Effect of chitosan characteristics and solution conditions on gelation temperatures of chitosan/2-glycerophosphate/nanosilver hydrogels

Article abstract of DOI:10.1016/j.carbpol.2011.01.035

In this study, we explored the effect of nanosilver, chitosan characteristics, and solution conditions on gelation temperatures of chitosan/2-glycerophosphate thermosensitive hydrogel. The gelation temperatures of hydrogels with or without 12 ppm nanosilver were insignificantly different regardless of the molecular weight (MW) of chitosan (degree of deacetylation, DD, 80% or 88%) and glycerophosphate concentration. The gelation temperature of hydrogel decreased with increasing DD (65-88%) of chitosan, increasing concentration of glycerophosphate (2-8%), and increasing pH value (6.5-6.8). It also decreased with decreasing chitosan's MW (DD88: 113-345 kDa, DD80: 145-900 kDa), but increased with increasing concentrations of NaCl (0-2%). By adjusting above conditions, the hydrogel gelation can be induced with a temperature range from 24.3 to 91.7 °C. Chitosan chains with higher flexibility and/or smaller hydrodynamic volume resulted in lower gelation temperature. Hydrogel became white turbidity in 2% NaCl below 38 °C because of its lower solubility at higher ionic strength.

Full text of DOI:10.1016/j.carbpol.2011.01.035

Carbohydrate Polymers 84 (2011) 1337–1343
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Effect of chitosan characteristics and solution conditions on gelation
temperatures of chitosan/2-glycerophosphate/nanosilver hydrogels
Min-Lang Tsai∗, Hsiang-Wei Chang, Hui-Chuan Yu, Ya-Shen Lin, Ying-Die Tsai
Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we explored the effect of nanosilver, chitosan characteristics, and solution conditions on
gelation temperatures of chitosan/2-glycerophosphate thermosensitive hydrogel. The gelation temper-
atures of hydrogels with or without 12 ppm nanosilver were insignificantly different regardless of the
molecular weight (MW) of chitosan (degree of deacetylation, DD, 80% or 88%) and glycerophosphate
concentration. The gelation temperature of hydrogel decreased with increasing DD (65–88%) of chitosan,
increasing concentration of glycerophosphate (2–8%), and increasing pH value (6.5–6.8). It also decreased
with decreasing chitosan’s MW (DD88: 113–345 kDa, DD80: 145–900 kDa), but increased with increas-
ing concentrations of NaCl (0–2%). By adjusting above conditions, the hydrogel gelation can be induced
with a temperature range from 24.3 to 91.7 ◦C. Chitosan chains with higher flexibility and/or smaller
hydrodynamic volume resulted in lower gelation temperature. Hydrogel became white turbidity in 2%
NaCl below 38 ◦C because of its lower solubility at higher ionic strength.
Received 1 November 2010
Received in revised form 14 January 2011
Accepted 20 January 2011
Available online 28 January 2011
Keywords:
Chitosan
2-Glycerophosphate
Nanosilver
Hydrogel
Gelation temperature
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
capacity of protein adsorption and good biocompatibility; in addi-
tion, hydrogel films were a promising candidate for biomaterial
Chitosan/glycerophosphate hydrogel showing sol–gel transi-
tion at a physiological pH and temperature has gained attention.
Applications of the hydrogel had reported as follows: Chenite et
al. (2000) reported that chitosan/glycerophosphate hydrogel was
used to deliver growth factors in vivo and encapsulated living
chondrocytes for tissue engineering applications. Ruel-Gariépy,
Chenite, Chaput, Guirguis, and Leroux (2000) encapsulated chlor-
pheniramine maleate, dextran, methylene blue, and albumin with
chitosan/glycerophosphate hydrogels, respectively, and evaluated
the in vitro release profiles of the above model compounds.
Crompton et al. (2007) reported that chitosan/glycerophosphate
hydrogel provided a suitable 3D scaffolding environment for
neural tissue engineering. Richardson, Hughes, Hunt, Freemont,
and Hoyland (2008) seeded human mesenchymal stem cells
in chitosan/glycerophosphate hydrogels; the stem cells could
be differentiated from nucleus pulposus-like cells. Zhou et al.
