable contents of trimers and higher oligomers [27]. In this research, the montmorillonite was used as a clay catalyst to acquire cyclic compounds. According to a previous study, dimer acids could be synthesized up to 89.6% conversion yield (mass of synthesized dimer acids/mass of unsaturated fatty acids in vegetable oil) using an acidic clay catalyst at 250 °C for 4 h reaction [10].
Fig. 1. Flow chart and schematic diagram of fatty acid dimerization system.
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K.J. Park et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 402–407
400 MHz 54 mm NMR spectrometer DD2 (Agilent Technologies, USA). The instrument operated at a field strength of 9.4 T. The 5 mm length of a liquid automated triple broadband (ATB) probe (2channel-1H/19F, X-nuclei) was used to obtain automated and hands-off analysis environments for sample change without cleaning any mechanical parts throughout the experiment. A spectral width of 6410.26 Hz and 32 scans were used to collect 32,768 data points with an acquisition time of 3.41 s and a relaxation delay of 30.0 s. All the chemical shifts were referenced to maleic acid (d = 6.2). The integration of peaks was performed using an automatic integration option provided by ACD NMR Processor (ACD/Labs, Inc., Toronto, ON, Canada). Results and discussion
Fig. 3. Possible chemical structures of dimer acids obtained from oleic and linoleic acids.
Dimer acid synthesis The main reaction of oleic and linoleic acids dimerization is illustrated in Figs. 2 and 3. After oligomerization, the mixture of fatty acids was converted to cyclic dimer as a desired product (Fig. 2), acyclic dimer (Fig. 3) trimer, oligomer, and unreacted monomer fatty acid as undesired products. By Diels–alder cyclization, CDA was synthesized from unsaturated fatty acids since the reaction requires double bonds. These double bonds are adsorbed by the unique structures of the clay catalyst (tetrahedral substitution sites) and this phenomenon helps to dimerize the fatty acids [28]. Products were analyzed by MS (Fig. 4) and presented peaks at around 320, 600, and 880 m/z for monomer, dimer, and oligomer fatty acids, respectively. However, due to the many possible combinations of oleic, linoleic acids and its isomers, each product group (Fig. 3) consists of numerous compounds, as observed from the MS spectrum. To confirm and determine the content of CDA among the products, further analysis of dimer acids was performed using 1H NMR. 1
H NMR analysis
For the analysis of CDA, a 1H NMR spectra of fatty acids, dimer acids, mesitylene, and maleic acid are illustrated in Fig. 5a. Generally, highly pure forms of analytic target materials for a quantitative analysis are required and used as standard materials. However, it is difficult to obtain some kind of reagents, such as dimer acids and CDA in a pure form. An inexpensive and readily available reference standard for those materials including CDA is therefore essential. For this purpose, mesitylene, a molecule that has a similar cyclic structure with that of CDA, was adopted for the verification and quantification of the CDA content in a mixture of dimer acids. The intention of adopting mesitylene is resemblance of not only the chemical structure but also the chemical shift with cyclic structure of CDA. Typically, mesitylene is used as an internal standard for cyclic compounds and it is known that two chemical equivalences appear in 1H NMR [29,30]. The chemical shift of 6.9 ppm originates from three identical protons attached to an aromatic ring and the other (2.3 ppm) is from the methyl groups. Furthermore, this chemical shift is similar to the
Fig. 2. Synthetic scheme of cyclic dimer acid using oleic and linoleic acids.
Fig. 4. LC–MS spectrum of reacted fatty acid containing monomer, dimer, and oligomer acids.
chemical shift of CDA (near 6.8 ppm). For this reason, mesitylene can serve as a pseudo-standard material for CDA. The term, pseudo-standard, was intended as not the highly pure form of analyte but the similar chemical structured material with target analyte. Additionally, as an IS, maleic acid is utilized in 1H NMR quantitative analysis techniques [31,32], because it supplies a separated signal without any overlapping peaks from the signals of the analyte. It is known that the maleic anhydride can react with oleic and linoleic acids. However, this reaction can be happen at more than 150 °C [33–35]. To avoid possible reactions with maleic acid and fatty acids, all the NMR samples with maleic acid were kept at room temperature during the analysis. Moreover, the NMR spectrum of fatty acids and dimer acids did not changed when the maleic acid was used as IS. Based on the NMR spectrum and controlled reaction condition, we can conclude that the maleic acid shows no interaction with other component. The detailed dimer acid and a representative CDA spectrum are presented in Fig. 5b. All the discussed peaks with their assigned protons are listed in Table 1. After the cyclization reaction, the vinyl proton in the unsaturated fatty acid at 5.6 ppm and allylic protons in linoleic acid at 2.0 ppm disappeared, as shown in Fig. 5. On the other hand, signals attributed to the protons of the cyclic structure gradually appear between 6.5 and 7.5 ppm. It is well documented that chemical shifts of 6.5–8.0 ppm represent the protons attached to the cyclic ring [36]. Furthermore, benzylic hydrogens appear at 2.5 ppm after dimerization. These peaks representing the unsaturated cyclic structure confirm the successful synthesis of the CDA. Other peaks may correspond to other isomers of the dimer acid. Signals between 0.8 and 3.0 ppm are characteristic aliphatic hydrogen resonances in dimer acids and other fatty acids. The peaks at 3.57–3.68 ppm and 4.0–4.1 ppm (too low to observe in Fig. 5b) represent the ester methyl group located next
K.J. Park et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 402–407
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to the carbonyl carbon and the proton on the glycerol residues, respectively. The specific peak of CDA does not overlap with any peak of the fatty acid, as shown in Fig. 5a. Therefore, the content of CDA in the dimer acid mixture can be determined using the specific signal of the proton in the cyclic structure.
