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Keigo Aoi, Akinori Takasu, and Masahiko Okada. Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya ...
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Anal. Chem. 1998, 70, 7-12

Determination of the Degree of Acetylation of Chitin/Chitosan by Pyrolysis-Gas Chromatography in the Presence of Oxalic Acid Hiroaki Sato, Shin-ichi Mizutani, and Shin Tsuge*

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-01, Japan Hajime Ohtani

Center for Integrated Research in Science and Engineering, Nagoya University, Nagoya 464-01, Japan Keigo Aoi, Akinori Takasu, and Masahiko Okada

Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-01, Japan Shiro Kobayashi,† Toshitsugu Kiyosada, and Shin-ichiro Shoda

Department of Materials Chemistry, Graduate School of Engineering, Tohoku University, Sendai 980-77, Japan

A new method to determine directly and rapidly the degree of acetylation of chitin/chitosan was developed based on reactive pyrolysis-gas chromatography in the presence of an oxalic acid aqueous solution. The degree of acetylation was precisely evaluated on the basis of peak intensities of the characteristic products such as acetonitrile, acetic acid, and acetamide originating from the N-acetyl group of N-acetyl-D-glucosamine units of chitin/ chitosan. The observed values were in good agreement with those obtained by 1H NMR and the other methods. Moreover, the proposed technique was applicable to any kinds of chitin/chitosan samples over the whole range of acetylation including insoluble chitin/chitosan and perfectly acetylated artificial chitin having higher crystallinity to which 1H NMR had been inapplicable. Chitin is a natural polysaccharide found particularly in the shell of crustaceans such as crab and shrimp, the cuticles of insects, and the cell walls of fungi. Because chitin is one of the most abundant biopolymers next to cellulose, much interest has been paid to its biomedical, biotechnological, and industrial applications.1 Chitin is substantially composed of 2-acetamide-2-deoxyD-glucopyranose (N-acetyl-D-glucosamine, GlcNAc) units linked by β-(1f4) linkage. Chitosan obtained from chitin mainly by N-deacetylation with an alkaline hydrolysis is chiefly composed of 2-amino-2-deoxy-D-glucopyranose (D-glucosamine, GlcN) units. Chitin and chitosan are, however, conceptual copolymers composed of GlcNAc and GlcN units. In these copolymers, the composition of GlcNAc units having an N-acetyl group is termed † Present address: Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan. (1) Chitin in Nature and Technology; Muzzarelli, R. A. A., Jeuniaux, C., Gooday, G. W., Eds.; Plenum Press: New York, 1986.

S0003-2700(97)00668-9 CCC: $14.00 Published on Web 01/01/1998

© 1997 American Chemical Society

degree of acetylation. It is well-known that the degree of acetylation of chitin/chitosan influences various properties such as solubility, chemical reactivity, and biodegradability. Recently, chitin/chitosan-based graft copolymers were synthesized, which enable one to make miscible blends with common synthetic polymers such as poly(vinyl chloride) and poly(vinyl alcohol).2-4 In our previous paper, it is clarified that not only the graft chain but also the chitin/chitosan main chain take part in the fairly denser intermolecular interaction with PVC in these chitin/ chitosan-based polymer hybrids.5 Thus, the degree of acetylation of the base chitin/chitosan should also affect miscibility and other properties of chitin/chitosan-based polymer hybrids. Determination of the degree of acetylation of chitin/chitosan has been attempted by various chemical analyses such as titrations,6-9 amino group determination with picric acid,10 and enzymolysis.11 However, these chemical analyses often require tedious sample preparation and/or measurement as well as a long measuring time and a large amount of the sample. Moreover, the applicability of most chemical methods is rather limited mainly due to highly deacetylated chitin/chitosan because of their high crystallinity and insolubility. Furthermore, observed results are not necessarily reliable mainly due to poor solubility and/or less chemical reactivity of chitin/chitosan. (2) Aoi, K.; Takasu, A.; Okada, M. Macromol. Chem. Phys. 1994, 195, 3835. (3) Aoi, K.; Takasu, A.; Okada, M. Macromol. Rapid Commun. 1995, 16, 53. (4) Aoi, K.; Takasu, A.; Okada, M. Macromol. Rapid Commun. 1995, 16, 757. (5) Sato, H.; Tsuge, S.; Ohtani, H.; Aoi, K.; Takasu, A.; Okada, M. Macromolecules 1997, 30, 4030. (6) Terayama, H. J. Polym. Sci. 1952, 8, 243. (7) Sannan, T.; Kurita, K.; Iwakura, Y. Makromol. Chem. 1976, 177, 3589. (8) Domard, A.; Rinaudo, M. Int. J. Biol. Macromol. 1983, 5, 49. (9) Raymond, L.; Morin, F. G.; Marchessault, R. H. Carbohydr. Res. 1993, 246, 331. (10) Neugebauer, W. A.; Neugebauer, E.; Brzezinski, R. Carbohydr. Res. 1989, 189, 363. (11) Nanjo, F.; Katsumi, R.; Sakai, K. Anal. Biochem. 1991, 193, 164.

