147 Progress on Chemistry and Application of Chitin and Its ...

, Volume XVI,
2011
147 Progress on Chemistry and Application of Chitin and Its ..., Volume XVI,
2011
STATE OF WATER IN NONCROSSLINKED AND CROSSLINKED HYDROGEL
CHITOSAN MEMBRANES DSC STUDIES
Jadwiga Ostrowska-Czubenko, Milena Pierg,
Magdalena Gierszewska-Druyska
Chair of Physical Chemistry and Physicochemistry of Polymers,
Faculty of Chemistry
Nicolaus Copernicus University
ul. Gagarina 7, 87-!! "oru#, Poland
$-mail% &oc'(chem.uni.torun.pl
Abstract
Modified chitosan hydrogel memranes !ere prepared using glutaraldehyde "#A$ and sodium
citrate "%aCI&$ as crosslin'ing agents. Molecular and supermolecular structure analyses
of unmodified and modified chitosan memranes ha(e een conducted y )&I* and X+ray
spectroscopy. )&I* results sho!ed co(alent and ionic crosslin's formation et!een chitosan
"Ch$ and #A or simultaneously Ch, #A and %aCI&. &he state of !ater in noncrosslin'ed and
crosslin'ed chitosan memranes !ere analysed y differential scanning spectroscopy ",-C$.
&hree types of !ater in hydrogel memranes !ere found. non+free/ing ound !ater, free/ing
ound !ater and free/ing free !ater, !hile there !ere (ariations in the amount of non+
free/ing
ound !ater in these polymers. &he effect of ionic crosslin'ing on !ater state, mainly on the
non+
free/ing !ater content, !as discussed.
Key words: chitosan memranes, crosslin'ing, ,-C, states of !ater, glutaraldehyde,
trisodium citrate.
0. 1stro!s'a+C/uen'o, M. Pier2g, M. #iers/e!s'a+,ru3y4s'a -tate of 5ater in %oncrosslin'ed and Crosslin'ed 6ydrogel Chitosan Memranes 7 ,-C -tudies
1 !ntrodu"tion
In recent years hydrogel membranes formed from natural polymers arouse a big
interest. A special attention has been given to such membrane material as chitosan.
Recently, chitosan is widely used as membrane material for ultrafiltration, reverse
osmosis, pervapo- ration and another membrane processes.
It is well nown, that e!uilibrium water content as well as state of water
influence on properties of hydrogel membranes. "enerally, the state of water in hydrogels
is catego- ri#ed into three different types. $he e%perimentally determined separate states of
water can be defined as follows &1, '() *i+ free !ater - water that is not intimately bound to
the polymer chain and behaves lie bul water, i.e. undergoes thermal transition at
temperature analo- gous to bul water *at , -.+, *ii+ free/ale ound !ater - water that is
wealy bound to the polymer chain or interacts wealy with nonfree#ing water and
undergoes a thermal phase transition at a temperature lower than , -. and *iii+ ound
!ater *non+free/ing !ater+ / wa- ter tightly bound to the polymer, which does not e%hibit a
first order transition over the tem- perature range from /7, to , -. &1(. $wo first types of
water mentioned above are so-called free#ing water. $here is a variety of techni!ues for
the study of water binding in polymers. 0ifferential scanning calorimetry *01.+, used by
us to characteri#e water state in chitosan hydrogel membranes, is in many ways the most
convenient and informative method &2(.
In our previous studies the ionically crosslined membranes *.h3.I$+ were pre-
pared of chitosan *.h+ and trisodium citrate *4a.I$+ and the characteristics of the state of
water in these membranes with different water content was performed by 01. &4(. $he
present wor deals with a 01. study of the state of water in doubly crosslined
.h3"A3.I$ membranes that were obtained by covalent crosslining of chitosan with
glutaraldehyde *"A+ and ne%t by ionic crosslining with 4a.I$.
# Materials and $et%ods
2.1. Materials
5edium molecular weight chitosan *5
v
672, 0a determined by viscometry &7(,
degree of deacetylation 00A678.29 determined by potentiometric titration method &8(+
and glutaraldehyde *"A, '7 wt.9 solution in water+ were analytical grade and were pur-
chased from 1igma-Aldrich *"ermany+. $risodium citrate *4a.I$+, sodium hydro%ide and
acetic acid were analytical grade and were purchased from :;.h *:oland+. :otassium bro-
mide for spectroscopy was purchased from 5erc *"ermany+.
