T H E SOLVATIOK OF GELATIN E. HEYMANN Department of Chemistry, University of Melbourne, Melbourne, Australia Received July 16, 1940
Several methods are available for determining “bound” water in gelatin gels, depending on the manner in which this nater is defined. The following definitions have been utilized: I, water that does not act as a solvent (3, 11); 11, water that cannot be frozen (10); 111, water incapable of hydrating cobaltous chloride dihydrate to form a higher hydrate (4). To these may be added a method (IV) which is based on the following principle: Gelatin, like all hydrophilic colloids, causes a contraction of the total volume when swelling in water (13). This can be readily determined in a dilatometer. It is due to the fact that the water molecules are polarized by the hydrophilic groups of the gelatin. The amount of volume contraction will decrease when the gelatin has been preriously hydrated in an atmosphere of water vapor. On increasing the initial water content of the gelatin, a point will be reached a t which no contraction of the total volume occurs when the gelatin is allowed to swell in liquid water. The amount of initial water content which is just sufficient to bring the volume decrease to zero may be regarded as the water of hydration. I n this case, the water of hydration is defined as the water which has a higher density than the free water. Compared with methods I and 111, this method estimates hydration in a system which is free from solutes, Le., from substances which may influence hydration. I n this respect, it more closely resembles method 11,which is also concerned with solute-free systems. There is, however, an objection against method IT-, based on volume contraction (Av). If Av is plotted against the initial water content of the gelatin, it approaches the zero value asymptotically; hence no very definite value can be obtained. This is, however, not a peculiarity of any experimental method, but is characteristic of the phenomenon of hydration itself. Although the polarizing influence of the gelatin particle on the water molecules presumably decreases with a high power of the distance from the particle, there is no hard and fast boundary between the water of hydration and the free water. The discrepancy which is generally found between hydration values obtained by various methods may reflect the varying 1143
1144
E. HEYMANN
extent of the influence on the hydration of gelatin of the solutes used in methods I and I11 or of the physical conditions in methods I1 and IV. I1
The contraction of the total volume on swelling was measured, using the previously described (7) dilatometer in a well-protected thermostat a t 23.3OC. The gelatin was Eastman purified gelatin, which contained 0.025 per cent of ash and in a 1 per cent solution gave a pH of 4.9, Le., it was almost isoelectric. It was used for the experiments in small blocks, suitable for the dimensions of the dilatometer. These were obtained by cutting concentrated jellies into rectangular pieces which were afterwards dried. Samples containing various amounts of water were obtained from
Au:::kL b
__
0.02
0.0I 0.1 a2
a3
w
a5 0.6 0.7
w
Fro. 1. Volume contraction on swelling (Av in milliliters per gram of dry gelatin) plotted against the initial water content per gram of dry gelatin ( w ) . Curve I, swelling in water; curve 11, swelling in 40 per cent ammonium sulfate.
the air-dry pieces, either by dehydrating in an oven a t a suitable temperature-for complete dehydration at least 105°C. is necessary-ar by allowing them to take up water vapor in a “moist chamber”. After swelling, the blocks contained 80-90 per cent water. I n figure 1, curve I, the volume contraction on swelling in milliliters per gram of dry gelatin is plotted against the initial water content per gram of dry gelatin (w). It is seen that Av = 0.058, which is in good agreement with Svedberg’s value (14). I t is seen further that Av decreases with increasing initial water content and approaches zero when the initial water content rises to 0.6-0.7 g. If the hydration ( h ) be defined as grams of bound water per gram of dry gelatin, we conclude from this result that h = 0.6-0.7. A few preliminary experiments with methylcellulose gave a considerably smaller hydration value,-namely, h g 0.25. In another series of experiments, 40 per cent ammonium sulfate solution
SOLVATION O F GELATIN
1145
was used insteaa VI water (figure 1, curve 11). I t is seen that the absolute value of the volume contraction is considerably reduced (Av = 0.039). Moreover, Av approaches zero a t an initial water content of about 0.25 g. per gram of dry gelatin. Hence it may be concluded that, in a 40 per cent ammonium sulfate solution, the hydration of gelatin is only about h = 0.25. This demonstrates well the dehydrating action of this well-known protein precipitant. Addition of 40 per cent ammonium sulfate to a gelatin solution causes precipitation. Hence, even in the precipitated state the hydration is still more than one-third of the hydration in the salt-free system. It is assumed, in these conclusions, that the volume of the system is not affected by secondary processes, such as the adsorption of ammonium sulfate on gelatin. $uch an influence, however, can be only slight, since Docking and Heymann (3) have shown that the adsorption of sulfates, if any, is certainly very small.