(2008) found that the chitosan/glycerophosphate hydrogel was
an ideal sustained release system especially for hydrophilic
drugs, due to the release rate of adriamycin (hydrophilic)
being slower than 6-mercaptopurine (hydrophobic) from chi-
tosan/glycerophosphate hydrogels. Zhao et al. (2009) reported the
chitosan/glycerophosphate hydrogel was suitable for the deliv-
ery of drugs, proteins and enzymes due to its high porosity, high
for pharmaceutical and tissue-engineering applications. Kim et
al. (2010) investigated the in vitro ellagic acid release rate from
chitosan/glycerophosphate hydrogel and the chitosan degradation
rate for cancer treatment. Moreover, a chitosan/glycerophosphate
system containing collagen in different proportions also showed
sol–gel transition at a physiological pH and temperature. The pres-
ence of collagen in chitosan-collagen materials was associated with
increased cell spreading and proliferation, as well as increased gel
compaction and a resulting stiffer matrix (Wang & Stegemann,
2010). Chitosan/glycerophosphate salt formulations were com-
bined with inorganic nanoparticles in order to prepare novel
injectable thermo-responsive hydrogels for orthopaedic applica-
tions (Muzzarelli, 2010). Molinaro, Leroux, Damas, and Adam
(2002) reported that a chitosan/glycerophosphate hydrogel formed
with higher degree of deacetylation (DD) chitosan was desirable in
regard to superior biocompatibility.
The addition of polyol- or sugar-phosphate to the chitosan
solution transformed pH-gelling solutions into thermal-sensitive
pH-dependent gel-forming aqueous solutions (Chenite et al., 2000).
The gelation of the chitosan/glycerophosphate hydrogel system
might involve several interactions as follows: At low tempera-
ture, glycerophosphate can increase the pH of chitosan solutions
to around neutrality. It might screen the electrostatic repulsion
between chitosan molecules and theoretically lead to gelation
behaviour because it is pH-induced. However, due to an attraction
+
between the phosphate moieties of glycerophosphate and –NH3
∗
Corresponding author. Tel.: +886 2 2462 2192x5122; fax: +886 2 2463 4203.
E-mail address: tml@mail.ntou.edu.tw (M.-L. Tsai).
groups of chitosan, hydroxyl groups of glycerophosphate could
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.01.035

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M.-L. Tsai et al. / Carbohydrate Polymers 84 (2011) 1337–1343
increase stability and hydrophilicity in the chitosan chain, as well
as maintain its solubility at low temperatures for a period of time.
When the temperature increased, (1) it reduced the polarity of the
chitosan chain and glycerol moiety of glycerophosphate increased
the hydrophobicity. (2) This caused chitosan chain dehydration
and increased an interchain hydrophobic attraction. (3) H+ was
removed from –NH3+ and was accepted by –PO42−, thereby further
reducing both the chitosan chain charge density and the attraction
of chitosan and glycerophosphate; this allowed the hydrophobic
and hydrogen-bonding between chains to predominate, and upon
heating of the chitosan/glycerophosphate solution a hydrogel was
formed (Chenite et al., 2000; Chenite, Buschmann, Wang, Chaput,
& Kandani, 2001; Cho, Heuzey, Bégin, & Carreau, 2006; Kim et al.,
2010; Ruel-Gariépy et al., 2000).
The characteristics of chitosan such as DD and molecular weight
(MW) and the preparation conditions include concentration of
chitosan, type and concentration of glycerophosphate, pH value of
the solution, concentration of urea, and so on, affect the gelation
temperature and gelation rate of chitosan/glycerophosphate
hydrogel (Chenite et al., 2000, 2001; Cho et al., 2006; Kim et
al., 2010; Ruel-Gariépy et al., 2000; Wang & Stegemann, 2010;
Wu, Su, & Ma, 2006; Zhou et al., 2008). Factors, such as the DD
and MW of chitosan, pH value of solution, concentration of urea,
and so forth, influence the hydrodynamic behaviour and chain
flexibility of chitosan (Chen & Tsaih, 1998, 2000; Chen, Lin, &
Lin, 1994; Chen, Tsaih, & Lin, 1996; Chen, Chen, Wang, Hsu, &
Tsai, 2009; Tsaih & Chen, 1997, 1999). Therefore, studying the
relationships between gelation temperature or gelation rate of
chitosan/glycerophosphate hydrogel and hydrodynamic behaviour
and chain flexibility of chitosan is worthwhile.
The antimicrobial activity of nanosilver has been well estab-
lished and is great interest. Nanosilver has several biotechnological
applications including wound dressings, nanosilver coated or
impregnated ceramic water filters, activated carbon air filters,
and catheters (Kora, Sashidhar, & Arunachalam, 2010; Maneerung,
Tokura, & Rujiravanit, 2008; Sharma, Yngard, & Lin, 2009). Adding
nanosilver to chitosan/glycerophosphate hydrogel enhances the
antimicrobial activity of the hydrogel and expands the hydrogel’s
applications.