2.2. Membrane preparation
:ure chitosan membranes *.h+ were prepared by casting and solvent evapora-
tion techni!ue, as described in detail elsewhere &4(. $wo-component chitosan3glutaralde-
hyde *.h3"A+ membranes were prepared as follows) first, 19 *w3v+ chitosan solution in
'9 *w3v+ acetic acid and ,.'79 *w3v+ glutaraldehyde solution in water were mi%ed and
stirred at room temperature for at least 8 hrs to obtain a homogeneous solution and then
the solution was cast as a film on a clean glass plate and evaporated to dryness at 27 <..
$he content of glutaraldehyde in casting solution was '.7 wt.9. =inally, the prepared
mem-
branes were washed repeatedly with bidistilled water, immersed in '5 sodium hydro%ide
solution for 7 min and again washed repeatedly with water and dried in air. $hree-com-
ponent chitosan3glutaraldehyde3sodium citrate *.h3"A3.I$+ membranes were prepared by
immersing two-component .h3"A membranes in a!ueous 4a.I$ solution for '4 hrs. $he
following crosslining conditions were applied) concentration of 4a.I$ solution c 6 79
*w3v+, temperature $
crosslin.
6 4 -., p> of 4a.I$ solution p> 6 7., *initial 4a.I$
solution
acidified with >.l+. After crosslining the membranes were repeatedly washed with
water,
thoroughly dried in air and then under vacuum at 8, -. for several days to obtain
completely dry films.
2.3. FTIR spectroscopy analysis
=$IR spectra of .h, .h3"A and .h3"A3.I$ in ?@r disc form were recorded on
:erin-Almer ',,, =$IR spectrometer from 4,,, to 4,, cm
-1
with a resolution 4 cm
-1
and
1,, scans.
2.4. Wide anle !"ray di##raction st$dies
Bide angle C-ray diffraction patterns of unmodified and modified chitosan mem-
branes were measured by an C-ray diffractometer *C-:ert :ro 1ystems, :hilips, 4ether-
lands+. C-ray diffraction was performed on powdered samples by e%posing them to .u?
D
radiation and scanned from 'E 6 4< to 4,< at a step si#e of ,.,'<.
2.%. &i##erential scannin calorimetry '&()* meas$rements
$he state of water in chitosan hydrogel membranes was analysed by 01. meas-
urements, as described elsewhere &4, 7(. A :olymer Faboratories Ftd. *Apsom, Gnited
?ingdom+ differential scanning calorimeter e!uipped with a li!uid nitrogen cooling acces-
sory was used to monitor both bound as well as free water in membranes. $he temperature
scale of the 01. cell was calibrated using water. 0ry membrane sample *about 7 mg+ was
weighed in an aluminium pan designed for volatile samples and a nown amount of water
was added by a micro-syringe. $he pan was sealed hermetically to prevent water loss
during 01. scanning, e!uilibrated for '4 hours at room temperature and then weighed.
After that the pan was first cooled from room temperature to /14, <. at a rate of 1,
<.3min, under constant purging of nitrogen at '.7 mF3min, allowed to stay at that
temperature for 1, min and then heated at the same rate up to 7, <.. After the 01.
measurement the pan was weighed in order to chec that it had been properly sealed and
that no water had evaporated. $he phase transition of water in the hydrogel membrane
during heating was recorded as the endothermic pea, which was later integrated using
01. software.
$he amount of water able to crystalli#e *free#able water+, 5
f
, defined as
5
f
"g8g$ 9 "!ater, g$8"dry polymer, g$ was calculated after integration of the melting en-
dotherm, using double distilled water as a reference and assuming both melting enthalpies
for free#ing free water *5
ff
+ and free#ing bound water *5
f
+ to be the same as that of bul
water *H6
0
9 ::; 08g+. $he amount of free#able water was calculated from the following
e!uation)
5
f
9H6
m
8H6
0
where H6
m
*I3g+ is the melting enthalpy for free#able water in hydrogel membrane
obtained from the 01. thermogram and H6
,
is the melting enthalpy of pure water. $he
total amount of non-free#ing bound water, 5
nf
, was obtained from the difference between
the amount of sorbed water, 5
c
, and the total amount of free#able water 5
f
)
5
nf
9 5
c
7 5
f
9 5
c
7"5
ff
<5
f
$
where 5
ff
is the amount of free#ing free water.