0.04 0.03
PER
CENT (”&%
FIG.2. Volume contraction on swelling of dry gelatin in 40 per cent ammonium sulfate solution (Au in milliliters per gram of dry gelatin) plotted against the concentration of ammonium sulfate (in grams per 100 g. of solution).
The dehydrating action of ammonium sulfate is also demonstrated in figure 2. Here Av, obtained with dry gelatin, is plotted against the concentration (in grams per 100 g. of solution) of the ammonium sulfate solution. Av decreases with increasing salt concentration. Obviously the extent of polarization of water molecules by the gelatin particles is reduced by the presence of this electrolyte. I11
An important factor has been stressed by Briggs (1). He, as well as Moran (lo), has shown experimentally that hydration, defined by one of the above methods, increases with increasing activity (a)of the water in the system. However, the following comparison will show that thevarying values of h obtained by different methods cannot be explained merely by the fact that the values of a are different. Hydration, defined by Hatschek (4) as water which is unable to hydrate
1146
E. HEYMANN
cobaltous chloride dihydrate further, is determined at an activity a = 0.31, corresponding to the dissociation pressure ( p = 5.4 mm. of mercury) of the system CoCl?.GHzO = CoClz.2H20 4Hz0 at 2OOC. Its value is h = 0.43-0.50. On the other hand, hydration defined by our method (IT’) in a 40 per cent ammonium sulfate solution is considerably lower,-namely, h = 0.25,-although the activity of the nater in this system is much higher ( a = 0.88). hloreover, hIoran’s value of h = 0.51-0.58 (xater that cannot be frozen a t - 1SOC.) is determined at a similarly high activity, but is actually not much higher than Hatschek’i value, which corresponds to a much lower activity. Our value, defined as water that has a higher density than free nater and determined in distilled water ( h = 0.6-0.7), is not very much higher than ?\Loran’s value, although the activity in the systems concerned approaches unity. The same value for h has been obtained by Docking and Heymann (2) from the negative adsorption in N/l ammonium sulfate at an activity of 0.96, but Sewton and Gortner (11) find h = 0.96 for a 5.4 per cent gelatin solution, and h = 2.05 for a 0.93 per cent gelatin solution a t an activity of 0.98 by the non-dissolving space method, using cryoscopic data. This procedure is essentially identical with the method based on negative adsorption; in both cases the amount of water which does not act as solvent is measured. Hence it is obvious that vanous methods m a y lead to w r y dzfferent values of h even at the same activzty. Hydration values calculated by Kunitz (9) from the deviation of the osmotic pressure from T-an’t Hoff’s law or using Schulz’ more general equation lead to much higher values ( h > 5 ) . These values are also determined at a water activity approaching unity, but they are very much higher than those obtained from other procedures at the same activity of the x-ater. Quite irrespective of the criticism which may be put forward against methods based on osmotic pressure, it is conceivable that they are sensitive to a kind of hydration which is different from the “adsorption-solvation” with which the other methods are concerned. Whereas with all other methods that amount of water is measured whichto a greater or smaller extent-is polarized by the gelatin particle, in the osmotic method the “immobilized” water (12) may also be included, since it virtually forms part of the particle in its thermal motion. The activity of the water has been calculated in all these cases as p / p o , where p is the partial pressure of the water in the sol or gel and I)O is the vapor pressure of pure mater a t the same temperature.
