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Structural characterization of wild rubbers, microstructure of the rubber extracted from ficus elastica by 13C-NMR
Polymer Testing 19 (2000) 429–434
Material Characterisation
Structural characterization of wild rubbers, microstructure of the rubber extracted from ficus elastica by 13C-NMR Jean R.D. Marinhoa, Elisabeth E.C. Monteirob,* a
Department of Physics and Chemistry, UNESP-Ilha Solteira, PO Box 31, CEP 15385-000, Ilha Solteira, SP, Brazil Instituto de Macromole´culas Professora Eloisa Mano/UFRJ, PO Box 68525, CEP 21945-970, Rio de Janeiro, RJ, Brazil
b
Received 23 December 1998; accepted 18 February 1999
Abstract Low molecular weight fractions of polyisoprene extracted from Ficus elastica Hoxb. ex Hornem. were studied by 13C-NMR. The identification of 2–3 trans terminal units at the end of the polymer chain needed the acquisition of more than 17 000 transients. 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction Biochemical research suggests that the biosynthesis of polyisoprene structure occurs by continuous addition of the isopentenyl pyrophosphate to the dimethylallyl pyrophosphate [1–7]. Recent 13 C-NMR spectrometry studies have allowed the observation of the chemical shifts attributed to terminal units of polyisoprene obtained by extracting from natural sources. The description [8– 13] of the polyisoprene chain suggests that this type of macromolecule is composed of a dimethylallyl group (), two or three trans isoprenoid structures, a great sequence of cis isoprenoid structure and an ␣-terminal hydroxylated isoprenoid or ester group as shown in Fig. 1. The presence of trans groups was also detected in laticiferous mushrooms [14]. The biogenetic reaction of rubber is supposed to be composed of a four-step sequence [1]: the first step is the synthesis of mevalonate ion. The second consists of the formation of isopentenyl
* Corresponding author. e-mail: [email protected] 0142-9418/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 4 1 8 ( 9 9 ) 0 0 0 1 5 - X
430
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
Fig. 1. Polyprenoid structure proposed for the natural rubber [13].
pyrophosphate. Farnesyl or geranylgeranyl pyrophosphate is obtained in the third step and the rubber structure is complete at the fourth step. The polyisoprene studied in this work was extracted from Ficus elastica, which belongs to the Moraceae family and possesses a laticiferous system similar to Hevea brasiliensis that furnishes cis polyisoprene of high molecular weight [15].
2. Experimental The latex was collected from mature trees existing in the Department of Agronomy of the Faculty of Engineering of Ilha Solteira-UNESP, coagulated in saturated NaCl solution, washed and extracted for 12 h with acetone in a Soxhlet apparatus. The product was dried under vacuum and dissolved in CHCl3 at 3.5–5 w/v% and centrifuged for 10 min at 13 000 rpm using a MLW centrifuge, model K-24, operating at 5°C to remove impurities. The polymer was precipitated in methanol, stored under nitrogen and protected from light. The low molecular weight fractions were obtained by selective precipitation using hexane/isopropanol as solvent/non-solvent pair [16]. This procedure allowed 8–10 fractions to be obtained with a polydispersion between 1.4 and 2. The 13C-NMR spectra were registered using a Bruker AC-200 and followed the method described by Tanaka and co-workers [8,13]. The number-average molecular weight values were obtained by using a Toyo Soda HLC 803A chromatograph, operating with G 2000 HXL and G 4000 HLX columns of type TKS gel at 30°C and toluene as solvent (0.1% solution) and calibrated with polystyrene standards. The molecular weight values were corrected by the equation [17]: [KMa]PS = [KMa]PI where K and a are the viscometric constants [18] for polystyrene (PS) and polyisoprene (PI) in toluene at 30°C shown in Table 1.
