Biomacromolecules 2000, 1, 746-751
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Solid State NMR for Determination of Degree of Acetylation of Chitin and Chitosan L. Heux,*,† J. Brugnerotto,† J. Desbrie` res,† M.-F. Versali,‡ and M. Rinaudo† Centre de Recherches sur les Macromole´ cules Ve´ ge´ tales (CNRS), Affiliated to Joseph Fourier University, BP 53, 38041 Grenoble Cedex 9, France; and Chitin-Chitosan Research Group, Department of Botany (B22), University of Lie` ge, Sart Tilman,B-4000 Lie` ge, Belgium Received July 17, 2000; Revised Manuscript Received September 15, 2000
The degree of acetylation (DA) of various samples has been evaluated by 1H liquid-state NMR and 13C and 15 N CP-MAS solid-state NMR over the whole range of DA. A good agreement has been found for all the experiments. The 13C and 15N CP-MAS experiments have permitted the evaluation of the chitin DA and content in the structural polysaccharides in a fungus named Aspergillus niger. The fungus structural carbohydrates mainly consist in pure chitin associated with glucans. Comparison of the 13C CP-MAS spectra with standard (1f3)-β-D-glucans strongly suggests that chitin and glucans are linked via covalent bonds. Introduction Chitin is one of the most important natural polymer constituting the shells of crustaceans and also the structure of many fungi. Chitin can be found with various average degree of acetylation (DA) ranging from the fully acetylated to the totally deacetylated products. With the higher degree of acetylation, this polymer is soluble in very few solvents, which limits its applications. But, after partial deacetylation, when DA is lower than 50%, it becomes water-soluble under aqueous acidic conditions; then it is called chitosan.1 The determination of the degree of acetylation remains up to now under discussion, but much work has been performed due to the large importance of this characteristic on the physical properties of the polymer. In addition, the determination of the chitin content in natural sources is important to evaluate a substrate in the objective to produce chitin. In particular, some fungi are known to contain chitin which may be exploited.2 Different techniques have been proposed to evaluate the average degree of acetylation of chitosans, including infrared,3-6 13C solid-state NMR,7,8 ultraviolet spectrometry,9 potentiometric titration,10,11 1H liquid-state NMR12-14 or elemental analysis.15 However, only infrared,3-6 13C solidstate NMR,7,8 and elemental analysis15 may be also used to determine the average degree of acetylation of chitins as these techniques do not require the solubilization of the polymer. However, some experimental difficulties arise in each technique. For instance, the deconvolution of the amide band (1655 cm-1) in the infrared spectrum is rather difficult and is described in a separate paper.3 Among these techniques, 13C solid-state NMR appears to be the most reliable for the evaluation of the acetyl content. The degree of acetylation (DA) is usually calculated by measuring the integral of the carbonyl or methyl group divided by the integral of all the * To whom all correspondence should be addressed. † Centre de Recherches sur les Macromole ´ cules Ve´ge´tales (CNRS). ‡ University of Lie ` ge.
