Abstract. Key words: Biopolymers; Vernonia galamensis; Soya bean seed oil; NMR spectroscopy. Research Article

Transcription

1 THE SCITECH JOURNAL ISSN ISSN Online Towards Sustainable Polymers and Plastics: NMR Spectroscopic Analysis and Characterisation of Vernonia Seed (Vernonia Galamensis) Oil and Epoxidised Soya Bean Seed (Glycine Max) Oil Darshil U. Shah Polymer Composites Group, Faculty of Engineering, University of Nottingham, University Park, Nottingham, NG7 RD, UK Present address: Oxford Silk Group, Dept. of Zoology, University of Oxford, Oxford OX 9JY, UK Abstract Owing to their low cost, wide variety, and abundance, plant seed oils and their derivatives have emerged as interesting sources for the development of sustainable polymers and plastics. While much research so far has been on soya bean seed (Glycine max) oil, oils from the seeds of exotic plants, such as Vernonia galamensis, naturally offer useful functionalities such as epoxide groups. As a precursor to considering vernonia seed oil for polymer development through established processes used with soya bean seed oil, here the structure and composition of vernonia seed oil is studied, in comparison to epoxidised soya bean seed oil, using NMR spectroscopy. Key words: Biopolymers; Vernonia galamensis; Soya bean seed oil; NMR spectroscopy Introduction Plant seed oils for sustainable polymers and plastics In recent decades, a tremendous amount of research has been conducted in finding alternative sources to polymers and plastics to the conventional petrochemical sources, primarily due to environmental and economical concerns. Research has primarily focussed on polymers derived from plant seed oils, polysaccharides and its derivatives (like starch, cellulose, sugar), and synthetic polymers derived from natural monomers (Chum 99; Chielline 000; A.K. Mohanty 005; V. Flaris 009). Due to the low cost, wide availability and abundance of plant oils, it is only sensible to investigate their viability further (K.D. Carlson 985; H.L. Bhardwaj 007; M.A.R. Meier 007) Plant oils, such as soybean oil, linseed oil and vernonia oil, are generally in the form of triglycerides where three long chain fatty acids are joined at a glycerol juncture. Due to the oil being a mix of several fatty acids, it is understood that several types of triglycerides may exist in the oil. However, genetic engineering has allowed better control in the variation of unsaturation, for instance (R.P. Wool 00). Triglycerides have been widely used in the food industry. There extensive applications in the paint, coating, cosmetic, ink, plasticizer, polymer and lubricant industry is also well known (C.F. Krewson 966; ; G.W. Bussell 974; L.E. Hodakowski 975; D.J. Trecker 976; ; Komii 980; ; C.G. Force 988; D.K. Salunkhe 99; R.P. Wool 000; R.P. Wool 00; A. Adhvaryu 00; P.G.J. McClory 007) Due to their relatively low viscosities, plant oils are good diluents (both reactive and unreactive) and eliminate the need for a solvent. Mixing a proportion of plant oil into the base resin reduces resin viscosity, thus improving processibility of highly filled laminates and composites. However, this generally depresses the glass transition temperature and lowers mechanical properties (R.P. Wool 00; M. Misra 00). Also reactive diluents (like epoxidised soybean oil) participate in the curing reaction and reduce the crosslink density, activation energy required and cure kinetic parameters. Diluents also reduce amine concentration and processing times. Plant oils are also useful tougheners and induce resistance to crack propagation in resins such as epoxy (Chielline 000; R.P. Wool 00; R.P. Wool 005). Plant oils have found various applicability as stabilizers and plasticizers (increase free volume, reduce glass transition, improve processability) in polymers, additives in lubricants (reduce viscosity), or as components in plastics (C.F. Krewson 966; G.W. Bussell 974; L.E. Hodakowski 975; D.J. Trecker 976; D.J. Trecker 976; Komii 980; U. Sasaki 98; R.P. Wool 000) Considerable research (L.H. Sperling 983; S. Qureshi 983; L.H 984; K.D. Carlson 985; L.W. Barrett 993; V. Kolot 004; S.S. Narine 007) has also looked at the possibility of producing interpenetrating networks, mechanical blend polymers (between functionalized oils and thermosetting resins like epoxy) and copolymers. However, in these works, the plant oil is only a minor component whose purpose is to modify the physical properties of the main matrix. Some acids in the triglycerides either naturally contain other functionalities (like epoxies, hydroxyls, and cyclic groups) or have active sites amenable to functionalisation. The epoxidation of unsaturated sites to produced epoxides (and later attach hydroxyl, acrylate and methacrylate functionalities) and the functionalisation of ester groups to produce mono-glycerides or esters (of starch/cellulose) have also been investigated (Chielline 000; S.N. Khot et.al. 000; ; R.P. Wool 00; A.K. Mohanty 005; R.P. Wool 005; M.A.R. Meier 007). These functionalized sites can be used to produce thermosetting resins by using ring-opening polymerization (for epoxies), free-radical polymerization Received: November04 Accepted: November 04 *Corresponding Author darshil.shah@zoo.ox.ac.uk 3 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

