T H E STRUCTURE OF GELATIN
SOLS AND GELS*
I. The Viscosity of Gelatin Solutions BY S. E. SHEPPARD AND R. C. HOUCK
The study of the “viscosity” of gelatin solutions has been complicated by the fact that such solutions below a certain temperature level show a dependence of the rate of flow upon the shearing stress. This phenomenon, very general with colloids and “high molecular” bodies, has been entitled variously “plastic flow,” “structure viscosity,” and the like. I n the case of gelatin solutions, this behavior appears to be definitely related to gelation, as shown by the correlation of increase of apparent viscosity with time, or aging, of solutions below the so-called setting temperature. Davis and Oakesl in an important memoir, brought evidence to show that this increase of “apparent viscosity” with time ceased at a certain temperature, a t which the viscosity became independent of the time. They considered their data gave evidence for a definite critical “transition temperature” of sol-gel forms of gelatin, at 38.03OC. It was shown by R. H. Bogue? and independently by S. E. Sheppard, F. A. Elliott and S. S. Sweet3that the temperature at which plasticity or structure viscosity effects disappeared depended upon the concrntration of the gelatin solution. They concluded from this that there is no critical transition temperature of sol form to gel jorm but only a temperature zone of transition from sol state to gel state. The possibility, however, of temperatures existing for which the viscosity is independent of the time, or age of the solution, is of great importance, even if that temperature should prove dependent upon the concentration and other factors. Such invariant temperatures would be of great importance for the comparative testing and standardizing of gelatins, even if their theoretical significance were doubtful. Our investigation was directed, therefore, primarily to ascertain: a. Whether a definite temperature exists at which the viscosity becomes independent of the time. b. If such an invariant temperature exists, whether independent of pH, concentration, origin of gelatin, etc. I n following through these questions, particularly in regard to the relation of viscosity to pH, certain interesting relations of the rate of change of viscosity to pH were discovered, which appeared to link up the behavior of gelatin solutions more definitely with that of known homogeneous systems.
Experimental The materials used were a good grade commercial calf-skin gelatin, and two lots of Eastman “de-ashed” gelatin brought to close approximation to
* Communication
Xo. 395 from the Kodak Research Laboratories.
274
S. E. SHEPPhRD AND R.
C. HOUCK
the tentative specifications for Standard Gelatin which were proposed at the American Chemical Society meeting in September, 1928.~The moisture contents were determined, and the solutions made up on a dry weight a t 105°C. basis. The procedure for preparing the solutions was as follows: After the gelatin sheet had been cut in small pieces, they were placed in a vohmetric flask, water equal to five times the weight of gelatin was added, and the gelatin allowed to soak for one hour at 2 5 T . Fifty cubic centimeters of water a t 52°C. were then added and solution finished a t j2OC., this being apparently complete in thirty minutes. The water necessary to make solution 6.9 percent on a weight normal, 7.0 percent on a volume normal basis, was then added, and the solution transferred to a thermostat at the temperature indicated. After a lapse of thirty minutes for the solution to come to temperature, 5.0 C.C. were transferred to an Ostwald type viscosity pipette, and the time of flow was measured. From this, together with the density of the solution and the calibration constant of the pipette, the viscosity was calculated. This was the “apparent viscosity,” assuming the validity of the Poiseuille-Hagenbach formula. For lower temperatures, where gelation is occurring, and the “apparent viscosity” is rising, this assumption is incorrect, but does not invalidate the consideration of the trend of the value with time. The solutions used in time studies were kept sterile by addition of various antiseptics, as thymol, isoeugenol, chloroform, and toluol. I n certain experiments, the solutions were sterilized by heating in steam for twenty to thirty minutes, incubating, and reheating. A number of antecedent conditions were examined for their possible effect on the viscosity of a solution prepared according to the procedure detailed.
Time of Soaking Experiments showed that increasing the time of soaking in water a t 25°C. above one hour did not change the viscosity.