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
431
Table 1 Viscometric constants K and a for polymers in toluene [18] at 30°C Polymer
K (ml/g) ⫻ 103
a
PS PI
9.2 15.0
0.72 0.74
3. Results and discussion The carbon atoms of the polyisoprene chain are numbered as follows.
Fig. 2 shows the expanded 13C-NMR spectra of two low molecular weight fractions of polyisop¯ n is 29 000. The terminal rene. Spectrum A corresponds to the fraction where the value of M groups of the polymer chain were assigned at 16.03 and 39.76 ppm and were attributed to the trans C-5 and trans C-1, respectively. Spectrum B shows the register obtained from the sample where the number-average molecular weight is 60 000. It can be noted that the spectrum exhibits the same peaks.
Fig. 2. 13C-NMR spectra of two fractions of polyisoprene extracted from Ficus elastica expanded in the region ¯ n ⫽ 60 000. ¯ n ⫽ 29 000; (B) M between 0 and 60 ppm. (A) M
432
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
Table 2 shows the chemical shifts observed in the spectra of polyisoprene extracted from Ficus elastica compared to the polymers obtained from the species Solidago altissima and Hevea brasiliensis and using the chemical shifts registered for ficaprenol-11 as a reference [8]. The values calculated by group contribution are also shown [19–21]. It can be noted that the data attributed to the trans groups are in good accordance. However, the signal corresponding to the group was not detected for F. elastica and S. altissima. The signal observed at 17.34 ppm in Fig. 2 cannot be attributed to a free C-5 atom. Recent studies on this matter indicate that the latex produced by far-reaching species like Hevea brasiliensis can present a protein soluble in the latex and a protein linked to the polyisoprene chain [22]. These proteins can be almost completely removed by deproteinization using an alkaline protease in the purification step [22,23]. The remaining peptide structure, assumed to be linked to the group [12], might promote a displacement of the corresponding NMR signal to another region of the spectrum. The absence of the signal attributed to the α terminal group similar to polyprenols indicates that the termination reaction of the polymer chain biosynthesis is more complex than the simple hydrolysis of the terminal pyrophosphate group [12]. Quantitative NMR data suggest the presence of 2–4 trans units in each polyisoprene molecule extracted from natural rubber [22]. These values were estimated from spectra similar to that depicted in Fig. 2, after discounting the Overhauser effect [10]. The relative intensities between the signals attributed to cis carbon atoms and trans C-1 were calculated and are shown in Table 3. The results obtained for the two fractions of polyisoprene suggest a good correlation between the values of molecular weight given by SEC and NMR assuming two trans units for each polyisoTable 2 13 C-NMR chemical shifts of polyisoprenes extracted from natural sources and calculated by group contribution [8,19–21] Type of carbon nucleus
Chemical shift (ppm) F. elastica
Trans C-5 C-5 Cis C-5 Trans C-4 C-1 Cis C-4 M from [M– C(⫽O)OR] Cis C-1 Trans C-1 ␣ C-4 [OH or M from M– OC(⫽O)R] a b c
Ficaprenol-11
S. altissima
H. brasiliensis
Calculated
16.03 a 23.42 c c 26.38 29.53
16.02 17.70 23.40 26.70 25.60 26.40 a
16.00 17.67 23.41 b b 26.43 29.60
16.02 a b b a b 29.50
15.90 18.00 23.40 26.80 24.90 26.40 29.70
32.18 39.76 a
32.29 39.76 58.80
32.23 39.74 59.00
b 39.80 a
32.20 39.80 60.50
Non-existent. Non-commented in the literature. Overlapped signal.
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434 Table 3 Molecular weights of the polyisoprene extracted from F. elastica obtained by SEC and Fraction
A
B
Type of carbon nucleus Trans C-1 Cis C-5 Cis C-4 Cis C-1 Trans C-1 Cis C-5 Cis C-4 Cis C-1
Intensity
3.42 645.25 607.41 596.12 6.49 1330.32 1364.12 1453.43
433
13
C-NMR
¯n Relative Intensity* Molecular weight, M
3 (2)** 593 (396) 500 (333) 504 (336) 3** 647 593.5 649
By SEC
By NMR
29 000
36 000 (24 000)
60 000
43 000
* Values obtained after discounting the Overhauser effect. ** Relative intensities considering three trans groups at the end of the chain; values in parentheses consider two trans groups.