Estimation of CDA content A 1H NMR analysis of CDA was performed to estimate the content among the dimer acids mixture. To establish the feasibility of the proposed method, a quantitative analysis of mesitylene was also performed with maleic acid. A known quantity of maleic acid (0.5 mg) was added to the NMR solvent with various amounts of mesitylene. A standard curve of the mesitylene concentration was constructed by these results and it showed high linearity (from 0.1 to 40 lg of mesitylene), as presented in Fig. 6a. The ratio of the peak area between mesitylene and IS was determined by the peak of mesitylene at 6.90 ppm to the peak of IS at 6.20 ppm. A regression analysis of the data from the mesitylene shows an R2 value of 0.995, indicating a highly linear relationship between the mass and the average integral of the peak in the 1H NMR spectra. Based on this curve, we compared the quantity of mesitylene in the prepared sample and confirmed that the predicted value was consistent with the added amount. This result indicates that cyclic
Fig. 5. 1H NMR results of (a) fatty acid (monomer), dimer acid, mesitylene, maleic acid, and (b) detailed dimer acid spectrum with unreacted fatty acid residues and magnified specific peak of CDA.
Table 1 H NMR chemical shift ranges for selected types of protons.
1
Chemical shifts (d, ppm)
Selected types of protons
0.8–0.9 1.2–1.3 1.5–1.6 1.9–2.0 2.3–2.4
2.5–2.6
3.5–4.1 5.6–5.7 6.2–6.3 6.8–6.9
Fig. 6. Measured signal area ratio of (a) mesitylene and (b) CDA to maleic acid by 1H NMR analysis versus mass of mesitylene and dimer acids mixture, respectively.
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compounds can be reliably quantified using NMR. Furthermore, this curve could also be applied to quantify the CDA level in dimer acid mixtures due to the structural similarity of mesitylene with CDA. The spectra of various dimer acid mixtures are similar to those in Fig. 5 except for the peak intensity from the cyclic proton due to the different concentration of CDA. A series of samples with identical IS and various CDA concentrations were analyzed. Similar with the mesitylene case (Fig. 6a), the curve in Fig. 6b represents the integrated area of CDA specific peak (6.8 ppm) referenced to IS. The intensity ratio of CDA to IS was obtained by averaging the peak area of the CDA signal at 6.81 ppm to the specific signal of the IS at 6.20 ppm. A linear regression with a correlation coefficient of 0.995 implied an excellent linear relationship between the concentration of IS and the integrated area of CDA peaks, as presented in Fig. 6b. With the linearity of the standard curve, NMR spectra of a dimer acid mixture containing CDA were evaluated. The intensity area ratio value of CDA from Fig. 6b was assigned to Fig. 6a and therein the CDA content was estimated via constructed mesitylene mass of standard curve. The estimated contents of CDA by the curve were presented in Table 2. Furthermore, general quantification technique using proton NMR was adopted to show the feasibility of proposed method based on pseudo-standard material. For this purpose, the CDA content (lg/g dimer acids mixture) in each sample was calculated from a previously suggested equation [19–21]:
ICDA HIS MWCDA M IS Total CDA content ¼ IIS HCDA MVIS M DA
ð1Þ
ICDA = average intensity area of CDA peak, IIS = intensity area of IS, HIS = number of protons of IS, HCDA = number of protons of CDA, MWCDA = molecular weight of CDA, MWIS = molecular weight of IS, MIS = mass of IS, and MDA = mass of dimer acid mixture. In order to calculate the CDA content from the Eq. (1), we should start with the obtaining of the values for each variable. The intensity ratio between CDA and IS (ICDA/IIS) is obtained from the Fig. 4b and the NMR spectrums of CDA. The molecular weight of CDA (MWCDA) and the number of protons of CDA (HCDA) as calculated based on the presented chemical structure of CDA in Fig. 2. Molecular weight and number of protons of IS are 116 g/mol and 4 atoms, respectively. Mass of IS (MIS) is fixed of 0.5 mg and dimer acid mixture (MDA) is also obtained from the Fig. 6. After the gathering of all values, we substitute every variables in the Eq. (1) with corresponding values. Finally, the content of CDA in an unknown sample was calculated and presented in Table 2. The predicted concentrations of CDA from both the standard curve and Eq. (1) show an average of 13% difference. For verification, a known quantity of mesitylene was also estimated using both techniques. The mass evaluation of mesitylene showed 40.49 lg by standard curve and 38.15 lg by Eq. (1) for the case of 40 lg. The estimation difference between the two methods was 5.8%. According to a previous report [23], the quantitative NMR method can show a 95% confidence level due to insignificant systematic error. This systematic error may contribute to the difference in the estimated value between the two quantification methods. Another possible cause of this difference could be derived from the molecular weight of CDA. In the equation, molecular weight of CDA was required to obtain the content but the CDA
Table 2 Comparison of CDA content determined by standard curve and equation.
