International Journal of Biological Macromolecules 121 (2019) 1101–1108

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Dissolution behavior and conformation change of chitosan in

concentrated chitosan hydrochloric acid solution and comparison with
dilute and semidilute solutions
Jianfeng Wu, Liyan Zhang ⁎
School of Food Science and Engineering, South China University of Technology, Wushan Road, 381, Guangzhou, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Viscosity, pH and electrolytic conductivity of dilute, semidilute and concentrated chitosan solution with hydro-
Received 18 July 2018 chloric acid adding were compared and the change in conformation of chitosan in concentrated solution was
Received in revised form 11 October 2018 discussed. The dissolution behavior of chitosan in concentrated chitosan hydrochloric acid solution was investi-
Accepted 15 October 2018
gated through determination of solution turbidity, microstate and size of chitosan particle. It was found that the
Available online 16 October 2018
concentration, degree of deacetylation (DD) of chitosan and content of hydrochloric acid in solution showed sig-
Keywords:
nificant effects on the changes in viscosity, pH, and electrolytic conductivity of chitosan solution as hydrochloric
Chitosan acid added. And the transforms of above properties of chitosan solution during dissolution were corresponding to
Dissolution behavior the proportion of hydrochloric acid to chitosan from which we could deduced that the confirmation of chitosan
Conformation changed from entangling with each other to becoming compact coils due to electrostatic screen of Cl− and hydro-
Concentrated solution phobic aggregation of chitosan with high concentration. In concentrated suspension solution, chitosan particles
agglomerated first and then expended before disintegrating and dissolving with hydrochloric acid adding,
resulting in solution turbidity, size and microstate of chitosan particle changed obviously during dissolution.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction influences of DD, Mw, acid concentration in concentrated chitosan solu-

Chitosan is a copolymer of glusamine and N-acetyl glucosamine and
prepared by alkaline N-deacetylation of chitin. It has some excellent
properties such as biocompatibility, biodegradability, low toxicity, and
antimicrobial activity and so on [1–4], which attract wide attentions in
the fields of food, textile, agriculture, medicine, and industrial anti-
corrosion etc. [5–9]. Chitosan is quite often used in the form of solution
and its applications are strongly affected by the properties of chitosan
solution like stability, viscosity of solution, and the molecular structure
or conformation of chitosan in solution [10,11]. Those properties are de-
pendent on the types of acids, pH, ionic strength, degree of
deacetylation (DD), molecular weight (Mw) and concentration of chito-
san [10,12,13].
Usually, chitosan can dissolve in strong hydrochloric acid and weak
carboxylic acids like formic acid, acetic acid and propionic acid [13,14],
but the properties of chitosan solution and structure of chitosan have
obvious difference in those two types of solvent. Until now, most of
studies were focused on the solution behaviors of dilute chitosan aque-
ous solution using rheology and light scattering methods [13,15,16],
while the solution behavior, conformation of chitosan and the
⁎ Corresponding author.
E-mail address: liyanzh@scut.edu.cn (L. Zhang).
tion are still unclear and under debate. Nowadays, concentrated chito-
san solution was often used in preparation of chitosan films, artificial
skin and electrospun fibers by chitosan. Oliveira et al. (2012) suggested
that coil dimensions of chitosan in solution decreased and macromolec-
ular overlapping increased as concentration of chitosan increased,
which resulted in higher intermolecular interactions and leading to
more rigid macromolecular systems by studying semidilute and con-
centrated chitosan acetic acid solutions (c = 10 g·L−1 and 20 g·L−1)
through dynamic light scattering (DLS) [17]. Wang et al. (1994) studied
the rheological properties of chitosan formic acid solutions (c =
20 g·L−1, 35 g·L−1 and 50 g·L−1) and the results showed that the
counter-ion concentration around NH3+ moieties of chitosan in solution
increased with increasing the concentration of chitosan and the viscos-
ity of solution decreased due to electrostatic repulsion of the polyelec-
trolyte in acidic solution [10]. There is few reports, however, on
dissolution behavior of concentrated chitosan in hydrochloric acid solu-
tion and the solution behavior characteristics of chitosan - hydrochloric
acid solution with the concentration of chitosan up to 80 g·L−1, which
was often used in chitosanase hydrolysis of chitosan in order to increase
the yield of hydrolysates like chito-oligosaccharides [18,19].
For a better understanding of the properties and rational use of chi-
tosan for preparing artificial skin, medical gauze and separation films
etc., a solid knowledge of the dissolution procedure and solution

