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Calorimetric study of gelatin gels melting
Article · October 1991
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PolymerScienceVol. 33, No. 10, pp. 2112-2118, 1991
0965-545X/91 $15.00+.00
I~11992Pergamon Press Ltd
Printed in Great Britain.
CALORIMETRIC STUDY OF THE MELTING OF GELATIN
GELS*
G. I. TSERETELI and O. I. SMIRNOVA
Research Institute of Physics at the Leningrad State University
(Received 28 December 1990)
The temperature dependence of excess heat capacity was studied by the DSC method in the melting range
of aqueous gelatin gels, with gelatin concentrations from 2 to 95%. Gelatin samples with various contents of
interchain crosslinks were examined. In the course of gelation, metastable collagen-like structures were
formed. Temperatures Tm and heats of melting Qm were determined for gels formed under various
conditions. The thermodynamic parameters of the melting of gelatin gels and of collagen denaturation were
compared. Differences in these parameters can be used to evaluate defect formation in collagen-like
structures in gels.
FOR A long time, gelatin was used by various authors as a model compound for studying the
processes of gel formation. Nevertheless, to this day the heat properties of its gels have not been
adequately studied [1-4]. Thus particularly the heats of melting published by various authors differ
several fold [5]. Various authors have studied non-comparable samples, with a complex prehistory,
so that it is hard to make a comparative analysis of literature data. Moreover, the general principles
of gel formation is denaturated proteins in general and in gelatin in particular are still being
discussed.
The aim of this work was a calorimetric study of the gel formation processes in gelatin with
various contents of interchain crosslinks, in a broad concentration range: from dilute solutions to
dried films. As known, gelatin is the product of processing the natural fibrillar protein collagen, and
the so-called gelatin molecule appears to be one of the chains forming the triple helix collagen
molecule. We have also hoped that a comparison of the data on the melting of gelatin gels with our
previously obtained data on thermal denaturation of collagen [6-8] might yield new information on
the structure and properties of gelatin gels.
Studies of the temperature dependence of excess heat capacity in the melting range of gelatin gels
were made with the differential scanning calorimetric DSC-111 of "Setaram Co," (France), with a
sensitivity of 3 x 10 -5 J/s. The sample mass was 50-100 mg, the error in temperature measurement
did not exceed +0.2 K, in determination of the heat effect +5%. For determination of the bound
water content, the studied samples were kept in vacuum to constant weight at 105°C.
Three types of gelatin samples were used, differing in the number of interchain crosslinks which
are known to strongly affect the process of gel formation [1]: I--gelatin prepared by thermal
denaturation of native collagen in a hermetic calorimetric ampoule, the same in which the gel
formation process was subsequently studied. In such gelatin, practically all natural interchain
crosslinks are preserved that existed in the original collagen. II--a-gelatin, consisting of completely
isolated chains of M -- 9.2 x 104; this was a preparation produced by the "Serva" Co. (F.R.G.) as a
*Vysokomol. soyed. A33: No. 10, 2243-2249, 1991.
2112
Study of melting of gelatin gels
2113
A Co, J/g K
7
ZO
~0
T, °C
FIG. 1. Dependence of the melting curves of gels of gelatin I on the time of gel formation: 30 min (1), 3 h
(2), 18 h (3) and 7 days (4). Temperature of gel formation 20°C, rate of heating 1 K/min.
calibrating substance for laboratory molecular mass determination; III--industrial gelatin of
"Sigma" Co. (U.S.A.), of structure intermediate between those of gelatins I and II, with a part of
the natural crosslinks disrupted.
At first we shall consider some general rules discovered by us in the gelatin gelation process. We
shall discuss them in detail using the example of gelatin I. In Fig. 1, the curves of the temperature
variation of excess heat capacity of sample I (gelatin concentration c = 20%) are shown, after
keeping at 22°C in a thermostat for various periods of time. The maximum of heat absorption
corresponds to melting of the gels. The curves Fig. 1 reflect the development of the gel formation
process in time. With increasing time of gel formation, there occurs a transformation of the gel
melting curves: the heat of melting, Qm, increases, as well as the melting temperature Tin, while the
width of the AT curve decreases. Let us note that the Qm value for the gel formed during the first
~30 min in the thermostat, amounts to - 1 / 2 of the maximum value attainable at this temperature.
