EFFECT OF IONIC CROSSLINKING AGENTS ON SWELLING BEHAVIOUR OF CHITOSAN HYDROGEL MEMBRANES

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Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
75
EFFECT OF IONIC CROSSLINKING AGENTS ON SWELLING BEHAVIOUR

OF CHITOSAN HYDROGEL MEMBRANES
Milena Pieróg, Magdalena Gierszewska-Drużyńska,

Jadwiga Ostrowska-Czubenko
Chair of Physical Chemistry and Physicochemistry of Polymers,

Faculty of Chemistry
Nicolaus Copernicus University
ul. Gagarina 7, 87-100 Toruń, Poland
e-mail: mili@doktorant.umk.pl
Abstract
Physically crosslinked chitosan hydrogels were prepared by treating chitosan
with sulfuric acid, trisodium citrate, sodium tripolyphosphate and sodium
alginate, respectively. The chemical structure of modified chitosan hydrogels
as well as unmodified one was analysed by FTIR spectroscopy and conclusions
on the formation of the ionic crosslinks between protonated amino groups of
chitosan and anionic functional groups of crosslinking agents were drawn out.
The swelling behaviour of the membranes formed from modified and unmodified
chitosan was studied in buffer solutions at various pH at 37 ºC. It was observed
that the swelling degree of chitosan hydrogel membranes depends both on pH of
buffer solution as well as on the type of crosslinking agent.
Key words:
chitosan membrane, hydrogel, ionic crosslinking, swelling.
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
76
M. Pieróg, M. Gierszewska-Drużyńska, J. Ostrowska-Czubenko
1. Introduction
In general, hydrogels are materials composed of three-dimensional hydrophilic
polymeric network and water that fills the free spaces inside this network. Hydrogels are
able to absorb and retain 10‑20% up to thousand times water or biological fluids than their
dry weight. Hydrogels are called as “intelligent materials”, because they respond reversibly
to slight changes of the properties of surrounding media. This ability causes that hydrogels
found numerous applications in industry and pharmacy, for example as controlled drug
release systems [1].
Recently much attention has been focused on the development of hydrogels based
on natural, biodegradable and biocompatible polymeric materials such as chitosan. Chitosan
is a deacetylated derivative of chitin. It is a copolymer of β‑(1→4)‑linked 2‑acetamido‑2‑de
oxy‑β‑D‑glucopyranose and 2‑amino‑2‑deoxy‑β‑D‑glucopyranose (
Figure 1
) [2].
Figure 1.
Chemical structure of chitosan (DDA-degree of deacetylation),
It is well known that pure chitosan hydrogel membranes exhibit a small resistance
to acidic media ‑ they undergo dissolution or disintegration. To avoid this processes chitosan
modification through crosslinking is widely used. Among all known chitosan crosslinking
methods ionic crosslinking is the simplest and mildest one. In ionically crosslinked hydrogels
a network is formed in the presence of negatively charged crosslinking agents (CAs), which
form bridges between the positively charged chitosan polymeric chains. Both low molecular
as well as high molecular ions can be used as CA [1, 3].
Among other significant properties of hydrogels the one of the most importance is
their swelling and dehydration behaviour [3]. Schematic representation of hydrogel swelling
and deswelling processes is shown in
Figure 2
.
Figure 2.
Schematic representation of hydrogel swelling and deswelling processes
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
77
Effect of Ionic Crosslinking Agents on Swelling Behaviour of Chitosan Hydrogel Membranes
The swelling behaviour of the ionic hydrogels is unique. It is affected by the
ionization of functional groups along the polymer chains and the ionization of crosslinking
agent molecules. Other factors that affect the swelling of these materials are: hydrophilicity
of materials used in hydrogel network formation, degree of crosslinking, pH, ionic strength
and nature of counterions of swelling medium [4].
In the present studies sulfuric acid, trisodium citrate, tripolyphosphate and sodium
alginate were used as chitosan crosslinking agents. The chemical structure of uncrosslinked
and ionically crosslinked chitosan hydrogel membranes were analysed by FTIR
spectroscopy. The influence of chemical structure of crosslinking agent on swelling kinetics
and equilibrium swelling degree of chitosan based hydrogel membranes were studied.
2. Materials and methods
2.1. Materials
Commercially available chitosan (Ch) from crab shells of medium molecular
weight, sodium alginate (NaAlg) and sodium tripolyphosphate (TPP) were purchased
from Sigma Aldrich (Germany). Sulfuric acid (H
2
SO
4
) and trisodium citrate (CIT) were
purchased from POCh (Gliwice). In all swelling experiments the following buffer solutions
of constant ionic strength I = 0,145 M were used: hydrochloric buffer (pH 1.2), acetic buffer
(pH 7.4) and TRIS buffer (pH 8.5). Acetic acid (HAc), sodium acetate (NaAc), sodium
chloride (NaCl), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were analytical
grade and were purchased from POCh (Poland) and TRIS of analytical grade was purchased
from Sigma Aldrich (Germany). Potassium bromide (KBr) for spectroscopy was purchased
from Merck (Germany).
2.2. Polymer characterization
Degree of deacetylation, DDA, of chitosan was determined by potentiometric
titration method. The viscosity average molecular weight of chitosan and sodium alginate,
M
v
, was determined by viscometry. The details of above measurements have been presented
elsewhere [5]. Degree of deacetylation was equal to 75.72 ± 3.82%. M
v
was equal to
730 kDa for chitosan and 100 kDa for sodium alginate.
2.3. Membrane preparation
Pure chitosan (Ch) membranes