https://doi.org/10.1016/j.ijbiomac.2018.10.128
0141-8130/© 2018 Elsevier B.V. All rights reserved.
1102 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108
behavior of concentrated chitosan is useful. In this paper, we investi-
gated the changes in viscosity, pH, and conductivity of chitosan solution
with various DDs, Mws and concentrations during HCl aqueous solution
added dropwise into the suspension solution of chitosan, and observed
the chitosan particles in solution by optical microscope and analyzed by
Particle Sizer so as to explore the dissolution behavior of concentrated
chitosan HCl aqueous solution. Additionally, structure or conformation
of chitosan in solutions was analyzed by fluorescence spectroscopy.
2. Materials and methods
2.1. Materials
Four chitosan samples were purchased from Changlong biological
technology Co. Ltd. (Huizhou, China). Chitosan #1: deacetylation degree
(DD), 75.57%; molecular weight (MW), 1.64 × 105 g/mol; intrinsic vis-
cosity [η], 0.372 L/g. Chitosan #2: DD, 79.25%; MW, 1.41 × 105 g/mol; in-
trinsic viscosity [η], 0.351 L/g. Chitosan #3, DD, 83.24%; MW, 1.37
× 105 g/mol; intrinsic viscosity [η], 0.343 L/g. Chitosan #4: DD, 89.98%,
MW, 1.18 × 105 g/mol; intrinsic viscosity [η], 0.321 L/g. DD of the sam-
ples was determined by UV spectrophotometric method [20] and the
Mw was determined using Mark-Houwink-Sakurada equation [17].
The viscometric parameters were measured at 25 ± 0.05 °C using a
Ubbelohde C529 capillary viscometer [13]. Hydrochloric acid was ana-
lytic grade and purchased from Guangzhou, China. Pyrene was pur-
chased from Sinopharm Chem. Reagent Co. Ltd.
2.2. Preparation of chitosan solutions with various concentrations
Chitosan (8 g, 3 g, 1 g and 0.2 g) was added respectively to 92 mL of
deionized water, then the chitosan-water suspension were stood in
water bath at 45 °C ± 1 °C for 20 min and stirred constantly. 1.0 mL of
6, 2.25, 0.75, 0.15 M HCl aqueous solutions was dropped respectively
per 5 min to the chitosan suspension with constant stirring and a total
of 8 mL HCl solution was added for 40 min.
2.3. Measurement of physicochemical parameters of chitosan solution
The turbidity of solutions was measured at 600 nm using ultraviolet-
visible spectrophotometer (Model 752, Guanke, China). Viscosity of so-
lutions was determined with a digital rotary viscometer (SNB-1A,
Fangrui, China). The determination of pH was performed by a digital
pH meter (PHS-3C, Lei-ci, China), and the conductivity was carried out
directly through a digital conductivity meter (DDS-307A, Lei-ci,
China). All the measurements were maintained within temperature
range of ±0.05 K. Repeat runs were carried out at least three times.
The reproducibility of the data was within ±0.5%.
2.4. Microstructure observation of chitosan particles during dissolution
A certain amount of chitosan-water mixture or chitosan HCl solution
with suspended chitosan particles was dropped on a glass slide and cov-
ered by a cover slide. The microscopic structure of chitosan particles in
solution was observed using a optical microscopy (CX31, OLYMPUS,
Japan) and photos were taken.
2.5. Measurement of particle size of chitosan in solution
Particles size of chitosan in solution was determined by a particle
size analyzer (Mastersizer3000,Malvern, UK) at a temperature of 25 °C
± 0.5 °C. All these experiments were repeated 3 times.
2.6. Measurement of fluorescence spectrum
Fluorescence spectrum of chitosan molecules in solution was run on
a fluorescence spectrometer (F-7000, Hitachi, Japan). Data acquisition
parameters were adjusted as: final concentration of pyrene, 6.0
× 10−7 mol/L; excitation wavelength of pyrene, 337 nm; excitation
spectrum band width, 5 nm; emission spectrum band width, 2.5 nm
[21]. The peak height of I1 and I3 was measured and the proportion of