Temperature of Dissolution The best temperature would very reasonably be one at which the rate of peptization and solution is as high as possible compatible with slight actual chemical breakdown or hydrolysis. Since hydrolysis is always proceeding in the solutions, and increases rapidly with rise of temperature, the choice is somewhat arbitrary. Manning has stated that if a gelatin solution is made up at the temperature at which its viscosity is to be measured, there is a rapid initial juZ1 in its viscosity, measured at this temperature, whereas if dissolved a t a higher temperature and cooled down to this lower operating temperature the viscosity will not fall but remain constant for several hours. We found this to be relatively correct as regards the initial fall in viscosity, provided the operating temperature is not too low, but did not secure the constant viscosity. For an operating temperature of 40°C., a gelatin solution made up a t 4 o T . shows a
THE STRUCTURE OF GELATIN SOLS AND GELS
275
greater initial fall of viscosity than one made up a t jz°C. (see Fig. I ) but both rapidly approach to the same rate of fall of viscosity, not to a constant viscosity. As the operating temperature is higher, the initial falls become nearer each other. The nature of the process of solution of gelatin a t given temperatures still requires much study, in order that the parts played therein by swelling, dispersion, true solution, and hydrolytic decomposition may be more definitely dist,inguished. From the point of view furnished by the present DE-ASHED LOT ‘36
I
\
\
‘\
EFFECT O F TEWPERATURE OF MAKLHG SOLWIOH *
MADE UP AT 40.C
0 MADE. UP AT 52°C
ma1.6b-
zso u
7.40-L
6
730-Q u)
720 7.IO
-5 -
TIME IN HOURS
S. E. SHEPPARD AND R. C. KOUCK
276
CYPYES
VISCOSITV-TIME
ODiN GELITIN AT V&RIQUS PW 40%
15.
PM $ 3 OPU
14. I3
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-
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ie
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THE STRUCTURE O F GELATIN SOLS A S D GELS
LOT
-37
VISCOSITY OF GLLATIN VISCOSITY-TIHL CURVES
FIQ.4
V ~ ~ S I T Y - T I M : -La DC-AWED GELATIN LOT*Sb nrnw AS ANTISEPTIC M 4.9 40.C A J6.C 0 36.C x 3S.C m 34-C TlVE IN HOURS FPOM THE ADDITION O F WATER 6' m ' I+ . 14 " IC 16 " zo z' 2 24 ' 26 '
.
5.3'
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277
S. E . SHEPPARD AND R. C. HOUCX
LOT *Sb VISCOSITY or OUZiTlN VmCT OC ANTISEPTICS
.
3'1% THYnOL
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n e I N HOURS FROM m n w w C
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34.C A N T ; 34':
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TIWE IN HOURS FROM THE ADDITION O F WLTEP If
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28
32
FIQ.8
$6
40
44
46
60
THE STRUCTURE OF GELATIN SOLS AND GELS
279
first requirement. I n view of the tendency to select a higher concentration of gelatin for viscosity specifications,18the experiments were carried out a t a concentration, aa already noted, of 7 . 0 percent. It appeared desirable to make an extensive study of the phenomena a t one concentration beforc varying this factor. Incidentally, the data bear on the question of the existence of a critical transition temperature for sol+gel forms of gelatin, as deducible from the existence of a temperature a t which the viscosity does not change with time. The data are given in Tables I to VI, and shown graphically in Figs. ec z to 8. These experiments indicate that comparable results for the viscosities of gelatin solutions are possible only with gelatin sols of definite pH of the same age at constant temperature. The data do not support the view that a definite temperature exists for which the viscosity is independent of time. With these solutions, at 7 percent, sols of the same p H kept below a certain temperature zone showed a steady increase of “apparent viscosity” with time, which we regard aa due to gelation.’g For temperatures above this zone, they showed steady fall of viscosity with time. In an intermediate zone, which also depends upon the pH, the viscosity 6rst rises, and then falls. Hence, there did not appear to be any temperature a t which the viscosity is truly independent of time. It has been suggested that a rise’in viscosity, followed by a steady fall, is due to bacterial attack. However, in our experiments we found this phenomenon to occur, fairly reproducibly, in presence FIQ.9 of different antiseptics, and with no evidence of Combined ViRoometer and bacterial decomposition (cj. Figs. 7 and 8). To Conductivity Cell check the matter more conclusively, we took more concentrated solutions (to allow for hydrolytic breakdown), enclosed these completely sealed in a combined viscometer and conductivity cell (cj. Fig. 9). These solutions were sterilized by successive heatings in steam followed by incubations, and both viscosity and conductivity measured at different periods of time at given constant temperatures. These were considerably lower than for solutions kept sterile with antiseptics, owing to th.e accelerated hydrolysis during steaming having destroyed considerable gelatin. However, the curves (Figs. I O and 1 1 ) show that here again there is a region of temperature for which there is 6rst a rise of viscosity to a maximum, followed by a steady fall. Yet there was no change in conductance such as would be produced by bacterial attack liberating ammonia.” The nature of the process or processes affordingthis rise to a maximum is a matter of interest. We consider that this represents the combination of gelation and hydrolysis effects. Although the rate of hydrolysis decreases rapidlywith fall of temperature (vide
280
S . E. SHEPPARD A K D R . C. HOUCK
infra) it does not disappear. At the temperatures and pH values in question, hydrolysis will be proceeding at a certain rate. If no gelation occurred, the viscosity would fall steadily toward a limiting value. But since there is initially a certain concentration of unhydrolyzed gelatin, there is an initial moment of gelation the temperature being sufficiently low. This implies aggregation of the gelatin molecules and micelles, and binding of water, increasing the viscosity.21 The gelation, however, is not sufficient to offset the hydrolysis, so that after an initial rise the viscosity then falls off.