Standard curve Eq. (1)
Sample 1 (lg)
Sample 2 (lg)
Sample 3 (lg)
Mesitylene (lg)
0.86
0.22
0.11
40.49
0.74
0.23
0.10
38.15
represents whole possible cyclic compounds in dimerization. However, representative value of possible molecular weights (560 g/mol for CDA in Fig. 2) was substituted. Conclusion In this study, we proposed a method for determining the CDA content in a mixture of dimer acids using a 1H NMR analysis. The CDA content was estimated by the standard curve of mesitylene with the CDA to IS signal area ratio and compared to an equation proposed previously [20]. The reliability of the proposed method was demonstrated by quantifying mesitylene, an easy accessible cyclic standard material. Based on the verification results using mesitylene, the accuracy of our method and the equation were 98% and 95%, respectively. The accuracy is defined as the mass difference ratio between the input mass and the estimated mass of mesitylene. The key advantage of this method is that it does not require a high purity analyte standard but an easily accessible pseudo standard and IS for the content determination. Moreover, a quantitative NMR analysis provides quantification of CDA on as low as a micro-gram scale. The proposed method can be applied in various fields that require quantification of specific cyclic compounds in mixtures of pharmaceuticals or processed food. Acknowledgements This study was supported by the R&D Center for Valuable Recycling (Global-Top Environmental Technology Development Program) funded by the Ministry of Environment – South Korea (Project No.: GT-11-C-01-250-0). References [1] M. Kouze, T. Kasuno, M. Tajika, Y. Sugimoto, S. Yamanaka, J. Hidaka, Fuel 87 (2008) 2798–2806. [2] L.C. Meher, D.V. Sagar, S.N. Naik, Renew. Sustainable Energy Rev. 10 (2006) 248–268. [3] A.E. Atabani, A.S. Silitonga, H.C. Ong, T.M.I. Mahlia, H.H. Masjuki, I.A. Badruddin, H. Fayaz, Renew. Sustainable Energy Rev. 18 (2013) 211–245. [4] M. Desroches, M. Escouvois, R. Auvergne, S. Caillol, B. Boutevin, Polym. Rev. 52 (2012) 38–79. [5] K. Yao, C. Tang, Macromolecules 46 (2013) 1689–1712. [6] A. Talebian-Kiakalaieh, N.A.S. Amin, H. Mazaheri, Appl. Energy 104 (2013) 683–710. [7] P. Kadam, P. Vaidya, S. Mhaske, Int. J. Adhes. Adhes. 50 (2014) 151–156. [8] A. Li, K. Li, ACS Sustainable Chem. Eng. 2 (2014) 2090–2096. [9] Y. Li, B.A.J. Noordover, R.A.T.M. van Benthem, C.E. Koning, Eur. Polym. J. 59 (2014) 8–18. [10] K.-H. Ko, H.R. Park, J.-S. Kim, Y.-W. Kim, J. Appl. Polym. Sci. 129 (2013) 2443– 2450. [11] S. Li, X. Yang, K. Huang, M. Li, J. Xia, Prog. Org. Coat. 77 (2014) 388–394. [12] S.J. Lee, Y.-W. Kim, S.-H. Yoo, N.-K. Kim, J.H. Shin, B.-T. Yoon, Appl. Chem. Eng. 24 (2013) 530–536. [13] K.S. Robbins, Y. Ma, M.L. Wells, P. Greenspan, R.B. Pegg, J. Agric. Food Chem. 62 (2014) 4332–4341. [14] R.L. Veazey, J. Am. Oil Chem. Soc. 63 (1986) 1043–1046. [15] J. Zhao, S.V. Olesik, Anal. Chim. Acta 449 (2001) 221–236. [16] M. Heimrich, M. Bönsch, H. Nickl, T.J. Simat, Food Addit. Contam. 29 (2012) 846–860. [17] J.P. Nelson, A.J. Milun, J. Am. Oil Chem. Soc. 52 (1975) 81–83. [18] R. Haselberg, V. Brinks, A. Hawe, G.J. de Jong, G.W. Somsen, Anal. Bioanal. Chem. 400 (2011) 295–303. [19] T. Ohtsuki, K. Sato, N. Sugimoto, H. Akiyama, Y. Kawamura, Talanta 99 (2012) 342–348. [20] T. Ohtsuki, K. Sato, N. Furusho, H. Kubota, N. Sugimoto, H. Akiyama, Food Chem. 141 (2013) 1322–1327. [21] T. Ohtsuki, K. Sato, N. Sugimoto, H. Akiyama, Y. Kawamura, Anal. Chim. Acta 734 (2012) 54–61. [22] J. Pan, X. Gong, H. Qu, J. Pharm. Biomed. Anal. 85 (2013) 28–32. [23] A.A. Salem, H.A. Mossa, B.N. Barsoum, Spectrochim. Acta, Part A 62 (2005) 466–472. [24] B.S. Somashekar, O.B. Ijare, G.A.N. Gowda, V. Ramesh, S. Gupta, C.L. Khetrapal, Spectrochim. Acta, Part A 65 (2006) 254–260. [25] A.A. Salem, H.A. Mossa, Talanta 88 (2012) 104–114. [26] R.F. Paschke, L.E. Peterson, D.H. Wheeler, J. Am. Oil Chem. Soc. 41 (1964) 723– 727.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Quantitative analysis of cyclic dimer fatty acid content in the dimerization product by proton NMR spectroscopy Kyun Joo Park a, Minyoung Kim a, Seunghwan Seok a, Young-Wun Kim b, Do Hyun Kim a,⇑ a b
Department of Chemical & Biomolecular Engineering, KAIST, Daejeon, Republic of Korea Green Chemistry Research Division, Surfactant & Lubricant Research Team, KRICT, Daejon, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The cyclic dimer acid (CDA) in a
complex mixture was quantified using proton NMR. Quantification of CDA was achieved by using structurally similar compound. Content of CDA is estimated by established standard curve and reported equation. Proposed NMR analysis provides a quantification of CDA as low as micro-gram scale.
a r t i c l e
i n f o