prene chain, where farnesyl pyrophosphate was considered as the initiating species. The high molecular weight fraction exhibits a larger difference that can be attributed to sample degradation or to differences that can arise on correlating the molecular weight and hydrodynamic volume, as the molecular weight becomes high. The presence of only two or three trans units per polyisoprene chain is not evident and still remains a matter of discussion. It is possible that both occur in different amounts and the possibility of the geranylgeranyl pyrophosphate (responsible for the presence of three trans units) acting as initiating species together with farnesyl pyrophosphate is not discharged. The existence of four trans units in the polyisoprene chain can be considered improbable because it arises from the geranylfarnesyl pyrophosphate acting as initiating species. The occurrence of this mechanism is very rare in nature [8] while the biosynthesis processes where farnesol and geranylgeraniol structures participate are very common. 4. Conclusion The NMR data of low molecular weight fractions of the polyisoprene extracted from F. elastica showed the presence of 2–3 trans units at the end of the polymer chain indicating farnesyl and geranylgeranyl pyrophosphate as probable initiating species. Signals attributed to and ␣ terminal groups were not detected. Acknowledgements The authors would like to thank to CNPq, FINEP, FAPESP and FUNDUNESP for financial support; Prof. Yasuyuki Tanaka for his suggestions; Prof. Marı´lia P. de Noronha for her help in the botanical systematic; and the Chemistry Department of USP-Sa¯o Carlos for the 13C-NMR spectra.
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J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
[19] [20] [21] [22] [23]
Johnston JA, Racusen DW, Bonner J. Proc Natl Acad Sci 1954;40:1031. Fournier P, Tuong CC. Rubber Chem Technol 1961;34:1229. Archer BL, Audley BG, Cockbain EG, McSweeney GP. Biochem J 1963;89:565. Archer BL, Audley BG. Adv Enzymol 1963;21:221. Lynen F. J Rubb Res Inst Malaya 1969;21:389. Benedict CR. In: Porter JW, Spurgeon SL, editors. Biosynthesis of isoprenoid compounds, vol. 2. New York: John Wiley & Sons, Inc., 1983. p. 355. Archer BL, Audley BG. Bot J Linnean Soc 1987;94:181. Tanaka Y, Sato H, Kageyu A. Rubber Chem Technol 1983;56:299. Tanaka Y. Proceedings of the International Rubber Conference, Kyoto, 1985. p. 141. Tanaka Y. Prog Polym Sci 1989;14:339. Tanaka Y. J Appl Polym Sci Appl Polym Symp 1989;44:1. Tanaka Y. Proceedings of the International Rubber Conference, Kobe, 1995. p. 27. Tanaka Y, Sato H, Kageyu A. Polymer 1982;23:1087. Tanaka Y, Mori M, Ute K, Hatada K. Rubber Chem Technol 1990;63:1. ´ teis do Brasil. Rio de Janeiro: Servic¸o de Informac¸a˜o Agrı´cola do Ministe´rio Correˆa MP. Diciona´rio de Plantas U da Agricultura, 1981. Elias HG. Theta solvents. In: Brandrup J, Immergut EH, editors. Polymer handbook, 3rd ed. New York: John Wiley & Sons, Inc., 1989. p. VII-211. Ouano AC, Barrall HEM, Johnston JF. Gel permeation chromatography. In: Slader Jr PE, editor. Polymer molecular weights—part II. 1975. p. 306 [Chapter 6]. Kurata M, Dsunashima Y. Viscosity–molecular weight relationships and unperturbed dimensions of linear chain molecules. In: Brandrup J, Immergut EH, editors. Polymer handbook, 3rd ed. New York: John Wiley & Sons, Inc., 1989. p. VII-5/VII-15. Levy GC, Nelson GL. Carbon-13 nuclear magnetic resonance for organic chemists. New York: Wiley & Sons, Inc., 1972. p. 5 [Chapter 3]. Stothers JB. Carbon-13 NMR spectroscopy. New York: Academic Press, 1972. p. 57–59. Beebe DH. Polymer 1978;19:231. Eng A-H, Tanaka Y. Trends in Polym Sci 1993;3:493. Eng A-H, Ejiri S, Kawahara S, Tanaka Y. J Appl Polym Sci Appl Polym Symp 1994;53:5.