I1/I3 was calculated. Any parameter calculated was the result of the av-
erage of six experiments and the experimental error was the standard
deviation.
2.7. Statistical analysis
The single factor variance analysis (one-way ANOVA) of SPSS19.0
software was used to analyze significance of the data, and Graphpad
Prism 5 was used to calculate mean and standard deviation of the data
and draw figures.
3. Result and discussion
3.1. Solution viscosity during chitosan dissolution
We referred V.A.V. de Oliveira's study (2012) and used the dimen-
sionless concentration c⁎ = [η]c, where c was the chitosan absolute con-
centration in solution and [η] was the intrinsic viscosity of chitosan. The
values of c⁎ and c for chitosan samples were list in Table 1. From the re-
sultant values of c⁎ and c in Table 1, it was exhibited that there were
three dimensionless concentrations [22]: c⁎ b 0.8, the dilute regime,
for the 2 mg/mL chitosan hydrochloric acid solution; 0.8 b c⁎ b 6, the
semidilute regime, for the 10 mg/mL chitosan hydrochloric acid solu-
tion; c⁎ ≥ 6, the concentrated regime, for the 30 mg/mL and 80 mg/mL
chitosan hydrochloric acid solutions. The dimensionless concentrations
implicated the difference in interactions between chitosan molecules at
various chitosan concentrations [17].
When HCl solution was dropped slowly into chitosan suspension so-
lution, chitosan dissolve gradually and the solution became viscous. The
changes in viscosity of chitosan hydrochloric acid solutions with the
proportion of HCl to chitosan increasing were shown in Fig. 1. It was
shown that concentration, DD and Mw of chitosan had significant ef-
fects on solution viscosity and the viscosity curve of chitosan hydrochlo-
ric acid solution showed obviously different tendencies at various
concentrations of chitosan. When chitosan concentration was
2 mg/mL, which was in dilute regime (Table 1), the addition of hydro-
chloric acid led to significant increasing of solution viscosity (p b 0.05)
at the proportion of HCl to chitosan below 6 mM/g, suggesting a chain
expansion of chitosan [12]. While the viscosity of chitosan solution
maintained almost constant (Fig. 1(a)) at the proportion of HCl to chito-
san over 6 mM/g. The result implicated that all chitosan dissolved in so-
lution at the proportion of HCl to chitosan of 6 mM/g within 40 min and
more hydrochloric acid led to no obvious changes in conformation of
chitosan molecule or interaction between chitosan molecules in such a
dilute chitosan hydrochloric acid solution within 80 min. On the other
hand, we could explain the result in Fig. 1(a) by stoichiometric method.
Chitosan concentration of 2 mg/mL stoichiometricly equaled to about
0.0093 M, 0.0098 M, 0.0103 M and 0.0112 M amino groups for chitosan
sample #1, #2, #3 and #4 respectively. Hydrochloric acid solution con-
centration was 0.012 M at the proportion of HCl to chitosan of 6 mM/g
in solution, in which there was enough hydrions to make chitosan lose
its lattice structure and dissolve totally [10]. This was also agreement
with the suggestion by Mazzarelli [23].
Fig. 1(a) also showed that there was no significant difference be-
tween the viscosity of chitosan #1, #2 and #3 HCl solution (p N 0.05),
which implicated that DD b85% had no obvious effect on the viscosity
of dilute chitosan HCl solution. Chitosan #4 solution exhibited signifi-
cantly lower viscosity (p b 0.05) compared with the other three chitosan
solutions. This could be explained by the fact that sample 4# had more
-NH2 groups (DD = 89.98%) and bound more free hydrions on molecu-
lar chains and the chitosan chains became more extended coils with a
smaller number of entanglements, making it stiffer due to electrostatic
 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108 1103

