ISSN 0965545X, Polymer Science, Ser. A, 2010, Vol. 52, No. 8, pp. 828–834. © Pleiades Publishing, Ltd., 2010.

Original Russian Text © V.V. Nikonorov, R.V. Ivanov, N.R. Kil’deeva, L.N. Bulatnikova, V.I. Lozinskii, 2010, published in Vysokomolekulyarnye Soedineniya, Ser. A, 2010,
Vol. 52, No. 8, pp. 1436–1443.

GELS

Synthesis and Characteristics of Cryogels of Chitosan Crosslinked

by Glutaric Aldehyde1
V. V. Nikonorova, R. V. Ivanovb, N. R. Kil’deevaa, L. N. Bulatnikovab, and V. I. Lozinskiib
a
Moscow State Textile University, Malaya Kaluzhskaya ul. 1, Moscow, 119071 Russia
b
Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
ul. Vavilova 28, Moscow, 119991 Russia
email: kildeeva@mail.ru
Received September 24, 2009;
Revised Manuscript Received February 4, 2010

Abstract—The mechanism of cryotropic gelation in moderately frozen solutions of chitosan crosslinked by

glutaric aldehyde is studied. Chitosan cryogels with large pores are synthesized at a low content of crosslink
ing agent in the reaction mixture or under conditions that do not lead to gelation at temperatures above 0°C.
The dependences of the yield of gel fractions, the degree of swelling of the polymer phase of cryogels, and the
hydrodynamic characteristics of cryogels on the temperature of synthesis are shown to be extremal. This
result may be explained by the competition between the cryoconcentration of reagents in the nonfrozen liquid
microphase, which assists the development of a crosslinked polymer network, and such factors as an increase
in the viscosity of the reaction mixture and a reduced reagent mobility, which prevent gelation. The morphol
ogy of chitosan cryogels is studied, and the character of the macroporous structure of the samples prepared
at different temperatures is shown to exert a stronger effect on the hydrodynamic characteristics of a cryogel
than the degree of swelling of crosslinked polymers in the walls of its macropores.
DOI: 10.1134/S0965545X10080092

1
INTRODUCTION
Chitosan is the Ndeacetylated derivative of the
natural polymer chitin [1]. Owing to such characteris
tics as biocompatibility and low toxicity, chitosan is
widely used in medicine, in the food industry, for envi
ronmental protection, etc. [2–5]. The presence of a
reactive amino group in the anhydropyranose mono
mer unit makes it possible to use this polymer to pre
pare covalently crosslinked hydrogels, which are able
to retain up to 10000% of water [3, 6]. Crosslinking of
chitosan and other aminocontaining polymers is
done with various crosslinking agents, including glu
taric aldehyde (GA), the most widely used among
these agents [7].
As follows from the literature data [8, 9], the
dynamics of gelation, i.e., the time to the gelpoint, in
the system of an aqueous acidic solution, chitosan,
and GA can be varied in a broad interval. However, at
room temperature, relatively robust crosslinked gels
can be prepared only at a sufficiently high content of
crosslinking agent in the initial solution [10]. It was
shown in [9–11] that GA is capable of aldol and cro
ton condensation, a process that leads to the forma
tion of toxic unsaturated derivatives. This circum
stance restricts the use of gels based on
1 This work was partially supported by the Russian Foundation for
Basic Research, project no. 090490403Ukrfa, and the tar
get program Development of the Scientific Potential of Higher
Institutes of Learning (2009–2010), project no. 2.1.1/2859.
GAcrosslinked chitosan in medical and related appli
cations. In this context, the development of new
methods for the preparation of sufficiently robust
hydrogels of a given poly(aminosaccharide) at low
concentrations of GA is an important problem.
In solvent–polymer–crosslinkingagent systems,
gelation processes are allowed not only at positive
temperatures but also in a moderately frozen medium,
at temperatures several tens of degrees below the crys
tallization point of a pure solvent [12, 13]. Under such
conditions, a frozen substance has the appearance of a
macroscopic solid, but, at the microscopic level, this
system is heterogeneous since it includes polycrystals
of freezedried solvent and a socalled unfrozen liquid
microphase [13, 14]. In this microphase, components
of the initial solution and reaction products are con
centrated; i.e., chemical processes happening in the
unfrozen liquid microphase are liquidphase pro
cesses. Because of the effects of cryoconcentration, an
apparent decrease in the critical concentration of gela
tion is observed when a polymer gel (in this case, a so
called cryogel [13]) can be formed at appreciably lower
initial concentrations of precursors than those for the
gelation at temperatures above the freezing point of
the system.
Earlier, it was demonstrated [12, 15] that gelation
may be performed by GA assisted crosslinking of chi
tosan dissolved in aqueous acetic acid in a moderately
frozen system at ⎯8°С. It was shown that chemically