Material Characterisation
Structural characterization of wild rubbers, microstructure of the rubber extracted from ficus elastica by 13C-NMR Jean R.D. Marinhoa, Elisabeth E.C. Monteirob,* a
Department of Physics and Chemistry, UNESP-Ilha Solteira, PO Box 31, CEP 15385-000, Ilha Solteira, SP, Brazil Instituto de Macromole´culas Professora Eloisa Mano/UFRJ, PO Box 68525, CEP 21945-970, Rio de Janeiro, RJ, Brazil
b
Received 23 December 1998; accepted 18 February 1999
Abstract Low molecular weight fractions of polyisoprene extracted from Ficus elastica Hoxb. ex Hornem. were studied by 13C-NMR. The identification of 2–3 trans terminal units at the end of the polymer chain needed the acquisition of more than 17 000 transients. 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction Biochemical research suggests that the biosynthesis of polyisoprene structure occurs by continuous addition of the isopentenyl pyrophosphate to the dimethylallyl pyrophosphate [1–7]. Recent 13 C-NMR spectrometry studies have allowed the observation of the chemical shifts attributed to terminal units of polyisoprene obtained by extracting from natural sources. The description [8– 13] of the polyisoprene chain suggests that this type of macromolecule is composed of a dimethylallyl group (), two or three trans isoprenoid structures, a great sequence of cis isoprenoid structure and an ␣-terminal hydroxylated isoprenoid or ester group as shown in Fig. 1. The presence of trans groups was also detected in laticiferous mushrooms [14]. The biogenetic reaction of rubber is supposed to be composed of a four-step sequence [1]: the first step is the synthesis of mevalonate ion. The second consists of the formation of isopentenyl
* Corresponding author. e-mail: [email protected] 0142-9418/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 4 1 8 ( 9 9 ) 0 0 0 1 5 - X
430
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
Fig. 1. Polyprenoid structure proposed for the natural rubber [13].
pyrophosphate. Farnesyl or geranylgeranyl pyrophosphate is obtained in the third step and the rubber structure is complete at the fourth step. The polyisoprene studied in this work was extracted from Ficus elastica, which belongs to the Moraceae family and possesses a laticiferous system similar to Hevea brasiliensis that furnishes cis polyisoprene of high molecular weight [15].
2. Experimental The latex was collected from mature trees existing in the Department of Agronomy of the Faculty of Engineering of Ilha Solteira-UNESP, coagulated in saturated NaCl solution, washed and extracted for 12 h with acetone in a Soxhlet apparatus. The product was dried under vacuum and dissolved in CHCl3 at 3.5–5 w/v% and centrifuged for 10 min at 13 000 rpm using a MLW centrifuge, model K-24, operating at 5°C to remove impurities. The polymer was precipitated in methanol, stored under nitrogen and protected from light. The low molecular weight fractions were obtained by selective precipitation using hexane/isopropanol as solvent/non-solvent pair [16]. This procedure allowed 8–10 fractions to be obtained with a polydispersion between 1.4 and 2. The 13C-NMR spectra were registered using a Bruker AC-200 and followed the method described by Tanaka and co-workers [8,13]. The number-average molecular weight values were obtained by using a Toyo Soda HLC 803A chromatograph, operating with G 2000 HXL and G 4000 HLX columns of type TKS gel at 30°C and toluene as solvent (0.1% solution) and calibrated with polystyrene standards. The molecular weight values were corrected by the equation [17]: [KMa]PS = [KMa]PI where K and a are the viscometric constants [18] for polystyrene (PS) and polyisoprene (PI) in toluene at 30°C shown in Table 1.
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
431
Table 1 Viscometric constants K and a for polymers in toluene [18] at 30°C Polymer
K (ml/g) ⫻ 103
a
PS PI
9.2 15.0
0.72 0.74
3. Results and discussion The carbon atoms of the polyisoprene chain are numbered as follows.
Fig. 2 shows the expanded 13C-NMR spectra of two low molecular weight fractions of polyisop¯ n is 29 000. The terminal rene. Spectrum A corresponds to the fraction where the value of M groups of the polymer chain were assigned at 16.03 and 39.76 ppm and were attributed to the trans C-5 and trans C-1, respectively. Spectrum B shows the register obtained from the sample where the number-average molecular weight is 60 000. It can be noted that the spectrum exhibits the same peaks.