+
IV
A few years ago, the author attempted to determine the change of hydration during the sol-gel transformation of gelatin by measuring the change of total volume ( A d ) in a sensitive mercury dilatometer (6). The volume decreases on gel formation, suggesting that the amount of hydra-
SOLVATIOS OF G E L A T I S
1147
tion increases during this process. Heller and Vassy (5) have visualized the processes occurring during sol-gel transformation in more detail. They quite rightly emphasize that in the isothermal measurement the gelatin sol is not in equilibrium initially and that actually the transition is nieasured from a hydration value, corresponding to the higher temperature when the sol is stable, to that at the loirer temperature \\hen the gel is formed. They investigate the sol-gel transformation by measuring the light absorption during this process (5). The curves obtained are almost identical in shape with our h’-curves, but the sign of the effect suggesta a process opposite to that deduced fiom the volume change. The optical density, i.e., the scattering power, increase5 on gel formation, suggesting that the hydration decreases. How& er, Heller used gelatin solutions containing 28 volume per cent of alcohol, n hereas the measurements of At%’11-ere carried out in aqueous solutions. Hence the two system- are not strictly comparable. Heller therefore suggests that the s’pteni; containing alcohol should be investigated by the dilatometric method. The sol-gel transformation of 6 per cent gelatin solutions, containing 28 volume per cent of alcohol, vaq investigated in a sensitive mercury dilatometer a t 25OC. The instrument used was actually more accurate than the one described previously. I t nas made of fused silica and its capillary wa4 of 0.016 mm. bore. Here alvo the 1-olume decreases on gel formation. Hence the sign of the volume change is the same in aqueous solutions and in systems containing alcohol. The absolute value of Av‘ is slightly greater in the latter case than in the former (0 068 ml., compared with 0.057 ml. per 100 g. of gelatin). This may, honever, be due t o experimental reasons, foi. the amount of transformation which is measured isothermally need not be the same in both cases. In the actual experiment the system is heated to a temperature a t which the sol is stable and is then placed in a thermostat a t a temperature a t which the gel is stable. While the system reaches thermal equilibrium, i.e., before the actual measurement can bestarted, the processes which accompany gel formation will begin to take place. The extent to which this occurs may be different in the two cases. It is thus seen that the dilatometric measurements give no indication that the sign of the change in hydration is different in the two systems. Hence the apparent contradiction between the results of the dilatometric and of the optical measurements cannot be explained on the supposition that systems containing alcohol show, nith regard to hydration, a behavior opposite to that of the purely aqueous gelatin solutions. V
Heller and S’assy also discuss another possibility of explaining the optical effects observed by them. Solvation may not be limited to water, but
1148
E. HEYMANN
alcohol molecules may also take part in it. The authors realize that such an assumption would necessitate a modification of Kruyt’s theory of sensitization (8), which assumes that, on addition of alcohol, the particles are dehydrated, whereas according to Heller’s hypothesis they would be simultaneously “alcoholized”. An attempt has been made to subject Heller’s hypothesis to a direct experimental test. This is done by investigating the complete isotherm of the apparent adsorption of dry gelatin in alcohol-water mixtures in order to show which of the constituents is preferentially adsorbed. Such an isotherm can be investigated by measuring the density (d) of alcohol-water mixtures before and after contact with gelatin. The measurements were carried out a t 0°C. in a heavily lagged ice-water thermostat. Five grams of gelatin and 100 ml. of solution were left in contact for a t least 3 days, a time which is more than sufficient to attain equilibrium. The gelatin had been dried previously a t 105°C. for several days. Xot only does this procedure ensure that the gelatin is absolutely dry; it makes it, in addition, virtually insoluble in water. It is essential for the applicability of the method, based on density determinations, that the amount of solid material which passes from the gelatin into the liquid be kept very small. However, even with dried gelatin, small amounts (up to l per cent) of solid material may pass into the liquid