Makromol. Chem.

178,2595-2602 (1977)

Department of Industrial Chemistry, College of Technology,

Seikei University, Musashino-shi, Tokyo, Japan

Studies on Chitin, 3*)

Preparation of Pure Chitin, Po~y(N-acetyl-D-glucosamine),from the Water-Soluble Chitin
Keisuke Kurita, Takanori Sannan, and Yoshio Iwakura
(Date of receipt: December 28,1976)
SUMMARY:
Pure chitin, poly(N-acetyl-D-glucosamine), was successfullyprepared by acetylating the partially deacetylated water-soluble chitin with acetic anhydride-pyridine or with acetic acid-dicyclohexylcarbodiimide.
Although enough selectivity between amino and hydroxyl groups was not shown, acetylation with
acetic anhydride-pyridinegave rise to the complete acetylation of amino groups and thereby 0-,N-acetylated chitin was formed. The ester groups were then selectively transformed to hydroxyl groups by
means of either hydrolysis or transesterification to give the pure chitin. Acetic acid-dicyclohexylcarbodiimide system was found to enable one step complete and selective acetylation of amino groups under
very mild conditions and it appeared to be superior to the others for the purpose.
Introduction
Chitin is generally considered to consist of N-aCetyl-D-glUCOSamine units, but it is well
known that chitin is found in close association with other organic and inorganic materials
such as protein and calcium carbonate in crustacean shells. Drastic procedures are hence
required to remove these accompanying substances. However, these methods also degrade
chitin, and thus, isolated chitin can not strictly be regarded as a natural chemical entity,
but has undergone degradation including deacetylation2! In fact chitin isolated by Hackmans
method3 is partially deacetylated by hydrolysis, and we have reported that the degree of
deacetylation was found to be 15%4. Most studies of chitin have been based on such a
sample. Hence obtaining pure chitin, poly(N-acetyl-D-glucosamine), appeared to be very significant for every field of studies relating to chitin.
Concerning the pure chitin, the only report found in the literature was that by McLachlan
et aL5, who claimed the isolation of the pure chitin from a kind of alga by a complicated
procedure. No attempt has been made hitherto to prepare poly(N-acetyl-D-glucosamine) by
the acetylation of the patially deacetylated chitin, although acetylated chitosan was briefly
mentioned in the literature with no detailed information about the extent of acetylation6!
Schorigin et al.) had investigated in detail the acetylation of chitin. They found that it
could not be acetylated in its inner region of the solid by the usual methods except a drastic
one with dry hydrogen chloride and acetic anhydride. As can be seen from the facts, it has
been by no means easy to acetylate chitin by the conventional heterogeneous methods.
We recently reported that chitin became water-soluble by an alkaline treatment under appropriate conditions and that this interesting solubility phenomenon was observed for only the
*I Part
I f

2: ~ f . ~ .

Revised manuscript of February 15, 1977.

K. Kurita, T. Sannan, and Y. Iwakura

2596

samples with the degree of deacetylation of about 50%4'. This water-solubility has enabled
us to study the acetylation of the partially deacetylated chitin under homogeneous or almost
homogeneous conditions to obtain the po~y(hi-acetyl-D-glucosamine).
The present study has sought to prepare poly(L~-acetyl-D-ghcosamine)
from the water-soluble
chitin by acetylation with three different agents: 1 ) acetic anhydride with pyridine, 2) acetic
acid with dicyclohexylcarbodiimide (DCC), and 3) acetyl chloride in interfacial condensation.
Results and Discussion
Acetylation with acetic anhydride-pyridine
Chitin and chitosan swell little in organic solvents including pyridine, which has been one
of the barriers in their acetylation and made it difficult to acetylate uniformly. The water-soluble
chitin, however, was found to be able to form a highly swollen gel by pouring its aqueous
solution into pyridine. The gel was considered to be easily accessible for the reaction and
it was, therefore, treated with acetic anhydride at room temperature.
The acetylation reaction appeared to proceed rapidly and almost homogeneously as the
gel remained in a highly swollen state during the reaction. Fig. 1 shows the IR spectra
of the acetylated chitin samples. All the spectra of the samples obtained after various length
of time indicated the presence of 0-acetyl group in addition to N-acetyl group. The absorption
bands due to the 0-acetyl group at 1735 and 1235 cm-' were found even in the spectrum
of the sample obtained after 3min reaction. As it is well known that the amino group is
much more reactive than the hydroxyl group towards acetylation, 3 min was considered to
be sufficient to complete the acetylation at the amino group. However, the enough selectivity
to acetylate only at the amino group was not attained under these conditions, and it was
found to be necessary to remove the 0-acetyl group selectively without harming the N-acetyl
group from the resulting acetylated chitin to obtain the pure poly(N-acetyl-D-glucosamine).
These acetylated chitin samples were highly swollen in reaction mixture after the reaction,
and even the dried samples showed good swelling property in polar solvents such as N,N-dimethylformamide (DMF),N,N-dimethylacetamide(DMAc), N-methyl-2-pyrrolidone, m-cresol, benzyl
alcohol, and pyridine, although they became insoluble in water and diluted hydrochloric acid
which were good solvents for the water-soluble chitin. Thus, hydrolysis and transesterification
under the swollen state were employed to regenerate the hydroxyl group.