In the course of the isothermic formation of the gel (7 days), its melting temperature increases by
7-8°C and reaches the limit of ~35°C. At the same time, the half-width of the melting curve
decreases about ~2 times (from 13 to 6°C). These results clearly demonstrate that the structures
formed in the gel are metastable, as their thermostability changes in the course of the gel formation
process.
As revealed in further studies, the above described transformation of the melting curves is
observed at various temperatures of gel formation and concentrations of initial solutions. At the
same time, actual changes in the values of Tm and Qm during gelation naturally depend on the
structure of the gelatins, temperature in the thermostat and concentration of the initial solution. For
example, as was to be expected, for the crosslinked gelatins (I and III) and for a-gelatin, the data on
the kinetics of gel formation differ considerably. While in the crosslinked gelatins at concentrations
c = 3-20% and at 22°C, gel is formed already during the first tens of minutes, with a Qm amounting
to --1/2 of the maximum value, then in a-gelatin gelation sets in only after several hours in the
2114
G . I . TSERETELI and O. I. SMImqOVA
aco, J/0~
il
aF
at
3O
6O
T, °C
FIG. 2. Dependence of the melting curves of gels of gelatin I (1-4) and gelatin II (5) on the temperature of
gel formation: 17 (1), 22 (2, 5), 27 (3) and 30°C (4). Time of gel formation 7 days. Rate of heating 1 K/min.
thermostat (at the same concentrations and temperatures). Moreover, in solutions of crosslinked
gelatine (c = 3-20%), the gelation process is practically terminated within 2-3 days, while in moist
films the same process takes several weeks. Nevertheless, in all cases studied (with gelatin
concentrations within 2-80%, and gelation temperatures within 15-35°C), the heat and temperature
of melting increase in the course of gel formation. This signifies that gel formation in gelatin leads to
the generation of metastable structures.
Using once more the example of gelatin I, we shall discuss the observed variation of the limiting
(maximum) values of the heats and temperatures of gel melting with the temperature of gel
formation. In Fig. 2, the melting curves of various gels prepared by long thermostating at 22°C of
solutions with c = 20% are shown. With increasing temperature of gel formation, the limiting Tm
values increase, while the values of Qm and the half-widths of the melting intervals decrease. The
demonstrated data are in good agreement with the results of reference [9]. Thus the obtained data
on the changes in the thermostability of gels in the process of their isothermic formation, and on the
temperature variation of gel formation bring compelling evidence that the rules of the gelation
process in gelatin parallel those of the crystallization process of synthetic polymers [10, 11].
Below we shall present the results of our studies of the effect of concentration of gelatin solutions
on the gelation parameters. Assuming that the character of the processes studied depends
considerably on the concentration of the initial solutions, we shall separately discuss the results
obtained for gels with c < 20%, and for those with higher concentrations.
As experimentally observed (Figs 2 and 3), on changing the gel concentration from 2 to 20% (gel
formation temperature 22°C), the limiting values of Tm practically do not depend on concentration
and are approximately the same for all gelatins, with deviations only slightly exceeding experimental
error (35.0+2°C). The relation between the values of Qm and the concentration of the initial
Study of melting of gelatin gels
2115
T.°¢
120
[
8O
~.0
×
×
×
i
20
GO
I
c,%
FIG. 3. Dependence of the temperatures of gel melting Tm and collagen denaturation Td, on concentration.