Table 1 Chitosan with DD over 80%, however, adopts the properties of a cationic
Chitosan absolute (c) and dimensionless (c⁎) concentrations used in this work. polyelectrolyte, whose behavior is governed by the charge density.
c (mg/mL) c⁎ For the solution with chitosan concentration of 30 and 80 mg/mL,
#1 #2 #3 #4
which were in concentrated regime, solution viscosity exhibited an ob-
viously different tendency compared to the solution with 2 and
2 0.74 0.70 0.69 0.64
10 mg/mL chitosan as shown in Fig. 1. As HCl solution was added
10 3.72 3.51 3.43 3.21
30 11.16 10.53 10.29 9.63 dropwise into chitosan suspension as the proportion of HCl to chitosan
80 29.76 28.08 27.44 25.68 was below 3.75 mM/g for sample #1, #2 and 4.5 mM/g for sample #3,
#4, chitosan dissolved gradually and chitosan segments occupied an in-
creasingly higher volume fraction in the solution. It led to counter-ion
concentration around increasing, and made polyelectrolyte effect less
repulsion [14], resulting in less inter-molecular interactions between important, decreased the distance between chitosan segments, and
chitosan molecules and a thinner solution. made easier the hydrophobic interactions to occur resulting in higher
At chitosan concentration of 10 mg/mL, viscosity changes of chitosan virtual crosslinking and macromolecular coils overlapping [17,25], and
HCl solutions with proportion of HCl to chitosan increasing in Fig. 1 solution viscosity increasing dramatically (Fig. 1(c) and (d)). Especially,
(b) showed a similar tendency as Fig. 1(a). Chitosan sample 1# HCl so- chitosan concentration of 80 mg/mL showed more significant effect on
lution, however, had the highest viscosity and there was no significant solution viscosity and the highest valve of viscosity was up to over 1.3
difference in solution viscosity between sample #2, #3 and #4. Chitosan × 105 cP resulting in that it was difficult to stir the solution and measure
concentration of 10 mg/mL is in semidilute regime (Table 1), and mac- the viscosity, although there were still some chitosan particles in
romolecular coil overlapping begins to play an important role [17] and solution.
results in a more viscous solution compared with chitosan concentra- It was worth noting that the solution viscosity decreased sharply in
tion of 2 mg/mL. Moreover, DD region of chitosan also has effect on in- concentrated chitosan hydrochloric acid solution at the proportion of
teractions between chitosan chains. Schatz et al. [24] suggested that for HCl to chitosan over 3.75 mM/g for sample 1#, 2# and 4.5 mM/g for
DD of chitosan below 80%, hydrophobic interactions associated with hy- sample 3#, 4# (Fig. 1(c) and (d)). Some researchers attributed the de-
drogen bonding became preponderant and enhanced the cationicity of creasing in solution viscosity to acidic hydrolysis of chitosan [26], but
chitosan in solution that increased the solution viscosity of sample 1#. we measured the reducing sugar content in solution during chitosan