The goal of this study was to investigate the effect of nanosil-
ver, MW and DD of chitosan, concentration of glycerophosphate,
and pH value and ionic strength of solutions on gelation temper-
atures of chitosan/2-glycerophosphate hydrogel. The relationships
between the gelation temperature of hydrogel and the hydrody-
namic behaviour as well as the chain flexibility of chitosan were
also discussed.
immersed in 500 ml of 1 M of hydrochloric acid solution overnight.
The sample was washed to neutrality and drained. Then, the sample
was soaked in 500 ml of 2 M of sodium hydroxide at an ambient
temperature overnight, washed and drained. Subsequently, sample
was reacted in 500 ml of 2 M of sodium hydroxide solution at 100 ◦C
for 4 h, washed to neutrality and dried. ␤-Chitin was added to a
50% (w/w) sodium hydroxide solution at a ratio of 1 (g solid):10
(ml solution). The deacetylation reaction took place at 100 ◦C for
1 h, 100 ◦C for 4 h and 140 ◦C for 4 h, respectively. Then the chitosan
was washed to neutrality and freeze-dried (Tsai, Bai, & Chen, 2008).
2.3. Preparation of same DD but different MW chitosans
The chitosans were prepared by a modified method proposed
by Feng, Gong, Du, and Huang (2009). Chitosan was dissolved in
1% (v/v) aqueous acetic acid. The pH value of the final solution was
5.5, which was regulated with 2 N NaOH at the optimum pH value
for cellulase. The cellulase (100 U/g) was used to hydrolyze chi-
tosan for 3, 6, 12, 18, 24, 36 and 48 h at 50 ◦C. At the end of the
reaction, cellulase was denatured quickly, by heating for 10 min at
100 ◦C. The supernatant was isolated by 10,000 rpm centrifugation
for 30 min at 4 ◦C. Then the supernatant was precipitated by adding
2 N NaOH, was washed with de-ionized water until neutrality and
freeze-dried.
2.4. Measurement of DD of chitosan
Infrared spectrometry was used to determine the DD of the chi-
tosan (Baxter, Dillon, Taylor, & Roberts, 1992). Chitosan powder was
sieved through a 200 mesh and then mixed with KBr (1:100), dried
at 60 ◦C for 3 days to prevent interference of the –OH group in FTIR
measurements, and pressed into a pellet. The absorbance of amide
1 (1655 cm−1) and the hydroxyl band (3450 cm−1) were measured
using a Bio-Rad FTS-155 infrared spectrophotometer (Hercules, CA,
USA). The band of the hydroxyl group at 3450 cm−1 was used as
an internal standard to correct for disc thickness and for differ-
ences in chitosan concentration when making the KBr disc. The
percentage of the amine group’s acetylation in a sample was given
by (A1655/A3450) × 115. Here, A1655 and A3450 were the absorbances
at 1650 cm−1 and 3450 cm−1, respectively. Every sample measure-
ment was repeated three times.
2.5. Determination of MW of chitosan
The size exclusion high performance liquid chromatography
(SE-HPLC) method of Tsai et al. (2008) was followed. A column
(7.8 mm × 30 cm) packed with TSK gel G4000 PWXL and G5000
PWXL (Tosoh Co. Ltd., Japan) was used. The mobile phase consisted
of 0.2 M of acetic acid/0.1 M sodium acetate and 0.008 M of sodium
azide. A sample concentration of 0.1% (w/v) was loaded and eluted
with a flow rate of 0.6 ml/min by an LDC Analytical ConstaMetric
3500 pump. The elute peak was detected by an RI detector (Gilson
model M132, USA). The data were analyzed by Chem-Lab software
(Scientific Information Service, Taipei, Taiwan). Pullulan standards
(Shodex, Kawasaki, Japan) with different MW values were used as
markers. The MW values of the samples were calculated from the
pullulan calibration curve with Chem-Lab software.
2. Experimental
2.1. Materials
Squid (Illex argentinus) pens were donated as a gift from Shin Dar
Bio-Tech. Co. Ltd. (Taoyuan, Taiwan). 2-Glycerophosphate, acetic
acid, sodium acetate, sodium azide, sodium chloride, silver nitrate,
sodium borohydride and potassium bromide were purchased from
the Sigma–Aldrich Co. (MO, USA). Hydrochloric acid and sodium
hydroxide were purchased from Merck & Co., Inc. (Darmstadt, Ger-
many). Cellulase was purchased from the Challenge Bioproducts
Co., Ltd. (Yunlin, Taiwan). Pullulan standards (for SE-HPLC calibra-
tion) were purchased from Showa Denko (Tokyo, Japan).