(1) sample prepared by drying of a gel with c = 5% (method A), gelatin III; (2) sample formed at high
concentration (method B), gelatin III; (3) collagen.
solution is less unambiguous (Fig. 4). In the crosslinked gelatins I and III, the value of Qm is
practically independent of concentration for changes within 2-20%, and amounts to 54.3 and 36.4
J/g for gelatins I and III, respectively. At the same time for a-gelatin, by reducing the concentration
from 20 to 2%, the value of Qm increases from 21.3 to 48.9 J/g. Let us note the following: from our
data on the rate of gel formation it follows that in a-gelatin at c = 1.5, even after 10 days gel
o,J/~
I
I
20
60
I
c,%
FIG. 4. Dependence of the heats of melting of gelatin gels Qm, and of collagen denaturation Qa, on
concentration. (1) gelatin I, (2) gelatin [L (3) gelatin III (3a--gel prepared by method A; 3b-~gel prepared
by method B), (4) collagen.
2116
G . I . TSERETELIand O. I. SMIRNOVA
formation is far from complete, and the presented values of am are the maximum, but not the
limiting ones, in contrast to the data at other concentrations.
In considering the obtained results, there arises the obvious question about what is the nature of
the structures formed in the gelation process. In references [12] it has been established that in dilute
aqueous collagen solutions (c < 0.2%) after denaturation, a collagen-like structure is restored, and
the rules of its recovery are similar to the rules governing crystallization of synthetic polymers from
solution. Moreover it was shown that in such solutions, no intermediate ordered structures
consisting of single collagen chains are formed, as had been previously suggested [1]. On the basis of
reference [12] it may be assumed that even in concentrated collagen solutions, cooled after
denaturation, collagen-like structures are generated in the course of gelatin gel formation. These
structures also lead to the build-up of the supermolecular gel network, which can be destroyed by
subsequent" heating. Also various collagen-like elements contain segments of various gelatin
molecules (collagen chains) as partners. From this it follows that the limiting (though in reality not
attainable) values of the thermodynamic parameters of gelatin gels can be equal to the values of the
temperature and heat of collagen denaturation in solutions of the same concentration. For a
collagen solution with c = 2 0 % , the temperature and heat of denaturation are: Td =43°C,
Qd = 78.6 J/g [6]. At the same concentration, for all the gelatins studied Tm = 35°C, while the heats
of melting for gelatins I, II and III are 54.3; 21.3 and 36.4 J/g, respectively. For comparison it should
be noted that the maximum heat of melting for the structures generated in collagen during cooling
after thermal denaturation, 65.2 J/g, was observed by us for collagen fibres crosslinked with
benzoquinone [7].
The ratio of the observed heats of gel melting to the heat of collagen denaturation can be used to
evaluate the degree of perfection of the structures formed in the gel, i.e. of their similarity to
collagen. In this respect, the nearest to collagen structure is the gel of gelatin I (ratio 0.70), the most
defective is the structure of the a-gel (0.27), while the structure of gel III is intermediate (0.46). As
previously stated, the natural interchain valency crosslinks are fully preserved in gelatin I, and
partly in gelatin III. It is particularly this circumstance that determines the high rate of gel formation
and the high limiting value of Qm of these gelatin gels. When the formation of collagen-like
structures is compared with the recrystallization of polymers, then it may be stated that in solutions
of crosslinked gelatins, nucleation is heterogeneous, and not homogeneous. At the same time, the
recrystallization of crosslinked fibrils appears as a limiting case of heterogeneous nucleation.
In the case of the a-gel, where interchain crosslinks are absent and nucleation appears to be
homogeneous, a gel with high Qm, approaching the values of gelatin I, is only formed at sufficiently
low concentrations. At the same time, at such relatively high concentrations as 20%, the initially
generated structures apparently prevent the formation of a large number of collagen-like structures.
Thus our studies of the effect of concentration on the gel formation process indicate that the
formation of collagen-like structures in the gelatin gels has kinetic, rather than thermodynamic
limits.
Let us note that the heats of gel melting were calculated per 1 g of dry substance, as customary in
determinations of the heats of denaturation. Heats of melting calculated per 1 g of real gel (or the
initial gelatin solution) for the a-gel are: c = 1.5%. Qm = 0.73 J/g (instead of 48.9 J/g); for c = 20%,
Qm = 4.2 J/g (instead of 21.3 J/g). Particularly the Qm values calculated in this way and
corresponding to the amount of ordered structures in one unit of gel mass can be compared with
other properties of the gels and should reflect the circumstance that at low concentrations a weak
gel, and at high concentrations, a strong gel is formed.