(Ac = COCH3)

2597

Studies on Chitin, 3

\
\

Fig. 1. IR spectra of the water-soluble chitin (A) and the acetylated chitin
samples prepared by acetylating the
water-soluble chitin with acetic
anhydride-pyridine for 3 min (B), 10
rnin (C), and 24 h (D)

1800

\
I

1600

1400

1200

900

Wave number in cm-

Hydrolysis
The hydrolysis of the ester group was carried out with a weak base such as sodium hydrogen
carbonate in order to avoid the unfavorable effect on the amide group. The swollen acetylated
sample, which was obtained after 3min reaction, was added to an aqueous sodium hydrogen
carbonate solution. The mixture was kept at room temperature for a given time and the extent
of the hydrolysis of the ester group was followed by IR spectroscopy.

(Ac = COCH3)

K. Kurita, T. Sannan, and Y. Iwakura

2598

The absorbances of the bands at 1735 and 1 235 cm-' decreased gradually as the reaction
progressed and the change was shown in Fig. 2. In the spectrum of the sample obtained
after 32 h, these bands became small shoulders. Finally even these shoulders disappeared completely in the spectrum of the sample hydrolyzed over 141 h. The spectrum of the resulting

1800

1600

1400

'

1200

900

Fig. 2. IR spectra of the samples

obtained by hydrolysis of the acetylated chitin in sat. aq. sodium hydrogen carbonate solution: Samples hydrolyzed for 17 h (A), 32 h (B), and 141 h
(C)

Wave number in cm-'

sample showed strong bands at 1650 and 1550cm-' of the amide and a distinct peak at
1llOcm-' as shown in Fig. 2. The assignment of the peak at l l l O c m - ' was not clear,
but it was found to have something to do with the content of the acetylamino groups or
the degree of deacetylation (DD): a distinct peak (DD<20%), a shoulder (DD=30=60%),
and almost n o band (DD>90%). The shape of the peak, therefore, appeared to be a rough
indicator for the degree of deacetylation and in the pure chitin, it should be a distinct one
instead of a shoulder.

Studies on Chitin, 3

2599

The samples obtained after hydrolyzing for 141 h were insoluble in water and diluted hydrochloric acid. Their degrees of deacetylation were determined by Elek and Harte's method')
and they were found to be 0 and 1 % for the two different samples, revealing that these
samples were the poly(N-acetyl-D-glucosamine). These results indicate that the amino groups
in the water-soluble chitin were completely acetylated with acetic anhydride-pyridine in the
swollen state under the conditions where a part of the hydroxyl groups were acetylated,
and the ester groups formed were then selectively and fully hydrolyzed to the hydroxyl groups
without affecting the acetylamino groups by the above manner.
7iaiisestelifictrtiorl

The ester interchange reactions of the dried acetylated chitin samples were first attempted
with dry methanol. They were, however, found to be unsuccessful even with the swollen
samples in DMF, DMAc, or rn-cresol as only a little decrease in the absorbances of the
ester bands was observed in the IR spectra of the products. The extent of the reaction seemed
to be quite limited probably because methanol was a poor solvent for the acetylated chitin
and it suppressed the swelling of the samples in the polar solvents.
Benzyl alcohol itself was a good swelling agent and the ester interchange reaction was
then tried in the solvent. The dried acetylation product was swollen in benzyl alcohol and
the reaction was carried out at 100C for 24h with the use of a small amount of sodium
metal. The product isolated was insoluble in water and diluted hydrochloric acid and slightly
colored. Its IR spectrum was the same as that of the samples obtained by the hydrolysis
method. Moreover, the degree of deacetylation was determined to be 4 %, which was considered
to be almost the same value as that obtained by the hydrolysis method within the experimental
error. At lower temperature, for example at 50"C, the product had weak bands owing to
the ester group, although the coloration was negligible. High temperatures seemed to make
the reaction rapid, but also caused the heavy coloration. Hence the reaction appeared to
be carried out successfully at IOO'C, but the slight coloration was always inevitable perhaps
due to some decompositions of chitin and/or benzyl alcohol.
Consequently, selective 0-deacetylation was confirmed to be accomplished by either the
hydrolysis method or the transesterification method, but the former was found to be more
clean and effective than the latter.
Acetylation with acetic acid-DCC
DCC has been widely used in the preparation of amide compounds from amines and carboxylic
acids under mild conditions. In a recent patent, Yaku et al.9' reported the acetylation of
chitosan films with acetic acid and DCC in aqueous DMF, but the acetylation was insufficient
so as to regard the product as pure chitin as the reaction was conducted heterogeneously
on the films. They examined the effect of the constitution of the solvent on the extent of
the reaction and found that when the water content was over 40%, excess acetic acid, functioning
as a strong acid, stimulated the reverse deacetylation.
Direct one step selective conversion of the water-soluble chitin to the pOly(N-aCetyl-D-glUCOSamine) was anticipated to be achieved by the acetic acid-DCC system, and the acetylation of
the aqueous solution of the water-soluble chitin was undertaken.