2.6. Intrinsic viscosity measurement
A capillary viscometer (Cannon-Fenske, No 75) was used to mea-
sure the passage time of solutions flowing through the capillary.
Solutions of chitosan (80% DD, 225 kDa) in 1% acetic acid contain-
ing 0.1%, 0.5%, 1.0%, and 2.0% glycerophosphate, were prepared. The
capillary viscometer was filled with 5 ml of the sample and equili-
brated in a water bath (Tamson TMV 40, Holland) at 30 0.1 ◦C for
2.2. Preparation of chitosan
␤-Chitin was prepared from squid pens. In brief the squid pens
were ground to a 40–60 mesh size. Each 100 g batch of powder was

M.-L. Tsai et al. / Carbohydrate Polymers 84 (2011) 1337–1343
1339
95
85
75
65
55
45
35
25
10 min. The sample was passed through the capillary once before
the running time was measured. The running time was used to cal-
culate the relative viscosity, specific viscosity and reduced viscosity.
Then, the reduced viscosity was plotted against the concentration,
with the intercept being the intrinsic viscosity (Chen & Tsaih, 1998;
Tsaih & Chen, 1997).
a
DD80/900kDa
DD80/900kDa/Ag
DD80/145kDa
DD80/145kDa/Ag
2.7. Preparation of thermosensitive hydrogel solutions
The chitosan/glycerophosphate hydrogel solution was prepared
by a modified method proposed by Chenite et al. (2001). Chitosan
with different DDs and MWs (200 mg) was dissolved in 1% (v/v)
aqueous acetic acid (9 ml) and stirred for 12 h until complete dis-
solution was achieved. Glycerophosphate (200–800 mg) and NaCl
(0–200 mg) were dissolved in distilled water and stirred for 15 min
into a clear solution. Both the chitosan solution and glycerophos-
phate/NaCl solution were chilled at 4 ◦C for 1 h, respectively, then
these solutions were mixed and stirred for 2 min under an ice bath.
Next, the hydrogel solution was shaken for 40 s by a vortex shaker.
Finally, the hydrogel solution was obtained and the pH value of the
solution was adjusted within the range of 6.5–6.8. The hydrogel
solution eventually was made up of 2% chitosan, 2–8% glycerophos-
phate, and 0–2% NaCl.
1
2
3
4
5
6
7
8
9
2-Glycerophosphate (%)
55
50
45
40
35
30
25
b
DD88/345kDa
DD88/345kDa/Ag
DD88/113kDa
DD88/113kDa/Ag
2.8. Preparation of thermosensitive hydrogel containing silver
nanoparticles
The nanosilver solution was prepared by a modified method
proposed by Lee and Meisel (1982). The 30 ml of 0.002 M sodium
borohydride solution was chilled in an ice bath for 20 min, and
6 ml of 0.001 M silver nitrate solution was slowly dropped into
the sodium borohydride solution. Then the silver nanoparticle was
formed and easily observed thanks to a visible colour change of the
solution to pale yellow. The size of the nanoparticles was deter-
mined by scanning electron microscopy (SEM) (Hitachi, S-4800,
Tokyo, Japan). Then the nanosilver solution was added to the chi-
tosan/glycerophosphate solution. The concentration of nanosilver
in the hydrogel solution was determined by an atomic absorption
spectrophotometer (PerkinElmer 510 OPC, Norwalk, CT, USA).
1
2
3
4
5
6
7
8
9
2-Glycerophosphate (%)
Fig. 1. Change of gelation temperature of chitosan/2-glycerophosphate hydrogels
formed with or without silver nanoparticles at different 2-glycerophosphate con-
centrations in pH 6.5 solution.
nanosilver will insignificantly affect the gelation temperature of
chitosan/glycerophosphate hydrogel.
3.2. Effect of DD on gelation temperature
2.9. Determination of hydrogel’s gelation temperature
Table 1 shows the effect of the DD and MW of chitosan and
the concentration of glycerophosphate on the gelation tempera-
ture of chitosan/glycerophosphate/nanosilver hydrogels formed in
a pH 6.5 solution. Results show that the gelation temperatures of
hydrogels which were prepared with chitosan that had different
DDs but similar MWs, decreased with the increase in DD. These
results are similar to reports by Chenite et al. (2000) and Ruel-
Gariépy et al. (2000). They considered that a higher DD of chitosan,
which had more amine groups, could form more cross-links with
the phosphate group of glycerophosphate. This led to an increase
in the gelation rate and consequently decreased the gelation tem-
perature. However, Zhou et al. (2008) reported that the viscosity of
chitosan/glycerophosphate hydrogel, prepared with a chitosan DD
of 75.4%, increased quickly at 37 ◦C, while others increased either
slowly or variedly. Therefore, the optimal DD for prepared hydrogel
was 75.4%.
