[acs symposium series] biopolymers volume 723 (utilizing nature's advanced materials) || alkali...

Chapter 7

Alkali Induced Polymorphic Changes of Chitin J. Li, J.-F. Revol, and R. H. Marchessault

Department of Chemistry, Pulp and Paper Research Center, McGill University, 3420 University Street, Montreal, Quebec H3A 2A7, Canada

By treatment with 50% NaOH and subsequent washing in water, α-chitin and ß-chitin undergo polymorphic transformations that have been followed by X-ray diffraction and solid state NMR. In both cases, during swelling in NaOH, the original lateral structure is destroyed and an alkali chitin complex is formed, in which the general orientation of the chitin chains remains parallel to the microfibrils axis. After washing in water, the alkali chitin from both polymorphs is converted to the α-chitin crystal structure, although the crystallinity remains poor. In the case of the ß-chitin polymorph, the original parallel arrangement of the chains is changed into an antiparallel arrangement during conversion to the α-form. A mechanism of polymorphic transformation involving chain interdigitation, similar to the one for the mercerization of cellulose, is also proposed. The classical deacetylation of α-chitin in the presence of 50% alkali to produce chitosan is a permutoid reaction where alkali saturated crystallites behave as individual reactors.

Chitin, a linear polysacharride consisting of N-acetyl-D-glucosamine, is widely distributed in nature, e.g., in the shells of crustaceans and insects, and in the cell wall of bacteria (/). Chemically, chitin is similar to cellulose, differing only in the fact that chitin has an aminoacetyl group instead of hydroxyl group at C-2 (2). In nature, chitin is biosynthesized by chitin synthetases with near simultaneous crystallization to form microfibrils (3,4). The polymorphic forms of chitin in nature is determined by chitin synthetases (5). Chitin with an antiparallel chain packing is referred to as a-chitin and is that found in crab, lobster and shrimp shells (1,6). Chitin with a parallel chain packing is referred to as p-chitin and occurs in squid pen orpogonophore tube (7,8).

In spite of the similarity in structure with cellulose, the chemical and physical properties of a-chitin are significantly different from those of cellulose. In particular, chitin is much less reactive to many chemicals due to the peptide-like hydrogen bonds between chains (6). Chitin is insoluble in most of the organic and inorganic solvents

88 © 1999 American Chemical Society

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In Biopolymers; Imam, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

89

known for cellulose (9). Many kinds of chitin derivatives, including benzyl chitin, carboxylmethyl chitin, etc., have been prepared via alkali chitin which is a soda-chitin complex (10,11). Preparation of alkali chitin was first reported in 1940 by Thor and Henderson, who steeped chitin in excess of 50% aqueous sodium hydroxide (12). Their results show that alkali chitin has similar properties to alkali cellulose in terms of reactivity with reagents of the type which react with alkali cellulose. However, compared with alkali cellulose, which has been extensively investigated, alkali chitin has received relatively little attention. It is generally accepted that native cellulose, cellulose I, which has a parallel chain structure, is converted to an antiparallel chain structure, cellulose II, after alkali treatment (13-19). It is expected that p-chitin, which also has a parallel packing, will behave in the same fashion. In the present study, x-ray diffraction, CP-MAS , 3 C NMR and polarized light microscopy have been used to investigate some structural features of alkali chitin and water regenerated chitin, which is referred to as "recovered chitin" in this paper. A comparison of crystalline order and polymorphism between untreated chitin and the recovered chitin was interpreted to provide further understanding in terms of the familiar mercerization process for native cellulose.

Experiment

Materials. Crab a-chitin, purchased from Fluka, was purified as previously described (20). Squid P-chitin was prepared from squid pen as described below. Approximately 100 g of dry squid pen was first pulverized in a Wiley mill to pass a 20 mesh sieve. Then, the ground squid pen was deproteinized by treating with 1 N NaOH for 1 hr at 100°C., followed by demineralization using 0.5 N HC1 at 100°C (1 hr). For both treatments, the ratio of solid to liquid was 1 g per 10 ml. Finally, the purified squid chitin was washed using deionized water and acetone, followed by air-drying in a fumehood.

Preparation of alkali-chitin. Alkali treated a-chitin was prepared by immersing 5 g of the purified chitin materials in a beaker with 25 ml of 10 N NaOH. To avoid deacetylation, this mixture was kept in a refrigerator at 4°C for 3 hr. For CP MAS , 3 C NMR measurements, 1 g of alkali treated a-chitin was pressed free of excess alkali solution with filter papers. The recovered chitin was obtained by repeatedly washing 1 g of alkali chitin with deionized water, followed by filtration until the filtrant reached neutrality. Subsequently, the wet sample was pressed free of excess water with filter paper, followed by air-drying in a fumehood. Squid pen P-chitin was alkali-treated as above, followed by washing-filtration cycles. Finally, the sample was dried by filter press, followed by air-drying in a fumehood. Samples of the alkali-treated chitins were examined after they had been pressed free of excess alkali.