Analytical Chemistry, Vol. 70, No. 1, January 1, 1998 7

Spectroscopic analyses such as infrared (IR),12-16 near-infrared (NIR),17 nuclear magnetic resonance (NMR),9,18-21 and ultraviolet (UV)22 spectroscopies are also widely used for determination of the degree of acetylation. However, for these spectroscopic analyses, both precision and accuracy of the resulting data are often unsatisfactory because of insufficient spectral resolution and less sensitivity. In addition, most of the spectroscopic methods are inapplicable to the highly acetylated insoluble chitin/chitosan. Chromatography combined with acid hydrolysis has also been applied to determine the degree of acetylation.23-25 These methods are commonly based on determining acetic acid liberated from chitin/chitosan by acid hydrolysis. Holan et al.23 and Stove and Velichkov24 proposed a method using gas chromatography (GC) for the determination of acetic acid liberated from N-acetyl groups of chitin/chitosan by acid hydrolysis with hydrochloric acid. Since the use of hydrochloric acid significantly reduces the lifetime of the GC column, Niola et al.25 proposed an alternative method using high-performance liquid chromatography (HPLC) to overcome the constraint of column longevity. In this method, acetic acid liberated from N-acetyl groups of chitin/chitosan in the presence of a mixture of oxalic acid and sulfuric acid was used as a key product to determine the degree of acetylation. Although both studies by use of GC and HPLC provided a new method applicable to the whole range of the degree of acetylation of the chitin/chitosan samples, these methods involved off-line sample digestion which needed at least 3 h or so prior to GC or HPLC separation. Moreover, these techniques required ∼10 mg of the samples which might be a constraint when it is to be applied to the chitin/chitosan in small biological samples such as microorganisms. Pyrolysis-gas chromatography (Py-GC) has also been applied to the determination of the degree of acetylation. Hayes et al.26,27 showed that the amine content of chitosan can be correlated with the ratio of the peak intensities of specific products such as acetic acid and various nitrogen-containing products obtained from GlcNAc and GlcN units. Although this method needed no tedious sample preparation and was applicable to the whole range of the degree of acetylation of the samples, well-defined chitin/chitosan standards are required for calibration. Furthermore, differences in their crystallinity or the distribution of GlcNAc units in the (12) Sannan, T.; Kurita, K.; Ogura, K.; Iwakura, Y. Polymer 1978, 19, 458. (13) Moore, G. K.; Roberts, G. A. F. Int. J. Biol. Macromol. 1980, 2, 115. (14) Miya, M.; Iwamoto, R.; Yoshikawa, S.; Mima, S. Int. J. Biol. Macromol. 1980, 2, 323. (15) Domszy, J. G.; Roberts, G. A. F. Makromol. Chem. 1985, 186, 1671. (16) Baxter, A.; Dillon, M.; Taylor, K. D. A.; Roberts, G. A. F. Int. J. Biol. Macromol. 1992, 14, 166. (17) Rathke, T. D.; Hudson, S. M. J. Polym. Sci.: Part A: Polym. Chem. 1993, 31, 749. (18) Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26, 87. (19) Va˚rum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smidsrød, O. Carbohydr. Res. 1991, 211, 17. (20) Va˚rum, K. M.; Anthonsen, M. W.; Grasdalen, H.; Smidsrød, O. Carbohydr. Res. 1991, 217, 19. (21) Pelletier, A.; Lemire, I.; Sygusch, J.; Chornet, E.; Overend, R. P. Biotech. Bioeng. 1990, 36, 310. (22) Aiba, S. Int. J. Biol. Macromol. 1986, 8, 173. (23) Holan, Z.; Votruba, J.; Vlasakova, V. J. Chromatogr. 1980, 190, 67. (24) Stoev, G.; Velichkov, A. J. Chromatogr. 1991, 538, 431. (25) Niola, F.; Basora, N.; Chornet, E.; Vidal, P. F. Carbohydr. Res. 1993, 238, 1. (26) Lal, G. S.; Hayes, E. R. J. Anal. Appl. Pyrolysis 1984, 6, 183. (27) Davies, D. H.; Hayes, E. R. In Methods in Enzymology; Wood, W. A., Kellogg, S. T., Eds.; Academic Press: San Diego, 1988; Vol. 161, Chapter 52, p 442.