A
chitosan/glycerophosphate hydrogel solution (2 ml) was
poured into a 10 ml tube to determine the gelation temperature in
a water bath at 25 ◦C. We determined the temperature in the test
tube using a temperature detector (SUNTEX, SP-701, Taipei, Tai-
wan), and kept the temperature constant for 1 min after the tube
temperature was the same as the set temperature. The sol–gel tran-
sition temperature was determined by a flow or no-flow criterion
over 30 s with the test tube inverted (Chung, Simmons, Gutowska,
& Jeong, 2002).
3. Results and discussion
3.1. Effect of nanosilver on gelation temperature
The particle size of the nanosilver was measured by
SEM and found to be 21.8 nm. The nanosilver concentration
of chitosan/glycerophosphate hydrogel solution was deter-
mined by atomic absorption spectrophotometer and found
A lower DD of chitosan has more –NCOCH3 on the molecular
chain. It forms a large three-dimensional spatial barrier for rotation
of the ␤-1,4 glycoside, so the chitosan chain is more rigid. Con-
versely, a higher DD of chitosan has less –NCOCH3 on the molecular
chain. It induces easy turning of the glycosidic bond, so the chitosan
chain is more flexible (Chen et al., 1996). In the gelling course of chi-
tosan/glycerophosphate hydrogel, the hydrated chitosan molecular
chains were gradually dehydrated; the chitosan chains interacted
with each other and then rearranged chains to form crystalline
regions and gel formation. Conformation and chain flexibility in
to be 12 ppm. Fig.
1 shows the gelation temperature of
chitosan/glycerophosphate hydrogel with or without 12 ppm
nanosilver. Results show that the gelation temperatures of
hydrogels with or without nanosilver were insignificantly dif-
ferent regardless of the MW of chitosan (DD 80% or 88%) and
concentration of glycerophosphate. This indicates that adding

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M.-L. Tsai et al. / Carbohydrate Polymers 84 (2011) 1337–1343
Table 1
Effect of degree of deacetylation and molecular weight of chitosan and concentration of 2-glycerophosphate on gelation temperature (◦C) of chitosan/2-
glycerophosphate/nanosilver hydrogels formed in pH 6.5 solution.
DD (%)
MW (kDa)
2-Glycerophosphate (%)
SLa
SH
SL/SH
2%
3%
4%
5%
6%
8%
88
345
326
270
225
204
160
146
113
52.0 0.0b
51.0 0.0
51.0 0.0
49.7 1.2
52.7 2.3
49.0 1.0
48.3 2.3
47.0 4.0
44.0 1.0
44.3 0.6
43.3 0.6
42.7 0.6
43.0 1.0
41.3 2.9
41.3 0.6
40.3 0.6
40.3 0.6
40.0 0.0
39.7 0.6
39.0 1.0
37.3 1.5
37.3 1.5
37.3 1.2
37.3 1.2
38.3 0.6
38.0 0.0
38.0 0.0
37.3 0.6
32.3 0.6
32.3 1.5
32.3 2.1
32.0 1.0
37.3 0.6
37.3 0.6
37.0 0.0
37.3 0.6
31.7 0.6
32.0 0.0
31.7 0.6
32.7 0.6
36.0 1.0
36.0 1.0
36.0 1.0
36.0 1.0
30.3 0.6
30.3 0.6
30.0 0.0
30.0 0.0
−4.5
−0.6
−0.5
−0.6
−0.4
−0.6
−0.6
−0.7
−0.7
7.5
8.6
7.2
10.3
11.2
9.0
−4.3
−4.3
−4.1
−6.7
−5.4
−5.2
−4.8
7.4
6.9
80
900
497
407
335
198
161
145
88.3 3.5
60.7 1.2
55.0 0.0
55.0 2.0
54.7 2.1
55.0 2.0
51.7 0.6
77.7 2.5
55.7 2.5
46.3 2.3
44.7 1.5
44.7 1.5
44.0 2.0
43.3 1.2
58.0 3.6
46.3 8.4
45.3 3.8
41.7 2.1
41.3 1.2
41.0 1.0
40.3 1.2
56.3 2.3
43.0 5.3
38.7 0.6
40.0 1.0
39.3 1.2
39.3 1.2
39.3 1.2
51.7 3.5
39.0 1.0
37.3 2.1
39.0 1.0
38.3 0.6
38.3 0.6
37.0 0.0
50.0 1.0
39.0 1.0
36.3 1.5
36.5 0.6
36.3 2.9
36.3 0.6
36.7 1.2
−11.6
−6.3
−5.0
−4.8
−5.0
−5.0
−4.0
−1.8
−1.3
−0.8
−1.1
−1.0
−1.0
−0.8
6.4
4.8
6.3
4.4
5.0
5.0
5.0
65
530
91.7 2.3
81.0 1.7
83.7 1.2
76.7 7.5
75.0 6.9
65.7 3.1
−4.8
−3.8
1.3
a
SL and SH express the slopes of plots in 2-glycerophosphate concentration of 2–5% and 5–8%, respectively.