In the following we shall present our studies of melting in gels at high concentrations (c > 20%). It
Study of melting of gelatin gels
2117
is well known that such gels can be prepared by two procedures: either by drying of gels of relatively
low concentration (method A), or by gel formation in highly concentrated solutions (method B).
The variation of thermodynamic parameters of gels prepared by method A (concentration of the
initial gel 5%) and by method B (formation and melting of the gel proceeds at the same
concentration) with the concentration of gelatin is shown in Figs 3 and 4. First of all let us note that
on reducing the concentration of water, aqueous gelating gels are transformed into so called
crystalline gelatins, with various amounts of bound water. The curves 1 and 2 (Fig. 3) and 3a and 3b
(Fig. 4) indicate that this transition is gradual, without any perturbation of the regular course of the
variation of the heats and temperatures of the formed structures with the content of water. From
that it may be concluded that in all cases studied, both in solutions of gelatin in water, and in
solutions of water in gelatin, collagen-like structures of a single type are generated.
Let us consider our data on the melting of gels prepared by method A. As seen from Fig. 3, the
temperature of gel melting increases with decreasing water content. This dependence is the sharper,
the lower the concentration of water. At the same time the value of Qmremains practically constant
with the water content decreasing down to - 2 5 % , and it drops steeply later on.
It seemed interesting to compare the thermodynamic parameters of the melting process of the gel
and of crystalline gelatin with the denaturation parameters of native collagen, obtained in reference
[6]. These are also shown in Figs 3 and 4. It appears that the variation of the thermodynamic melting
parameters of the gel and of crystalline gelatin parallels the analogous variation of the denaturation
parameters of collagen, with the only difference that for gelatin the corresponding values are always
lower than for collagen (at the same water content). These results indicate that the studied
structures generated in gels at high concentrations and in crystalline gelatins actually appear to be
collagen-like.
The lowering of the heat of melting observed in gelatin at low water contents is caused, similarly
as in collagen, by the loss of bound water which stabilizes the helical structure. The increase of the
melting temperature with decreasing concentration of water is evidently caused by an extropy
effect: a reduction of AS, owing to an increased degree of order generated in the course of melting
[6].
Let us now present our data on the melting of gels actually formed at high concentrations
(method B). For such gels, with increasing gelatin concentration, a very small increase in Tm is
observed, together with a substantial lowering of Qm. Thus by increasing the concentration of
gelatin from 20 to 80%, the value of Tm increases by 10-12 K, while Qmdrops from 33.4 to 10.8 J/g.
From the presented data it appears that the thermodynamic parameters of two gels prepared by
various procedures, differ considerably. The differences in the values of the heats and temperatures
of melting become greater with decreasing water content. Thus at a water content of 20%, the
difference between the melting temperatures amounts to - 4 0 K, while the Qm values differ
three-fold. This comparison once more demonstrates that the studied structures are metastable, as
the values of their thermodynamic parameters strongly depend on their thermal and concentration
prehistory.
The observed difference in the thermodynamic parameters of two highly concentrated gels and
crystalline gelatins can be explained by means of the well-known dependence of the melting
parameters of small systems on their dimensions and defectiveness. Separate segments of the
collagen-like helices, responsible for the generation of the network, can be regarded as small
systems. Particularly by the disruption of these small systems in the course of melting, the
macroscopic network of the supermolecular gel is transformed into an isotropic solution, or
crystalline gelatin becomes amorphous. Evidently at small water contents (method B), because of
2118
G . I . TSERETELI and O. I. SMIRNOVA
kinetic limitations in highly concentrated gels, more defective structures of smaller dimensions are
formed than in gels of moderate concentrations (method A). Consequently such small defective
structures exhibit lower values of heats and temperatures of melting [13].
Translated by D. DOSKO~ILOVA
REFERENCES
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