2.+. (wellin meas$rements
A!uilibrium water content *=5C+ of the membrane was measured by the gravi-
metric method. $he preweighed, completely dried membrane sample was immersed in wa-
ter at temperature 27 <. for '4 hrs, which was determined to be sufficient to reach an e!ui-
librium state. $hen membrane was taen out, wiped !uicly with filter paper and weighed.
AB. was calculated using the following e!uation)
5
s
+5
d
=5C 6
5
d
J1,,>
where 5
s
is the weight of the swollen membrane and 5
d
is the weight of the dried mem-
brane.
& 'esults and dis"ussion
3.1. Membrane c,aracteri-ation by FTIR and !"ray spectroscopy
Figure 1 shows =$IR spectra of hydrogel chitosan membrane before and after
chemical and physical modification. In the spectrum of noncrosslined chitosan absorption
bands situated at 1878 cm
-1
*.6; stretching in amide group, amide I vibration+, 17KL cm
-1
*-4>
'
bending in non-acetylated '-aminoglucose primary amine+ and 178, cm
-1
*4/>
bending in amide group, amide II vibration+ can be seen &L - 1,(. 1ome changes can be
observed after chitosan modification - crosslining with glutaraldehyde. $he pea at 1878
cm
-1
shifts to the lower wavenumber, i.e. to 1841 cm
-1
. $his band is most probably com-
posed of amide I band of chitosan, that as we discussed above appears at 1878 cm
-1
, and
the .64 stretching band of 1chiffMs base, that according to literature appears at the
fre!uency range 18',-188, cm
-1
&11(. 5oreover, it is not observed any band at N1717
cm
-1
, related to the free aldehyde group &1'(. $wo main crosslining mechanisms,
involving formation of 1chiffOs base structures or 5ichael-type adducts, have been
proposed for the reaction of chitosan and glutaraldehyde &12(. ;ur findings seem to
indicate that under e%perimental reaction conditions "A completely reacted with chitosan
and imines *1chiff bases+ were formed, as presented in Figure 2. $he formation of
crosslins between .h and "A was also confirmed visually by the change of membrane
color from transparent to deep yellow &14(. .omparison of the =I$R spectra of .h3"A and
.h3"A3.I$ indicates that these spectra are
!uite similar in the fre!uency range of 187,-17,, cm
-1
. In both mentioned spectra two
main bands can be seen) the first at 1841 cm
-1
*.h3"A+ or 1827 cm
-1
*.h3"A3.I$+ and the
second at 1772 cm
-1
*.h3"A+ or 17L4 cm
-1
*.h3"A3.I$+. 5oreover, some differences in
relative intensity of these bands can be observed. It can be supposed that these bands
represent en- velope of several bands, because both protonated amine groups as well as
amine, acetamide and imine groups absorb in these fre!uency region. :rotonated amines
show an antisymmet-
Figure 1. )&I* spectra of noncrosslin'ed and crosslin'ed chitosan memranes.
O
OH OH
O
O H
O
. O O
+ OH
H
H
H
H
H
H
OH
H
H
O
.

H
H
O
.
OH
H
H
H
H NH
2
H NH
+
H NH
H
O
-
HO
OH
O
H
O
.
- H O
OH
O .
H
O
.
CHO
NH
2
OH
H
H
H
H
O
O H
CH
2
OH
OH
O
H
O .
OH
H
H
H
H N
OHC
OH
H
H
.
H
H NH
H2O
+
.
H2O
H
H N
N OH
H
H
H
H
O
OHC
OHC
.
O
H CH
2
OH
Figure 2. *eaction mechanism et!een amino groups of chitosan and caronyl groups of
glutar+ aldehyde for the formation of -chiff ase.
ric and symmetric 4-> deformation vibrations in the 18'7-178, cm
-1
and 177,-17,7 cm
-1
range, respectively &1'(. In conclusion we can say that the absorption band at 1827 cm
-1
and 178, cm
-1
in the spectrum of .h3"A3.I$ derive mainly from the antisymmetric 4/>
deformation vibrations in protonated amines, but the initial amide-I, amide-II and imine
bands are possibly overlapped by these vibrations. In the spectrum of .h3.I$ membrane,
presented and discussed elsewhere &4(, analogous peas were observed at 1824 cm
-1
and
.
2
. .
. .
.