h condition such that the viscosity of the solution was independent of time would occur: a. Because both aggregation (gelation) and hydrolysis were entirely absent. b. Because the aggregation balanced the hydrolysis. Condition (a) is excluded, since hydrolysis will not be entirely absent. Condition (b) is not perhaps impossible, but seems unlikely to be realized. Pos-
281
T H E STRUCTURE O F GELATIN SOLS AND GELS
sibly the conditions would be more favorable, the less the fluctuations of temperature in the system. I n our experiments the temperature was regulated only to 0.05 to 0.1o’C. Further work is planned on the viscosimetry and pyknometry of gelatin solutions with much closer regulation of temperat ure. Shape of Viscosity: pH Curve From our experiments it appears that the shape of the viscosity:pH curve is largely determined by the age of the solution. This is illustrated in Fig. 1 2
100-
9.0
a.
a0-g
> k 7.0
- 2u
6.0
-F
5.0
-
4.0
-
DLASHED GELATIN L O T No 36
-
VISLOSITV P n CURVES AT I N C R L C I S I N G AGL
ems
OAGC . 1 I* * 44 r I, , a x I. 6
.
om&
IOYRS.
O uY 1 1 0 44 . rII II E 4 .I
a
A/ [
c. PH
I
I
A
1
4
3
Frc.
I
I
I
6
8
3
I 10
1
11
12
TABLE I Effect of Time on the Viscosity of Odin* Gelatin Viscosity in Centipoises at Different pH and Age 11.65 10.85
11.35
11.30
11.35
12.05
4
11.00
11.00
11.00
11.75
6
10.15
10.70
10.80
10.90
11.50
12.20
12.75
8
9.5
10.36
10.62
11.35
11.90
12.40
10.00
11.18
11.65 11.44
12.10
18
8.95 8.50 8.10 7.75 7.40
20
7.10
22
6.80
24
6.55
2
IO 12
14 16
13.25 12.60
13.75 13.45
10.45
10.70 10.50
9.70 9.40
10.30
10.35
11.03
10.10
10.18
10.90
9.10
9.95 9.72 9.60
10.00
10.77
11.24 11.05
9.85
10.65
10.87
9.65
10.55
10.70
10.80
9.40 9.26
9.40 9.33
10.45
10.50
10.35
10.42
10.55 10.30
8.75 8.45 8.15 7.90
*A commercial calf-skin gelatin.
11.80 11.55 11.30 11.05
282
S. E. SHEPPARD AND
R. C. HOCCK
C P
E v 0
9
@
.t
d v If
3
I I :
4 c
d
T c;
om I.0
rm 3
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h h h h W W W W
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283
THE STRUCTURE OF GELATIN SOLS AND GELS
TABLE I11 Viscosity-time Curves for Gelatin No. 36 a t Different Temperatures pH 4.9. Antiseptic-Thymol Viscosity in Centipoises Age
in hours
34OC.
35°C.
36°C.
40O"C.
9.96 10.42 I O .68 10.83
9 .os 9 .os 9.05 9.05 9 .os 9.03 9.03 9.02 9.01 8.99 8.92
7.57 7 .so
IO
10.87
I2
I O .90
I4 16
10.86 10.83
9.32 9.50 9.58 9.63 9.65 9.63 9.60 9.55
I8
10.75
9.50
20
10.65
22
10.57
24
IO.50
9.45 9.40 9.35
2
4 6
8
in
Hours
PH 2.3 38°C. 40°C.