2 THE SCITECH JOURNAL ISSN ISSN Online (acrylates, maleate half esters) and polycondensation (hydroxyl group containing triglycerides) ) (S.N. Khot et.al. 000; E. Can 00; A.E. Gerbase 00; Ahamed 004; ; A.K. Mohanty 005; M.A.R. Meier 007; N. Mann 008; Z. Liu 00). Although the research conducted has led to the discovery of many new products; their high relative costs (due to low-volume production) and poor mechanical performance (especially low glass transition temperature and low stiffness/strength) have been major barriers to their wide-scale application as a primary component for a resin system. Thus, they are only used as up to 30% mixes with petrochemically derived resins, particular where structural applications are being considered. Much of the research so far has been done on soya bean seed (Glycine max) oil due to its relative abundance, low cost, and multiple accessible unsaturation sites. Typically, as a first step, it is converted into epoxidised soya bean seed oil (ESO). Vernonia oil (VO) is an exotic plant oil derived from the seeds of the Vernonia galamensis plant which are found in Ethiopia. This oil can be highly rich in vernolic acid (up to 80%) (F.O. Ayorinde 990), which has a naturally occurring epoxide group. The epoxide functionality of commercial ESO is epoxide groups per triglyceride (R.P. Wool 00), while that of vernonia oil is between.4-.8 (R.P. Wool 00). VO also has a lower degree of saturated acids and viscosity, making it an ideal candidate for polymer synthesis. The possibility of transferring ESO processing techniques to VO through standard processes (such as ring opening polymerization using amine curing agents), requires as a first step to appreciate the structure and composition of VO (in comparison to ESO). Here, a thorough NMR spectroscopic analysis is carried out on VO and ESO. 3 H-NMR, C-NMR, DEPT-35 (Distortionless Enhancement by Polarisation Transfer NMR using a proton pulse flip angle of 35 ) 3 and D, H- C- HMQC/HSQC (Heteronuclear Multiple-Quantum Correlation/Heteronuclear Single-Quantum Coherence) have been effectively used this purpose. While it is not uncommon to 3 investigate fatty-acids with H-NMR and C-NMR, the use of more advanced NMR techniques is demonstrated here. The DEPT-35 spectrum helps identify the multiplicity of CH, as signals from n methylenes (CH ) will be negative, while signals from methines and methyls (CH and CH ) will be positive. The HMQC and HSQC 3 3 experiments allow the dispersion of the proton spectra along the C dimension. Hence, overlapping multiplets in the proton spectrum 3 can be spread apart by differences in the C chemical shifts, which makes it possible to recognize H chemical shifts of each multiplet. Materials and Methods Materials Vernonia seed oil (VO) Vernonia seed oil (VO) was obtained from Addis Ababa University (Ethiopia). Epoxidised soya bean seed oil (ESO) was supplied by PolyBlend UK Ltd (UK). Deuterated chloroform, CDCl3 was purchased from Sigma-Aldrich (UK). NMR spectroscopy Test parameters for H-NMR, 3C-NMR, DEPT-35 and D, H- 3C- HMQC/HSQC spectroscopy are presented in Table. Chemical shifts were automatically (and when required, manually) referenced by the software (ACD/Labs, UK) using the resonance frequency of the solvent (CDCl3) with the internal standard (tetramethylsilane, Si(CH3)4). Quantified H-NMR is used by integrating peaks and equating an integration value of to one proton. Results and Discussion H-NMR of ESO Soybean oil is a triglyceride that is a mixture of linoleic acid (53.