were prepared by casting solution and solvent
evaporation technique. 1 wt.% chitosan solution prepared by dissolving chitosan powder in
2 wt.% acetic acid was cast as film on clean glass plate, evaporated to dryness at 37 ºC and
further dried under vacuum at 60 ºC.
Chitosan/sulfuric acid (Ch/H
2
SO
4
) membranes

were prepared by immersing of
pure chitosan membranes in 2 M sodium hydroxide solution for 5 min to remove of acetic
acid residues, thoroughly washed with deionized water and air‑dried. Then the films were
immersed in 0.5 M sulfuric acid solution at room temperature for 60 min. The crosslinked
membrane was thoroughly washed with deionised water and dried as pure chitosan
membrane.
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
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M. Pieróg, M. Gierszewska-Drużyńska, J. Ostrowska-Czubenko
Chitosan/trisodium citrate (Ch/CIT) membranes

were obtained by immersing
of pure chitosan membranes in aqueous 5.0% (w/v) sodium citrate solution for 60 min.
Following crosslinking conditions were applied: T
crosslink.
= 4 ºC, pH = 5.0 (initial solution
acidified with HCl). After crosslinking membranes were repeatedly washed with deionised
water and dried as pure chitosan membranes.
Chitosan/sodium tripolyphosphate (Ch/TPP) membranes

were prepared by
immersing of pure chitosan membranes in aqueous 1.3% (w/v) phosphate solution for
60 min. Applied crosslinking conditions were as follows: T
crosslink.
= 4 ºC, pH = 5.5.
Chitosan/sodium alginate (Ch/NaAlg) membranes

were prepared by casting
solution and solvent evaporation technique. 1 wt.% chitosan solution in 2 wt.% acetic acid
and 1 wt.% sodium alginate solution were mixed in the 3 : 1 volume ratio, respectively
(pH
solution mixture
= 3.5). The crosslinked membranes were additionally thoroughly washed
with deionised water and dried as pure chitosan films.
2.4. FTIR spectroscopy analysis
FTIR spectra of chitosan and ionically crosslinked chitosan (ICCh) samples in KBr
disc form were recorded on Perkin‑Elmer 2000 FTIR spectrometer from 400 to 4000 cm
-1

with a resolution 4 cm
-1
and at 100 scans. Ch and ICCh polymers were thoroughly powdered
and powders dried under vacuum at 60ºC for 24 hours before milling with anhydrous KBr.
2.5. Kinetics swelling experiments
Ch and ICCh membranes ability to swell was determined by kinetics swelling
experiments in acidic, inert and basic buffer solutions at constant ionic strength I = 0.145 M
and at 37 ºC. Solution media of pH 1.2, pH 7.4 and pH 8.5 were used, simulate gastric fluid,
plasma blood and small intestinal fluid, respectively. The swelling behaviour was measured
by immersing a dry membrane sample of a known weight in buffer solution on definite time
period. Membrane was weighed periodically after carefully drying its surface with a filter
paper. The degree of swelling (
S
) was calculated according to the formula:
S
= (
W
t
-
W
0
)/
W
0
where:
W
0
- weight of the dry sample in g,
W
t