4 6
9.42 8,77 8.25
8
7.80
IO
7.40 7.03 6.67 6.35 6.00 5.65 5.30 4.95
2
I2
I4
16 18 20
22
24
8.95 8.30 7.65 7.03 .,6.45 5.92 5.45 5.05 4.72 4.50 4.40 4.35
PH 4.3 35°C. 36°C.
9.90 9.92 9.90 9.84 9.75 9.62 9.50 9.36 9.24 9.10 8.95 8.85
7.45 7 .?I
7.37 7.34 7.30 7.25
7.20
7.16 7.12
8.8j
PH 4.5
7.07
PH 6.9
34°C.
35°C.
36°C.
38°C.
9.45 9.40 9.32 9.27 9.12 9.00 8.90
10.20
11.65 11.90 12.05
10.37 10.32 10.26
10.80 10.80 10.77 10.70
11.42 11.39 11.35 11.31
12.14 12.14
11.27
10.15
11.24
10.10
12.10
11.20
10.05
8.80 8.70
10.60
12.05
11.17
10.00
10.50
12.00
11.15
8.60 8.48 8.38
10.38 10.25 10.13
11.93
11.10
11.87
11.05
11.82
11.00
10.55
10.65
12.10
10.20
9.94 9.89 9.83 9.75
S . E. SHEPPARD AND R. C. HOUCK
TABLE V Viscosity-time Curves for Gelatin No. 3 7 for Increasing Temperature Antispetic: Thymol except a t 34OC, when isoeugenol was used. pH 4.7 Viscosity in Centipoises Age in Hours
34OC.
35OC.
36°C.
38°C.
40°C.
2
9.02 9.04 9.02 9.00
8.15 8.03
7.58 7.48
7.20
4 6
8.40 8.52 8.55
9.92 9.85 9.75 9.68 9.60
8.45 8.36 8.25 8.15 8.02
7.35 7,25 7.15 7 .os 6.95 6.85
9.54 9.45 9.37
7.90 7.80
7.95 7.85 7.75 7.65 7.57 7.48 7.40 7.30 7.20
7.70
7. IO
8 IO
I2
I4
16
18 20 22
24
8.51
7 .oo 6.85 6.70 6.60 6.50 6.40 6.30 6.22 6.15 6.08 6.00
6.75 6.65 6.55 6.45
TABLE VI Viscosity-time Curves Gelatin No. 36, Effect of Antiseptics Temperature 3 4 T . pH 4.9 Viscosity in Centipoises A.ge in
Thymol as Antiseptic
Thymol and CHClj &S Antiseptic
lsoeugenal as Antiseptic
2
9.93 10.45 10.70 10.82 10.87 I O .87 10.85
9.90 10.42 10.68
IO.50
Hours
4 6 8 IO I2
I4 16 18
10.80
22
10.74 I O .7 0 10.60
24
10.55
20
10.81 I O . 85 10.85 10.80 10.75
9.95 .80 10.98 11.08 1 1 .08 1 1 .07 IO
11
.03
IO.
70 10.64
10.95 10.90
10.57
10.82
10.49
10.73
THE STRUCTURE OF GELATIN SOLS AND GELS
285
The change with time appeared to take place in a quite regular manner (see next section). Johlin22has shown that with such solutions-gelatin above 4ooC., dilute soap solutions-the surface tension varies with time in a regular manner, and suggests that this occurs only in the case of colloidal solutions in which the solute is highly dispersed in a manner similar to that of true solutions.
FIG. 14 Fluidity-time Curves
De-ashed Gelatin No. 36
Hydrolysis at 40°C.
Hydrolysis and pH In solutions above a certain temperature when the viscosity falls steadily with time, it is probable that this fall is entirely due to hydrolysis. I n order to follow the rate of change, it appeared desirable to plot fluidities, Le., the reciprocals of viscosities, against time, for different pH values. These curves
2 86
S . E. SHEPPARD AND R. C. HOUCK
of 4 = I / q against time (cf. Figs. 1 3 and 14)were found to give fairlystraight lines for the initial period of change. We deemed it reasonable, therefore, to take the slopes A$,/At as a measure of the velocity of change of fluidity.