%), oleic acid (3.4%), linolenic acid (7.8%), palmitic acid (%) and stearic acid (4%) (S.N. Khot et.al. 000). Some variations, however, may be observed; Can et.al. (E. Can 00) show that no linolenic acid is present in their soybean oil sample, while oleic acid content is up to 3%. ESO would have a similar composition of these epoxidised acids. Figure shows one of the possible triglycerides of ESO, and shall be used as a generic ESO triglyceride for H-NMR assignment. Table gives the H-NMR assignments and chemical shifts of the different functional groups; the H-NMR spectrum is presented in Figure. The H-NMR spectrum of ESO (Figure, Table ) shows large expected signals for the terminal methyl of the acids, ω (labelled A) between ppm. If the total proton count of the terminal methyl groups is referenced as 9, the H-NMR spectrum confirms that the commercially obtained ESO is present purely in the form of triglycerides as the primary and secondary glycerol hydroxyl peaks (labelled H, I) at ppm and ppm have a proton count very close to the expected values of 4 and while the proton counts of the CH groups at positions C and C3 (α and β to the acid group; labelled F, D) are both close to the expected value of 6. From the H-NMR, several other pieces of information can be obtained (G. Knothe 004). The epoxide content of ESO can be calculated as 8./ = 4. epoxide groups per triglyceride (Table ). This lies within the widely quoted values of epoxide groups per triglyceride (R.P. Wool 00). The contributing acids are oleic (9,0C), linolenic (9,0C;,3C) and linolenic (9,0C;,3C; 5,6C). The oxirane hydrogen's are observed between ppm, in the form of 3 distinct peaks. Using the HMQC in 3 conjunction with the intense peaks observed in the C-NMR spectrum does suggest that the corresponding peak between ppm is due to the epoxide group(s) in linolenic acid. The degree of unsaturation of the oil can also be derived from the H- NMR spectra. On the H-NMR spectrum (Figure, Table ), the CH groups α to the epoxide groups (labelled C, E', E'') are observed in the range of.4-.57ppm and ppm. The contribution to CH groups α' to the epoxide groups (labelled C) in a triglyceride comes from all the epoxidised unsaturated acids and has a proton count of 0.3 (Eq. ). Therefore, the degree of unsaturation of ESO is 0.3/ = 86%. This value agrees with several quoted literature values (S.N. Khot et.al. 000; P. Kiatsimkul 006). H-NMR spectra (Figure, Table ) can also be used to determine the composition of acids in the oil. Firstly, the terminal methyl peak ω of linolenic acid (labelled A',.0-.5ppm) is downfield compared to the other acids (labelled A, ppm) due to electron withdrawing and deshielding effect of the oxygen in the 5,6C epoxide group. Using the proton count of the terminal methyl ω, the content of linolenic acid can therefore be calculated as 0.4/9 = 4.4% 4 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