Value of gelation temperature represents mean S.D (n = 3).
b
the gelling process play an important role. Chitosan chain flexibil-
indicator of hydrodynamic volume: the greater the intrinsic viscos-
ity, the more extended the conformation of the molecular chain.
The phosphate group of glycerophosphate is the counter-ion for
chitosan. When the concentration of glycerophosphate is increased,
it enhances the neutralization charge on the –NH3+groups of chi-
tosan chains (Chen et al., 1996; Tsaih & Chen, 1999). Therefore,
reducing both the chitosan chain charge density and the elec-
trostatic repulsion between the –NH3+groups (Chen et al., 1996)
increases the chain flexibility. Then the chitosan chains are easier
to close, entangle, interact and gel.
Table 1 also shows that the gelation temperatures of chi-
tosan/glycerophosphate/nanosilver hydrogels prepared with
the same DD but different MW chitosans, decreased with
the decreasing MW of chitosan. However, this trend, with
an increasing concentration of glycerophosphate, becomes
imperceptible; for example, the gelation temperature of chi-
ity increased as DD increased (Chen et al., 1996). Molecular chains
were more flexible, creating easier turning of the glycosidic bond,
and it became easier to change the original molecular conformation
in the gelation process. Then chitosan chains approached, entan-
gled and interacted with each other; the result was a rearrangement
of chains and crystalline regions formed, resulting in decreased
gelation temperature.
3.3. Effect of MW and glycerophosphate concentration on
gelation temperature
Table 1 also indicates that the gelation temperature of chi-
tosan/glycerophosphate/nanosilver hydrogel decreased with the
increased concentration of glycerophosphate. This result was sim-
ilar to the reports by Chenite et al. (2001), Wang and Stegemann
(2010) and Wu et al. (2006). Fig. 2 shows that the concentration of
glycerophosphate affected the intrinsic viscosities of chitosan (80%
DD, 225 kDa) at 30 ◦C. Fig. 2 indicates that the intrinsic viscosities
of chitosan decreased from 11.5 to 4.7 dl/g with increasing con-
centrations of glycerophosphate (0.1–2.0%). Intrinsic viscosity is an
tosan/glycerophosphate/nanosilver
hydrogels
which
were
prepared with a DD of 88% and MWs of 113–204 kDa chi-
tosans mixed 4% glycerophosphate was 37.3 ◦C. Zhou et al. (2008)
reported that the viscosity of chitosan/5% glycerophosphate hydro-
gels prepared with different MW chitosans (75.5% DD) increased
as the MW increased from 88 to 1360 kDa when incubated at
37 ◦C. Therefore, the increase of MW was favourable for sol-to-gel
transition, and a high MW chitosan was optimal for hydrogel
preparation. However, Chenite et al. (2000) reported that the effect
of the chitosan’s MW on the gelation temperature of chitosan/5.6%
glycerophosphate hydrogel (pH 7.15) prepared with 81% DD and
124–846 kDa chitosan was insignificant.
12
10
8
The effect of the chitosan’s MW on the gelation tempera-
ture of chitosan/glycerophosphate hydrogel became less obvious
with increasing concentration of glycerophosphate, which may be
related to the hydrodynamic behaviour and chain flexibility of chi-
tosan. When the concentration of glycerophosphate was lower,
the electric shielding effect of glycerophosphate on the chitosan
6
+
was weaker, and the chitosan chain had more –NH3 groups.
Consequently, the intrinsic viscosity was greater (Fig. 2), i.e. the
hydrodynamic volume was larger. Therefore, the effect of the chi-
tosan’s MW on the hydrodynamic volume was more evident and
caused the gelation temperature to differ more. However, when the
concentration of glycerophosphate was higher, the electric shield-
ing effect of the phosphate group on the –NH3+ group of a chitosan
chain was stronger, and the chitosan chain had fewer –NH3+ groups.
4
0.0
0.4
0.8
1.2
1.6
2.0
2-Glycerophosphate (%)
Fig. 2. The 2-glycerophosphate concentration affected the intrinsic viscosities of
chitosan (80% DD, 225 kDa) in 1% acetic acid solution at 30 ◦C.