:
t
, ,
PH 5
6
7
9
0
FL",mT"-"cLoclT~-w
10
II
J
It
cuwr
LOT 'Jb 40.C
FIQ.1 5
\
\
rLUlDlTY - V C L O C I T Y - P ~ L O T H. 36
8
9
IO
CURVE
30. c
Frc.
16
On plotting this value $k = A$/At against pH, the following graphs were obtained ( c j . Tables VI1 to XIII, and Figs. 1 5 to 1 7 ) : It will be seen that the graphs consist of inclined straight lines, which meet when protracted (at 40'C.) at pH 6 , but are intercepted by a horizontal portion between pH 5 to p H 8. It is evident that this graph is very similar, first and specifically, to the graphs obtained by N ~ r t h r o p ?when ~ plotting log K as a function of
THE STRUCTURE OF GELATIN SOLS AND GELS
35OC.
34OC.
PH 9 E&% TCHClr hymaand
PH 4.3. Thymol
in
88
How
Antiseptic
88
Antiseptic
287
Antiseutlc
PH 4.9
Isoeugenol 88
as
Antiseptic
Antiseptic
11.60
2
10.20
9.90
9.95
4
10.57
IO.45
10.42
11.95
6 8
10.75
IO.70
10.68
IO. 50 10.80
10.85
10.82
10.81
10.98
12.10
IO
10.90
10.87
10.85
11.08
12. IO
I2
10.95
10.87
10.85
11.08
12.07
I4
10.93
10.85
10.80
11.07
I2
16 I8
10.90
10.80
10.75
I1 .oj
12.03
IO.85
10.74
10.70
10.95
I2
20
10.77
I O .70
I O .64
10.90
11.95
22
10.68
10.60
10.57
10.82
1 1 .90
24
10.57
10.55
10.49
‘0.73
I1
9.95
12.10
.os .oo
.8;
Skrabal points out t h a t for such reactions t h e generalized equation holds:
+ kl [A] + kl [OH] (a - x) K = k, + kl[k] + kl [&€I]
dx/dt = k, xifa -x =
I
where k, = velocity constant for neutral water. This is a generalized form of the “catalytic catenary” of Hudson.= If values are taken such that
- log [&]= pH log K = pk and pk is plotted as a function of pH, a graph is obtained of intersecting straight lines; this may be regarded as consisting of a left limb corresponding to hydrion catalysis, a right limb corresponding to hydroxyl ion catalysis, and an intermediate flat “stability” or iso-electric region of neutral water catalysis. The fact that the data for +k as a function of pH follow the same general law as the chemical logarithmic velocity coefficient Pk = log K certainly indicates a close connection between them. It may be noted that our data for
(bk
at
40°c. give
an intersection point of the[&] and [&H]linesat
288
S. E. SHEPPARD AND R. C. HOUCK
W
m
i
0
*
I
I N H I
m 0d aZ m W h2
3
h
h m 0
W
h m 0x0
N N
. . . . . . . . . . . .
0
H
U
I
r
r
l
I
N
THE STRUCTURE OF GELATIN SOLS AND GELS
0 0 h h d N O h m O t 0 0 r9row N e 0 . N W 0 " ) h
2. 2. 2. 2. e.: :. :: . .: . 2. 2. 2. 0
--
P - N roo roo roo r o r w N N m m d d m 0.0 0 I
90 . . 2 . 2 .2 .2 .2 2. 2. 2. 2. 2. 2 . 0
S. E. SHEPPARD AND R. C . HOUCK
FIG. 1 7
5 4
3 $
2
0
1
P
o -1
-2 -3 -4
+ 3 t E *I
0
I
2
3 4
5 6 7 8 9 10 I1 12 I3 14 pH 40'
FIG. 18 (Northrop: J. Gen. Physiol.. 3, 715 (1921)).