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polymer sequence influenced the associated degradation mechanisms. Therefore, the yields of the specific products obtained from GlcNAc and GlcN units changed as a function not only of the composition but also of the sequence distribution. In order to improve the accuracy of the Py-GC method, a specific pyrolysis that yields characteristic products reflecting the N-acetyl group of the GlcNAc units selectively and quantitatively has to be developed. Here, reactive Py-GC in the presence of some specific reagents might provide a key to solve this problem. It has been shown that reactive Py-GC of condensation polymers in the presence of an organic alkali such as tetramethylammonium hydroxide (TMAH) results in a simplified pyrogram which consists of characteristic peaks of methyl derivatives reflecting the components of the original polymers.28,29 This technique has successfully been applied to structural characterization of various synthetic polymers,30-32 rosin sizing agent in paper,33 and lipids in zooplanktons.34 Another reactive pyrolysis in the presence of inorganic solid acids such as cobalt sulfate has been successfully applied to the sequential analyses of copolymer polyacetals.35 In this work, reactive Py-GC in the presence of oxalic acid, which was used in the previous HPLC work as a constituent of hydrolysis reagents for chitin/chitosan in solution,25 was investigated to develop a new, direct, and rapid method to determine the degree of acetylation of chitin/chitosan over the whole range of acetylation. EXPERIMENTAL SECTION Samples. Various chitin/chitosan samples (CH-1-CH-8) with different degrees of acetylation were used in this study. Most of these samples were prepared through deacetylation of naturally available chitin/chitosan to different degrees. Among these, CH1-CH-4 are commercially available deacetylated chitin/chitosan samples, CH-1, CH-2, and CH-4 were purchased from Katokichi Co., and CH-3 was from Katakura Chikkarin Co. of which degree of acetylation determined by colloidal titration was reported to be 20%. Partially deacetylated chitin/chitosan samples CH-5 and CH-6, prepared from natural chitin (CH-7) purchased from Sigma Chemical Co., were obtained by the method of Sannan et al.12 The “pure” chitin (CH-8) was synthesized by the ring-opening polyaddition of a di-N,N′-acetylchitobiose oxazoline derivative promoted by a hydrolysis enzyme of chitin, chitonase.36 Its highly stereoregular polysaccharide structure having a β-(1f4) linkage and perfect N-acetylation was characterized by CP/MAS 13C NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) measurements. N-acetyl-D-glucosamine, the monomer of chitin, purchased from Sigma Chemical Co., was also used as a reference sample. All of the samples were desiccated for 12 h at room temperature under reduced pressure. (28) Challinor, J. M. J. Anal. Appl. Pyrolysis 1989, 16, 323. (29) Challinor, J. M. J. Anal. Appl. Pyrolysis 1991, 20, 15. (30) Ohtani, H.; Fujii, R.; Tsuge, S. J. High Resolut. Chromatogr. 1991, 14, 388. (31) Ito, Y.; Ogasawara, H.; Ishida, Y.; Tsuge, S.; Ohtani, H. Polym. J. 1996, 28, 1090. (32) Ishida, Y.; Tsuge, S.; Ohtani, H.; Inokuchi, F.; Fujii, Y.; Suetomo, S. Anal. Sci. 1996, 12, 835. (33) Ishida, Y.; Ohtani, H.; Kato, T.; Tsuge, S.; Yano, T. Tappi J. 1994, 77, 177. (34) Ishida, Y.; Isomura, S.; Tsuge, S.; Ohtani, H.; Sekino, T.; Nakanishi, M.; Kimoto, T. Analyst 1996, 121, 853. (35) Ishida, Y.; Ohtani, H.; Abe, K.; Tsuge, S.; Yamamoto, K.; Katoh, K. Macromolecules 1995, 28, 6528. (36) Kobayashi, S.; Kiyosada, T.; Shoda, S. J. Am. Chem. Soc. 1996, 118, 13113.

