the velocity of ice crystallisation through...

307

The Velocity o f Ice Crystallisation through Supercooled Gelatin Gels.

By E rnest H arold Callow .

(A Report to the Food Investigation Board of the Department of Scientific and Industrial Research.

Communicated by Sir W. B. Hardy, Sec.R.S.-—Received March 7, 1925.)

(From the Low Temperature Research Station, Cambridge.)

I ntroduction .

It was observed that gelatin gels could be kept for several days at — 3° C. in a supercooled condition. A test-tube containing such a supercooled gel was seeded with a minute piece of ice and, from this point, ice crystals were observed to separate out downwards through the gel. The following preliminary experiment shows that this process occurs at a regular ra te :—A solution of gelatin (about 3 per cent.) was poured into twelve test-tubes, of approximately equal internal diameter, which were supercooled by leaving them at — 3° C. for sixteen hours. They were then seeded with ice and, after definite intervals of time, the distance to which the ice crystals had penetrated was measured with a ruler. For each reading the average of the twelve results, was taken, and a curve was constructed (see fig. 1).

6 ■

4 0 6 0T I M E IN M I N U T E S

Fig. 1.—The relation between the time, and the distance to which the ice face hadpenetrated.

The distance was plotted against time, and it will be seen that a straight line resulted. The velocity of ice crystallisation in these gels was_5* 1 cm./hr.„ whilst that for distilled water under similar conditions was 1800 cm./hr.

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308 E. H. Callow.

Experiments with other concentrations of gelatin, both in the presence and absence of electrolytes, confirmed the conclusion that crystallisation of ice from supercooled gels occurs a t regular rates. Since further experiments also showed that the velocity of ice crystallisation at — 3° C. was affected by the concentration of gelatin, the hydrogen-ion concentration, and the salt content of the gels, it was considered that a detailed investigation of these points might elucidate some of the problems of gel structure.

-Methods.

“ Ash-free ” gelatin, prepared by an electrolytic process, was obtained from the Eastman Kodak Company. The ash content was found to be 0*05 per cent, of the dry weight. All stock gelatin solutions were made up on the basis jim

of dry weight, the moisture content of the gelatin being determined by drying in an electric oven at 110° C.

Owing to the possibility of error being introduced by the presence of traces f ijfl of electrolytes, hard-glass test-tubes were used. They were carefully selected so that the maximum variation in internal diameter did not exceed 3 per cent, of the average diameter.

Gelatin was weighed out and soaked in distilled water overnight. It was then melted by heating to about 80° C., and diluted with distilled water to the required volume. The solution was thoroughly mixed, and given volumes were pipetted into beakers, enough distilled water being added to give the required percentage of gelatin. In order to vary the hydrogen-ion concentration, definite volumes of standard acid or alkaline solutions were used instead of water. Similarly, standard salt solutions were used to vary the concentration of salts. The beakers were heated on a water-bath, and their contents thoroughly mixed. For each determination, six test-tubes were taken and filled to within 2 cm. of the top with the hot solution of gelatin. They were placed in a rack and allowed to cool for three hours at room temperature.The racks were then put into a chamber at — 3° C. Since falling particles are apt to cause separation of ice at this temperature, the tubes were always covered with paper caps. After sixteen hours at — 3° C., the gels were seeded with a crystal of ice. It was found necessary to puncture the surface of the gels to a depth of 1 mm. before seeding, because contact with the air caused a hard surface to be formed on the gel. At intervals, measurements were made of the distance to which the ice crystals had penetrated. Each value finally adopted for the velocity of ice crystallisation was the average of all the readings obtained from the six test-tubes.



Hydrogen ion concentrations were estimated by means of the quinhydrone electrode at 18° C. It was found that this method gave readily reproducible results on the acid side of pH 6-5. On the alkaline side of pH 6 • 5, the hydrogen- ion concentration was determined by means of indicators.

E x per im en ta l .

I. The Effect o f varying the Concentration of Gelatin.

Gels containing from 1 to 12 per cent, of gelatin were made up with “ ash-free ” gelatin and distilled water. The hydrogen-ion concentration was found to be at pH 4-75, except in the case of 1 and 1*5 per cent, gels, which were at pH 4*80. Another series containing from 1 to 8 per cent, of gelatin was made up at pH 2*70 by adding the appropriate amount of HC1. Similarly, other series at pH 2*6 and pH 1*5 were made up. The velocity of ice crystallisation was measured at — 3° C. in the usual way, and the percentage of gelatin was plotted against the time (in hours) taken by the ice face to advance 10 cm. Typical curves are shown in figs. 2 and 3. In order to compare the results for pH 4*75 with the others, a portion of the curve (shown completely in fig. 3.) is included in fig. 2.