4
3
+I
4
I 2 3 4 5 6
pH
7 8 910I1I213I4
-(log CH) 65'
FIQ.19
(Xorthrop: J. Gen. Physiol., 3, 712 ( 1 9 2 1 ) )
THE STRUCTURE OF GELATIN SOLS AND GELS
3.9 4.13 4.9 5.5 5.9 6.1
2.15
I .I2
0.2907 0.162 0.164 t
104
7.9 8.3 8.7 9.0 9.3 9.7
291
.300 ,381 .450
,630 .76 I .02
S. E . SHEPPARD AND R . C. HOUCK
THE STRUCTURE OF GELATIN SOLS AND GELS
0
0
m
N
h
-
0 0 “ r h r n r n r n t - 0 0 0 “ra o\ N v l f f i rncc
Zd d d d w m m a W ? . . . . . . . . . . . .
m
I
I
H
w
I
H
3
I
r
i
0
0 “r
i d
.d
0
. . . . . . . 0
0 - 0 m o 0 h O N d o d N d v i W 0 0 m o N m m m d d d d d d d m m m
a a -
. . . . . . . . . . . .
+
l
I
I
l
l
l
-
I
I
I
H
I
6. E. SHEPPARD AND R. C.
294
HOUCK
t o deduce this relation theoretically, and perhaps in view of the uncertain relation of the fluidity (or viscosity) of solvated colloids to the concentration of solid colloida1this is not t o be expected. But the relation does not seem of improbable type. The viscosity is some function of the striction or immobilization of water. As the large colloid molecules are broken down by hydrolysis, the striction of the water decreases, and the fluidity increases. That over a certain interval the rate of fluidity increase should be proportional to the logarithm of the rate of molecular loosening is a very reasonable conclusion. These results are in agreement with the view that in such solutions gelatin is molecularly dispersed, at any rate in the sense that every part of the molecule is exposed to the solvent.J2 I t may be pointed out that this is in
TABLE XI Velocity-pH Curve-Gelatin No. 36. so°C. l h e
Incresse
in
in Fluidity
PH
Hours
3.4 4.0 4.6 4.9
6 I8 I8 I8 I8 I8 18 I8 I8 I8 I8 I8 I8 I8 I8
5.1
5.3 5.6 5.9 6.7 7.1
7.5 7.9 8.3 9 .O 9.4
0.071
.103 ,064 .0290 .02 I 5
% Incresse 4
47.7 64.4 40.3 16.70 12.45
.OIZO
7.50
.OISO
9.50 6.42 6.67 g .08 9.56 11.95 14.60
,0095 * 0094 ,0125 .OIjO
.or65 .020
% increase Time in HOW 7.95 3.58 2.23
0.930 .692
.0270
20.01
1.114
.Os40
25.7
= .430
agreement with the conclusions of Simms on the gelatin molecule from a study of the ionic activity of gelatin.” He concludes that “The gelatin molecule is large;that the dielectric constant of the medium between these [ionizable] groups is not greatly different from water, and that the free ionic able groups are all functioning and are acoessible to the inorganic ions in solution. We conclude that the protein molecule is spongy or arborescent in shape, with molecules of solvent and of other solutes invading the interstices.’’ There are, however, certain recent results which lead to some modification of this picture. The experiments of Gorter and GrendeP on the spreading of proteins in water gave a value of about 7.0 b.U. for the molecular thicknesa, and our work in this Laboratory56 has confirmed this result when spreading dilute solutions of gelatin on mercury. This indicates that
295
THE STRUCTURE O F GELATIS SOLS AND GELS
0
lobar
N m l o l n v l O
c ) l o P - O . I
c ) m W
0 0
N
b
W
N
I
rolomwwwww - h h h h m m m m a 0
. . . . . .
N
N
N
N
N
N
N
N
. . .
N
N
N
N
?
4
N
N
l
?
0
v)
0 N mm 0 0 0 0 0 I
c) CI
m m loo m m 0 m l o w rr)m I
-N
N
N
N
mer)