828
 SYNTHESIS AND CHARACTERISTICS OF CRYOGELS
crosslinked chitosan cryogels can be prepared with the
use of lower amounts of GA than those in the case of
chitosan gelation in an unfrozen medium. However, in
earlier studies, the temperature dependence of the
efficacy of cryotropic gelation in a given system and
the corresponding characteristics of asprepared gels
were not investigated, because the importance of this
information for understanding basic factors affecting
cryotropic gelation was revealed much later [13].
The objective of this study is to investigate the char
acteristics of a highly porous morphology of cryogels
synthesized at different temperatures below 0°С and
at different ratios between the initial components, chi
tosan : GA.
EXPERIMENTAL
In this study, the following reagents were used: chi
tosan (ZAO Bioprogress, Russia) with М = 46.7 × 104
and a degree of deacetylation of 84.6%; a 25% solution
of GA (Merck, Germany); glacial acetic acid, ace
tone, and ethyl ether (Khimmed, Russia); and
Rhodamine 6G dye (Reakhim, Russia) without any
purification. For the preparation of solutions, bidis
tilled water was used.
For dissolution of chitosan, 2% acetic acid was
added to the dry polymer (2 g), and the polymer was
allowed to stay for 1 h; then, the mixture was stirred for
2 h with a magnetic stirrer at a rotation speed of
400 rpm. Then, to the asprepared solution, a
crosslinking agent was added so that, in the final mix
ture, the formal molar ratio between NH2 groups of
chitosan and CHO groups of GA was 2.5 : 1, 5 : 1,
10 : 1, 15 : 1, 20 : 1, or 25 : 1. In all cases, the concen
tration of chitosan was kept constant, 1.6%. This
cocktail was quickly stirred and collected in disposable
plastic 5ml syringes; then, this composition was fro
zen at a given temperature in the chamber of an
FP 45 HP precision programmable cryostat (Julabo,
Germany).
The samples were allowed to stay for 24 h in a fro
zen state; then, they were released from the cryostat
and thawed for 25 s in an M1712NR microwave oven
(Samsung, South Korea) at a power of 800 W. Then,
through each syringe, 50 ml of a 1% solution of acetic
acid (to remove the sol fraction) and 50 ml of water
were permeated. The flow rate of a liquid through the
cryogel was estimated from the volume of distilled
water permeated through the syringe for 1 h at a pres
sure gradient of 100 cm H2O.
To estimate the yield of the gel fraction, the gel
sample was ground in a porcelain mortar; placed onto
a glass filter; washed with a 1% solution of acetic acid
(three times with 15 ml), distilled water (three times
with 15 ml), a 50% aqueous solution of acetone
(2 times with 15 ml), acetone (2 times with 15 ml), and
ether (2 times with 15 ml). Then, the sample was dried
in an SNOL 24/200 (AB Utenos Elektrotechnika,
Lithuania) air thermostat at 50°С until a constant