Ice Crystallisation through Supercooled Gelatin Gels. 309

5 2 J0 • PORTION OF CURVE U FOR | P h 4 .7 5 X Ph 1.50

Q P h 2 .70

PERCENTAGE OF GELATIN PERCENTAGE OF GELATIN

F ig. 2. F ig. 3.

Fig. 2. The relation between concentration of gelatin and time of crystallisation atpH 1*50, 2*60 and 2*70.

Fig. 3. The relation between tbe concentration of gelatin and the time of crystallisationat pH 4*75.

The curves in fig. 2 show that at pH 1*5, 2*6 and 2 • 7 for concentrations of gelatin above 1 per cent., increases in the concentration of gelatin cause per



310 E. H. Callow.

fectly regular increases in the time taken by the ice face to advance through a given distance. This increase of time is different for each pH value. For lower concentrations of gelatin, however, increases in the concentration have much less effect. There are thus two distinct portions to each curve. Owing to the scale employed, the first portions of all three curves appear to be super- | imposed. Table I, however, shows that even here the three curves are quite i distinct.

T able I.

Percentage of Gelatin. p H .

Time taken by Ice Face to

advance 10 cm.Turbidity. Velocity of Ice

Crystallisation.

Hours. Units. Cm./hr.i 4-80 0-010 4 1,000H 4-80 0-011 5 9302 4-75 0-014 6 71024 4-75 1-43 6 7-0

1 2-70 0-008 Nil. 1,200

1 2-60 0-006 Nil. 1,680

1 1-50 0-010 Nil. 960

Ill]I nI i i

I'iitoi

mThe data recorded in fig. 2 and Table I show that for every concentration of

gelatin the time taken is least (i.e.the velocity of ice crystallisation is greatest) at pH 2-6, and that the velocity is least at pH 4*75, which is the iso-electric tu., point of gelatin.

The complete curve for pH 4-75 (see fig. 3) is also composed of two distinct portions. The first portion is, however, prolonged to 2 per cent, of gelatin.All gels represented on this portion of the curve exhibited a marked turbidity (see Table I), whilst all gels at pH 1*5, 2-6 and 2 • 7 were perfectly clear. The second portion of the curve for pH 4-75 is a straight line at first (see figs. 2 and 3), but when the time taken for crystallisation becomes very great, the slope of the curve becomes less steep as the percentage of gelatin increases.

II. The Effect of varying the Hydrogen-Ion Concentration.Gels containing 2 and 4 per cent, of gelatin were made up at various hydrogen-

ion concentrations over a range of pH 1 to 10 by the addition of the requisite amount of HC1, H2S04 or NaOH. The rate of ice crystallisation was measured at — 3° C. in the usual way, and the results, expressed as velocity in centimetres per hour, were plotted against pH (see fig. 4).

I si s\ \u




A marked parallelism between the 2 and 4 per cent, curves is evident, except for a zone around the iso-electric point (pH 4-75). The minimum velocity on

Fig. 4.- -The effect of varying the hydrogen-ion concentration by means of HC1, H2S04 and NaOH for 2 and 4 per cent. gels.

the 4 per cent, curve lies at pH 4*60, whilst on the 2 per cent, curve the velocity increases suddenly between pH 4-50 and pH 4-95, attaining 920 cm./hr. at pH 4-80. All the gels which exhibited this remarkably increased velocity also showed varying degrees of turbidity, the maximum being reached at pH 4-80. Between pH 4*55 and pH 4-85, 4 per cent, gels were also turbid, but only to a very slight degree, the maximum turbidity being only half that of the corresponding 2 per cent. gel.

On the acid side of pH 4-75, the HC1 curves both have a maximum at pH 2 • 6. When H2S04 is used instead of HC1 for a 4 per cent, gel, the velocities of ice crystallisation are greatly reduced. In the case of the H2S04 curve the maximum velocity is approximately half that of the HC1 curve. This interesting relationship also holds for the swelling of gelatin {see fig. 5). Moreover, the

Fig. 5.—Loeb’s curves for the swelling of gelatin.

curves obtained by Loeb for the swelling of gelatin at different hydrogen-ion concentrations, using HC1, H2S04 or NaOH, show a striking similarity to the curve for the velocity of ice crystallisation through 4 per cent, gels at varying hydrogen-ion concentrations.

VOL. c v iii .— A. Y



312 E. H. Callow.

The curves in fig. 6 show that similar results were obtained for 3, 5 and 8 per cent. gels. They resemble the 2 and 4 per cent, curves in that the maxima

P h

Fig. 6.—The effect of varying the liydrogen-ion concentration by means of HC1 in thecase of 3, 5 and 8 per cent. gels.

appear to be at pH 2 • 60, but it will be noticed that flatter curves result when the gelatin concentration is increased beyond 4 per cent.