3 THE SCITECH JOURNAL ISSN ISSN Online Figure. Example triglyceride arrangement for ESO, with an oleic acid (8:), linoleic acid (8:) and linolenic acid (8:3) component, that is used for H-NMR assignment Figure. H-NMR spectrum of ESO. Refer to Figure for the triglyceride schematic. 5 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

4 THE SCITECH JOURNAL ISSN ISSN Online of the total composition (Gunstone 007; Gunstone 007). Secondly, the contribution to CH groups α'' to the epoxide groups (labelled E', E'') in a triglyceride comes from epoxidised unsaturated acids with multiple epoxide groups (that is, linoleic and linolenic acid) and has a proton count of As the content of linolenic acid is known, the content of linoleic acid can be calculated to be 5% (= (3.59 *0.044)/6). Thirdly, it is known that the unsaturated acids are oleic, linoleic and linolenic acid and the level of unsaturation is 86%. As the content of linoleic and linolenic acids are known, the content of oleic acid can be calculated as 3% (=86% 5% 4.4%). In summary, the determined ESO composition is 5% linoleic acid, 3% oleic acid, 4% saturated acids (palmitic and stearic) and 4% linolenic acid. This composition would yield a theoretical value for the oxirane hydrogen content per triglyceride content of 8.77, which is comparable to the observed value of 8.0. Also, the calculated epoxide content for the composition falls within the literature value of epoxide groups per triglyceride. Furthermore, the determined composition is very close to the widely quoted composition (S.N. Khot et.al. 000; E. Can 00). Although such an elaborate study on the proton spectra of ESO has not been performed, work by Mann et.al. (), Adhvaryu et.al. (), Akintayo et.al. () and Gunstone et.al. (Gunstone 007; ) on the proton NMR characterization of ESO and other oils are in agreement with the deductions made here. 3 C-NMR and DEPT-35 of ESO 3 The C-NMR of ESO is assigned to it with the aid of DEPT-35 analysis. Figure 3 shows one of the possible triglycerides of ESO where the symbols correspond to the assignments on the carbon spectrum. Table 3 gives the assignments and chemical shifts of the different functional groups, while the spectrum is presented in Figure 4. The spectrum has 59 signals, which is in agreement with Gunstone's study of the carbon spectra of ESO and other epoxidised oils (Gunstone 993). The spectrum shows expected signals for C-3, ω-3 and glycerol. The most intense peaks have been taken to be from linoleic acid, the next intense peaks to be from oleic acid followed by saturated acids (palmitic and stearic), and the almost insignificant peaks to be from linolenic acid. Twelve epoxide signals are observed in the range of ppm: three major ones, which are associated with linoleic acid, six moderate ones (two of which are associated with oleic acid) and three very small ones, which are perhaps associated with linolenic acid. All of the epoxide peaks observed are in agreement with Gunstone's investigation (Gunstone 993). Twelve signals are observed between ppm which correspond to CH groups α' or α'' to the epoxide groups. This is confirmed from the HMQC of ESO. These peaks are the most difficult to assign and effort has not been made to perform any further analysis on this. The methylene envelope between ppm also provides valuable information about ESO. Theoretically, a total of 4 (or more) signals should be observed: 6 from oleic acid (at C4-6 and C3-5), 3 each from linoleic and linolenic acids (at C4-6) and from stearic acid (at C4-5). However, many of these will overlap. Due to the insignificant levels of linolenic acid, no peaks are observed from it. A total of 5 signals are observed, 3 of which have been assigned to linoleic acid and 6 to oleic acid, by looking at the signal intensities. The 6 other signals have either been left unassigned or designated to the saturated acids. The methylene peaks give a better understanding of the unsaturated acids than the saturated acids. It should again be pointed out that just like the proton spectrum, no olefinic signals are seen, indicating that epoxidation is virtually complete. The assignments and conclusions Table. Test parameters for NMR spectroscopy. Spectra NMR spectromete r H-NMR Bruker AV400 Operating frequency [MHz] Spectral width [khz] RF pulse width [μs] at flip angle [ ] Recovery delay [μs] Data acquisition duration [s] Relaxation delay [s] μs at Total scans 3 C-NMR Bruker AV μs at DEPT-35 Bruker AV C: 0 µs at 90, 0 µs at 80 H:.7 µs at 90, 3.4 µs at HMQC/HS QC Bruker AV(III)400 (inverted probe) C: 4.4 µs at 90 H: 7µs at THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

5 THE SCITECH JOURNAL ISSN ISSN Online Table. H-NMR spectrum of ESO. Refer to Figure for the triglyceride schematic. Assignment Chemical shift, δ Protons (multiplicity) Symbol Terminal methyl, R-CH (ω ) (multiplet) A R-CHOCH-CH -CH, (ω of linolenic acid) (triplet) A' R-CH -R' (multiplet) B α'-ch of epoxy group (multiplet) C R-O-CO-CH -CH -R' (C3) (broad singlet) D Α''-CH of epoxy group (multiplet) E', E'' R-O-CO-CH -R' (C) (triplet) F Epoxy group, R-CHOCH-R' (multiplet) G (broad singlet) G' (multiplet) G'' 3 Linoleic acid (from CNMR and HMQC) (multiplet) G''' primary glycerol hydroxyl group, R-CH -O-CO-R' (two doublet of doublets, J =.9, 4.3) H secondary glycerol hydroxyl group, R-CH-O-CO (pentet) I Figure 3. Example triglyceride arrangement for ESO, with an oleic acid (8:), linoleic acid (8:) and linolenic acid (8:3) component, that 3 is used for C-NMR assignment. 7 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

6 THE SCITECH JOURNAL ISSN ISSN Online 3 Figure 4. C-NMR spectrum of ESO: a) full spectrum, b) methylene region, c) epoxide carbon region. Refer to Figure 3 for the triglyceride schematic. 8 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