M.-L. Tsai et al. / Carbohydrate Polymers 84 (2011) 1337–1343
1341
60
55
50
45
40
35
50
DD80/407kDa
45
DD80/671kDa
DD80/145kDa
DD88/146kDa
y = -4.99 x + 63.79
2 = 0.93
R
40
35
30
y = -0.75 x + 42.20
R
2 = 0.92
25
20
1
2
3
4
5
6
7
8
9
6.4
6.5
6.6
6.7
6.8
6.9
2-Glycerophosphate (%)
pH
Fig. 3. The 2-glycerophosphate concentration affected the gelation temperature of
hydrogel formed with 80% DD, 407 kDa chitosan.
Fig. 4. The pH of the solution affected the gelation temperature of chitosan/6% 2-
glycerophosphate hydrogel. The pH differences were manipulated by 2 N NaOH or
1 N HCl solution.
Furthermore, the chain flexibility of chitosan with a higher MW
was greater and conformation was more contracted. In contrast,
the chain flexibility of a lower MW was more rigid and had more
extended conformation (Chen et al., 2009; Tsai et al., 2008; Tsaih
& Chen, 1997). For these reasons, the higher and lower MWs of
chitosan had little effect on the hydrodynamic volume with higher
concentrations of glycerophosphate, and caused little difference to
the gelation temperature. Chenite et al. (2000) indicated that the
effect of the chitosan’s MW on the gelation temperature of chi-
tosan/glycerophosphate hydrogel was insignificant. This may be
attributed to the hydrogel solution having a higher glycerophos-
phate concentration and higher pH value.
3.4. Effect of pH on gelation temperature
Fig.
4
shows
the gelation
temperature
of
chi-
tosan/glycerophosphate hydrogel with different pH values.
Results show that the gelation temperature of the hydrogel
decreased with the increase in the solution’s pH value (Fig. 4).
These results were similar to the reports by Chenite et al. (2000,
2001) and Kim et al. (2010). These results may be due to the
higher pH value of hydrogel causing a lower degree of chitosan
protonation. Therefore, glycerophosphate had fewer opportunities
+
to combine with –NH3 groups, and the chitosan hydration was
poor (Chenite et al., 2000, 2001). In addition, the pH value of
the hydrogel was higher and the degree of chitosan protonation
was lower, indicating decreased solubility of the chitosans. In an
endothermic process, the protons are released more easily and
hydrating water is more easily removed from the chitosan, with
increased hydrophobic interactions between chitosan molecules
(Kim et al., 2010). Furthermore, the chitosan molecular charge
density was lower, which while reducing the charge repulsion
As shown in Fig. 1, the influence trend of glycerophosphate
concentrations on the gelation temperature with different chi-
tosan MWs was not consistent. For instance, Fig. 1a shows that
the gelation temperature of two different chitosan MWs decreased
as glycerophosphate concentrations increased at a lower concen-
tration of glycerophosphate and the decreasing range was not
consistent. But in the higher concentrations of glycerophosphate,
the decreasing range was almost the same. So gelation tempera-
ture versus glycerophosphate concentration was plotted for each
chitosans, and the SL and SH expressed the slopes of the plots in
glycerophosphate concentration of 2–5% and 5–8%, respectively
(Fig. 3). The SL and SH indicated the effect of glycerophosphate
concentration on the gelation temperature, and are listed in Table 1.
Results in Table 1 indicate that the SL of chitosan with a DD of
80% and 88% was more evident than the SH (SL/SH > 4.4), while the
chitosan with 80% DD and 900 kDa was the greatest among these
SL data. However, the SH of a chitosan with 65% DD was similar to
the SL (SL/SH = 1.3), and the SH was greater than the other SHs of
different DDs for chitosan. The influence trend of glycerophosphate
concentration on gelation temperature for different MWs for chi-
tosan was not consistent, which may relate to the hydrodynamic
behaviour and chain flexibility of chitosan. As the DD of chitosan
decreased (DD 65%), the molecular chain was more rigid; the chi-
tosan did not decrease rapidly in hydrodynamic volume and had a
contracted conformation by charge neutralization, so the SL/SH was
small. The 80% DD and 900 kDa chitosan had a large hydrodynamic
volume due to a large MW, but the molecular chain was more flex-
ible (Chen et al., 2009; Tsai et al., 2008; Tsaih & Chen, 1997). The
hydrodynamic volume of chitosan decreased rapidly, the electric
shielding effect of glycerophosphate created a contracted confor-
mation, and the SL/SH was large. Moreover, the SL/SH of 88%, 80%,
and 65% DD for chitosan were 6.9–11.2, 4.4–6.4, and 1.3, respec-
tively. This indicates that the SL/SH is related to the chitosan DD.