carbon atoms in the backbone.8 As the chemical shifts of these groups are well separated, this measurement does not require any peak deconvolution. However, chitin is frequently associated with other polysaccharides like (1f3)-β-D-glucans, which makes the evaluation of the acetyl content more problematic and, in some cases, impossible. The need for a technique which can provide an evaluation of the acetyl content is obvious in the case of complex assembly of natural polysaccharides including chitin or chitin derivatives. In a recent communication,16 Yu et al. showed that the evaluation of the acetyl content is possible through the 15N CP-MAS NMR technique. As in polysaccharides the 15N nucleus is only present in chitin and chitosan, this technique appears very promising to evaluate the acetyl content, without any strong purification processes. The aim of this work is to compare systematically the DA evaluated from 1H liquidstate NMR and 13C and 15N solid-state NMR in the whole range of acetyl content from 0 to 100%. The two latter techniques will be used to measure the chitin proportion and its acetyl content in the structural polysaccharides of the Aspergillus niger fungus. Material and Methods Crab chitin in the R-form (sample A) was delivered by Pronova Biopolymer (Trondheim, Norway). A second sample with a high acetyl content, obtained by homogeneous reacetylation of chitosan, was provided by G. A. F. Roberts17 (sample B). The A. niger fungus was given by M. F. Versali (Gesval, Liege, Belgium). The two original chitosans (samples C and D) are commercial products from Pronova Biopolymer (Trondheim, Norway) and Aber Technologies (Plouvien, France), respectively. The study has been realized on purified chitosans. For their purification, the chitosan samples were first solubilized in acetic acid (0.5 M). The solutions were filtered through membranes respectively with 8, 3, 1.2, and 0.8 µm pore diameters and, then, neutralized with sodium hydroxide 10%
10.1021/bm000070y CCC: $19.00 © 2000 American Chemical Society Published on Web 11/10/2000
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Determination of Degree of Acetylation
(w/w) up to pH 8. The precipitates were then filtered and washed successively with water and ethanol/water mixtures from 70/30 to 100/0 (v/v). Finally, they were dried at ambient temperature. The chitin sample was used as received. The A. niger fungus was washed with two successive (1 × 0.1 M + 1 × 0.5 M) NaOH solutions during 2 h at 25 °C, then with chloroform, a chloroform/methanol mixture (2/1 (v/v)), dichloromethane, dimethyl sulfoxide, and water. These treatments are adapted to extract lipids, proteins, and free glucans. The residual yield after this treatment is 45% (w/ w). Sugar analysis concluded on the presence of only glucose and N-acetyl-D-glucosamine in the residual sample obtained after purification. Liquid-State NMR. The acetylation degree (DA) of chitosans was first determined by 1H NMR, considered to be the most sensitive technique,12 using an AC 300 Bruker spectrometer. The samples were dissolved at a concentration of 10 mg.mL-1 in deuterated water with HCl (pH 4) and freeze-dried three times to exchange labile protons by deuterium atoms. The spectra were performed at 353 K. The DA was determined, with a precision of 5%, from the integral of the CH3 signal at 1.97 ppm compared with the integral of H-1 protons considered as an internal standard. Solid-State NMR. The NMR experiments were performed on a Bruker MSL spectrometer operating at a 1H frequency of 200 MHz using the combined techniques of proton dipolar decoupling (DD), magic angle spinning (MAS), and crosspolarization (CP). Field strengths corresponding to 90° pulses of 4 and 7.5 µs were used for the matched spin-lock crosspolarization transfer for the 13C and 15N, respectively. The contact time was 1 ms, the acquisition time 70 ms, the sweep width 29 400 Hz, and the recycle delay 4 s for the 13C. A typical number of 10 000 scans were acquired for each spectrum. The chemical shifts were externally referred by setting the carbonyl