7 THE SCITECH JOURNAL ISSN ISSN Online are consistent with the results of several investigations on epoxidised oils and acids (; P. Kiatsimkul 006; Gunstone 007; Gunstone 007; Gunstone 007; N. Mann 008). H-NMR of VO Figure 5 shows one of the possible triglycerides of VO, and shall be used as a generic ESO triglyceride for H-NMR assignment. Table 4 gives the assignments and chemical shifts of the different functional groups. The actual spectrum is presented in Figure 6. Quantified H- NMR is used by integrating peaks and equating an integration value of to one proton. The H-NMR spectrum of VO is fairly similar to that of ESO, where expected peaks for the terminal methyl (labelled A) are observed between ppm. Although the secondary glycerol hydroxyl peaks are easily recognizable (about ppm; labelled J), the primary glycerol hydroxyl peaks coincide with the olefinic hydrogen peaks. An interesting point to note is that both ESO and VO show a J coupling constant of about and 4.3 Hz for the secondary glycerol hydroxyl peaks. If the total proton count of the terminal methyl groups is referenced as 9, the H-NMR spectrum shows that the VO sample is not purely in the form of triglycerides; that is free fatty acids and mono-, diglycerides are present. This is because the integrals of the primary and secondary glycerol hydroxyl peaks are 3.36 and 0.45, rather than the expected values for a triglyceride of 4 and. This was expected, however, as vernonia oil was extracted in-house, rather than industrially. Triglycerides can biochemically convert to diglycerides and monoglycerides by subsequent release of fatty acids, leaving hydroxyl functional groups on the remaining positions of the glycerol moiety. The presence of mono- and diglycerides can be confirmed if characteristic peaks of the carboxylic acid and hydroxyl groups are found. As the H-NMR spectrum is limited to 7.8 ppm, the carboxylic acid peak is not observed in the 3 H-NMR, but can be seen in the C-NMR spectrum (at about 78 ppm). However, small peaks in the range of ppm are observed on the H-NMR spectrum, signalling the presence of some hydroxyl groups. Epoxide peaks are observed between ppm. The epoxide content of the oil is.345 (=.69/) epoxide groups per triglyceride. As vernolic acid is the only acid present with an epoxy group, it can be said that 45% (=.345/3) of the oil is composed of vernolic acid. This value is consistent with the proton count for the α-ch of oxirane group observed between.4-.57ppm. Vernolic acid also has a unique characteristic peak between.3-.5ppm due to the CH amid the double bond and epoxy ring. Using that integral value of. suggests that 37% (= (./)/3) of the oil is composed of vernolic acid. It can be said that 37-45% of the oil consists of vernolic acid. This content of vernolic acid is in agreement with literature (; T. Baye 005). A characteristic peak of linoleic acid is also observed between ppm due to the CH amid the two double bonds. Using the integral value of 0.35, it can be said that 5.8% (=(0.35/)/3) of the oil is linoleic acid. Additionally, olefinic and allylic peaks are observed between ppm and ppm. Using rigorous analysis, these could be used to determine the saturated acid content of the oil (G. Knothe 004). The three primary unsaturated acids present in VO are vernolic, linoleic and oleic acid. These have, and carbon double bonds, respectively. Hence, per triglyceride they would have 6, and 6 hydrogens, respectively. The proton count of the olefinic peaks is obtained as Hence, it can be deduced that the content of oleic acid is obtained as approximately 0%. As vernolic, linoleic and oleic acid account for approximately 45%, 6%, and 0% of the oil, the total content of unsaturated acids is 7%. Unsaturated acids (palmitic, stearic and archidic acid) comprise of the remaining 9%. This can be verified by counting the protons in the allylic position (E). The actual proton count is 5.3. For each Figure 5. Example triglyceride arrangement for VO, with a vernolic acid, linoleic acid and oleic acid component, that is used for H-NMR assignment. 9 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

8 THE SCITECH JOURNAL ISSN ISSN Online Figure 6. H-NMR spectrum of VO. Refer to Figure 5 for the triglyceride schematic. 3 Figure 7. Example triglyceride arrangement for VO, with a vernolic acid, linoleic acid and oleic acid component, that is used for C-NMR assignment. acid group, 4 allylic protons come from oleic acid and linoleic acid (the bis-allylic protons are included in H rather than E) and allylic protons come from vernolic acid (the bis-allylic-oxirane protons are included in F rather than E). Hence, per triglyceride the allylic proton count is obtained as This theoretical value of 5.78 compares reasonably with the observed proton count of 5.3. It is suggested in literature, that vernonia oil is a mixture of vernolic acid (75%), oleic acid (6%), linolenic acid (3%), palmitic acid (3%) and stearic acid (3%) (F.O. Ayorinde 990; T. Baye 005). The epoxy functionality of VO is quoted to be around.8 (R.P. Wool 00). Significant variations, however, may be observed; variations of vernolic acid between 34-87% have been recorded depending on age of seed, location of crop, and soil and environmental conditions (T. Baye 005; T. Baye 005). Clearly, the VO used in this study falls on the lower scale, in terms of vernolic acid content. Notably, if unsaturation sites in our VO were to be epoxidised, epoxidised VO would have an epoxide content of 3.7. This would still be lower than 0 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