POLYMER SCIENCE Series A Vol. 52 No. 8 2010
829
weight was attained. In other experimental runs, the
samples washed from the sol fraction were subjected to
lyophilic drying with a Freezone 1L freeze drier (Lab
conco, United States) and were further dried in an air
thermostat until constant weights were attained. The
content of residual moisture in the dried samples was
estimated through the coulometric method with the
use of Karl Fischer reagent.
The yield of the gel fraction was calculated through
the equation
Yield = (mexp/mtheor) × 100, (1)
where mexp is the weight of dry polymer after the
extraction of the sol fraction and drying and mtheor is
the weight of the polymer in the case of 100% gelation.
The degree of swelling was estimated via weighing.
To the weighed amount of the dried cryogel sample,
10 ml of distilled water was added, and the mixture was
allowed to stay for 48 h at room temperature under
periodic stirring. (A longer duration did not affect the
results.) Then, the swollen sample was placed onto a
glass filter, and the free solvent was removed in vacuum
(water pump, 5 min) and weighed. The degree of
swelling was calculated according to the equation
Degree of swelling = (msw – mdr)/mdr, (2)
where msw is the weight of the swollen cryogel and mdr
is the weight of the dry polymer.
The morphology of chitosan cryogels was studied
on an Eclipse 55i optical microscope (Nikon, Japan)
equipped with a digital image recorder. The samples of
thin sections were prepared with a Shandon Cryotome
freezing microtome (Thermo Electric Corp, United
Kingdom). The sections were colored with
Rhodamine Red (excess dye was removed with water).
Then, the sample was placed onto a glass slide, excess
moisture was removed with a gauze sponge, and the
sample was coated with one drop of a “coating
medium” (a solution of 1 g of gelatin in 12 ml of 50%
aqueous glycerol containing 0.2 g of phenol as a bacte
riostat) and pressed with a cover slip. The samples for
studies were stored at 4°С in a sealed vessel.
RESULTS AND DISCUSSION
One of the main approaches to the preparation of
covalent gels is crosslinking of polymer precursors by
lowmolecularmass crosslinking agents in a solution
[16]. The characteristics and structure of the formed
chemically crosslinked network are controlled by the
nature of the polymer and the nature of the crosslink
ing agent, by their concentrations, by the solvent used,
and by the reaction conditions (temperature and dura
tion) [17]. The above factors govern cryotropic gela
tion as well, and the temperature of the frozen reaction
system controls the porous morphology of the formed
cryogel because crystals of the frozen solvent serve as a
porogen, while their dimensions and shape primarily
depend on the regime of cryogenic treatment [13, 18].
Therefore, in this study, we investigated the effect of
830 NIKONOROV et al.