- weight of the swollen sample in g at time
t
.
3. Results and discussion
FTIR spectra of unmodified (Ch) and modified chitosan (ICCh) are shown in
Figure 3
. All spectra exhibit a strong and broad nonsymmetric band at about 3430 cm
-1

that results from overlapping of the O‑H and N‑H stretching vibrations of functional groups
engaged in hydrogen bonds [6]. The spectrum of Ch exhibits characteristic absorption bands
at 1656 cm
-1
(C=O stretching in amide group, amide I vibration), 1598 cm
-1
(N‑H bending
in nonacetylated 2‑aminoglucose primary amine) and 1560 cm
-1
(N‑H bending in amide
group, amide II vibration). Absorption bands at 1153 cm
-1
(antisymmetric stretching of the
C‑O‑C bridge), 1083 cm
-1
and 1031 cm
-1
(skeletal vibrations involving the C‑O stretching)
are characteristic of chitosan saccharide structure [7].
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
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Effect of Ionic Crosslinking Agents on Swelling Behaviour of Chitosan Hydrogel Membranes
Figure 3.
FTIR spectra of chitosan and ionically crosslinked chitosan membranes
After crosslinking process in the spectra of all ICCh samples appears a new band
at 1635 cm
-1
. This band, that can be assigned to antisymmetric deformation N‑H vibrations
in NH
3
+
ion [6], was observed earlier by us and others for ionically crosslinked chitosan
membranes [6, 8 ‑ 10]. Moreover, additional changes in the spectra of chitosan after its ionic
crosslinking can be observed: (i) in the spectrum of Ch/H
2
SO
4
the new absorption band at
617 cm
-1
appears that corresponds to S‑O bending vibration in sulfuric

ions [6], (ii) in the
spectrum of Ch/CIT the new band at 1380 cm
-1
appears. It corresponds to C‑O symmetric
vibrations in COO
-
ions [6], (iii) in the spectrum of Ch/TPP the new band at 1223 cm
-1

appears that corresponds to ‑P=O stretching vibrations in phosphate ions [6], (iv) in the
spectrum of Ch/NaAlg the new absorption band at 1411 cm
-1
appears that corresponds to
C‑O antisymmetric vibrations in COO
-
ions [5, 6].
Spectral changes characterized above indicate the formation of ionic crosslinks
between chitosan and crosslinking agents as presented
Figure 4
.
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
80
M. Pieróg, M. Gierszewska-Drużyńska, J. Ostrowska-Czubenko
(A)
O
H
H
NH
2
H
OH
CH
2
OH
O
H
H
NH
3
+
H
OH
CH
2
OH
O
H
H
H
OH
CH
2
OH
O
O
O
H
OH
O
H
H
H
CH
2
OH
NH
2
H
O
H
H
H
CH
2
OH
HNH
3
+
H OH
O
H
H
H
CH
2
OH
HNH
2
H OH
O
O
O
H
S
O
-
O
O
-
O
C
CH
3
NH
O
(B)

O
H
H
NH
2
H
OH
CH
2
OH
O
H
H
NH
3
+
H
OH
CH
2
OH
O
H
H
H
OH
CH
2
OH
O O
O
H
OH
O
H
H
H
CH
2
OH
NH
2
H
O
H
H
H
CH
2
OH
HNH
3
+
H
OH
O
H
H
H
CH
2
OH
HNH
2
H OH
O
O
O
H
C
COO
-
COO
-
HO
H
2
C
H
2
C COO
-
C
CH
3
NH
O
(C)