Fig. 2. 13C-NMR spectra of two fractions of polyisoprene extracted from Ficus elastica expanded in the region ¯ n ⫽ 60 000. ¯ n ⫽ 29 000; (B) M between 0 and 60 ppm. (A) M
432
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
Table 2 shows the chemical shifts observed in the spectra of polyisoprene extracted from Ficus elastica compared to the polymers obtained from the species Solidago altissima and Hevea brasiliensis and using the chemical shifts registered for ficaprenol-11 as a reference [8]. The values calculated by group contribution are also shown [19–21]. It can be noted that the data attributed to the trans groups are in good accordance. However, the signal corresponding to the group was not detected for F. elastica and S. altissima. The signal observed at 17.34 ppm in Fig. 2 cannot be attributed to a free C-5 atom. Recent studies on this matter indicate that the latex produced by far-reaching species like Hevea brasiliensis can present a protein soluble in the latex and a protein linked to the polyisoprene chain [22]. These proteins can be almost completely removed by deproteinization using an alkaline protease in the purification step [22,23]. The remaining peptide structure, assumed to be linked to the group [12], might promote a displacement of the corresponding NMR signal to another region of the spectrum. The absence of the signal attributed to the α terminal group similar to polyprenols indicates that the termination reaction of the polymer chain biosynthesis is more complex than the simple hydrolysis of the terminal pyrophosphate group [12]. Quantitative NMR data suggest the presence of 2–4 trans units in each polyisoprene molecule extracted from natural rubber [22]. These values were estimated from spectra similar to that depicted in Fig. 2, after discounting the Overhauser effect [10]. The relative intensities between the signals attributed to cis carbon atoms and trans C-1 were calculated and are shown in Table 3. The results obtained for the two fractions of polyisoprene suggest a good correlation between the values of molecular weight given by SEC and NMR assuming two trans units for each polyisoTable 2 13 C-NMR chemical shifts of polyisoprenes extracted from natural sources and calculated by group contribution [8,19–21] Type of carbon nucleus
Chemical shift (ppm) F. elastica
Trans C-5 C-5 Cis C-5 Trans C-4 C-1 Cis C-4 M from [M– C(⫽O)OR] Cis C-1 Trans C-1 ␣ C-4 [OH or M from M– OC(⫽O)R] a b c
Ficaprenol-11
S. altissima
H. brasiliensis
Calculated
16.03 a 23.42 c c 26.38 29.53
16.02 17.70 23.40 26.70 25.60 26.40 a
16.00 17.67 23.41 b b 26.43 29.60
16.02 a b b a b 29.50
15.90 18.00 23.40 26.80 24.90 26.40 29.70
32.18 39.76 a
32.29 39.76 58.80
32.23 39.74 59.00
b 39.80 a
32.20 39.80 60.50
Non-existent. Non-commented in the literature. Overlapped signal.
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434 Table 3 Molecular weights of the polyisoprene extracted from F. elastica obtained by SEC and Fraction
A
B
Type of carbon nucleus Trans C-1 Cis C-5 Cis C-4 Cis C-1 Trans C-1 Cis C-5 Cis C-4 Cis C-1
Intensity
3.42 645.25 607.41 596.12 6.49 1330.32 1364.12 1453.43
433
13
C-NMR
¯n Relative Intensity* Molecular weight, M
3 (2)** 593 (396) 500 (333) 504 (336) 3** 647 593.5 649
By SEC
By NMR
29 000
36 000 (24 000)
60 000
43 000
* Values obtained after discounting the Overhauser effect. ** Relative intensities considering three trans groups at the end of the chain; values in parentheses consider two trans groups.