Table 1. Content of residual water in the samples of cryogels cules are transformed into the reactive dialdehyde
after different procedures of drying form. Taking into account the fact that the mechanism
of this reaction is not a subject of this study, we do not
CDontent of residual water
Temperature in the samples after drying
present these data and assume that the formal molar
Molar ratio ratio between NH2 groups of chitosan and CHO
of cryogel
NH2 : СНО
synthesis, °C without lyo with lyo groups of GA can be used as a parameter.
philization philization Chitosan is a highly hydrophilic polymers; hence,
2.5 : 1 –15 7.9 ± 3.0 15.1 ± 2.0 chitosanbased hydrogels can effectively retain solvate
moisture, a factor that should be taken into account in
–25 9.9 ± 5.1 13.9 ± 0.3 the estimation of the yield of the gel fraction. In this
–30 13.2 ± 4.5 13.4 ± 2.0 context, we selected the optimum regime of drying for
10 : 1 –15 10.2 ± 0.1 12.8 ± 0.5 the crosslinked samples. The samples prepared at
NH2toCHO molar ratios of 2.5 : 1, 10 : 1, and 25 : 1
–25 7.0 ± 0.4 12.9 ± 1.6 were dehydrated by different methods, and the con
–30 3.1 ± 0.7 14.5 ± 1.5 tent of residual water was estimated via the Fischer
25 : 1 –15 9.6 ± 0.3 12.2 ± 0.6 method.
–25 5.3 ± 0.5 11.3 ± 1.6 Table 1 lists the data on the water content for vari
ous cryogels after their washing from the sol fraction,
–30 9.0 ± 4.5 12.4 ± 0.7 treatment with dehydrating organic solvents, and dry
ing at 50°С to a constant weight (this is the adopted
protocol for drying of polymer hydrogels) in the com
the concentration of the crosslinking agent in the ini parison with the data for the samples after lyophilic
tial system and the reaction temperature on the effi drying. In the first protocol, the scatter in the data on
cacy of gelation (the yield of the gel fraction) and such residual moisture was high (up to ~50%), as was the
characteristics of cryogels as the degree of swelling of case, for example, for cryogels synthesized at –25°С
the crosslinked network, the hydrodynamic character and at an NH2toCHO molar ratio of 2.5 : 1. More
istics of a macroporous polymer block, and its mor over, there is no evident correlation between the con
phology. ditions of gel synthesis and the content of residual
Let us take into account the wellknown fact that, moisture. Evidently, these results cannot serve as a
in aqueous solutions, GA exists in an equilibrium mix basis for the reliable estimation of the yield of the gel
ture of free aldehyde, monohydrate, dihydrate, and fraction. The cause of this marked scatter in the exper
cyclic cis and trans isomers [8]. However, according to imental data is likely related to a partial cornification
our data, during the reaction with amino groups of the of the surface regions of cryogels, which is induced by
polymer, the equilibrium in the reaction mixture is densification of the gel material upon drying. This
shifted toward the free aldehyde and most GA mole phenomenon prevents water removal from the sample.
In the case of lyophilic drying of the sample, more cor
rect results on moisture content were obtained (in all
Yield of the gel fraction, % cases, the scatter was always below 15%), even though
90 the absolute moisture content, on the whole, is higher
1 than that after the treatment with dehydrating organic
2 solvents. Therefore, the content of residual moisture
3 in gels after lyophilic drying was used for the calcula
4 tion of the yield of the gel fraction. Figure 1 presents
5 the calculation results for cryogels synthesized at a
6 constant polymer concentration but a varying content
of the crosslinkingagent–GA system, with the tem
perature being varied from –10 to –30°С.
80 First, let us mention that cryotropic gelation in the
system under study, even at the lowest temperature
within the above temperature interval and at the min
imum GA concentration, proceeds effectively because
the yield of the gel fraction always exceeds 75%. How
ever, at the same concentration of chitosan and GA
−30 −20 −10 but at 25°С, gelation ceases: The reaction solution
T, °C
does not lose its fluidity for 24 h, although this solution
has the visual appearance of being more viscous. This
Fig. 1. Yield of the gel fraction plotted against the temper
ature of cryogel synthesis. NH2toCHO molar ratios: result is a vivid manifestation of a decrease in the crit
(1) 2.5 : 1, (2) 5 : 1, (3) 10 : 1, (4) 15 : 1, (5) 20 : 1, and ical gelation concentration [12, 13, 15, 19], which is
(6) 25 : 1. provided by cryoconcentration of reagents in the