phase in the case of mixtures which are rich in water. Hence the experimental change in density which is observed when gelatin is left in contact with an alcohol-water mixture is due to two effects: (1) The change in molar fraction as a consequence of preferential adsorption of alcohol or water, causing a change in the density of the liquid phase (Ad). This is the effect we desire to measure. (8) Dissolved gelatin causes an increase in the density (Ad’). In the actual experiment, A = Ad Ad‘ is measured. Fortunately, the second effect occurs only to a small extent and a correction eliminating its influence on the results can be easily applied. The second effect is due to the affinity of gelatin for water, gelatin being completely insoluble in mixtures containing more than 60 mole per cent of alcohol. The correction Ad’ can be determined in the following way: The solid residue in the mixture after adsorption (2) is determined. Xoreover, a blank containing gelatin and pure water is left in the thermostat together with the experiment and for the same time. The density change (Ado) and the solid residue (y) are determined afterwards. The density change due to the dissolved gelatin in the experiment is then Ad’ = x.Ado __
+
Y When Ad‘ is known, Ad can be determined, as Ad = A - Ad‘. Knowing the molar fraction-density curves of alcohol-water mixtures, the change
1149
SOLVATION O F GELATIN
of molar fraction of alcohol (f - f’) as a consequence of preferential adsorption can be determined, where f is the molar fraction of alcohol before adsorption and f’ the molar fraction after adsorption. I n figure 3, f - f’ is plotted against f. I t is seen that a t 0°C. (curve I) f - f’ is negative a t all concentrations, i.e., the molar fraction of alcohol in the mixture increases when the mixture is left in contact with gelatin. Hence water is preferentially adsorbed a t all concentrations. However, the shape of the curve, especially at low molar fractions, shows clearly that the adsorption of alcohol is not zero. The figures of a few typical experiments are shown in table 1. The results of a series of experiments a t 22°C. are shown by curve 11. The experimental accuracy is considerably less in this case, since the Ad’ correction is greater than in the experiments a t 0°C. Nevertheless it is quite obvious that at 22°C. the negative adsorption of alcohol is much less + 0.005
f
I
FIG.3. Apparent adsorption of alcohol on dry gelatin from alcohol-water mixtures. f, molar fraction of alcohol before adsorption; f’,molar fraction of alcohol after adsorption.
marked than a t 0°C. At a molar fraction of 0.25 the molar fraction does not alter during contact with gelatin, Le., for 3 moles of water, 1 mole of alcohol is taken up by the gelatin. The curve indicates moderate positive apparent adsorption a t low molar fractions. However, this part of the curve has to be examined with caution, since the experimental accuracy is not great here, owing to the comparatively higher value of the Ad’ correction a t 22°C. in systems which are rich in water. I t is possible to draw only qualitative conclusions from the curves of apparent adsorption in liquid mixtures. The results show that the gelatin particles contain alcohol besides water in the solvation layer, and that the ratio of alcohol to water in the solvation layer increases with increasing temperature. The bzaring of these results on Heller’s hypothesis, mentioned above, and on Kruyt’s theory of sensitization is obvious. Sensitization by alcohol may be due partly to an “alcoholization” of the particles and not merely to dehydration. I t is known that the sol-gel transformation of gelatin is accompanied by particle aggregation. The ultramicroscopic investigations of Zsig-
1150
E. HEYMAKS
mondy and Bachmann (15; cf. also 2) are the most conclusive proof for this fact. It may be asked whether the volume contraction on gel formation is partly a result of this, rather than of an increase in hydration. This view is supported by the fact that formation of crystalline curd from the clear jelly of sodium oleate is accompanied by a decrease in volume, as a consequence of the transition from the less densely packed “liquid” spherical micelle to the more densely packed crystalline micelle ( 7 ) . It may be argued that a similar process may be responsible for the volume contraction during the sol-gel transformation of gelatin, although there is no experimental evidence for the existence of such a process. However, the interpretation of the complete adsorption isotherm in the mixed system gelatin-water-alcohol lends support t o the author’s original assumption. TrlBLE 1 Equtlabrza at 0 ° C .