Article history: Received 2 December 2014 Received in revised form 27 April 2015 Accepted 28 April 2015 Available online 6 May 2015 Keywords: Cyclic dimer acid Dimer acid Quantitative analysis Proton NMR spectroscopy
a b s t r a c t In this work, 1H NMR is utilized for the quantitative analysis of a specific cyclic dimer fatty acid in a dimer acid mixture using the pseudo-standard material of mesitylene on the basis of its structural similarity. Mesitylene and cyclic dimer acid levels were determined using the signal of the proton on the cyclic ring (d = 6.8) referenced to the signal of maleic acid (d = 6.2). The content of the cyclic dimer fatty acid was successfully determined through the standard curve of mesitylene and the reported equation. Using the linearity of the mesitylene curve, the cyclic dimer fatty acid in the oil mixture was quantified. The results suggest that the proposed method can be used to quantify cyclic compounds in mixtures to optimize the dimerization process. Ó 2015 Elsevier B.V. All rights reserved.
Introduction Vegetable oil has won attention as a renewable resource upon the background of environmental concerns and fossil fuel depletion [1–6]. Dimer acids, produced by the dimerization of unsaturated fatty acids from vegetable oil, are highly value-added products and are widely used as adhesives [7–9], plastic additives [10,11], and lubricants [12]. Several synthesis methods have been investigated to date to obtain dimer acids. However, the reported methods inevitably produce a mixture of several isomers of dimer ⇑ Corresponding author. Tel.: +82 42 350 3929. E-mail address: [email protected] (D.H. Kim). http://dx.doi.org/10.1016/j.saa.2015.04.099 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
acids including acyclic and cyclic dimer acids (CDAs) owing to the use of different raw materials. Due to several possible couplings, synthesized CDAs may have several chemically different side arms on the cyclic ring. Moreover, both the detection and separation of specific dimer acids from the product mixture are difficult due to the similar in molecular weights of the acids. The development of analysis techniques for such complex compounds is therefore required. To analyze the dimer acid, several methods including high-performance liquid chromatography (HPLC) [13–16], gas chromatography [17], and capillary electrophoresis [18] have been employed for the quantitative determination of dimer acids. However, in the case of complex mixtures, these methods require
K.J. Park et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 402–407
a complicated and time-consuming pre-treatment to extract and concentrate each target compound from the mixture to prepare high purity analytical grade target materials [15,16]. To determine the contents of different substances in complex mixture, quantitative 1H NMR spectroscopy can also be used. NMR spectroscopy provides high selectivity and absolute quantification of specific compound in a mixture by using an internal standard (IS) reference material [19–21]. The magnitude of the NMR peak is directly proportional to the number of nuclei and the molar concentration of the sample. Based on this intensity relationship, the concentration of the analyte can be measured by quantitative 1H NMR using the ratio between the integral value of the sample’s specific chemical shift and that of the IS [19–21]. Previous studies have demonstrated the advantages of quantitative 1 H NMR such as reduced measurement time, simple sample preparation, and a small volume requirement compared to conventional analysis techniques [19–24]. Additionally, NMR analysis has further advantages of containing chemical structure information and providing nondestructive analytic test [19–24]. Owing to these benefits, quantitative 1H NMR has been adopted to quantify the specific component of crude samples, such as urine, processed foods, and tablets [19–21,23,25]. Based on these strengths, we applied the NMR method to quantify the absolute content of cyclic component in a mixture. We developed a 1H NMR spectroscopic method to quantify CDAs by employing IS and a pseudo-standard material. Owing to the difficulty of obtaining the CDA in pure form and the resemblance of chemical structure and chemical shift with CDA, mesitylene was adopted as a pseudo-standard material. The intensity area ratio of sample to IS was constructed based on NMR spectrums. The relationship between the sample concentration and the peak intensity of 1H NMR showed a high linearity. With this linearity and the standard curve of mesitylene, the cyclic dimer fatty acid was quantified in a mixture containing side products such as acyclic, bicyclic dimer acid, unreacted monomer, overreacted trimer, and oligomers. For the verification of the proposed technique, a known amount of mesitylene was quantified by the curve and compared with the value from the reported equation.