CH 2OH
H
O
OH
CH 2OH
H
O
O
OH
CH
2OH
H
O
G(
O
OH O
H H
CH 2OH
H
O
H
OH O
H
H NH
CH 2OH
O
OH
H N
CH
2OH H
O
OH
H
H
O
O
H
NH2
H NH2
H NH2
C%
H NH
C O
CH3
acetic acid
solution
C O
CH3
.>
(CH2)3
. >
NH2 H
H
N H NH2 H
H
H
CH 2OH
O
CH
2
OH
H
O
CH
2OH
CH
2OH
H OH
H
H2
OH
H
O
H OH
H
H
O
OH
O
H
+
H3
H O
OH
O
OH
O
H
H
H NH
H N
OH O
H
H NH
2
O H
CH
2OH
O H
CH2O
H
O H
CH
2OH
C%)G(
>'.
>;
.
>'.
.;;-
.;;-
.;;-
C O
CH3
.>
(CH2)3
.>
+
H3 NH
2
H
H
H
N H
NH2 H
H H
H OH
H O
O H
H OH
H
O H
H OH
H O
O H
H OH
H
O
O H
CH 2OH
CH
2OH
CH
2OH
CH
2OH
C%)G()C!*
Figure 3. -ynthesis of Ch8#A and Ch8#A8CI& memranes.
17L4 cm
-1
. 5oreover, in the spectrum of .h3"A3.I$ membrane the characteristic band
at 12L, cm
-1
is observed. It corresponds to .-; symmetric vibrations in .;;
-
ions &1'(.
$he spectral changes in the =$IR spectra of chitosan membrane treated with glu-
taraldehyde and sodium citrate indicate the formation of covalent and ionic crosslins be-
tween chitosan and crosslining agents, as presented in Figure 3.
$o confirm the influence of modification on crystallinity of chitosan membrane
the wide C-ray diffraction patterns of unmodified and modified membranes were compared
*Figure 4+. C-ray pattern of unmodified chitosan shows three maPor crystalline peas) the
two weaer peas at 'Q R 1,- and 'Q R 17- and the strongest one at 'Q R ',-, characteristic
for the crystalline forms I, II and anhydrous form &17, 18(. $he same reflections can be
also observed on BAC1 diffraction patterns of .h3"A3.I$ and .h3"A membranes, but
in the case of .h3"A membrane the pea at 'Q R 17- practically disappeared. 5oreover, in
the case of crosslined membranes crystalline peas, mainly the pea at 'Q R ',-, became
wider and weaer. $hese results seem to indicate that crystallinity of the chitosan
decreased after its crosslining with glutaraldehyde and sodium citrate, but the crosslined
membranes retained their semicrystalline morphology.
3.2. Membrane swellin and state o# water
$he e!uilibrium degrees of swelling *AB.+ for noncrosslined and crosslined
chitosan membranes are presented in Figure 5. Salues of AB. decrease in the following
order) .h T .h3"A T .h3.I$ T .h3"A3.I$. $he e!uilibrium swelling of hydrogels is a
result of the balance of osmotic forces determined by their affinity to the solvent and the
net- wor elasticity. 1welling ability of the studied chitosan membranes depends
simultaneously on several parameters, such as hydrophilicity of the whole networ,
crosslining degree
H2
H
2
O
H
N H
H2
N H
O
H2
;
1+,
1#,
-,
.,
&,
,
C% C%)G( C

%)C!*
C%)G()C!*
Figure 4. X+ray diffraction patterns
of unmodified and modified chitosan
memranes.
Figure 5. Values of =5C for
noncrosslin'ed and crosslin'ed chitosan
memranes "data for Ch8CI& ta'en from
the earlier !or' ?;@$.
and polymer crystallinity. $hese parameters were different for .h, .h3"A and .h3"A3.I$
membranes.
Figure 6 shows the 01. melting
thermograms of fro#en water in .h3"A3.I$ membrane
with various wa- ter content 5
c
. $he other two studied
membranes *.h and .h3"A+ showed very similar
melting behaviour of wa- ter to .h3"A3.I$. =or all
analysed polymer-water sys- tems no peas are
observed at $ R , <. below certain water content. =or
e%ample, no pea is observed for .h3 "A3.I$
membrane with 5
c
6 ,.'7 *g
water
3g
polymer
+, 5
c
6 ,.4, *g
water
3g
polymer
+ *Figure 6+. $he absence of en-
dothermic peas above a water content threshold indi-
cates that this water is non-free#ing bound type. 5oreo-
ver, for each studied polymer at define water content
the broad endothermic pea appears, corresponding to
the melting of free#able water. 1uch pea appears on
ther- mogram of .h3"A3.I$ with water content 5
c
6
,.7L *g
water
3g
polymer
+. In all the samples melting of
water starts at temperature lower than that of pure water
*the 01. heating curve of pure water is shown in
Figure
6 by the dashed line+. "enerally, for all studied hydro-
gel membranes of higher water content the endothermic
peas are broad and structured. =or some water content
Figure 6. ,-C heating cur(es
for Ch8#A8CI& memrane !ith
different !ater content.