prene chain, where farnesyl pyrophosphate was considered as the initiating species. The high molecular weight fraction exhibits a larger difference that can be attributed to sample degradation or to differences that can arise on correlating the molecular weight and hydrodynamic volume, as the molecular weight becomes high. The presence of only two or three trans units per polyisoprene chain is not evident and still remains a matter of discussion. It is possible that both occur in different amounts and the possibility of the geranylgeranyl pyrophosphate (responsible for the presence of three trans units) acting as initiating species together with farnesyl pyrophosphate is not discharged. The existence of four trans units in the polyisoprene chain can be considered improbable because it arises from the geranylfarnesyl pyrophosphate acting as initiating species. The occurrence of this mechanism is very rare in nature [8] while the biosynthesis processes where farnesol and geranylgeraniol structures participate are very common. 4. Conclusion The NMR data of low molecular weight fractions of the polyisoprene extracted from F. elastica showed the presence of 2–3 trans units at the end of the polymer chain indicating farnesyl and geranylgeranyl pyrophosphate as probable initiating species. Signals attributed to and ␣ terminal groups were not detected. Acknowledgements The authors would like to thank to CNPq, FINEP, FAPESP and FUNDUNESP for financial support; Prof. Yasuyuki Tanaka for his suggestions; Prof. Marı´lia P. de Noronha for her help in the botanical systematic; and the Chemistry Department of USP-Sa¯o Carlos for the 13C-NMR spectra.
434
J.R.D. Marinho, E.E.C. Monteiro / Polymer Testing 19 (2000) 429–434
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
[19] [20] [21] [22] [23]
Johnston JA, Racusen DW, Bonner J. Proc Natl Acad Sci 1954;40:1031. Fournier P, Tuong CC. Rubber Chem Technol 1961;34:1229. Archer BL, Audley BG, Cockbain EG, McSweeney GP. Biochem J 1963;89:565. Archer BL, Audley BG. Adv Enzymol 1963;21:221. Lynen F. J Rubb Res Inst Malaya 1969;21:389. Benedict CR. In: Porter JW, Spurgeon SL, editors. Biosynthesis of isoprenoid compounds, vol. 2. New York: John Wiley & Sons, Inc., 1983. p. 355. Archer BL, Audley BG. Bot J Linnean Soc 1987;94:181. Tanaka Y, Sato H, Kageyu A. Rubber Chem Technol 1983;56:299. Tanaka Y. Proceedings of the International Rubber Conference, Kyoto, 1985. p. 141. Tanaka Y. Prog Polym Sci 1989;14:339. Tanaka Y. J Appl Polym Sci Appl Polym Symp 1989;44:1. Tanaka Y. Proceedings of the International Rubber Conference, Kobe, 1995. p. 27. Tanaka Y, Sato H, Kageyu A. Polymer 1982;23:1087. Tanaka Y, Mori M, Ute K, Hatada K. Rubber Chem Technol 1990;63:1. ´ teis do Brasil. Rio de Janeiro: Servic¸o de Informac¸a˜o Agrı´cola do Ministe´rio Correˆa MP. Diciona´rio de Plantas U da Agricultura, 1981. Elias HG. Theta solvents. In: Brandrup J, Immergut EH, editors. Polymer handbook, 3rd ed. New York: John Wiley & Sons, Inc., 1989. p. VII-211. Ouano AC, Barrall HEM, Johnston JF. Gel permeation chromatography. In: Slader Jr PE, editor. Polymer molecular weights—part II. 1975. p. 306 [Chapter 6]. Kurata M, Dsunashima Y. Viscosity–molecular weight relationships and unperturbed dimensions of linear chain molecules. In: Brandrup J, Immergut EH, editors. Polymer handbook, 3rd ed. New York: John Wiley & Sons, Inc., 1989. p. VII-5/VII-15. Levy GC, Nelson GL. Carbon-13 nuclear magnetic resonance for organic chemists. New York: Wiley & Sons, Inc., 1972. p. 5 [Chapter 3]. Stothers JB. Carbon-13 NMR spectroscopy. New York: Academic Press, 1972. p. 57–59. Beebe DH. Polymer 1978;19:231. Eng A-H, Tanaka Y. Trends in Polym Sci 1993;3:493. Eng A-H, Ejiri S, Kawahara S, Tanaka Y. J Appl Polym Sci Appl Polym Symp 1994;53:5.