Fig. 1. Changes in viscosity of chitosan solution with HCl adding (45 °C ± 1 °C). (a) chitosan solution with concentration of 2 mg/mL; (b) chitosan solution with concentration of 10 mg/mL;
(c) chitosan solution with concentration of 30 mg/mL; (d) chitosan solution with concentration of 80 mg/mL.
1104 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108

dissolution and found no obvious increase in reducing sugar content.
For one thing, the hydrochloric acid in aqueous solution formed simple
ion pairs that affects the ionic strength in solution like salts [27]. In con-
centrated chitosan solution, more hydrochloric acid was added and
caused great increase of ionic strength in solution resulting in that the
concentration of counter-ions rose, which screened the protonated
amine group and made the molecule contracted, and in turn the viscos-
ity decreased [28]. For another, viscosity decrease of chitosan solution
could be attributed to the intramolecular hydrophobic aggregation of
chitosan occurred at higher concentration which led chitosan to become
extended coils [29,30].
Fig. 1(c) and (d) also exhibited that DD and Mw of chitosan had sig-
nificant impact on the viscosity of concentrated chitosan solution (p b
0.05), which increased in the order of Mw of chitosan, while decreased
in the order of the DD. Furthermore, for chitosan DD b 80%, the trans-
form of viscosity of concentrated chitosan solution happened at lower
ratio of HCl to chitosan than that of chitosan of DD over 80%. There
were two possible reasons: chitosan of lower DD (#1 and #2) possesses
less -NH3+ groups in acid solution, as a result, less Cl− is needed to
screen the protonated amine group and make the molecule contracted;
chitosan of higher DD (#3 and #4) needs more to dissolve and form a
solution, in which chitosan molecule become contracted.
3.2. Changes in conductivity of solutions during chitosan dissolution
The changes in conductivity of chitosan solutions were shown in
Fig. 2. When chitosan dissolves in hydrochloric acid, chitosan is proton-
ated and carries positive charges, but cannot move orderly and trans-
port charges. Therefore in chitosan hydrochloric acid solution, only
chlorions and unbound hydrions transport charges [14]. As shown in
Fig. 2(a), chitosan #1 dilute solution had higher conductivity, while chi-
tosan of higher DD (chitosan #2, #3 and #4) had lower conductivity be-
cause of the higher amounts of N‑amino‑glucose units and more
hydrions bound on chitosan molecules. In semiconcentrated and con-
centrated solution, however, there was no obvious difference in solution
conductivity between four samples because more hydrochloric acid
added and the concentration of hydrochloric acid showed greater im-
pact on conductivity of chitosan solution than DD of chitosan. Fig. 2
(b), (c) and (d) exhibited that electrolytic conductivity of solutions in-
creased linearly with hydrochloric acid concentration rising when the
proportion of HCl to chitosan was below 4.50 mM/g for sample 1#, 2#
and 5.25 mM/g for sample 3#, 4#. When the proportion of HCl to chito-
san rose, there was a transform of conductivity of chitosan solutions,
which might be attributed to free hydrions increasing in solution after
most of chitosan dissolving. As a result, the transform of conductivity

Fig. 2. Change in conductivity of chitosan solution with HCl adding (45 °C ± 1 °C). (a) chitosan solution with concentration of 2 mg/mL; (b) chitosan solution with concentration of
10 mg/mL; (c) chitosan solution with concentration of 30 mg/mL; (d) chitosan solution with concentration of 80 mg/mL.
 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108 1105