X-ray diffraction. All X-ray powder diffractograms were recorded using a Siemens D-5000 diffractometer with a Cu K a (A.=1.5A) radiation source equipped with a scintillator counter and a linear amplifier. A typical scan range is from 0 to 50° (26).

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CP-MAS ! 3 C NMR. All CP-MAS 1 3C NMR spectra of chitin samples were recorded using cross polarization magic angle spinning at 75 MHZ on a Chemagnetics CMX-300 spectrometer. Powder samples of chitin were first inserted into a 7.5 zirconia rotor. A contact time of 2 ms and recycle delay of 2 s were employed. A magic angle spinning rate of 5 kHz was used.

Polarized Light Microscopy. Alkali chitin samples were transferred to glass slides and samples were examined using a Nikon Microphot-FXA polarized optical microscope. Micrographs were recorded with the sample between crossed polars.

Results and Discussion

The X-ray powder diffractograms of original and alkali-treated a-chitin samples are shown in Figure 1. The X-ray diffractogram of the original chitin shows a typical a-chitin pattern (see Figure la) in keeping with the CarlstrSm unit cell (6). Figure lb shows that alkali chitin is noncrystalline with only a small trace of the a-chitin diffraction. When alkali chitin is neutralized by washing with water, it regains the appearance of the original a-chitin. The X-ray diffractogram of the recovered chitin shows the diffraction pattern of a-chitin, although a general line broadening and amorphous background is observed compared with the original chitin (see Figure 3a). This is due to poor lateral ordering in the recovered chitin. These results, coupled with observations with the polarizing microscope, demonstrate that immersion of chitin in a concentrated aqueous alkali solution results in swelling and the destruction of the original lateral order. Alkali ions penetrate into the crystalline lattice and break the hydrogen bonds between polymer chains. Once hydrogen bonds are broken, the polymer chains are pushed apart by water and alkali. Physically, the crystallites are swollen and, according to Thor and Henderson (72), chitin forms complexes with alkali in which an alkali chitin could contain approximately 0.75 equivalents of combined sodium, or more, per acetyl hexosamine unit, depending on the ratio of alkali to chitin and the treatment time. By comparison, the alkali cellulose complexes contain more sodium (16) and, contrary to the alkali chitin, they exhibit relatively well defined crystalline structures (16,19). However, there is still a long range order existing in the alkali chitin samples as demonstrated by the observation of a strong birefringence (not shown here), which indicates a parallel arrangement of the molecular chains. When the alkali ions in the latices are washed away, the aligned polymer chains spontaneously reorder and interchain hydrogen bonds reform. Thus, the polymer chains are reorganized into the original crystalline packing, although not as perfectly as for native a-chitin. Because this reorganization process of microfibrils is fast, some disorder or defects are unavoidable.

The results from a CP-MAS , 3 C NMR study are consistent with those from X-ray diffraction. In particular, the spectrum of the recovered chitin is typical of the original a polymorph (see Figure 2b) (7,27). Typically, the spectrum consists of eight well defined resonances which are assigned to carbonyl groups (-174 ppm), methyl groups (-23 ppm) and six glucose carbons. The detailed assignments are given in Tanner's paper (7). For the alkali complex, the degree of order shown by the linewidth

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| ' i i i | i i i i | » i i i | i i i i | i i i i | i i i i | i i i i | i i i i | i i i i

5 10 15 20 25 30 35 40 45

Figure 1. X-ray diffractograms of native crab a-chitin (antiparallel chains) (a) and alkali treated crab chitin (b).

P P M I . I . I i I . I 1

200.00 150.00 100.00 50.00 -0.00

Figure 2. Solid state NMR spectra (CP-MAS l 3 C NMR) of the alkali complex from a-chitin (a) and the recovered chitin (b).

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of signals is poor (cf, Figure 2a). This confirms that a well-defined alkali chitin crystalline structure has not been achieved, but the chemical shift of the partially merged peaks leaves no doubt that an alkali chitin complex has formed.

The X-ray powder diffractograms of the squid P-chitin (not shown) indicate that p-chitin is, in general, less ordered than a-chitin. The diffraction pattern of the recovered chitin from the P-form (squid chitin) is shown in Figure 3b. It exhibits a very poor crystallinity, but the characteristics of the a-chitin can be identified and no trace of p-chitin can be detected. The CP/MAS 1 3C NMR spectra of the original squid p-chitin and corresponding recovered chitin are shown in Figure 4. They confirm that the recovered chitin from the P-form contains a larger amount of disordered material than the recovered chitin from the a-form (see Figure 2b). The typical splitting of the C3-C5 resonances, which is clearly observed in Figure 2b, is only marginally evident in Figure 4a, probably due to the lack of crystallinity. Similar behaviour was observed for the recovered material from squid P-chitin after a solid state conversion into the a-polymorph by treatment in 6N HC1 (7).