403
For the clay catalyzed synthesis, soybean based vegetable oil was used as a source for dimerization. It is composed of palmitic acid (C16, 15.9%), stearic acid (C18, 3.1%), oleic acid (C18, 44.9%), linoleic acid (C18, 31.5%), and some residues (4.6%) [10,12]. In this study, the fatty acids mixture consisted of 19.1% saturated fatty acid (C16) and 80.9% unsaturated fatty acids (C18) which are composed of oleic acid, linoleic acid, and their isomers [10]. The overall synthesis process is illustrated in Fig. 1. For the dimerization, the fatty acids mixture (93.7 wt%), catalyst (3.8 wt%), co-catalysts (0.2 wt% of lithium carbonate, 0.3 wt% of calcium carbonate and 0.1 wt% of hypophosphorous acid), and deionized (DI) water (1.9 wt%) were mixed in a 40 mL high-pressure stainless-steel gastight reactor and sealed tightly. The batch-reactor consists of separable upper and lower parts. The upper part has two bolts to introduce gas for pressurizing. The large chamber in the lower part is designed for the reaction. Once all components were assembled, these two parts were tightly joined and prepared as a gastight reactor to prevent gas or chemical leakage. To pressurize up to 10 bar, nitrogen gas was purged into the reactor 3 times and the reactor was filled until the desired pressure was obtained. After these preparation steps, the reactor was placed into a 250 °C furnace for 4 h. Cooling, filtration, and distillation steps were followed to obtain the dimer acids among the synthesized product. The reaction was terminated after 4 h by lowering the temperature to room temperature. Subsequently, product and catalysts were pulled out from the reactor to remove the solid catalyst. Through filtration, the clay catalyst was removed and the liquid product was prepared for collecting the dimer acids. Finally, the liquid product was distilled at 270 °C to separate the dimer acids. The formation of dimer fatty acids was confirmed by liquid chromatography mass spectroscopy (LC–MS) and 1H NMR. NMR analysis 1
H NMR spectra of the fatty acids mixture, dimer acids mixture, mesitylene, and maleic acid were obtained using an Agilent
Experimental Materials Vegetable oil for synthesizing the dimer acid was provided by the Korea Research Institute of Chemical Technology (KRICT). The catalyst, montmorillonite, was purchased from Dong Hae Chemical Co., Ltd. Lithium carbonate, calcium carbonate, and hypophosphorous acid as co-catalysts, chloroform-d, 1,3,5-trimethylbenzene (mesitylene), and maleic acid for NMR analysis were purchased from Sigma–Aldrich. All chemicals were used as provided without purification. Synthesis of dimer acid The synthesis of the dimer acid using the fatty acid in vegetable oil followed previous reports [10,12,26,27]. The clay catalyzed method is well known as a better strategy to synthesize the dimer acids than radical coupling method, which inevitably leads to greater amounts of undesirable contents of trimers and higher oligomers [27]. In this research, the montmorillonite was used as a clay catalyst to acquire cyclic compounds. According to a previous study, dimer acids could be synthesized up to 89.6% conversion yield (mass of synthesized dimer acids/mass of unsaturated fatty acids in vegetable oil) using an acidic clay catalyst at 250 °C for 4 h reaction [10].
Fig. 1. Flow chart and schematic diagram of fatty acid dimerization system.
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400 MHz 54 mm NMR spectrometer DD2 (Agilent Technologies, USA). The instrument operated at a field strength of 9.4 T. The 5 mm length of a liquid automated triple broadband (ATB) probe (2channel-1H/19F, X-nuclei) was used to obtain automated and hands-off analysis environments for sample change without cleaning any mechanical parts throughout the experiment. A spectral width of 6410.26 Hz and 32 scans were used to collect 32,768 data points with an acquisition time of 3.41 s and a relaxation delay of 30.0 s. All the chemical shifts were referenced to maleic acid (d = 6.2). The integration of peaks was performed using an automatic integration option provided by ACD NMR Processor (ACD/Labs, Inc., Toronto, ON, Canada). Results and discussion
Fig. 3. Possible chemical structures of dimer acids obtained from oleic and linoleic acids.