9 THE SCITECH JOURNAL ISSN ISSN Online 3 Table 3. C-NMR spectrum of ESO. Refer to Figure 3 for the triglyceride schematic. Assignment Chemical shift, δ [ppm] DEPT-35 Signal Intensity Symbol Terminal methyl, R-CH 3 Linolenic-8 (ω ) Linoleic-8 Oleic-8 Saturated* ( ) A R-CH -CH, (ω ) 3 Linolenic-7 Linoleic-7 Oleic-7 Saturated* ( ) B R-O-CO-CH-CH-R' (C3) Linoleic-3 Oleic-3 Saturated* ( ) C Α'-CH of epoxy group ( ) D Α''-CH of epoxy group ) E Α''-CH of epoxy groupα'- CH of epoxy group ) F R-CH -R' Oleic ( ) G THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

10 THE SCITECH JOURNAL ISSN ISSN Online 3 Table 3. C-NMR spectrum of ESO. Refer to Figure 3 for the triglyceride schematic. Assignment Chemical shift, δ [ppm] DEPT-35 Signal Intensity Symbol R-CH -CH -CH (ω ) 3 3 Linoleic-6 Oleic-6 Saturated* ( ) H R-O-CO-CH -R' (C) Linoleic- Saturated* Oleic- ( ) I Epoxy group, R-CHOCH-R' ( ) J Linolenic Linoleic Linoleic Linoleic J' J'' Oleic Oleic J''' J'''' Linolenic Linolenic R-CH -O-CO-R'' K R-CH-O-CO-R' * 0.48 L R-CO-O-CH-R' (C) 7.7 NONE 0.6 M R-CO-O-CH -R' NONE N Assignments C-3 and ω - 3 refer to carbon atoms at the acyl and methyl ends of the chain, respectively. * Saturated acids include palmitic and stearic acid. THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

11 THE SCITECH JOURNAL ISSN ISSN Online Table 4. H-NMR spectrum of VO. Refer to Figure 5 for the triglyceride schematic. Assignment Chemical shift, δ [ppm] Protons (multiplicity) Symbol Terminal methyl, R-CH (ω (multiplet) A R-CH -R' (multiplet) B Α-CH of epoxy group And β-ch of allylic group (multiplet) C R-O-CO-CH -CH -R' (C3) (multiplet) D R-CH=CH-CH -R' (α-ch of allylic group) (multiplet) E R-CH=CH-CH -CHOCH-R' (vernolic acid) (multiplet) F R-O-CO-CH -R' (C) (multiplet) G R-CH=CH-CH -CH=CH-R (Linoleic acid) (multiplet) H Epoxy group, R-CHOCH-R' (triplet), J = 4.6x() R-CH -O-CO-R' (two doublet of doublets, J =, 4.3) I J R-CH-O-CO-R' (pentet) K R-CH=CH-R' (multiplet)4.59 (multiplet) L (multiplet) (multiplet) (multiplet) that of ESO (epoxide content of 4. observed). 3 C-NMR and DEPT-35 of VO 3 The C-NMR of VO is assigned to it with the aid of DEPT-35 analysis. Figure 7 shows one of the possible triglycerides of ESO where the symbols correspond to the assignments on the carbon spectrum. Table 5 gives the assignments and chemical shifts of the different functional groups, while the spectrum is presented in 3 Figure 8. The C-NMR spectrum has 79 signals. The spectrum shows expected signals for C-3, ω and glycerol. The most intense -3 peaks have been taken to be from vernolic acid, the next intense peaks from oleic followed by saturated acids and then from linoleic acid. Two epoxide signals are observed in the range of ppm. The two signals have been identified as the vernolic acid peaks. Gunstone (Gunstone 993) noticed the presence of other peaks in this range but didn't discuss any possible sources of these extra signals other than mentioning the possibility of the presence of other natural epoxy acids in VO. Multiple peaks are observed for the secondary glycerol peaks suggesting again the possibility of the presence of mono- and diglycerides. Several peaks are observed in the range of 6-75 ppm. The HMQC shows that these peaks correspond to the ppm range on the H-NMR spectrum. This makes a strong case on the presence of mono- and di-glycerides and hydroxyl groups attached to the glycerol moieties. In addition, a strong signal is observed at about 78ppm. C is identified to be in the 7-73 ppm region. The signal at 78 ppm is suggested to be from an acid/ester formed during the biochemical formation of mono- and di-glycerides. Other than the epoxide peaks, the olefinic (N) and allylic (D-G) peaks are of interest. This is because the olefinic and allylic peaks are characteristic of the unsaturated acids. Specific peaks (like D and E) also give more information about particular acids in the oil. Using the signal intensities, many of the peaks have been assigned. The 3 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