The fact that only a very poor crystallinity is recovered during the solid state conversion from the P-form into the a-form is an indication that the recrystallization is inhibited. On the other hand, when alkali chitin from the a-form is washed in H 20, the return to the a-chitin crystalline structure is obviously far easier, although still not perfect (see Figure 3a). This behavior may be the direct consequence of the different chain polarity in the two polymorphs.

It is generally admitted that, in p-chitin, each microfibril contains chains of the same polarity (parallel chains) (#), whereas a-chitin contains chains of alternating polarity (antiparallel chains) (6). A reversal of the chain polarity must occur during the polymorphic transformation. When solid chitin is recovered from solutions as shown by Focher et al. (27), the reversal of the chains to form the a-structure is readily obtained by crystallization into this more favorable packing. On the other hand, during the solid state conversion, even though chitin is swollen, the molecules are not free to rotate as in solution and the conversion is severely restricted. In 1969, Rudall (22) proposed as possible mechanisms that the parallel piles of chitin chains, which are present in the P-form, separate in alkali and recombine with nearest antiparallel neighbors to give the a-chitin structure upon washing in water.

Such a concept has also been considered and discussed for the mechanism of mercerization of cellulose, in which a similar chain reversal occurs to convert, in the solid state, the parallel packing of cellulose I into the antiparallel packing of cellulose II (13-19). More specifically, the following has been proposed: Native cellulose microfibrils contain chains of the same polarity, but they point alternately up and down in the cell wall (23). Two neighboring microfibrils of opposite polarity may coalesce during swelling in alkali and allow the chains to adopt the antiparallel arrangement needed to form cellulose II. Crystallographic studies seem to indicate that an antiparallel arrangement is already present in the alkali cellulose (76*). Similarly to cellulose, the solid state phase transformation of P-chitin to a-chitin can be explained by the mingling of chains from adjacent and antiparallel P-chitin microfibrils to form a-chitin crystals of antiparallel chains. Perhaps more important than the p->a conversion mechanism, is the role played by the alkali intermediate in the deacetylation

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10 20 30 40 SO 60 70 80 90

Figure 3. X-ray diffractograms of recovered chitin from crab a-chitin (a) and from squid P-chitin (b).

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of chitin into chitosan. As discussed in our previous publication (24), each swollen crystallite in the alkali complex acts as a separate reactor in the kinetics of deacetylation, a phenomenon referred to as " permutoid" reaction in the early cellulose literature (25). Hence, deacetylation of chitin only depends on the concentration of acetamide groups since alkali is in excess and the reaction is pseudo first order. The first step is swelling, which makes the chains of each microfibril uniformly accessible to alkali. Deacetylation, therefore, proceeds from within at temperatures above about 25°C. Once this pervading swelling takes place, each swollen crystallite is one phase saturated by alkali. The reaction is considered as homogeneous, although there is still a boundary between swollen crystallites and alkali solution. Therefore, the reaction rate depends only on the concentration of acetamide, but not on the diffusion of alkali ions. In this case, the plot of log concentration of acetamide group versus time is linear as shown in our previous paper (24). On the other hand, if there would be no swelling and complexation, the reaction, if present, would proceed from the microfibril surface inward. The implication of that would be a heterogeneous reaction leading to a mixture of chitosan and chitin, which has never been observed.

Conclusions

It is clear that antiparallel chain, polymorphic a-chitin undergoes permutoid swelling in alkali and recovers the same polymorphic crystal structure upon neutralization. It requires much higher concentrations of alkali than cellulose for mercerization, presumably because its unique amide intermolecular H-bonding has higher cohesive energy. Conversion of p-chitin to a-chitin through the alkali chitin complex is similar to the conversion of cellulose I to cellulose II (mercerized cellulose). In both cases, crystals containing a parallel packing of the chains are changed into an antiparallel packing without dissolution of the polymer. An intermediate alkali complex forms in which coalescence of adjacent swollen microfibrils having opposite polarity allows the chains to interdigitate and to adopt the antiparallel arrangement. Tentatively, the following schematic states the conclusions outlined above:

a-chitin NaOH (50%)

H2O NaOH (50%)

Of greater significance at this time is the role of alkali chitin in the transformation from a-chitin to chitosan, a valuable water soluble polyelectrolyte. As discussed in our previous publication (24), the kinetics of deacetylation of a-chitin follow a pseudo first order law; i.e., a log plot has linear dependence on the acetamide concentration. This was interpreted as due to each alkali chitin microfibril acting as a separate reactor with a finite acetamido concentration, but an excess of alkali in the reaction.

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Acknowledgements

The authors thank Ms. Anne-Marie Lebuis and Dr. Fred Morin for obtaining the X-ray diffractograms and the solid state NMR spectra used in this paper.

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[acs symposium series] biopolymers volume 723 (utilizing nature's advanced materials) || alkali...

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