? ? ? ? ? ? ? ? ? ? ? ? ? ? 0
m
296
S. E. SHEPPARD AND R.
C. HOUCK
if gelatin has an arborescent molecuIe of branched chains, the arborescence is practically confined to two dimensions, since Simms shows that the distance betwezn ionizable groups in the gelatin molecule must be of the order of 17 to 18 A.U. or more. Again, from investigations to be published in the second paper of this series, on the anisotropy of gelatin gels, it is concluded that gelatin consists of markedly asynimetrical, flat or elongated, molecules, capable of parallel orientation under stress. It appears very possible, there-
TABLE XI11 Velocity-pH Curve-De-ashed Gelatin No. 36. 8 5 O C . PH
Time in Minutes
Increase in Fluidity
Increase
4.9 5.4 5.7
70
,089
26.88
80 70
,056 ,034
18.35 11.18
5.9
85
,042
6.7
I10
,046 .03 I
I
,I
55
%
14.91 18.67 11.45
k'
7,increase =Time in Hours 23.04 I I .26 9.72
10,30 9.97 12.40
fore, that the micelles of gelatin sols, at sufficient dilution and temperature, are macromolecules of elongated form but with some degree of branching of the chains, and with a strong tendency to %warming" and association, which increases with higher concentration and lower temperature. The Relation of Gelatin to Collagen With gelatin, hydrolysis involves reduction of water-binding power, and consequently decrease of viscosity. On the other hand, in the conversion of collagen to gelatin, also considered a process of hydrolysis, the opposite condition holds. If a suspension of hide powder (collagen) is digested at temperatures of 80°C. or upward in dilute acid or alkaline solution, the viscosity will rise a t first,-particularly if measured a t a lower temperature than that of digestion-then pass through a maximum, as the gelatin formed is itself broken down by hydrolysis. Now in some earler studies of the viscosity fall on hydrolysis a t 98°-1000C. of a number of commercial gelatins, it was found for a considerable number of these that relatively brief periods of hydrolysis produced a considerable rise of viscosity, then followed by a steady fall. Tables XIV-XVI illustrate this: If the value of A , increase of viscosity at 30OC. between 5 mins. and 2 4 hours be taken with Schroder36as an approximate measure of gelatinizing power, it will be seen that a relatively short period of hydrolysis at ioo"C. may increase the gelatinizing power of many commercial gelatins enormously, before finally destroying it. The explanation appears t o be that these gelatins, and probably most commercial gelatins, contain a certain amount of incompletely peptized
THE STRUCTURE O F GELATIN SOLS AND GELS
297
TABLE XIV Gelatin 6902 Hydrolyzed in Calfskin Gelatin 6902, 5 percent Hydrolyzed a t 9 8 ° - ~ 0 0 0 c .in N/rooo N / ~ o o oHC1 a t 98°-~000C. NaOH pH = 5.20 PH 5.07 q = viscosity a t 3oOC. after j mins. and 24 hours _3
Tim'e of Hydrolysis in mins. 0
8 15
30 75 198
11"
qj
4.80 530 4.15 3.79 3.14 2.46
5.01
4.97 11.48 5.01
3.21 2.24
A =Increase of Viscosity
Time of Hydrolysis in mms.
75
+0.21
.07
o 5 I5 30 45
5.98 5.34 4.32 4.60 3.80
I2
75
3.11
- .33 +7.33 +1.22
+ -
Hide Gelatin (Commercial) 5 percent Hydrolyzed a t 98°-~000C.in N/zoo NaOH 'rime of Hydrolysis in mins.
vi
1-
o
7.21
j
6.93 6.70 6.09 5.48 4.64 3.78 3.44
7.45 11.76 9.71 7.35 5.86 4.74 3.82 3.53
15 3j 80 140
280 320
A
!
S0.24
$4.83 +3.01 +1.26 .38
+ + +
of
Viscosity
7-
6.10 5.30 9.47
+o.12
- .04 f5.15 4-1.32 -k . 2 9 - .OI
5.92
4.09 3.10
Hide Gelatin (Commercial) Hydrolyzed at 98°-~000C.in K/4000 HCl
~
i1
A =Increase
H$$k
in mins.
11;
o 5
6.82 7.10 6.45 6.03 4,95 3.45 3.29
15 35 80
.IO
260
.04
320
A
11"
7.69
+0.87
12.15
+5.0j
7.60 6.55 5.28 3.54 3.36
+ I . I ~
+
+ .3; f
+
$. . I 1
Hydrolyzed a t 9 8 ° - ~ 0 0 0i ~n .N / ~ o o oNaOH Time of Hvdrolvsis il; mini. 0
6 '5 30 45 60
1
I
115
4.38 5.51
6.53 4.53 4.16 4.76
'I"
4.48 5.82 24.01 9.90 5.25
6.40
+
A 0.10
.31 +17.48 5.37 1.09 1.64 $.