O
H
H
NH
2
H
OH
CH
2
OH
O
H
H
NH
3
+
H
OH
CH
2
OH
O
H
H
H
OH
CH
2
OH
O
O
O
H
OH
O
H
H
H
CH
2
OH
NH
2
H
O
H
H
H
CH
2
OH
HNH
3
+
H OH
O
H
H
H
CH
2
OH
HNH
2
H
OH
O
O
O
H
P
O
-
O
O
-
O
PO
-
O
O
PO
-
O
O
-
C
CH
3
NH
O
(D)
O
H
H
NH
2
H
OH
CH
2
OH
O
H
H
NH
3
+
H
OH
CH
2
OH
O
H
H
H
OH
CH
2
OH
O
O
O
O
H
H
H
OH
COO
-
O
OH
O
H
H
H
OH
O
OH
O
H
H
OH
OH
O
COO
-
COO
-
H
H
H
H
C
CH
3
NH
O
Figure 4.
The chemical structure of ionically crosslinked chitosan membranes:
(A) Ch/H
2
SO
4
, (B) Ch/CIT, (C) Ch/TPP, (D) Ch/NaAlg.
The results obtained for the swelling kinetics at pH 1.2, 7.4 and 8.5 are reported
in
Figure 5
. As can be seen in
Figure 5.A
, all uncrosslinked and crosslinked chitosan
membranes swollen in acidic media underwent disintegration. It has been observed by us
that the time needed to membrane disintegration in acidic media increased in the following
order: Ch ≈ Ch/H
2
SO
4
≈ Ch/CIT < Ch/NaAlg << Ch/TPP.
In buffer solution of pH 7.4 (
Figure 5.B
) the studied membranes, except pure
Ch and Ch/H
2
SO
4
, were stable and reached constant swelling degrees. Degree of swelling
at equilibrium state decreased in the following order: Ch > Ch/H
2
SO
4
> Ch/CIT > Ch/
NaAlg > Ch/TPP. Thus, it can be stated that at pH 7.4 the membrane swelling ability changes
in the same order.
In buffer solution of pH 8.5 (
Figure 5.C
) all examined membranes were stable
and the constant
S
value was attained. In comparison to results presented in
Figure 5.B
, the
corresponding
S
values were lower. The decreasing values of equilibrium swelling degree
allow putting the Ch and ICCh membranes in following order: Ch < Ch/H
2
SO
4
< Ch/
NaAlg < Ch/CIT < Ch/TPP.
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
81
Effect of Ionic Crosslinking Agents on Swelling Behaviour of Chitosan Hydrogel Membranes
(A)
(B)
(C)
Figure 5.
Swelling kinetics of ionically crosslinked membranes at (A) pH 1.2, (B) pH 7.4 and (C)
pH 8.5 (* - moment of membrane disintegration).
The comparison of the swelling results presented above indicates the high
pH‑dependence of swelling process. The swelling behaviour of non‑modified chitosan
membranes and chitosan membranes crosslinked with low molecular ionic agents in all
studied buffer solutions is very similar. In general, Ch membrane exhibits the highest
swelling degree values and the lowest mechanical resistance while Ch/TPP membrane
exhibits the lowest
S
values and the highest mechanical resistance.
The swelling behaviour of chitosan membranes crosslinked with sodium alginate
is different from characterized above. It most probably results from difference in density
of ionic crosslinking. Ch/NaAlg membrane is polyelectrolyte complex membrane of high
ionic crosslinking density. Ch/H
2
SO
4
, Ch/CIT and Ch/TPP membranes have very similar
but not very high ionic crosslinking density.
It is well known, that swelling ability of ionically crosslinked membranes
depends strongly on hydrophilicity of the whole network [11]. Chitosan membrane after its
crosslinking becomes less hydrophilic as a result of loss of amino binding sites engaged in
reaction with CA. Simultaneously, the hydrophilicity of crosslinking agent used in network
formation influences the network hydrophilicity. The hydrophilicity of low molecular CAs
used in chitosan crosslinkig processes increases in order: TPP < CIT < H
2
SO
4
[12]. Results
Progress on Chemistry and Application of Chitin and Its ...
, Volume XIV, 2009
82
M. Pieróg, M. Gierszewska-Drużyńska, J. Ostrowska-Czubenko
presented in
Figure 5
indicate analogous relation between swelling degree values and
hydrophlicity of low molecular crosslinking agent.
4. Conclusions
The results reported above can be summarized as follows:
n
Modification of chitosan membranes by addition of low and/or high molecular
crosslinking agents leads to the formation of ionically crosslinked chitosan membranes.
n
FTIR spectroscopy indicates the formation of ionic crosslinks between protonated amino
groups of chitosan and anionic functional groups of crosslinking agents.
n
For all studied membranes the degree of swelling is pH‑dependent.
n
The type of crosslinking agent and its hydrophilicity and the crosslinking density are
important factors determining both the pH‑sensitive swelling behaviour as well as the
stability of chitosan modified hydrogel membranes.
5. References
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; (2001) Polysaccharides as Biomaterials. In: Dumitriu S (ed), Polymeric Biomaterials.
CRC Press, Boca Raton, pp. 1-62.
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; (2002) Chitin and chitosan. Part I. Properties and production. Polymer (War
-
saw) 47, pp. 316-325.
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; (2004) Fast Swelling Hydrogel Systems. In: Yui N., Mrsny R.J.,
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; (1998) Effect of crosslinker functionality on swelling
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; (2007) Synteza i właściwości membran
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