resonance of glycine to 176.03 ppm. According to Yu et al.,16 the contact time was 2 ms, the acquisition time 20 ms, the sweep width 10 000 Hz and the recycle delay 1 s for the 15N. A typical number of 100 000 scans were acquired. The chemical shifts were externally referred to NH4+ of enriched ammonium nitrate. The spinning speed was set at 3000 Hz for all samples. Biochemical Analysis. Chitin content was enzymatically determined according to the method proposed by Jeuniaux18,19 in the insoluble material left after the successive extractions performed on A. niger mycelium. The residue was incubated with purified chitinase (Sigma 6137; 1 mg‚mL-1) at 37 °C during 24 h. The supernatant was incubated with β-Nacetylglucosaminidase (diluted lobster serum) at 37 °C during 2 h. The N-acetylglucosamine monomers liberated by the combined enzymatic treatments were then determined by the colorimetric method of Reissig et al.20 Results and Discussion Evaluation of DA by 13C CP-MAS NMR. Special care was taken for the quantitative analysis of the NMR measurements. As aforementioned, the DA was evaluated from the relative integrals of methyl or carbonyl groups compared to the carbon integrals of the polysaccharidic backbone. However, the kinetics of the cross-polarization process should
Figure 1. 13C CP-MAS NMR spectra of samples A-D (with decreasing DA). Table 1. Chemical Shifts of Chitin and Chitosan Obtained by CP-MAS
13C
sample CdO C1 C4 C5 C3 C6 C2 CH3
A
B
C
D
173.8 104.1 83.0 75.7 73.3 60.8 55.2 22.8
173.7 103.5 82.4 74.7 74.7 60.3 56.6 23.1
173.6 104.7 82.4 75.0 75.0 60.1 57.6 23.2
ND 104.7 85.7-81.0 74.1 74.1 60.7-59.6 56.8 ND
Table 2. Degree of Acetylation of Chitin and Chitosan Obtained by Liquid State (1H) and Solid State (13C and 15N) NMR sample A
B
C
D
1H
DA from NMR (liquid state) insoluble 0.58 0.21 acetyl traces DA from 13C NMR (solid state) 0.99 0.61 0.20 0 DA from 15N NMR (solid state) 1 0.63 0.20 0
not be the same for each group. The contact time usually used in CP-MAS is 1 ms. The rise of the magnetization with contact time has been followed for the highly acetylated chitin sample. The attribution of the peaks was done according to Tanner et al.21 It has been found that, after 1 ms, the carbons of the methyl and polysaccharidic backbone magnetization reach a value of 88% of the theoretical maximum magnetization M0, whereas the carbonyl only reach 84%. By considering the carbonyl peak, a systematic error of a least 4% could be done. In the following, we will only consider the DA measured by integration of the methyl peak. Figure 1 displays the 13C CP-MAS spectra of four samples from the highest (sample A) to the lowest (sample D) degree of acetylation. Corresponding chemical shifts and DA appear in Tables 1 and 2, respectively. Sample A corresponds to pure chitin, whereas sample D consists of pure chitosan at the sensibility of the experiment. A detection level of 5% of either chitin or chitosan seems reasonable. The line assignment and chemical shifts for those samples are those previously found for R-chitin21 and chitosan from crab or shrimp shell22 for samples A and D, respectively.
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Figure 2.
15N
Heux et al.
CP-MAS NMR spectra of samples A-D (with decreasing DA).
Table 3. Full Width at Half-Height (fwhh) in the NMR Spectra
15N
CP-MAS
sample fwhh amide (Hz) fwhh amine (Hz)
Figure 3.
13C
A
B
C
D
140 ND
131 115
266 131
ND 117
CP-MAS NMR spectra of (a) raw and (b) purified fungus.
The two intermediate degrees of acetylation (sample B and C) led to a combination of the pure product spectra. However, the characteristic doublet for the C-4 signal of the chitosan around 86 ppm appears only in the less acetylated product (sample D) and cannot be observed for samples B and C. Another doublet is observed for the C-6 signal of sample D.
Determination of Degree of Acetylation
Biomacromolecules, Vol. 1, No. 4, 2000 749
Figure 4.
15N
CP-MAS NMR spectra of purified fungus.
Figure 5.
13C
CP-MAS NMR spectra: (a) purified fungus; (b) difference between purified fungus and R chitin; (c) curdlan.