+
between the –NH3 groups of chitosan led to a more flexible chain
(Chen et al., 1994, 1996; Tsaih & Chen, 1999). Consequently, it
was easier for the chitosan interchain to approach, entangle, and
interact, so the chitosan formed a gel easily in the endothermic
process.
65
60
55
50
DD88/146kDa
DD80/145kDa
45
40
35
30
0.0
0.5
1.0
1.5
2.0
2.5
NaCl (%)
Fig. 5. Effect of sodium chloride concentration on gelation temperature of chi-
tosan/6% 2-glycerophosphate hydrogel formed in pH 6.5 solution.

1342
M.-L. Tsai et al. / Carbohydrate Polymers 84 (2011) 1337–1343
Fig. 6. Changes of appearance of 88% DD, 146 kDa chitosan/6% 2-glycerophosphate hydrogel containing 2% sodium chloride formed in pH 6.5 solution.
3.5. Effect of NaCl concentration on gelation temperature
Fig. shows the gelation temperature of chi-
different no matter of the MW of chitosan (DD 80% or 88%) and
concentration of glycerophosphate. The gelation temperature of
the hydrogel decreased with increasing DD of chitosan, concen-
tration of glycerophosphate and pH value. It decreased as the MW
of chitosan decreased and increased with an increased concentra-
tion of NaCl. By adjusting above factors, the hydrogel gelation can
be induced with a temperature range from 24.3 to 91.7 ◦C. Effect
of chitosan’s MW on gelation temperature of hydrogel become
less obvious with increasing concentration of glycerophosphate.
The hydrodynamic behaviour and chain flexibility of chitosan were
affected by the DD and MW of chitosan, concentration of glyc-
erophosphate, pH value of the solution, and concentration of NaCl;
then the gelation temperature was affected. Briefly, the chitosan
chains with higher flexibility and/or smaller hydrodynamic volume
leaded to lower gelation temperature. The appearance of hydro-
gel solution containing 2% NaCl was white turbidity below 38 ◦C,
was different from hydrogel in lower NaCl concentration, due to its
lower solubility at higher ionic strength.
5
tosan/glycerophosphate hydrogel when different concentrations
of NaCl were added. Results show that the gelation temperature of
the hydrogel increased when the concentration of NaCl increased
(Fig. 5). This may be due to the fact that adding NaCl to hydrogel
causes the chloride ions to compete with the phosphate group
+
at the glycerophosphate to neutralize the chitosan –NH3 group
charge. Size and steric hindrance of the chloride ions were less
than for glycerophosphate, causing the chloride ions to interact
+
with a –NH3 group more predominantly than with the glyc-
erophosphate, thereby resulting in a more contracted chitosan
chain. However, these small ions formed diffuse double layers on
the surface of chitosan chains, limited the glycoside of a chitosan
rotation, caused the molecular chains to become more rigid (Chen
et al., 1994; Tsaih & Chen, 1999), and hindered the chitosan chains
from closing with one another. Therefore, for a chitosan interchain
it was more difficult to form a crystalline region and gel while the
temperature was rising. Consequently, a greater quantity of NaCl
was added to the hydrogel, and it was more difficult to form a gel,
resulting in a higher gelation temperature (Fig. 5).
At lower concentrations of NaCl (0.25%, 0.5%), the hydrogel solu-
tion’s appearances were transparent below gelation temperatures.
However, when the concentration of NaCl was 2%, the hydrogel
solution appearance was white turbidity below 38 ◦C (Fig. 6a),
and changed into more transparent at higher temperature (Fig. 6b
and c), then converted into white turbidity again due to gelation
(Fig. 6d). This may be due to the number of chloride ions being
about three times the number of amine groups. The concentration
of chloride ions was high and the charge neutralization was strong,
leading to a strongly contracted chitosan chain. The interaction
of the intrachain and interchain was strong, leading to decreasing
solubility. Therefore, the appearance of hydrogel solution showed
white turbidity below 38 ◦C. However, the conformation of chitosan
became more extended as the temperature rose (Chen & Tsaih,
1998). The molecular movement was more intense than it had been
at low temperature, so the interaction between an intrachain and
interchain was weaker. Sequentially the solubility of chitosan was
increasing and the appearance of solution was changed into more
transparent.
Acknowledgements
The authors wish to express their appreciation for the financial
support from National Science Council, ROC (NSC 97-2313-B-019-
007-MY3) and Stone & Resource Industry R&D Center (Taiwan)
[SRDC-770-A1-56(99)].
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