K. Kurita, T. Sannan, and Y. Iwakura

2600

The acetylation reaction was carried out by adding a solution of twenty-fold excess acetic
acid and DCC in D M F to the 1 % aqueous solution of the water-soluble chitin at room
temperature. When the ratio of water to D M F was 3 : 2 (water content 60%) or 6:5 (water
content 55%), the acetylated product was in the solution. The isolated product was insoluble
in water, but soluble in diluted hydrochloric acid, which insinuated the incomplete acetylation.
This result might be interpreted by the high water content as suggested by Yaku et aL9).
Then the ratio of water to D M F was reduced to 2:3 (water content 40%). The reaction
proceeded similarly except the precipitation of an almost homogeneous highly swollen gel.
The IR spectrum of the product isolated showed the strong amide I and I1 bands, the distinct
band at 1 IOOcm-', and no bands due to ester group. It was identical with those of the
pure chitin samples obtained by the hydrolysis or the transesterification of the 0-, N-acetylated
chitin. The sample was insoluble in water and diluted hydrochloric acid. Furthermore, the
degrees of deacetylation were found to be 0, 3, and 4%, respectively, for the three acetylated
samples. These values were considered to be 0% within the experimental error, and hence
it was concluded that the acetic acid-DCC system acetylated only the free amino groups
successfully without acetylating the hydroxyl groups to give the poly(N-acetyl-D-glucosamine)
in one step.
Although the acetylation was complete with twentyfold excess of acetic acid and DCC, smaller
amounts of acetic acid and DCC gave rise to the incomplete acetylation. For instance, the
reaction with five-fold excess acetic acid and ten-fold excess DCC gave the products with
the degrees of deacetylation of 16 and 20% by the two different acetylation runs.

(Ac = COCH,)

Tab. 1. Acetylationof the water-solublechitin with acetic acid and DCC or the water-soluble carbodiimide"'
Sample

Carbodiimide

Amount of excess
acetic

acid

'I111
IV
V

b,

''

20-fold

DCC
Water-soluble
carbodiimide

Band at
1 110 cm-'

- -A- (

5-fold
5-fold
3-fold
20-fold

carbodii mi de

20-fold
10-fold

20-fold

Peak

Solubilityb'in

DD
in

, -A- ,

water

dil. HCI

+
+

+
+

*+

%,'I

0, 3, 4
16,20
~

The reactions were carried out in a mixed solvent of DMF and water (3:2) (I-IV) or water (V)
at room temp. for 44 h.
(-): insoluble; (+): partially soluble; ( + ) : soluble.
DD=Degree of deacetylation, determined by Elrk and Harte's method.

Studies on Chitin, 3

2601

In place of DCC, the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, was tried as a condensing agent in order to carry out the reaction in water instead
of aqueous DMF. The product, however, was soluble in water and almost no acetylation
was found to have proceeded judging from its IR spectrum. This result indicates that the
acetylation of water-soluble chitin is hampered by high water content. The results are summarized
in Tab. 1.
Interfacial condensation
Cellulose xanthate was reported to be acetylated to some extent by p-nitrobenzoyl chloride
by the interfacial condensation method"). In the case of the water-soluble chitin, the free
amino groups in the partially deacetylated chitin were expected to be acetylated by acetyl
chloride quickly. The reactions were carried out according to the usual manner of the interfacial
condensation with the variations of the organic solvent, the acid acceptor, and the surface-active
agent. However, the lowest degree ofdeacetylation obtained was 35% and the extent of acetylation
by this procedure was found to be very low.
Sun et a1.I') examined the esterification of cellulose derivatives by the interfacial condensation
and found that the limitation in the extent of the acetylation was attributable to a steric
arrangement of the cellulose molecules at the liquid interface such that only 50% of the
hydroxyl groups were exposed to the organic phase and were thereby made accessible for
the reaction with the acid halide. The same reasoning appeared to be applicable to the present
case, i.e., the results seemed to suggest that only a part of the amino and hydroxyl groups
were exposed to the organic phase for the reaction as in the cellulose case.
Conclusion
Pure pOly(N-aCetyl-D-glUCOSamine)was prepared from water-soluble chitin by either 1) acetylation with acetic anhydride-pyridine and subsequent hydrolysis or transesterification or 2) by
selective acetylation with acetic acid-DCC. Between the two, however, the acetic acid-DCC
system was considered to be superior as the acetylation could be completely and selectively
achieved under mild conditions by one step simple reaction.