Evaluation of DA by 15N CP-MAS NMR. Figure 2 displays the 15N CP-MAS spectra obtained with the samples
used in the 13C experiments. Chitin and chitosan (samples A and D) display two unique peaks at respectively ca. 110
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ppm for the amide and ca. 10 ppm for the amine groups. Cross-polarization kinetics has been followed for the two peaks of sample B. This corresponds to the observation of Yu et al. on partially deacetylated chitin.16 For the two intermediate samples (B and C), the two peaks are present. The two groups share the same T1F(1H) of 5 ms. At a contact time of 2 ms, both groups reach 66% of the theoretical magnetization M0, so that the quantization by direct integration is reasonable. However, for sample C, the peak arising from the amide is very broad and weak. The full width at half-height (fwhh) is given in Table 3. The line broadening of the amido peak could come from the inhomogeneity arising from the heterogeneous deacetylation process. The weakness and broadness of this peak conjugated to the low signal-to-noise make the quantitative analysis of the DA delicate. However, reliable information can be obtained by fitting the lines by Gaussian functions. This strongly reduces the error arising from the poor signal-to-noise ratio. The results thus obtained are listed also in Table 2 together with DA obtained by the two other techniques. Although the confidence interval should not be greater than 5%, data from liquid-state 1H NMR and solid-state 13C and 15N CP-MAS are in good agreement in the whole range of DA. Additionally, due to the line broadening effect in 15N CP-MAS, one could not expect to detect an acetylation level lower than 10% with this technique. In that sense, solid-state 15N NMR is less sensitive than 13C. However, the line-widths in 15N should give a good indication of the crystallinity of the sample. Additionally, the lines are isolated and easier to treat in 15N NMR in the general case in which there is no other nitrogenated species. Application to the Characterization of A. niger Structural Polysaccharides. Like many other fungi, A. niger is a complex mixture of structural carbohydrates, proteins and lipids. The 13C CP-MAS spectrum shown in Figure 3a illustrates this complexity. These carbohydrates should consist in a mixing of (1f3)-β-D-glucans and chitin, more or less deacetylated.23 To remove the undesired signals arising from proteins and lipids, a mild treatment was adopted on the raw fungus as described in the Experimental Section. The final product is a white powder composed of the structural polysaccharides. The 13C CP-MAS spectrum of this product appears in Figure 3b. Although all lipids and proteins have been removed, the overall spectrum remains very complex. The two sharp lines at 174 and 23 ppm indicate that there is a large amount of chitin. However, due to the complexity of the spectrum, it is impossible to evaluate if this chitin is pure or partially deacetylated. A stronger purification process should alter the acetylation content of the native chitin. The 15N CP-MAS spectrum of this sample, shown in Figure 4, exhibits only one peak at ca. 110 ppm. This corresponds precisely to the chemical shift measured in the pure chitin sample. Moreover, no additional peak at 10 ppm is detected, which could arise from the amino group. This indicates that the chitin present in the fungus is not deacetylated (at the experimental sensibility). Then, we propose to subtract the contribution of the chitin (sample A) to the overall spectrum shown in Figure 5a. The
Heux et al.