I
d*
0.048 0.065 0.129 0.160 0.203 0.273 0.370 0,472 0.533 0.602 0.770 0.870 0.925
0.98152
* The
0.95315 0.94472 0.93302 0,91296 0.89028 0.86696 0.85472 0.84256 0.81710 0.80364 0,79728
A
I
I
000020 000019
1
~
o o m -0oooO9 -0 00050 -000114 -0 00148 -000190 -000182 -000184 -0 00156 -0 00076 -0 00068
, 1
Ad’
000027 ooO022 000019 000015 OoO022 ooooo9 0 00013 o m 8 OoooO5 OoooO3 0 00000 0 00000 0 00ooO
Ad
f-r
-0.00001
+0.000s
-0.ooOOs
-0.oo02 -0.oOOS -0.000s - 0.0024 -0.004S -0.0061 -0.0091 -0.009, -0.010s -0.0110 -0.0060 -0.0050
-0.00019 -0.00024 -0.00072 -0.00123 -0.00161 -0.0019s -0,00187 -0,00187 -0.0015e -0.00078 -0.0006s
densities are measured a t 0°C. (&:)
Since the ratio of water t o alcohol in the solvation layer increases with decreasing temperature, gel formation in gelatin solutions on cooling is accompanied by, though is probably not the result of, increasing hydration of the particles. SCllhlARY
1. The hydration of gelatin has been determined from the minimum amount of water present in the gelatin which causes the volume contraction on swelling in water to be reduced to zero. It is thus defined as that part of the water in the gel which has a higher density than “free” water. 2. The results of various methods for the determination of hydration are discussed. The discrepancies cannot be explained on the basis of the varying activity of the water in the systems concerned.
SOLV.\TIOK
OF GELATIK
1151
3. The volume change during the sol-gel transformation of gelatin in a n alcohol-water mixture has been measured. A decrease in the volume is observed, as in purely aqueous solutions. 4. The complete isotherm of the apparent adsorption on gelatin in alcohol-water mixtures has been investigated a t 0’ and a t 22°C. The solvation layer contains some alcohol besides water, the ratio of alcohol to mater increasing with increasing temperature. The bearing of this result in other directions is discussed. The author wishes to express his thanks to Professor H. Freundlich and Dr. TT’. Heller, of the University of Minnesota, Minneapolis, Minnesota, for having drawn his attention to the problems dealt with in sections IV and V of this paper and for valuable discussions. REFERENCES (1) BRIGGS, D . R . : J. Phys. Chem. 36, 367 (1932). K . : Proc. Roy. SOC. (London) 122, 76 (2) DOSSAN,F. G , , ASD KRISHSAMCRTI, (1929); 129, 490 (1930); Colloid Symposium Monograph 7, 1 (1930). (3) DOCKISG, A. R., AND HEYMASK, E.: J. Phys. Chem. 43, 513 (1939). (4) H A T S C H E K , E.: Trans. Faraday soc. 32, 787 (1936). (5) HELLER, W,, ASD VASSY, E . : Compt. rend. 207, 857, 991 (1938). ( 6 ) HEYMASK, E.: Trans. Faraday Soc. 31, 846 (1935): 32, 462 (1936). (7) H E Y M ~ NE.: N , Trans. Faraday SOC.34, 689 (1938). (8) KRIYT,€1.: Cf. H. Freundlich’s K a p i l l a d w n i e , Vol. 11, p . 366. Akademische Verlagsgesellschaft, Leipzig (1932j . (9) KUNITZ, 31.: J. Gen. I’hysiol. 10, 811 (1927). (10) >\IoR.4S, T.: Proc. Roy. SOC.(London) A l l 2 , 35 (1926); 136, 111 (1932); B107, 182 (1936). (11) SEWTOS, R . , ASD GORTSER, R . h.:Botan. Qaz. 74, 442 (1922). (12) OSTWALD, IVo.: Kolloid-2. 46, 259 (1928). (13) SCHULZ, G. V , : 2. physik. Chem. A168, 237 (1931); A169, 374 (1932): A160, 403 (1932). (14) SVEDBERG, T H E : J. Am. Chem. Soc. 46, 2672 (1924). W.: Kolloid-Z. 11, 115 (1912); 2. anorg. Chem. (15) ZEIGMOSDY,R . , ASD BACHMASS, 73, 125 (1912).