Experimental Part
Chitin: Chitin was isolated from shells of Penneirs japonicus and the water-soluble chitin with the
degree of deacetylation of about 50% was obtained according to the previously mentioned procedures4'.
Acetylation with acetic a,ih~cirirle-pyrirlirlc:A 0,30g of the water-soluble chitin sample was dissolved
by stirring with 30g of crushed ice. The aq. solution was poured into 200ml of pyridine to form
a highly swollen precipitate. I t was separated by centrifugation, washed well with pyridine repeatedly,
and squeezed to about log. It was then swollen in 20ml of fresh pyridine, and 60ml of acetic anhydride
was added to the mixture. After stirring for 3min at room temp., the 0-. N-acetylated chitin was
filtered, washed successively with water, methanol, and acetone, and squeezed. I t was used for the
subsequent hydrolysis and transesterification.
Hydrolysis of 0-,N-acetylated chitin: To a lOOml of sat. sodium hydrogen carbonate solution was
added the acetylated chitin obtained above without drying. The mixture was left standing at 20C
for 141 h. The resulting chitin was separated by filtration, and washed with water, methanol, and
acetone. Yield 0.28 g.

2602

K. Kurita, T. Sannan, and Y. Iwakura

The sample was strongly hygroscopic and attempts to obtain the reliable data of elemental analysis
were unsuccessful. The degree of deacetylation was determined by Elek and Hartes method on the
sample dried thoroughly in a vacuum oven and weighed in a breakable seal. It was found to be
0%.
Transesterification of 0-, N-acetglated chitin; The acetylated chitin obtained above was dried well
i. vac. at room temp., and allowed to swell in 60ml of dry benzyl alcohol for 1 2 h at room temp.
A small amount of sodium metal was added to the mixture. After heating at 100C for 24h, the
prod. was filtered and washed well with methanol and acetone. It weighed 0,32 g. The degree of deacetylation was determined to be 4 % by EIek and Haires method.
Acerylation with acetic acid-DCC: To an aq. solution of 0,434g of the water-soluble chitin in which
1,2mmol of glucosamine units were present in 40g of water was added 1,44g (24mmol; 20-fold excess)
of acetic acid and 4,94g (24mmol; 20-fold excess) of DCC in 60ml of D M F with stirring. The stirring
was continued for 44 h at room temp., and then the mixture was taken into a sat. aq. sodium hydrogen
carbonate solution for neutralization. The precipitate was filtered. washed with water and methanol,
and dried i. vac. to give 0,432g of chitin whose degree of deacetylation was O%, determined by
Elek and Hartes method*).

A. B. Foster, J. M. Webber, Adv. Carbohydr. Chem. 15, 372 (1960)

A. G. Richards, The Integument of Arthropods, University of Minnesota Press, Minneapolis,
Minn. 1951
3, R. H. Hackman, Austr. J . Biol. Sci. 7, 168 (1954)
4, T. Sannan, K. Kurita, Y. Iwakura, Makromol. Chem. 177, 3589 (1976)
5 , J. McLachlan, A. G. McInnes, M. Falk, Can. J. Bot. 43, 707 (1965)
P. Karrer, G. V. FranGois, Helv. Chim. Acta 12, 986 (1929)
I P. Schorigin, E. Hait, Ber. Dtsch. Chem. Ges. B 68, 971 (1935)
A. Elek, R. A. Harte, Ind. Eng. Chem., Anal. Ed. 8, 267 (1936); see also A. Steyermark, Quantitative
Organic Microanalysis, The Blackistone Comp., New York 1951, p. 244
9, Jpn. P. 7319213 (1973), invs.: T. Yaku, I. Yamashita; C. A. 80, 72291 v (1973)
l o ) T. Sun, V. K. Chang, Z. A. Rogovin, Vysokomol. Soedin. 3, 382 (1961); C. A. 55, 27881c(1961)
I

*)