+
+ +
.52
.09 .07
2 98
S. E. SHEPPARD AND R. C.
HOUCK
collagen particles. The transformation of these into gelatin on hydrolytic digestion first raises the viscosity and gelatinizing power, when this is overtaken by the destruction of the gelatin. The theory of gelatin formation which these results, and other work to be described, suggest is the following: We suppose, in agreement with Meyer and Mark’s X-ray i n ~ e s t i g a t i o n sthat ~ ~ the protein collagen consists of fibers built up of crystallites composed of primary valence chains of the anhydroamino-acids-the protein macromolecules. The formation of gelatin consists in the peptization of these fibers, whereby not only the crystallites, but the primary valency chains become disoriented and separated; complexes of these chains, possibly in a partly oriented or smectic ordering bind water by dipole orientation. On this view, the molecules of gelatin are fundamentally identical with those of collagen, the difference being only in t h e degree of association and orientation. The mutual attraction of these macromolecules, and their water-binding capacity, are reduced as hydrolysis progressively shortens the chains, until ultimately only a mixture of aminoacids is left.
References Davis and Oakes: J. Am. Chem. Soc., 44, 464 (1922). 2R.H. Bogue: J. Am. Chem. SOC.,44, 1343 (1922). Sheppard, Elliott, and Sweet: Trans. Faraday Soc., 19, 261 (1923). Cf. Ind. Eng. Chem. (Analytical Edition), 1, 56 (1929). Manning: Biochem. J., 18, 1085 (1924). J. Loeb: J. Gen. Physiol., 3, 827 ( 1 9 2 1 ) ;4 , 73, 97, 351 (1921-3). Cf. Bogue: loc. cit. a Sheppard: J. Phys. Chem., 29, 1224 (1925). ’R. H. Bo ue: J. Am. Chem. SOC.,44, 1343 (1922). lo Davis an! Oakes: loc. cit. H. Freundlich and Seukircher: Kolloid-Z., 38, 180 (1920). Loebel: J. Ph s. Chem., 32, 763 (1928). l 3 Davis and SaLbury: Ind. Eng. Chem., 20, 829 (1928). l4 Cf. E. Hatschek: “The Viscosity of Liquids, 120 (1928). I5E. Hatschek: “The Viscosity of Liquids,” 200 (1928). “Kunits: J. Gen. Physiol., 12, 289 (1929). Kunits and Northrop: J. Gen. Physiol., 12, 379 (1929). Briefer and Cohen: Ind. Eng. Chem., 19, 252 (1927). Cf. von Schroder: Z. physik. Chem., 45, 75 (1903). *OParsons and Sturgess: d. Bacteriology, 11, 177 (1926); Parsons, Drake and Sturgess: J. Am. Chem. Soc., 51, 166 (1929). 21 Or pseudo-viscosit Johliri: J. Phys. &em. 29 2 7 1 (1925). 2a Northrop: J. Gen. Phykoi.: 3, 715 ( 1 9 2 1 ) . 24 A. von Skrabal: 2. Elektrochemie, 33, 322 (1927). H. Hudson: J. Am. Chem. SOC.,29, 1571 (1907). 26 KO hrop: loc. cit. ,’C#H. M. Dawson: J. Chem. SOC.,107, I422 (1927). 28H. Dakin: J. Biol. Chem., 13, 357 ( 1 9 1 2 - 1 3 ) ;H. Dakin and H. Dudley: 13, 263 (1915);H. Dakin and H. Dale: Biochem. J., 13, 248 (1919). 29 D. J. Lloyd: Biochem. J., 14, 154 (1920). 30 Levene et al.: J. Biol. Chem., 6>, 661 (1925); 68, 287 (1926);74, 7 1 j (1927). E. Hatschek: o p . cit. 32 S. E. Sheppard: Xature, 107, 73 (1921); S. E. Sheppard and S. S. Sweet: J . Am. Chem. Soc., 44, 2797 (1922). as Simms: J. Gen. Physiol., 11, 629 (1928). 34 Gorter and Grendel: Trans. Faraday SOC., 22, 477 (1926). S. E. Sheppard and R. L. Keenan: Xature, 121, 982 (1928), Sheppard, Keenan and Siete: Ind. Eng. Chem., 21, 126 (1929). 35von Schroeder: loc. a t . : 21, 126 (1929). 37 K. Meyer and H. Mark: Ber., 61, 693 ( ~ 9 2 8 ) . A p r i l , 1929 Rochester, A’. Y. @