multipea with two well-defined subma%ima can be observed. =or e%ample, more than one
transition for .h3"A3.I$ with water content of 1.,L *g
water
3g
polymer
+ is evident. $his phe-
nomenon can be attributed to the presence of at least two types of free#able water &1() *i+
free#ing free water, which undergoes similar thermal transition to that of bul water and
*ii+ free#ing bound water, which undergoes a thermal phase transition at temperature
shifted to
lower temperatures with respect to that of bul water. $he formation of different states of
.
W
)

/
0
1
Figure 7. ,ependence of A6m per unit
sample !eight on !ater content.
Figure 8. &he amounts of free/ing !ater
"5f$ and non+free/ing !ater "5nf$ in
Ch8#A8 CI& memrane !ith different
!ater content.
water within a polymeric networ taes place in the following order) non-free#ing,
free#ing bound and free#ing free water.
$he area under the 01. pea represents the change in enthalpy associated with
the melting of free#ing water *free water and free#able bound water+. Figure 7 presents
a graph of the enthalpy of melting of free#ing water per gram of polymer versus the water
content 5
c
. $he slope of the linear plot represents the Uaverage apparentV value of the
melt- ing enthalpy associated with the free#ing water *A6
m
+. $he intercept with the
hori#ontal a%is corresponds to the ma%imum amount of non-free#ing water *5
nf,maB
+ in the
hydrogel membrane. 5
nf,maB
is defined as the ma%imum amount of water present in the
membrane, which is not associated with any endothermic pea &1(.
$he procedure described above gave A6
m
6217 I3g and 5
nf,maB
6,.87 g3g for .h
membrane, A6
m
6 2'8 I3g, 5
nf,maB
6 ,.47 g3g for .h3"A membrane and A6
m
6 'K8 I3g,
5
nf,maB
6 ,.72 g3g for .h3"A3.I$ membrane. According to literature data &17, 1L( bound
water content in different polymer-water systems depends on both chemical as well as
higher-order structure of a polymer. $hus, it can be supposed that differences in values of
5
nf,maB
for chitosan membranes result both from the changes in hydrophilicity or
hydropho- bicity as well as crystallinity of noncrosslined and crosslined membranes.
$o get more information on water state in hydrogel membranes effect of total
water content on free#able and non-free#able water content in membranes was analysed.
Figure 8 shows the dependencies of free#able water content 5
f
and non-free#able water
content 5
nf
on total water content, 5
c
, for the .h3"A3.I$ membrane. Analogous curves
were obtained for .h and .h3"A membranes. =or all membranes 5
f
increased linearly
with increasing B
c
, while 5
nf
increased with increasing 5
c
until it reached a characteristic
value and then remained constant.
/ Con"luding re$arks
5odified chitosan hydrogel membranes were prepared using glutaraldehyde *"A+
and sodium citrate *4a.I$+ as crosslining agents. =ourier transform infrared spectros-
copy of unmodified and modified chitosan membranes confirmed the formation of
covalent and ionic crosslining between chitosan *.h+ and "A or simultaneously covalent
and ionic crosslining between .h, "A and 4a.I$. It was found that crosslining
influenced both molecular and supermolecular structure of membranes as well as swelling
properties and state of water in studied membranes. A!uilibrium water content decreased in
the following order) .h T .h3"A T .h3"A3.I$. 01. studies showed the presence of
both free#ing and nonfree#ing water in noncrosslined and crosslined chitosan
membranes. $he formation of different states of water within a polymeric networ too
place in the following order) non-free#ing, free#ing bound and free#ing free water. =or all
membranes the free#able wa- ter content increased linearly with the water uptae and the
non-free#able water content remained constant beyond critical value *ranging from ,.47 to
,.87 g3g dry membrane+.
+ 'e0eren"es
1. Higuchi A, Komiyama J, Iijima T; (1984) The states of wate i! ge" ce""o#ha!e mem$a!es.
%o"ym &u"" 11, '()*'(8.