12 THE SCITECH JOURNAL ISSN ISSN Online Figure 9. HMQC spectrum of ESO. Figure 0. HMQC spectrum of VO. 5 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

13 THE SCITECH JOURNAL ISSN ISSN Online 3 Figure 8. C-NMR spectrum of VO: a) full spectrum; b) methylene region. Refer to Figure 7 for the triglyceride schematic. 4 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

14 THE SCITECH JOURNAL ISSN ISSN Online peaks observed and the assignments made are in agreement with other works (Gunstone 993; Gunstone 007; Gunstone 007). HMQC of ESO and VO The HMQC spectrum of an oil is perhaps the most important tool for quick characterization of the oil. Overlapping multiplets in the 3 proton spectrum can be spread apart by differences in the C chemical shifts, thus making it possible to recognize H chemical 3 shifts of each multiplet. Hence, an HMQC allows the H- and C- NMR spectra to be correctly assigned. The HMQC becomes even more useful when comparing two different oils together. This is because in an HMQC spectrum, bonds and functional groups are present in clusters that are easily identifiable. The HMQC spectra of ESO and VO are presented in Figure 9 and Figure 0, respectively. It can be seen that the spectra are very similar with the glycerol, epoxide, C-3, methylene and ω 3 peaks appearing in the same regions. There are 4 important differences in the two spectra. Firstly, the presence of olefinic and allylic clusters is observed for VO but not for ESO. This is why several clusters are noticed in the 5-5.5ppm region and.5-.5ppm region for VO, but not for ESO. Secondly, in ESO all carbon doubles bonds have been converted to epoxide groups. This is why in VO there is only one cluster for the epoxide group, whereas ESO shows three clusters. Thirdly, there is an absence of a downfield CH α-oxirane peak in VO, which is clearly visible for ESO. Finally, VO exhibits several clusters, showing that the oil is a mixture of mono-, di- and triglycerides. This is not the 3 Table 5. C-NMR spectrum of VO. Refer to Figure 7 for the triglyceride schematic. Assignment Chemical shift, δ [ppm] DEPT-35 Signal Intensity Symbol Terminal methyl, R-CH 3 (ω ) Vernolic-8 Linoleic-8 Oleic,Saturated* ( ) A R-CH-CH 3 (ω ) Linoleic-7 Vernolic-7 Oleic,Saturated* ( ) B R-O-CO-CH -CH -R' (C3) ( ) C R-CH=CH-CH - CH=CH-R' Linoleic D α-ch of oxirane group, R-CH=CH-CH - CHOCH-R' Vernolic-5 Vernolic- ( ) E R-CH=CH-CH -R' Oleic, Linoleic Vernolic-8 Vernolic-4 ( ) F Α-CH of oxirane group G 6 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

15 THE SCITECH JOURNAL ISSN ISSN Online 3 Table 5. C-NMR spectrum of VO. Refer to Figure 7 for the triglyceride schematic. Assignment Chemical shift, DEPT-35 Intensity Symbol δ [ppm] Signal R-CH -R' Vernolic Vernolic Vernolic ( ) H R-CH-CH-CH 3 (ω 3) Linoleic Vernolic-6 Oleic,Saturated* ( ) I R-O-CO-CH -R' (C) ( ) J Epoxy group, R-CHOCH-R' Vernolic-3 Vernolic- ( ) K R-CH -O-CO-R' Triglyceride Di-glyceride Mono-glyceride ( ) L R-CH-O-CO-R' * 0. M R-CH=CH-R' Vernolic-9 Linoleic Linoleic Oleic Linoleic, Oleic Linoleic Vernolic-0 ( ) * N 7 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