result thus obtained is shown in Figure 5b together with the spectrum of curdlan, a standard (1f3)-β-D-glucan (Figure 5c). The chitin of the fungus should be slightly different from the reference as there is a small remaining methyl signal at 22 ppm. However, the result of the subtraction resembles qualitatively to the spectrum of curdlan in peak position and amplitude at the notable exception of the C-4 peak and an additional peak arising near 166 ppm in the carbonyl region. This strongly suggests that there is a covalent linkage between chitin and (1f3)-β-D-glucan through a carbonyl linkage to the C-4 of the (1f3)-β-D-glucan as was shown before.24,25 The comparison between the carbonyl resonances indicates that there is 1,2 linkage for every chitin carbonyl. Quantitatively, the analysis of the integral of each spectrum indicates an approximate chitin weight content of 25%, so a chitin weight content of about 11% in the original fungus. This result is in good agreement with the quantitative estimations of chitin content obtained by the enzymatic method. The analysis of the isolate after treatment of fungal mycelium revealed a chitin content of 22%, corresponding to a total chitin content of about 10% of cell wall dry weight. Conclusion In this study, 13C and 15N CP-MAS NMR and 1H liquidstate NMR of chitin and chitosan samples have been compared over the whole range of acetylation degree. The three methods are in good agreement. The limitation of solidstate NMR relies in the detection threshold not higher than 5%. Nevertheless, 15N CP-MAS is particularly a powerful technique to evaluate the acetyl content in the case of complex association of chitin and other polysaccharides. The high degree of acetylation of chitin has been proven in the case of the A. niger fungus, for which it is also proposed that chitin is in strong association with (1f3)-β-D-glucans. Combination with 13C CP-MAS NMR allows also the determination of the chitin content in the structural polysaccharides of the fungus. Acknowledgment. The authors thank Gesval S.A. (Lie`ge, Belgium) for their financial support. References and Notes (1) Rinaudo, M.; Milas, M.; Desbrie`res, J. In Applications of chitin and chitosan; Goosen, M. F. A., Ed.; Technomic Pub. Co. Inc.: Lancaster, PA, 1997; p 89. (2) Muzzarelli, R. A. A. Chitin; Pergamon Press Ltd.: Oxford, England, 1977. (3) Brugnerotto, J.; Lizadi, J.; Goyocoolea, F.; Argu¨elles, W.; Desbrie`res, J.; Rinaudo, M. Polymer, in press. (4) Miya, M.; Iwamoto, R.; Yoshikawa, S.; Mima, S. Int. J. Biol. Macromol. 1980, 2, 323. (5) Baxter, A.; Dillon, M.; Taylor, K. D.; Roberts, G. A. F. Int. J. Biol. Macromol. 1992, 14, 166. (6) Domszy, J. G.; Roberts, G. A. F. Makromol. Chem. 1985, 186, 1671. (7) Pelletier, A.; Lemire, I.; Sygusch, J.; Chornet, E.; Overend, R. P. Biotechnol. Bioeng. 1990, 36, 310. (8) Raymond, L.; Morin, F. G.; Marchessault, R. H. Carbohydr. Res. 1993, 246, 331. (9) Muzzarelli, R. A. A.; Rochetti, R. Carbohydr. Polym. 1985, 5, 461. (10) Broussignac, P. Chim. ind.-ge´ nie chim. 1968, 99, 1241. (11) Hayes, E. R.; Davies, O. H. In Proceedings of the First International Conference on Chitin/Chitosan; Skja¨k-Braek; Gadmiund, A., Anhronen, T., Sandford, B., Eds.; Elsevier: Amsterdam, 1978.
Determination of Degree of Acetylation (12) Rinaudo, M.; Le Dung, P.; Gey, C.; Milas, M. Int. J. Biol. Macromol. 1992, 14, 122. (13) Varum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smisrød, O. Carbohydr. Res. 1991, 211, 17. (14) Desbrie`res, J.; Martinez, C.; Rinaudo, M. Int. J. Biol. Macromol 1996, 19, 21. (15) Roberts, G. A. F. Chitin Chemistry; MacMillan Press: London 1992. (16) Yu, G.; Morin, F. G.; Nobes, G. A. R.; Marchessault, R. H. Macromolecules 1999, 32, 518. (17) Maghami, G. G.; Roberts, G. A. F. Makromol. Chem. 1988, 15, 281. (18) Jeuniaux, C. H. In Chitine et chitinolyse: un chapitre de biologie mole´ culaire; Masson: Paris, 1963, p 181.
Biomacromolecules, Vol. 1, No. 4, 2000 751 (19) Jeuniaux, C. H. Bull. Soc. Chim. Biol. 1965, 47, 2267. (20) Reissig, J.; Strominger, J.; Leloir, L. J. Biol. Chem. 1955, 217, 959. (21) Tanner, F. S.; Chanzy, H.; Vincendon, M.; Roux, J.-C.; Gaill, F. Macromolecules 1990, 23, 3576. (22) Saitoˆ, H.; Tabeta, R.; Ogawa, K. Macromolecules 1987, 20, 2424. (23) Hearn, V. M.; Sietsma, J. H. Microbiology 1994, 140, 789. (24) Sietsma, J. H.; Wessels, J. G. H. J. Gen. Microbiol. 1979, 114, 99. (25) Sietsma, J. H.; Wessels, J. G. H. J. Gen. Microbiol. 1981, 125, 209.
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