Reactive Py-GC Conditions. Basically the same measuring system for reactive Py-GC described in our previous papers30-35 was used in this system. A vertical microfurnace pyrolyzer (Frontier Lab, PY-2010D) was attached to a gas chromatograph (Shimadzu GC-17A) equipped with a flame ionization detector (FID). In the case of quantitative analyses, ∼50 µg of a given sample taken in a platinum sample cup was immersed in 3 µL of oxalic acid aqueous solution (1.0 M) and allowed to stand for 10 min. Then the sample cup was dropped into the center of the pyrolyzer heated at 450 °C under helium carrier gas. The flow rate of 50 mL/min of carrier gas at the pyrolyzer was reduced to 1.0 mL/min at the capillary column by means of a splitter. A metal capillary column (Frontier Lab, Ultra Alloy-CW; 0.25 mm i.d. × 30 m long) coated with immobilized poly(ethylene glycol) (PEG) (0.25 µm thickness) was used. The column temperature was initially set at 35 °C for 5 min and then heated to 220 °C at a rate of 5 °C/min. Identification of the peaks on resulting pyrograms was carried out by use of a GC/mass spectrometer (MS) system (JEOL AM-II 150) to which the pyrolyzer was also attached. NMR Measurements. The degree of acetylation for soluble chitin/chitosan samples (CH-1-CH-6) was also determined by 1H NMR using a Bruker ARX-400 spectrometer at 400 MHz under the following conditions: spectral width of 6410 Hz, acquisition time of 2.55 s, and 90° plus width of 7.2 µs. About 10 mg of a sample dissolved in ∼1 mL of D2O/CD3COOD (95:5 v/v) in a 5 mm sample tube at ambient temperature for overnight was measured at 85 °C. The deuterium resonance was used as a fieldfrequency lock, and the chemical shifts were referenced to internal sodium 2,2-dimethyl-2-silapentane-5-sulfonate. Peak assignments was carried out by referring to reports by Hirai et al.18 and Varum et al.19 as follows; σ (ppm) ) 2.05 (CH3CdO), 3.17 (H-2 of GlcN units), 3.53-3.94 (H-2 of GlcNAc units, and H-3-H-6 of pyranose ring), 4.60 (H-1 of GlcN), and 4.86-4.88 (H-1 of GlcNAc units). The degree of acetylation (DA, %) was evaluated by use of the following equation:

DA )

IH1′ + IAC/3 × 100 IH1 + IH2 + IH1′ + IAC/3

(1)

where IH1, IH1, IH2, and IAC are peak intensities for H-1 or GlcN unit at 4.60 ppm, for H-1 of GlcNAc unit at 4.86-4.88 ppm, for H-2 of GlcN unit at 3.17 ppm, and for the acetyl group of the GlcNAc unit at 2.05 ppm, respectively. The dividing factor of 3 for IAC is associated with the proton number for the acetyl group. Here, peak intensities at 3.53-3.94 ppm (H-2 of GlcNAc units, and H-3-H-6 of pyranose ring) were excluded for the determination due to less resolution and overlapping of the HOD signal at 4.05 ppm from the solvent. RESULTS AND DISCUSSION Determination of the Degree of Acetylation by Conventional Pyrolysis. Figure 1 shows typical pyrograms obtained from a chitin/chitosan sample (CH-1), the artificial chitin (CH8), and N-acetyl-D-glucosamine monomer at 550 °C under conventional pyrolysis without adding an oxalic acid solution. The assigned common peaks in these pyrograms are listed in Table 1, where the elemental composition of unknown peaks was estimated by referring the previous report by means of pyrolysis

Figure 1. Typical pyrograms for (a) chitin/chitosan CH-1, (b) artificial chitin CH-8, and (c) N-acetyl-D-glucosamine obtained at 550 °C. The numbers correspond to those of the compounds in Table 1. Table 1. Peak Assignment in the Pyrograms of Chitin and N-Acetyl-D-glucosamine Obtained by Conventional Pyrolysis at 550 °C peak no.