POLYMER SCIENCE Series A Vol. 52 No. 8 2010

 SYNTHESIS AND CHARACTERISTICS OF CRYOGELS
unfrozen liquid microphase. Finally, a cryogel is
formed at a lower concentration of precursors than
that in the solution without freezing.
With allowance for the fact that scatter in the
experimental data in the estimation of the gel fraction
does not exceed ±4%, the extremal dependence of the
gel fraction yield on the temperature corresponding to
the formation of cryogels should be assumed to be
accurate. The maximum yield of the gel fraction is
observed at ⎯25°С at the above ratios between chito
san and GA. Similar extremal dependences of the effi
ciency of cryochemical reactions in moderately frozen
solutions have been described both for lowmolecular
mass compounds [14] (including monomer precursors
of polymerized or polycondensed highmolecular
mass compounds [20]) and for polymer reagents [21,
22] (including cryotropic gelation [13]). The main
cause of such dependences is the competition between
several oppositely directed factors. For example, when
the concentration of reagents in the unfrozen liquid
microphase during freezingout of a pure solvent
increases, chemical reactions speed up, the apparent
critical gelation concentration decreases, and the yield
of the gel fraction increases. On the other hand, the
efficiency of gelation decreases with a decrease in tem
perature owing to a strong increase in the viscosity of
the reaction medium, which leads to a marked reduc
tion in the mobility of reagents.
The thermal regime of the preparation of cryogels
exerts a marked effect on their characteristics, for
example, on the osmotic characteristics (swelling) of
the polymer phase of macroporous material and its
morphology. Figure 2 presents the degree of swelling of
chitosan cryogels plotted against the temperature of
their synthesis, and this dependence likewise shows
the extremal character. The maxima of the curves lie at
–15°С, rather than at –25°С, as in the case of the
temperature dependence of the yield of the gel frac
tion (Fig. 1). The maximum degree of swelling of the
samples synthesized at –15°С indicates that, in such
samples, crosslinking of the polymer network of the
gel phase (or the walls of macropores) is minimum.
Therefore, incorporation of polymer chains into the
threedimensional network proceeds with a higher
efficacy than that in the case of gelation at –25°С
(Fig. 1), and the frequency of crosslinks of macromol
ecules in the network is minimum at ⎯15°С. The
absence of any pronounced inflection points in the
temperature dependence of the yield of the gel frac
tion (Fig. 1) at ⎯15°С and in the temperature depen
dence of the degree of swelling (Fig. 2) at –25°C indi
cate that the effect of the temperature corresponding
to the cryotropic gelation in the chitosansolution–
GA system on the efficiency of incorporating the poly
mer into the threedimensional network and on the
degree of crosslinking is different.
As for the dependence of the degree of swelling of
the polymer framework of chitosan cryogels on the
concentration of crosslinking agent, the data show a
POLYMER SCIENCE Series A Vol. 52 No. 8 2010
831
Degree of swelling, g of H2O/g of dry polymer
16
6
14 5
4
12 3
2
1
10
−30 −20 −10
T, °C
Fig. 2. Degree of swelling of chitosan cryogels plotted
against the temperature of their synthesis. NH2toCHO
molar ratios: (1) 2.5 : 1, (2) 5 : 1, (3) 10 : 1, (4) 15 : 1,
(5) 20 : 1, and (6) 25 : 1.
wellexpected tendency: With a decrease in the GA
content or with variation of the NH2toCHO ratio
from 2.5 : 1 to 25 : 1 (Fig. 2), the degree of swelling of
the polymer phase progressively increases. Evidently,
this behavior is the direct consequence of a decrease in
the degree of crosslinking of the polymer phase of
cryogels.
In this context, the results on the hydrodynamic
characteristics of cryogels estimated from water flow
through sponge samples with similar volumes, which
are formed as piles inside plastic syringes, are very
illustrative. Since the rate of liquid flow (in the case
under study, water) through a porous medium is pro
portional to the inlet area of capillaries, a lower
crosslinking density in the network of wall of
macropores will lead to a decrease in the above rate.
A less crosslinked network experiences greater swell
ing and covers the inlet of capillaries to a greater
extent, thus decreasing the flow parameter owing to a
higher resistance to the flow. Moreover, in sponge
cryogels, the cross section of macropores depends on
the temperature of cryostructuring. The lower the
temperature, the smaller the polycrystals of a porogen
(a frozen solvent). As a result, the pore dimensions are
smaller and, hence, liquid flow decreases. Therefore,
studies on poly(acrylamide) gels showed for the first
time [23] that the results of relatively simple measure
ments of flow rates make it possible to investigate the
microstructure of the gel matrix in such heterophase
832 NIKONOROV et al.