of solution can be used as an indication which suggests the thorough
dissolution of chitosan in inorganic acidic solution. Moreover, the data
in Fig. 2(b), (c) and (d) implicated that DD region of chitosan had signif-
icant impact on the conductivity transform of semi-concentrated and
concentrated solution. For DD N 80%, chitosan needs more hydrions in
hydrochloric acid for protonation and dissolution than that of chitosan
of DD below 80%.
3.3. Changes in solution pH during chitosan dissolution
pH of solution depends on the concentration of free H+ and OH− in
the solution [31], therefore changes in chitosan solution pH showed the
amount of free hydrions in solution and implicated the protonation of
amino groups in chitosan. The results shown in Fig. 3 were coincident
with the suggestion of Hamdine et al. (2005): at constant proportion
of HCl to chitosan, increase in chitosan concentration resulted in higher
pH values due to chitosan protonation, while the increase in acid con-
centration at constant polymer content resulted in lower solution pH.
The solution pH of chitosan #1 and #2, however, decreased with chito-
san content increasing at proportion of HCl to chitosan of 5.25 mmol/g
and 6 mmol/g (Fig. 3(a) and (b)) due to their lower DD and the dilute
and semidilute concentration. (See Fig. 3.)
In addition, Fig. 3 also showed there were three phases for changes
in solution pH with hydrochloric acid adding: pH of solution dropped
quickly at the proportion of HCl to chitosan below 1.5 mmol/g, which in-
dicated that there were many free hydrions in solution because hydro-
chloric acid added and since most of chitosan didn't dissolve in this
time; when the proportion of HCl to chitosan was in the range from
1.5 mmol/g to 3.75 mmol/g for #1 and #2, and 4.5 mmol/g for #3 and
#4 (Fig. 3(b) and (c)), or 4.5 mmol/g for #1 and #2, and 5.25 mmol/g
for #3 and #4 (Fig. 3(d)), solution pH decreased insignificantly as hy-
drochloric acid added (p N 0.05) due to the interaction of hydrions
with amine groups in chitosan and buffering capacity of the glucos-
amine units in chitosan molecules [31]; pH of solution decreased signif-
icantly (p b 0.05) due to increasing of free hydrions in solution after
most of chitosan dissolving.
In dilute solution, characteristics of chitosan like DD and Mw had no
influence on solution pH during dissolution (Fig. 3(a)). At concentration
of 10 mg/mL and 30 mg/mL, however, chitosan of DD over 80% (sample
#3 and #4) needed more hydrochloric acid to dissolve and solution pH
decreased obviously at proportion of HCl to chitosan of 4.5 mmol/g,
while for chitosan of DD below 80% (sample #1 and #2), pH drop hap-
pened at 3.75 mmol/g (Fig. 3(b) and (c)). When concentration of chito-
san solution was 80 mg/mL, chitosan concentration exhibited more
significant effect on dosage of hydrochloric acid needed to make chito-
san dissolve (p b 0.05). Therefore, solution pH of chitosan with DD
over 80% dropped dramatically at higher proportion of HCl to chitosan
(5.25 mmol/g).