Dimer acid synthesis The main reaction of oleic and linoleic acids dimerization is illustrated in Figs. 2 and 3. After oligomerization, the mixture of fatty acids was converted to cyclic dimer as a desired product (Fig. 2), acyclic dimer (Fig. 3) trimer, oligomer, and unreacted monomer fatty acid as undesired products. By Diels–alder cyclization, CDA was synthesized from unsaturated fatty acids since the reaction requires double bonds. These double bonds are adsorbed by the unique structures of the clay catalyst (tetrahedral substitution sites) and this phenomenon helps to dimerize the fatty acids [28]. Products were analyzed by MS (Fig. 4) and presented peaks at around 320, 600, and 880 m/z for monomer, dimer, and oligomer fatty acids, respectively. However, due to the many possible combinations of oleic, linoleic acids and its isomers, each product group (Fig. 3) consists of numerous compounds, as observed from the MS spectrum. To confirm and determine the content of CDA among the products, further analysis of dimer acids was performed using 1H NMR. 1
H NMR analysis
For the analysis of CDA, a 1H NMR spectra of fatty acids, dimer acids, mesitylene, and maleic acid are illustrated in Fig. 5a. Generally, highly pure forms of analytic target materials for a quantitative analysis are required and used as standard materials. However, it is difficult to obtain some kind of reagents, such as dimer acids and CDA in a pure form. An inexpensive and readily available reference standard for those materials including CDA is therefore essential. For this purpose, mesitylene, a molecule that has a similar cyclic structure with that of CDA, was adopted for the verification and quantification of the CDA content in a mixture of dimer acids. The intention of adopting mesitylene is resemblance of not only the chemical structure but also the chemical shift with cyclic structure of CDA. Typically, mesitylene is used as an internal standard for cyclic compounds and it is known that two chemical equivalences appear in 1H NMR [29,30]. The chemical shift of 6.9 ppm originates from three identical protons attached to an aromatic ring and the other (2.3 ppm) is from the methyl groups. Furthermore, this chemical shift is similar to the
Fig. 2. Synthetic scheme of cyclic dimer acid using oleic and linoleic acids.
Fig. 4. LC–MS spectrum of reacted fatty acid containing monomer, dimer, and oligomer acids.
chemical shift of CDA (near 6.8 ppm). For this reason, mesitylene can serve as a pseudo-standard material for CDA. The term, pseudo-standard, was intended as not the highly pure form of analyte but the similar chemical structured material with target analyte. Additionally, as an IS, maleic acid is utilized in 1H NMR quantitative analysis techniques [31,32], because it supplies a separated signal without any overlapping peaks from the signals of the analyte. It is known that the maleic anhydride can react with oleic and linoleic acids. However, this reaction can be happen at more than 150 °C [33–35]. To avoid possible reactions with maleic acid and fatty acids, all the NMR samples with maleic acid were kept at room temperature during the analysis. Moreover, the NMR spectrum of fatty acids and dimer acids did not changed when the maleic acid was used as IS. Based on the NMR spectrum and controlled reaction condition, we can conclude that the maleic acid shows no interaction with other component. The detailed dimer acid and a representative CDA spectrum are presented in Fig. 5b. All the discussed peaks with their assigned protons are listed in Table 1. After the cyclization reaction, the vinyl proton in the unsaturated fatty acid at 5.6 ppm and allylic protons in linoleic acid at 2.0 ppm disappeared, as shown in Fig. 5. On the other hand, signals attributed to the protons of the cyclic structure gradually appear between 6.5 and 7.5 ppm. It is well documented that chemical shifts of 6.5–8.0 ppm represent the protons attached to the cyclic ring [36]. Furthermore, benzylic hydrogens appear at 2.5 ppm after dimerization. These peaks representing the unsaturated cyclic structure confirm the successful synthesis of the CDA. Other peaks may correspond to other isomers of the dimer acid. Signals between 0.8 and 3.0 ppm are characteristic aliphatic hydrogen resonances in dimer acids and other fatty acids. The peaks at 3.57–3.68 ppm and 4.0–4.1 ppm (too low to observe in Fig. 5b) represent the ester methyl group located next
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to the carbonyl carbon and the proton on the glycerol residues, respectively. The specific peak of CDA does not overlap with any peak of the fatty acid, as shown in Fig. 5a. Therefore, the content of CDA in the dimer acid mixture can be determined using the specific signal of the proton in the cyclic structure.