MW

structure

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

44 58 56 41 83 79 80 94 74 60 67 81 122 59 127 137 73 139 60 125

acetaldehyde propanol 2-propenal acetonitrile unknown pyridine pyrimidine methylpyrazine 1-hydroxy-2-propanone acetic acid pyrrole methylpyrroles 1-acetylpyrazine acetamide C6H9NO2 C7H7NO2 methylguanidine C7H9NO2 2-hydroxyacetaldehyde C6H7NO2

high-resolution field ionization mass spectrometry (Py-FIMS) by van der Kaaden et al.37 In the pyrogram of CH-1 (Figure 1a), in addition to characteristic products of the N-acetyl groups such as acetonitrile, acetic acid, and acetamide, various nitrogen-containing heterocyclic compounds such as pyridine, pyrimidine, pyrrole, and their derivatives are observed. The latter products would be formed mostly from the amino group of GlcN units through complicated inter- or intramolecular reactions. On the other hand, in the pyrogram of the artificial chitin, CH-8 (Figure 1b), fairly strong peaks characteristic of the N-acetyl groups are observed. (37) van der Kaaden, A.; Boon, J. J.; de Leeuw, J. W.; de Lange, F.; Schuyl, P. J. W.; Schulten, H.-R.; Bahr, U. Anal. Chem. 1984, 56, 2160.

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Table 2. Degree of Acetylation of Chitin/Chitosan Samples Determined by Conventional Py-GC Along with Those by 1H NMR Spectroscopy and by Other Methods degree of acetylation (%) sample code CH-1 CH-2 CH-3 CH-4 CH-5 CH-6 CH-7 CH-8

by

Py-GCa

13 ( 1 21 ( 1 24 ( 1 41 ( 1 41 ( 2 49 ( 4 75 ( 5 74 ( 5

by 1H NMR 10 16 20 37 44 51 c c

by other methods 20b

68d 100e

a For three measurements. b Determined by colloid titration offered from manufacturer. c Not applicable. d Determined by FT-IR using Sannan’s method.7 e Determined by CP/MAS 13C NMR (100 MHz).36

The pyrogram of CH-8 is similar to that of N-acetyl-D-glucosamine monomer (Figure 1c). Provided that the characteristic products (acetonitrile, acetic acid, acetamide) are quantitatively formed from both chitin/ chitosan and N-acetyl-D-glucosamine, their yields from chitin/ chitosan can be related with those from N-acetyl-D-glucosamine as follows:

ic/in )

mcDA/100

/

mn 221 203DA/100 + 161(100 - DA)/100

(2)

where DA (%) is the degree of acetylation of a given chitin/ chitosan sample, mc and mn are the sample weights for the chitin/ chitosan sample and N-acetyl-D-glucosamine monomer, respectively, ic and in are the sums of the intensities of the characteristic products (acetonitrile, acetic acid, acetoamide) from the chitin/ chitosan sample and N-acetyl-D-glucosamine, respectively, and 221, 203, and 161 are molecular weights of a monomeric N-acetyl-Dglucosamine, a GlcNAc repeating unit, and a GlcN unit, respectively. Here, it was confirmed that the relative molar sensitivities for FID of acetonitrile, acetic acid, and acetoamide were experimentally the same. Rearranging eq 2, the degree of acetylation can be expressed as

DA )

161Ic × 100 221In - 42Ic

(3)

where Ic ) ic/mc and In ) in/mn, respectively. Table 2 shows the calculated value of the degree of acetylation of each sample by conventional Py-GC together with those mainly by 1H NMR. For the deacetylated chitin/chitosan samples (CH-1-CH-6), the values obtained by conventional Py-GC are in good agreement with those obtained by 1H NMR. Furthermore, as for the insoluble chitin/chitosan sample (CH-7), which cannot be measured by 1H NMR, the value obtained by Py-GC is slightly higher than that estimated by IR using the Sannan method.7 However, for the perfectly acetylated artificial chitin sample (CH-8), the value obtained by Py-GC deviates unexpectedly. The observed low value of 74% suggests that the decom10

Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

Figure 2. Typical pyrograms for (a) artificial chitin CH-8 and (b) N-acetyl-D-glucosamine in the presence of oxalic acid obtained at 450 °C.

position efficiency and/or mechanisms of the highly acetylated chitin/chitosan might be different from those of its monomer. The reason may be mostly attributed to its high crystallinity. Actually as shown in Figure 1b for CH-8, the additional peaks appearing after 35 min, especially peaks 15, 16, and 18, assigned to the products originating from N-acetyl groups, suggested that more complicated degradation might occur for the Nacetyl group in CH-8 rather than simply to yield only acetonitrile, acetic acid, and acetamide. Furthermore, char residue remaining in the sample cup after conventional pyrolysis of the chitin/chitosan samples also suggested the less quantitative recoveries. Reactive Pyrolysis in the Presence of Oxalic Acid Aqueous Solution. In order to achieve more quantitative pyrolysis for chitin/chitosan, reactive pyrolysis in the presence of an oxalic acid aqueous solution as an acid hydrolysis reagent was examined rather than in the presence of inorganic acid such as hydrochloric acid or sulfuric acid, of which vapors or degradation products might damage the stationary phase of the separtion column. Figure 2 shows the pyrograms of (a) the artificial chitin (CH-8) and (b) N-acetyl-D-glucosamine observed by reactive pyrolysis in the presence of the oxalic acid aqueous solution at 450 °C. The simplified peak pattern on the pyrogram of the chitin sample (Figure 2a) is almost comparable to that of its monomer (Figure 2b). On these pyrograms, levoglucosenone (1,6-anhydro-3,4dideoxy-β-D-glycero-hex-3-enopyranose) is also observed as a common major product in addition to the characteristic products such as acetonitrile, acetic acid, and acetamide originating from the N-acetyl group. On the other hand, excess oxalic acid and its pyrolysates such as CO, CO2, H2O, and formic acid hardly interfere on the pyrograms due to their less or no sensitivity for FID. Furthermore, no residue was observed in the sample cup after the reactive pyrolysis of chitin/chitosan. Figure 3 shows a possible mechanism of the reactive pyrolysis of GlcNAc units in the presence of the oxalic acid aqueous solution. When the N-acetyl groups are subjected to hydrolytic pyrolysis, either acetic acid or acetamide would be liberated with remaining GlcN units or glucose units, respectively. Acetonitrile may be formed to some extent by the further thermal decomposition of acetamide. Glycoside bonds of the remaining polysaccha-

Table 3. Degree of Acetylation of Chitin/Chitosan Samples Determined by Py-GC in the Presence of Oxalic Acid Solution Along with Those by 1H NMR Spectroscopy and by Other Methods degree of acetylation (%) by PyGCa sample code

+ oxalic acid

- oxalic acid

by 1H NMR

CH-1 CH-2 CH-3 CH-4 CH-5 CH-6 CH-7 CH-8

11 ( 0.5 20 ( 1 22 ( 1 41 ( 1 46 ( 2 50 ( 4 72 ( 5 99 ( 5

13 ( 1 21 ( 1 24 ( 1 41 ( 1 41 ( 2 49 ( 4 75 ( 5 74 ( 5

10 16 20 37 44 51 c c

by other methods 20b

68d 100e

a For three measurements. b Determined by colloid titration offered from manufacturer. c Not applicable. d Determined by FT-IR using Sannan’s method.7 e Determined by CP/MAS 13C NMR (100 MHz).36

Figure 3. Possible mechanism of reactive pyrolysis of the Nacetyl-D-glucosamine unit in the presence of oxalic acid aqueous solution.