Fig. 3. Change in pH of chitosan solution with HCl addition (45 °C ± 1 °C). (a) chitosan solution with concentration of 2 mg/mL; (b) chitosan solution with concentration of 10 mg/mL;
(c) chitosan solution with concentration of 30 mg/mL; (d) chitosan solution with concentration of 80 mg/mL.
1106 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108
3.4. Fluorescence chromatography of chitosan in solution
We measured the hydrophobic index (I1/I3) of chitosan in solution,
the proportion of fluorescence intensity of 373 nm (I1) and 384 nm
(I3) by using pyrene as fluorescence probe [21,30,32] at chitosan #4
concentration of 80 mg/mL when the proportion of HCl to chitosan
was between 4.5 mmol/g and 6.0 mmol/g. The I1/I3 value depended
on polarity of molecule and the higher the I1/I3 value, the greater the po-
larity of molecule [32]. As shown in Fig. 4 and Table 2, the value of I1/I3
reduced with the proportion of HCl to chitosan rising, which was indi-
cated that the polarity of molecule decreased and hydrophobic
microzones formed at the proportion of HCl to chitosan above
4.5 mmol/g. As mentioned above, electrostatic repulsion of chitosan
molecules was screened and more chitosan dissolved in solution as
HCl added into chitosan aqueous solution. As a result, the conformation
of chitosan molecular chain changed into contracted coils and hydro-
phobic domains formed by intramolecular hydrophobic interactions be-
tween chitosan [29,30,33], which contributed to the viscosity decrease
of chitosan solution (Fig. 1(d)).
3.5. Changes in turbidity of solution during chitosan dissolution
Chitosan suspension is turbid and opaque, but after chitosan dis-
solves in acidic solution, the solution become uniform and transparent.
Therefore, we measured the turbidity (100-light transmittance) of solu-
tion with chitosan concentration of 80 mg/mL to investigate the state of
solution and process of chitosan dissolution, and results were shown in
Fig. 5. Although the conductivity and pH of chitosan solution showed
obvious changes at proportion of HCl to chitosan below 1.5 mmol/g,
the turbidity of solution was great and changed insignificantly (p N
0.05) indicating that there were still many chitosan particles
suspending in solution and hindering the light from transmitting and
scattering. For chitosan #1 and #2, turbidity of solution began to de-
crease at proportion of HCl to chitosan of 2.25 mmol/g and there was
a drop from ~100% to ~75% (chitosan #1) and 40% (chitosan #2) as pro-
portion of HCl to chitosan reached 4.5 mmol/g (Fig. 5), and then de-
creased slightly with the increase of hydrochloric acid. For chitosan #3
and #4, the turbidity of solution decreased at 3.75 mmol/g and achieved
the minimum ~40% (chitosan #3) and ~30% (chitosan #4) at
5.25 mmol/g. The above conditions at which chitosan solution showed
minimum turbidity corresponded with the transform point of solution
pH and conductivity with hydrochloric acid addition in Fig. 2(d) and
Fig. 3(d). From Fig. 5, we also found that solution turbidity decreased
with DD of chitosan increasing in the range of chitosan DD from about
75% to 80% and from about 83 to 90% respectively. The solution of chito-
san #2 and #3 had similar turbidity at the proportion of HCl to chitosan
of 6 mmol/g. #1 chitosan solution exhibited highest turbidity even at
higher proportion of HCl to chitosan due to the low solubility caused
Fig. 4. fluorescence chromatography of pyrene in chitosan solution with different HCl
concentration (Chitosan #4, 80 mg/mL, 45 °C ± 1 °C).
Table 2
Peak height fluorescence chromatography of pyrene.
Proportion of HCl to chitosan/mmol/g Peak height/A.U.
I1 I3 I1/I3
4.50 411.5 261.5 1.574
5.25 255.9 196.1 1.305
6.00 131.6 119.6 1.100
by the lowest DD, high concentration (80 mg/L) and solution viscosity
(Fig. 1(d)).
3.6. Observation of chitosan particles in solution by optical microscope
Microscopy photographs of particles of chitosan #2 and #4 in solu-
tions were taken by optical microscope and shown in Fig. 6. It was
showed that chitosan particles suspended evenly and densely in
water. However, when hydrochloric acid was added into the suspen-
sion, chitosan particles began to agglomerate into larger particles (pic-
ture at 0.75 mmol/g) and the agglomeration of chitosan particles
decreased gradually with chitosan dissolution (Fig. 6), which was coin-
cident with Shao and Shen's report [34]. As shown in Fig. 6, small parti-
cles dissolved first and the amounts of chitosan particles in solution
decreased apparently with hydrochloric acid adding at the proportion
of HCl to chitosan in range from 1.5 mmol/g to 3.00 mmol/g for chitosan
#2 or 3.75 mmol/g for chitosan #4, although Fig. 5 showed no obvious
change in turbidity of solution due to the hindrance of light scatter by
chitosan particles. At proportion of HCl to chitosan over 4.5 mmol/g
for chitosan #2 or 5.25 mmol/g for chitosan #4, there was no visible chi-
tosan particles seen in solution. The above changes in the microstate of
chitosan solution were corresponding to the tendency of solution con-
ductivity, pH and turbidity shown in Figs. 2(d), 3(d), and 5.
From Fig. 6, so called “dissolution” of chitosan, in fact, was disinte-
gration of chitosan particles in aqueous acid solution, which could be
observed in pictures at proportion of HCl to chitosan of 1.5 mmol/g,
2.25 mmol/g and 3 mmol/g for chitosan #2, and 3 mmol/g,
3.75 mmol/g for chitosan #4. Mazzarelli [23] and Wang [10] both sug-
gested that the broken of hydrogen bonds between the chitosan mole-
cules, destroy of its stable crystal structure and disintegration of
chitosan particles carried out only at the condition that there was
enough number of H+ in acid solution, which at least equaled to the
-NH2 groups content in chitosan. Chitosan #4 with higher DD and
lower Mw had higher content of -NH2 groups in molecule, therefore,
needed more hydrochloric acid to dissolve, resulting in lower light
transmittance (Fig. 5) and more chitosan particles in solution (Fig. 6)
compared with chitosan #2 at lower ratio of HCl to chitosan.
Fig. 5. Change in turbidity of concentrated chitosan solution with HCl addition (chitosan
#4, 80 mg/mL, 45 °C ± 1 °C).
 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108 1107