'. Ho+ge ,-, .+wa+ /H, 0imo! /%; (1991) %o"yme )2, 1)21*1)21.
). 3o4hi"" %H, Jo""y A-, 5g 3h6, Tighe &J; (1982) 0y!thetic hy+oge"s7 1. Hy+o8ya"4y" acy"ate
a!+ methacy"ate co#o"ymes 9 wate $i!+i!g stu+ies. %o"yme '8, 12:8*1211.
4. Ostrowska-Czubenko J, ier!" #$ ('(1() %tate o& water in citrate crosslinked c'itosan
(e(brane. I!7 Jawos4a -- (e+), %ogess o! 3hemisty a!+ A##"icatio! of 3hiti! a!+ its
;ei<ati<es. =o". >=, %o"ish 3hiti! 0ociety, ?@+A, ))*4(.
:. I"Bi!a A=, =a"amo< =%; ('((4) Hy+o"ysis of chitosa! i! "actic aci+. A##" &iochem
-ico$io"
4(, )((*)().
1. -uCCae""i ,AA, ,occhetti ,, 0ta!ic =, Dec48 -; (1992) -etho+s fo the +etemi!atio! of the
+egee of acety"atio! of chiti! a!+ chitosa!. I!7 -uCCae""i ,AA, %ete -/ (e+s), 3hiti! Ha!+*
$oo4, Atec .+iCio!i, /ottammae, 1(99119.
2. 6stows4a*3Cu$e!4o J, /iesCews4a*;uEyFs4a -; ('((9) .ffect of io!ic coss"i!4i!g o! the
wate state i! hy+oge" chitosa! mem$a!es. 3a$ohy+ %o"ym 22, :9(*:98.
8. %aw"a4 A, -ucha -; ('(()) Themoga<imetic a!+ GTI, stu+ies of chitosa! $"e!+s. Themo*
chim Acta )91, 1:)*111.
9. -o+Cejews4a H, ;oa$ia"s4a -, HaCyc4i ,, DojtasC9%ajI4 A; ('((9) The mecha!ism of
so#tio! of Ag
J
io!s o! chitosa! micoga!u"es7 I, a!+ 5-, stu+ies. I!7 Jawos4a -- (e+),
%ogess o! 3hemisty a!+ A##"icatio! of 3hiti! a!+ its ;ei<ati<es. =o". >I=, %o"ish 3hiti!
0ociety, ?@+A, 49*14.
1(. %easo! G/, -achessau"t ,H, Kia!g 3L; (191() I!fae+ s#ecta o cysta""i!e
#o"ysacchai+es. =. 3hiti!. J %o"ym 0ci 4), 1(1*111.
11. K!au" J, Hu+so! 0-, Creber )*#$ (1999) 3oss"i!4i!g of chitosa! fi$es with
+ia"+ehy+es7
#o#osa" of a !ew eactio! mecha!ism. J %o"ym 0ci %at &7 %o"ym %hys )8, 1(29*1(94.
1'. ,ao 35,; (191)) 3hemica" A##"icatio! of I!fae+ 0#ectosco#y. 5ew Lo4, Ko!+o!7
Aca+emic %ess.
1). 0chiffma! J;, 0chaue 3K; ('((2) 3oss*"i!4i!g chitosa! !a!ofi$es. +io(acro(olecules
,,
:94*1(1.
14. -o!teio 6A3, Aio"+i 3; (1999) 0ome stu+ies of coss"i!4i!g chitosa! 9 g"utaa"+ehy+e i!te*
actio! i! homoge!eous system. I!t J &io" -acomo" '1, 119*1'8.
1:. 6gawa K, Lui T; (199)) 3ysta""i!ity of #atia""y 5*acety"ate+ chitosa!s. &iosci &iotech &iochem
:2, 1411*1419.
11. 6gawa K, Hia!o 0, -iya!ishi T, Lui T, Data!a$e T; (1984) A !ew #o"ymo#h of
chitosa!. -acomo"ecu"es 12, 92)*92:.
12. Mu >, DisN! A, A"$etsso! A*3; ('((() 5o<e" #H*se!siti<e chitosa! hy+oge"s7 swe""i!g
$eha<io
a!+ states of wate. %o"yme 41, 4:89*4:98.
18. Hata4eyama H, Hata4eyama T; (1998) I!teactio! $etwee! wate a!+ hy+o#hi"ic #o"ymes.
Themochim Acta )(8, )*''.