ride chain may then cleave in monosaccharides or their homologues by acidic catalysis. Levoglucosenone may be formed from the glucose moiety through dehydration reaction in the presence of the acid catalyst.38 Further degradation of the GlcN units might yield highly polar components which would be trapped as a tar at the tar trap in the glass insert tube of the GC injection port. Thus, it is apparent that the addition of oxalic acid affects not only the deacetylation from the N-acetyl group but also cleavage of glycoside bonds. Furthermore, no char residue after pyrolysis suggests that quantitative recovery of N-acetyl group might be achieved on the observed pyrogram. Optimization of Conditions for Reactive Pyrolysis. In order to achieve quantitative pyrolysis, the effects of the pyrolysis temperature, the concentration and the amount of reagent, and prereaction time on the peak intensities of the characteristic products in the pyrogram obtained by the reactive pyrolysis were examined. Here, optimization was carried out mostly using the N-acetyl-D-glucosamine monomer. First, the effect of pyrolysis temperature on the intensities of the characteristic peaks per unit sample weight in the pyrogram of N-acetyl-D-glucosamine observed in the presence of 3 µL of 1.0 M oxalic acid aqueous solution was examined. At temperatures below 400 °C, the peak intensities became relatively low due to incomplete pyrolysis. At higher temperatures, around 450 °C, the peak intensities became the strongest. At temperatures above 500 °C, the peak intensities of the characteristic products became (38) Shafizadeh, F.; Furneaux, R. H.; Stevenson, T. T. Carbohydr. Res. 1979, 71, 169.

fairly low, while additional peaks were produced mainly due to the competitive contribution of conventional pyrolysis. These results suggest that the characteristic products are most efficiently formed through reactive pyrolysis in the presence of oxalic acid aqueous solution at ∼450 °C. Therefore, reactive pyrolysis for the following determination was carried out at 450 °C. Concentration and amount of the reagent are also key factors in the reactive pyrolysis. Stoichiometrically, 50 µg of 100% acetylated chitin and N-acetyl-D-glucosamine monomer require 0.0045 µL of water for complete hydrolysis of amide bonds. In this case, oxalic acid may act as a catalyst. However, since a large excess amount of the oxalic acid aqueous solution is ordinary needed for quantitative achievement of the rapid reactive pyrolysis at high-temperatures, 3 µL of the reagent in the concentration range of 0.1-1.5 M were added to 50 µg of the sample. The effect of the concentration of the oxalic acid aqueous solution on the peak intensities per unit sample weight of the characteristic products in the pyrogram of N-acetyl-D-glucosamine pyrolyzed at 450 °C was also examined. The relative peak intensities increased with an increase in concentration, and became almost constant above 1.0 M. Therefore, 3 µL of 1.0 M oxalic acid aqueous solution was used for the following determination. Owing to the higher crystallinity of chitin/chitosan, sufficient prereaction time is needed for the quantitative achievement of the reactive pyrolysis. The influence of prereaction time at ambient temperature on the peak intensity per unit sample weight of the characteristic products in the pyrogram of N-acetyl-D-glucosamine was also examined at 450 °C in the presence of 3 µL of 1.0 M oxalic acid aqueous solution. The relative peak intensities increased with an increase in prereaction time, showed a maximum at 10 min, and then slightly decreased at 15 min due to vaporization of the water and/or reactants. Therefore the sample with reagent was allowed to stand for 10 min at the top of the pyrolyzer at ambient temperature and was then dropped into the furnace at 450 °C. Determination of the Degree of Acetylation by Reactive Pyrolysis in the Presence of Oxalic Acid Aqueous Solution. The degree of acetylation of the chitin/chitosan samples measured by reactive pyrolysis according to eq 3 is summarized in Table 3 Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

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together with those determined by 1H NMR and the other methods. The values over a wide range of acetylation from ∼10100% obtained by reactive Py-GC are in fairly good agreement with those obtained by the other methods. This fact suggests that the reactive pyrolysis can be applied for over the whole range of acetylation including insoluble chitin/chitosan and the perfectly acetylated artificial chitin having higher crystallinity. Furthermore, both accuracy and precision were able to be improved not only for lower acetylated chitin/chitosan samples but also for higher ones. This technique has various advantages in that the measurement is not only very rapid and simple but also very sensitive and that it is applicable to any degree of acetylation of chitin/ chitosan samples regardless of properties such as solubility and crystallinity.

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ACKNOWLEDGMENT Financial support by the Grant-in-Aid for Scientific Research (A) (07555262 and 09305056), (B) (09555262), and (C) (09650888) of the Ministry of Education, Science, Sports and Culture, Japan, and by a Grant from the “Research for the Future” Program of the Japan Society for the Promotion of Science (JSPS-RFTF, 96R11601) are gratefully acknowledged.

Received for review June 25, 1997. Accepted October 8, 1997.X AC9706685 X

Abstract published in Advance ACS Abstracts, November 15, 1997.