0.00 mmol/g 0.75 mmol/g 1.50 mmol/g

2.25 mmol/g 3.00 mmol/g 3.75 mmol/g

4.50 mmol/g 5.25 mmol/g

(a)

0.00 mmol/g 0.75 mmol/g 1.50 mmol/g

2.25 mmol/g 3.00 mmol/g 3.75 mmol/g

4.50 mmol/g 5.25 mmol/g 6.00 mmol/g

(b)
Fig. 6. Microscopy photographs of chitosan particles in concentrated solution (×100, 80 mg/mL, 45 °C ± 1 °C). (a) Chitosan #2; (b) Chitosan #4.
1108 J. Wu, L. Zhang / International Journal of Biological Macromolecules 121 (2019) 1101–1108
Fig. 7. Change in particle size of chitosan in concentrated solution with HCl addition
(Chitosan #4, 80 mg/mL, 45 °C ± 1 °C).
3.7. Particle size of chitosan in solution during dissolution
As shown in Fig. 7, particle size of chitosan in solution increased sig-
nificantly from 50 μm to 60 μm at proportion of HCl to chitosan below
2.25 mmol/g due to the agglomeration of chitosan particles (Fig. 6(b))
(p b 0.05). While at the proportion of HCl to chitosan of 3 mmol/g,
only larger chitosan particles left in solution and expanded obviously
before disintegrating (Fig. 6(b)), which resulted in that particle size of
chitosan increased markedly to over 70 μm (p b 0.05). With proportion
of HCl to chitosan continually increasing, particle size of chitosan de-
clined sharply due to the disintegration and dissolution of chitosan.
Most of former researches on dissolution or properties of chitosan in
acidic solution were focused on the viscometric study and rheological
properties of chitosan solutions [13,35,36]. Equation of Mark–
Houwink was used as a conformation indicator of chitosan in dilute so-
lution [37]. Our research on dissolution behavior and conformation of
chitosan in semidilute and concentrated solution would stimulate fur-
ther development and application of chitosan.
4. Conclusions
In this paper, we measured and compared the viscosity, conductivity
and pH of dilute, semidilute and concentrated chitosan solution as hy-
drochloric acid solution dropped into chitosan suspension, and
discussed the difference in dissolution behavior of chitosan with various
concentrations in hydrochloric acid aqueous solution. It was found that
chitosan with various concentrations showed significant different
changing tendency of solution viscosity during dissolution, and the vis-
cosity of concentrated chitosan solution decreased sharply when the
proportion of HCl to chitosan was over 3.75 mmol/g or 4.5 mmol/g.
From above results, we can deduce that the confirmation of chitosan
changed from entangling with each other to becoming compact coils
due to electrostatic screen of Cl− and hydrophobic aggregation of chito-
san with high concentration. The result of fluorescence chromatography
of pyrene in chitosan solution also proved this deduction.
Through the study of changes in solution turbidity and the size, mi-
crostate of chitosan particle in concentrated solution (80 mg/mL), we
could suggest that with hydrochloric acid adding, chitosan particles in
suspension solution agglomerated first and then expended before
disintegrating and dissolving. The above behaviors of chitosan in solu-
tion were corresponding to the concentration of chitosan and hydro-
chloric acid solution. This is the first paper supplying detailed solution
data about concentrated chitosan solution and it will stimulate further
study and promote the application of chitosan.
From the results, the transform in electrolytic conductivity, pH of
chitosan solution with hydrochloric acid adding can be used to indicate
the endpoint of chitosan dissolution in hydrochloric acid solution, while
the changes in viscosity of solution can reflect the transform of chitosan
conformation during dissolution. The DD of chitosan showed important
effect on dissolution behavior of chitosan. Chitosan with DD over 80%
needed more hydrochloric acid to dissolve and the transform in electro-
lytic conductivity, pH of solution and conformation of chitosan hap-
pened at higher proportion of hydrochloric acid to chitosan.
Acknowledgement
We gratefully acknowledge for a financial support from Department
of Science and Technology of Guangdong Province of China
(2014B020204002).
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