Water flow rate, ml/h

400

300
1

200 2

3
100
4
(а) 100 µm

5
6
0
−30 −20 −10 0
T, °C

Fig. 3. Flow rate of water through 4.5ml columns with

chitosan cryogels synthesized at different temperatures.
NH2toCHO molar ratios: (1) 2.5 : 1, (2) 5 : 1, (3) 10 : 1,
(4) 15 : 1, (5) 20 : 1, and (6) 25 : 1.

materials and the specific features of their

macroporous morphology.
This reasoning suggests that, for chitosan cryogels
whose polymer framework have the maximum degree
of swelling, i.e., for the samples synthesized at –15°C
(Fig. 2), the liquid flow through the capillaries is char
acterized by the minimum rate, while the flow rate is
lower than that of less swollen cryogels prepared at dif
ferent temperatures. Figure 3 shows that, with varia
tion in the molar ratio between NH2 polymer groups
and CHO groups of GA from 2.5 : 1 to 25 : 1 or with a
decrease in the initial concentration of the crosslink
ing agent, the flow rate gradually decreases owing to
high swelling of the walls of macropores in cryogels.
However, the dependence of the hydrodynamic char
acteristics of cryogels on the preparation temperature
differs from the predicted curve. It was found that the
flow rate of water through the sample is maximum for
the cryogels prepared at –15°C (Fig. 3) or for the sam
ple with the maximum degree of swelling of the poly
mer phase (Fig. 2). In other words, a small difference
in the degree of swelling of the polymer framework
(about 2–3 g Н2О per 1 g dry polymer or 20–30% with
respect to the overall degree of swelling) (Fig. 2) can
not serve as the parameter that controls the hydrody
namic characteristics of cryogels (Fig. 3). The more
important parameter is the character of porosity of
cryogels.
Figure 4 presents, as typical examples, micrographs
of thin sections (in the transversal and longitudinal
(b) 100 µm
Fig. 4. Optical micrographs of thin sections: (a) transversal
cross section and (b) longitudinal cross section of chitosan
cryogel synthesized at –30°С; the NH2toCHO molar
ratio is 5 : 1.
directions) of the cylindrical sample of chitosan cryo
gel synthesized at –30°C at an NH2toCHO molar
ratio of 10 : 1. In Fig. 4, dark regions correspond to the
Rhodaminecolored gel phase (the walls of large
pores) of the cryogels; light regions are the pores of
capillary dimensions that are filled with water. In the
transversal cross section (Fig. 4a), it is possible to
observe an anisotropic morphology of this relatively
heterogeneous porous gel structure with large cells
with crosssectional dimensions varying from tens of
micrometers to ~200 µm. In the longitudinal cross
sections (Fig. 4b), it is possible to observe a certain ori
entation in the thin (the cross section is 5–10 µm)
walls of macropores along the main axis of the cylin
drical sample. This character of porosity is character