Estimation of CDA content A 1H NMR analysis of CDA was performed to estimate the content among the dimer acids mixture. To establish the feasibility of the proposed method, a quantitative analysis of mesitylene was also performed with maleic acid. A known quantity of maleic acid (0.5 mg) was added to the NMR solvent with various amounts of mesitylene. A standard curve of the mesitylene concentration was constructed by these results and it showed high linearity (from 0.1 to 40 lg of mesitylene), as presented in Fig. 6a. The ratio of the peak area between mesitylene and IS was determined by the peak of mesitylene at 6.90 ppm to the peak of IS at 6.20 ppm. A regression analysis of the data from the mesitylene shows an R2 value of 0.995, indicating a highly linear relationship between the mass and the average integral of the peak in the 1H NMR spectra. Based on this curve, we compared the quantity of mesitylene in the prepared sample and confirmed that the predicted value was consistent with the added amount. This result indicates that cyclic
Fig. 5. 1H NMR results of (a) fatty acid (monomer), dimer acid, mesitylene, maleic acid, and (b) detailed dimer acid spectrum with unreacted fatty acid residues and magnified specific peak of CDA.
Table 1 H NMR chemical shift ranges for selected types of protons.
1
Chemical shifts (d, ppm)
Selected types of protons
0.8–0.9 1.2–1.3 1.5–1.6 1.9–2.0 2.3–2.4
2.5–2.6
3.5–4.1 5.6–5.7 6.2–6.3 6.8–6.9
Fig. 6. Measured signal area ratio of (a) mesitylene and (b) CDA to maleic acid by 1H NMR analysis versus mass of mesitylene and dimer acids mixture, respectively.
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compounds can be reliably quantified using NMR. Furthermore, this curve could also be applied to quantify the CDA level in dimer acid mixtures due to the structural similarity of mesitylene with CDA. The spectra of various dimer acid mixtures are similar to those in Fig. 5 except for the peak intensity from the cyclic proton due to the different concentration of CDA. A series of samples with identical IS and various CDA concentrations were analyzed. Similar with the mesitylene case (Fig. 6a), the curve in Fig. 6b represents the integrated area of CDA specific peak (6.8 ppm) referenced to IS. The intensity ratio of CDA to IS was obtained by averaging the peak area of the CDA signal at 6.81 ppm to the specific signal of the IS at 6.20 ppm. A linear regression with a correlation coefficient of 0.995 implied an excellent linear relationship between the concentration of IS and the integrated area of CDA peaks, as presented in Fig. 6b. With the linearity of the standard curve, NMR spectra of a dimer acid mixture containing CDA were evaluated. The intensity area ratio value of CDA from Fig. 6b was assigned to Fig. 6a and therein the CDA content was estimated via constructed mesitylene mass of standard curve. The estimated contents of CDA by the curve were presented in Table 2. Furthermore, general quantification technique using proton NMR was adopted to show the feasibility of proposed method based on pseudo-standard material. For this purpose, the CDA content (lg/g dimer acids mixture) in each sample was calculated from a previously suggested equation [19–21]:
ICDA HIS MWCDA M IS Total CDA content ¼ IIS HCDA MVIS M DA
ð1Þ
ICDA = average intensity area of CDA peak, IIS = intensity area of IS, HIS = number of protons of IS, HCDA = number of protons of CDA, MWCDA = molecular weight of CDA, MWIS = molecular weight of IS, MIS = mass of IS, and MDA = mass of dimer acid mixture. In order to calculate the CDA content from the Eq. (1), we should start with the obtaining of the values for each variable. The intensity ratio between CDA and IS (ICDA/IIS) is obtained from the Fig. 4b and the NMR spectrums of CDA. The molecular weight of CDA (MWCDA) and the number of protons of CDA (HCDA) as calculated based on the presented chemical structure of CDA in Fig. 2. Molecular weight and number of protons of IS are 116 g/mol and 4 atoms, respectively. Mass of IS (MIS) is fixed of 0.5 mg and dimer acid mixture (MDA) is also obtained from the Fig. 6. After the gathering of all values, we substitute every variables in the Eq. (1) with corresponding values. Finally, the content of CDA in an unknown sample was calculated and presented in Table 2. The predicted concentrations of CDA from both the standard curve and Eq. (1) show an average of 13% difference. For verification, a known quantity of mesitylene was also estimated using both techniques. The mass evaluation of mesitylene showed 40.49 lg by standard curve and 38.15 lg by Eq. (1) for the case of 40 lg. The estimation difference between the two methods was 5.8%. According to a previous report [23], the quantitative NMR method can show a 95% confidence level due to insignificant systematic error. This systematic error may contribute to the difference in the estimated value between the two quantification methods. Another possible cause of this difference could be derived from the molecular weight of CDA. In the equation, molecular weight of CDA was required to obtain the content but the CDA
Table 2 Comparison of CDA content determined by standard curve and equation.