16 THE SCITECH JOURNAL ISSN ISSN Online 3 Table 5. C-NMR spectrum of VO. Refer to Figure 7 for the triglyceride schematic. Assignment Chemical shift, δ [ppm] DEPT-35 Signal Intensity Symbol R-CO-O-CH -R' 7.8 NONE 0.06 P R-CO-O-CH-R' NONE Q Acid, R-CO-OH NONE 0. R Assignments C-3 and ω-3 refer to carbon atoms at the acyl and methyl ends of the chain, respectively. * Saturated acids include palmitic and stearic acid. Conclusions NMR has been used as an effective tool to determine and compare the chemical structure and composition of ESO and VO. We find that the commercially-produced ESO comprises purely of triglycerides, while in-house extracted VO is a mix mono-, di- and tri-glycerides. We also find that ESO has a much higher unsaturation content and epoxide per triglyceride content (86% and 4.) than VO (7% and.345). If epoxidised VO were to be produced, the epoxide per triglyceride content would be 3.7, which is still lower than that of ESO. A low content of vernolic acid (<50%) was found in the VO. For VO to be considered as an alternative to ESO for polymer development, it is recommended that i) VO with higher vernolic acid content is used, ii) ensure that only triglycerides are present in VO, and iii) epoxidation of the unsaturation sites is carried out. Acknowledgements The author thanks Dr Peter Licence (University of Nottingham) and Dr Simon Puttick (University of Nottingham) and the Nottingham Innovative Manufacturing Research Centre for running some of the NMR samples and for discussions on the NMR spectra. The author also thanks Dr Tegene Desalegn (Addis Ababa University) for supplying the vernonia oil. For funding, the author thanks the University of Nottingham. References A. Adhvaryu, S. Z., Erhan 00. Epoxidized soybean oil as a potential source of high-temperature lubricants.industrial Crops and Products 5: A. Cunningham, A., Yapp 974. U.S. Patent 3,87,993: Liquid polyol compositions. USA. A.E. Gerbase, C. L., Petzhold, A.P.O Costa (00). Dynamic mechanical and thermal behavior of epoxy resins based on soybean oil. Journal of American Oil and Chemists Society 79(8): A.K. Mohanty, M., Misra, L.T., Drzal 005. Natural Fibers, Biopolymers, and Biocomposites, Taylor and Francis. Ahamed, S., et.al Dynamic mechanical characterization of soy based epoxy resin system. Proceedings of the SAMPE conference. Long Beach, CA. C.F. Krewson, G. R., Riser, W.E. Scott 966. Euphorbia and Vernonia seed oil products as plasticizer-stabilizers for polyvinyl chloride. Journal of American Oil and Chemists Society 43(6): C.G. Force, F. S., Starr 988. U.S. Patent 4,740,367: Vegetable oil adducts as emollients in skin and hair care products. Chielline, E., et.al 000. Biorelated polymers: sustainable polymer science and technology, Kluwer Academic / Plenum Publishers. Christie, W. W "GAS CHROMATOGRAPHY AND LIPIDS: PART : THE ANALYSIS OF FATTY ACIDS. R e t r i e v e d 8 / 0 / 0 0, 0 0, f r o m Chum, H. L. 99. Polymers from Biobased Materials, Noyes Data Corporation. D.J. Trecker, G. W., Borden, O.W. Smith 976. U.S. Patent 3, 9 3, : A c r y l a t e d e p o x i d i z e d s y b e a n o i l a m i n e compositions and method. D.J. Trecker, G. W., Borden, O.W. Smith 976. US Patent 3,979,70: Method for curing acrylated epoxidized soybean oil amine compositions. D.K. Salunkhe, J. K., Chavan, R.N. Adsule, S.S. Kadam 99. Worm Oilseeds: Chemistry, Technology, and Utilization. New York, Van Nostrand Reinhold. E. Can, S., Kusefoglu, R.P. Wool 00. Rigid, Thermosetting Liquid Molding Resins From Renewable Resources. I. Synthesis and Polymerization of Soy Oil Monoglyceride Maleates." Journal of Applied Polymer Science 8(69-77). E. T. A k i n t a y o, T., Z i e g l e r, A. O n i p e d e G a s chromatographic and spectroscopic analysis of epoxidised canola oil. Chemical Society of Ethiopia 0(): F.O. Ayorinde, B. D., Butler, M.T. Clayton 990. Vernonia galamensis: A rich source of epoxy acid. Journal of American Oil and Chemists Society 67(): G. Knothe, J. A., Kenar 004. Determination of the fatty acid profile by H-NMR spectroscopy. European Journal of Lipid Science and Technology 06: G.W. Bussell, G. W. (974). U.S. Patent 3,855,63: Maleinized fatty acid esters of 9-oxatetracyclo-4.4..,5O,6O8,0 undecan-4-ol. Gunstone, F. D The Study of Natural Epoxy Oils and 8 THE SCITECH JOURNAL VOLUME 0 ISSUE DECEMBER 04

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