POLYMER SCIENCE Series A Vol. 52 No. 8 2010

 SYNTHESIS AND CHARACTERISTICS OF CRYOGELS 833

istic of sponge cryogels with large pores [13, 18], and

similar orientation of microcapillaries was earlier
revealed for agarose cryogels formed in similar plastic
syringes [24]. This structural feature of cryogels can be
explained as follows: During freezing of the content of
a syringe immersed into a freezing agent in a cryostat
chamber, crystallization commences with the solvent
being in the volume of a thin drain tube where the
formed ice serves as a certain “nucleation site” from
which ice crystals grow upward in the syringe, thus set
ting the direction of the orientation of future
macropores in the cryogel.
Important factors controlling the “architecture” of
cryogel pores are the freezing temperature of the sys
tem as well as such phenomenon as the possible super
cooling of the system. Indeed, the lower the tempera
ture, the higher the rate of primary nucleation. In this
case, more crystallization sites are formed, and the (а) 100 µm
formed polycrystals of a frozen solvent themselves
have smaller dimensions [25, 26]. However, when
supercooling of the system is high (a phenomenon that
is often characteristic of aqueous solutions of polymers
[13, 27]), ice formation proceeds at a high rate and the
formed crystals can be even smaller than those formed
during freezing of the same system at a lower temper
ature. Note that, as the negative temperature
decreases, the system exists in the supercooled state
longer, but the crystallization rate increases. We pro
posed that this effect may be responsible for a higher
“permeability” of cryogels prepared at –15°С relative
to that of cryogels formed at –10°С (Fig. 3). To verify
this hypothesis, the samples of cryogels were prepared
at –10, –15, and –30°С (i.e., at the boundaries of the
temperature interval and at the point of maximum in
the curves shown in Fig. 3). The samples were pre (b) 100 µm
pared as a flat disks with a thickness of 2 mm, and their
morphology was studied by the method of optical
microscopy.
Figure 5 shows the corresponding micrographs,
and Table 3 lists the results of their quantitative analy
sis. Compared to the technique of thin sections
(Fig. 4), the above image collection procedure makes
it possible to obtain a better visualization of a three
dimensional structure of cryogels with large pores; the
collected images provide a correct presentation since
the samples in the equilibrium swollen state were not
distorted by the preparation procedure. In this case,
image contrast was limited by the resolution of a
microscope, a parameter that was lower than that
observed for the images of thin sections. This evidence
indicates that pores (capillaries) with the minimum
cross section are characteristic of the cryogel formed (c) 100 µm
at the minimum temperature, –30°С; the maximum
pore cross sections were observed for the chitosan
cryogel synthesized not at –10°C but at –15°С. This
result, first, correlates well with the data on the perme
ability of cryogels (Fig. 3) and, second, verifies the Fig. 5. Optical micrographs of 2mmthick disks of chito
hypothesis about the possible effect of supercooling san cryogels synthesized at (a) –10, (b) –15, and (c)
phenomena on the morphology of the porogen, that ⎯30°С. The NH2toCHO molar ratio is 5 : 1.

POLYMER SCIENCE Series A Vol. 52 No. 8 2010

834 NIKONOROV et al.

Table 2. Data on the quantitative analysis of microscopic images for chitosan cryogels formed at different temperatures
Temperature of cryogel synthesis, °C Dn, µm Dw, µm k
–10 57.4 70.1 1.22
–15 72.3 87.0 1.20
–30 44.3 56.0 1.26
1/3
3
Note: Numberaverage and weightaverage pore diameters, Dn and Dw, respectively, calculated through the equations Dn = ⎜ ∑
⎛ ( N i D i )⎞
⎟
⎝ ∑ Ni ⎠
6 1/3

and Dw = ⎜ ∑
⎛ ( N i D i )⎞
⎟ , where ΣNi is the overall number of test objects in the micrograph and k = Dw /Dn is the coefficient of poly
⎝ ( N D 3 )⎠
∑ i i
dispersity. The NH2toCHO molar ratio is 5 : 1.

is, ice polycrystals, during cryotropic gelation in the 10. N. R. Kil’deeva, P. A. Perminov, L. V. Vladimirov, et al.,
systems containing highmolecularmass precursors. Bioorg. Khim. 35, 397 (2009).

Therefore, this study has revealed the effects char
acteristic of cryotropic gelation in general—a high
efficacy of gelation at a low content of crosslinking
agent and an extremal temperature dependence of the
gel fraction induced by cryoconcentration of reagents
in the unfrozen liquid microphase—as well as the spe
cific features typical of the chitosansolution–GA sys
tem. These features include different positions of max
ima in the temperature dependences of the yield of the
gel fraction and the degrees of swelling as well as a
complex dependence of the flow rate of water through
cryogels on the degree of swelling, a result that may be
related to the effect of supercooling phenomena on the
morphology of ice crystals, which produce a gel struc
ture with large pores during cryotropic gelation.
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