Standard curve Eq. (1)
Sample 1 (lg)
Sample 2 (lg)
Sample 3 (lg)
Mesitylene (lg)
0.86
0.22
0.11
40.49
0.74
0.23
0.10
38.15
represents whole possible cyclic compounds in dimerization. However, representative value of possible molecular weights (560 g/mol for CDA in Fig. 2) was substituted. Conclusion In this study, we proposed a method for determining the CDA content in a mixture of dimer acids using a 1H NMR analysis. The CDA content was estimated by the standard curve of mesitylene with the CDA to IS signal area ratio and compared to an equation proposed previously [20]. The reliability of the proposed method was demonstrated by quantifying mesitylene, an easy accessible cyclic standard material. Based on the verification results using mesitylene, the accuracy of our method and the equation were 98% and 95%, respectively. The accuracy is defined as the mass difference ratio between the input mass and the estimated mass of mesitylene. The key advantage of this method is that it does not require a high purity analyte standard but an easily accessible pseudo standard and IS for the content determination. Moreover, a quantitative NMR analysis provides quantification of CDA on as low as a micro-gram scale. The proposed method can be applied in various fields that require quantification of specific cyclic compounds in mixtures of pharmaceuticals or processed food. Acknowledgements This study was supported by the R&D Center for Valuable Recycling (Global-Top Environmental Technology Development Program) funded by the Ministry of Environment – South Korea (Project No.: GT-11-C-01-250-0). References [1] M. Kouze, T. Kasuno, M. Tajika, Y. Sugimoto, S. Yamanaka, J. Hidaka, Fuel 87 (2008) 2798–2806. [2] L.C. Meher, D.V. Sagar, S.N. Naik, Renew. Sustainable Energy Rev. 10 (2006) 248–268. [3] A.E. Atabani, A.S. Silitonga, H.C. Ong, T.M.I. Mahlia, H.H. Masjuki, I.A. Badruddin, H. Fayaz, Renew. Sustainable Energy Rev. 18 (2013) 211–245. [4] M. Desroches, M. Escouvois, R. Auvergne, S. Caillol, B. Boutevin, Polym. Rev. 52 (2012) 38–79. [5] K. Yao, C. Tang, Macromolecules 46 (2013) 1689–1712. [6] A. Talebian-Kiakalaieh, N.A.S. Amin, H. Mazaheri, Appl. Energy 104 (2013) 683–710. [7] P. Kadam, P. Vaidya, S. Mhaske, Int. J. Adhes. Adhes. 50 (2014) 151–156. [8] A. Li, K. Li, ACS Sustainable Chem. Eng. 2 (2014) 2090–2096. [9] Y. Li, B.A.J. Noordover, R.A.T.M. van Benthem, C.E. Koning, Eur. Polym. J. 59 (2014) 8–18. [10] K.-H. Ko, H.R. Park, J.-S. Kim, Y.-W. Kim, J. Appl. Polym. Sci. 129 (2013) 2443– 2450. [11] S. Li, X. Yang, K. Huang, M. Li, J. Xia, Prog. Org. Coat. 77 (2014) 388–394. [12] S.J. Lee, Y.-W. Kim, S.-H. Yoo, N.-K. Kim, J.H. Shin, B.-T. Yoon, Appl. Chem. Eng. 24 (2013) 530–536. [13] K.S. Robbins, Y. Ma, M.L. Wells, P. Greenspan, R.B. Pegg, J. Agric. Food Chem. 62 (2014) 4332–4341. [14] R.L. Veazey, J. Am. Oil Chem. Soc. 63 (1986) 1043–1046. [15] J. Zhao, S.V. Olesik, Anal. Chim. Acta 449 (2001) 221–236. [16] M. Heimrich, M. Bönsch, H. Nickl, T.J. Simat, Food Addit. Contam. 29 (2012) 846–860. [17] J.P. Nelson, A.J. Milun, J. Am. Oil Chem. Soc. 52 (1975) 81–83. [18] R. Haselberg, V. Brinks, A. Hawe, G.J. de Jong, G.W. Somsen, Anal. Bioanal. Chem. 400 (2011) 295–303. [19] T. Ohtsuki, K. Sato, N. Sugimoto, H. Akiyama, Y. Kawamura, Talanta 99 (2012) 342–348. [20] T. Ohtsuki, K. Sato, N. Furusho, H. Kubota, N. Sugimoto, H. Akiyama, Food Chem. 141 (2013) 1322–1327. [21] T. Ohtsuki, K. Sato, N. Sugimoto, H. Akiyama, Y. Kawamura, Anal. Chim. Acta 734 (2012) 54–61. [22] J. Pan, X. Gong, H. Qu, J. Pharm. Biomed. Anal. 85 (2013) 28–32. [23] A.A. Salem, H.A. Mossa, B.N. Barsoum, Spectrochim. Acta, Part A 62 (2005) 466–472. [24] B.S. Somashekar, O.B. Ijare, G.A.N. Gowda, V. Ramesh, S. Gupta, C.L. Khetrapal, Spectrochim. Acta, Part A 65 (2006) 254–260. [25] A.A. Salem, H.A. Mossa, Talanta 88 (2012) 104–114. [26] R.F. Paschke, L.E. Peterson, D.H. Wheeler, J. Am. Oil Chem. Soc. 41 (1964) 723– 727.
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