Even 1 per cent, gels, which invariably exhibited very high velocities of ice crystallisation, gave the same type of curve with a maximum about pH 2-60 (see fig. 7). The 1 per cent, curve, like the 2 per cent., shows an anomalous

Fig. 7.—The effect of varying the hydrogen-ion concentration by means of HC1 in the caseof a 1 per cent. gel.

zone around the iso-electric point. Here also the gels that gave rise to abnormally high velocities of ice crystallisation always exhibited some degree • of turbidity.



III. A Preliminary Investigation of the Effect of the Concentration ofNeutral Salts.

Varying concentrations of NaCl were added to 3 per cent, gels at pH 4 • 75 and at pH 2-0, the latter containing HC1 in requisite amount.

Fig. 8 shows that, at the iso-electric point, small quantities of NaCl up to 0-01 Molar have no effect on the velocity of ice crystallisation. All the gels on this part of the curve exhibited a uniform turbidity. Between 0*01 M. and

Ice Crystallisation through Supercooled, Gelatin Gels. 313

— •— • —

Ph 2.0

•02 -03 -04 -05 -06 *07 -08 *09 -10MOLARITY o f SODIUM CHLORIDE

Fig. 8.—The effect of NaCl on gelatin at pH 4-75 and on gelatin chloride at pH 2-0.

•02 'OS -O* *05 *06 *07 •(M O L A R I T Y o f SODIUM CHLORIDE.

Fig. 9.—Loeb’s curve, showing the effect of different concentrations of NaCl on the relative viscosity of a 0-8 per cent, solution of gelatin chloride at pH 4*0.

0*05 M. an increase of the concentration of NaCl caused an increase of velocity of ice crystallisation, and a corresponding decrease of turbidity. A clear gel was obtained when the concentration of NaCl reached 0*04 M., and above this concentration further increases of NaCl up to 0 • 1 M. did not materially affect the velocity of ice crystallisation.

With a 3 per cent, gel of gelatin chloride at pH 2*0, however, the case is entirely different. Addition of even 0*001 M. NaCl exerts a marked influence, the velocity of ice crystallisation being decreased. The presence of 0*04 M.

v 2



314 E. H. Callow.

NaCl is sufficient to reduce the velocity of ice crystallisation to one-third of its initial value. This antagonistic effect of NaCl on such systems of gelatin and hydrochloric acid is a well-known phenomenon, which is well illustrated by the curve, obtained by Loeb, for the effect of varying concentrations of NaCl on the relative viscosity at 24° C. of gelatin and hydrochloric acid at pH 4-0 (see fig. 9).

IV. Preliminary Experiments with Opaque Gels.

(a) The Effect of the Concentration of Gelatin.—Varying concentrations ofm■w

gelatin solutions were made up with 4 4 ash-free ” gelatin and distilled water inthe usual way. They were allowed to cool at room temperature and examined after sixteen hours. Concentrations of 1 per cent, and over gave gels (i.e. they could not be poured out of test-tubes), \yhilst concentrations of less than 0*8 per cent, gave gelatinous or normal sols. The degree of turbidity increased with increasing concentration of gelatin up to 2-5 per cent, and then decreased, the 7 per cent, gel being quite free from turbidity. Turbidities expressed in terms of an arbitrary standard, the maximum being defined as 6 units.

Fig. 10 shows that the alteration of turbidity under these conditions is surprisingly regular.

(b) The Effect of Electrolytes.—Varying quantities of electrolytes were added to 2 and 3 per cent, gels and the resultant turbidities and velocities of ice crystallisation were measured. The more important results are given in

turbidity and concentration of gelatin.

Table II.Table II.

Percentage of Gelatin. Electrolyte. Concentration. Turbidity.

Velocity of Ice Crystallisation

in cm./hr.

2 NaCl Nil 6 920 02 99 M./250 3 3-92 99 M./25 Nil 3*4

3-13 99 Nil 53

BaCl2Na2C03

M./10 Nil 3*43 M./250 99

3*4 6*1 A A3 M./750 99

3 HC11

M./250 994*0




)0.

¥

It lias already been shown that, around the iso-electric point, 2 per cent, gels exhibit a zone of turbid gels, which have remarkably high velocities of ice crystallisation. These abnormalities can both be eliminated by the addition of sufficient HC1. It is of great interest to find that sodium chloride also has this power (see Table II), but whereas M./500 HC1 is enough to give a clear gel, M./25 NaCl is required for this purpose.

In Table II the amount of electrolyte recorded for 3 per cent, gels is approximately the least amount necessary for the production of a clear gel. It will be noticed that this value depends entirely on the type of electrolyte. The fact that the solubility of gelatin is known to be increased by the electrolytes used is doubtless of great significance. With 3 per cent, gels the addition of a sufficient quantity of an electrolyte causes an increased velocity of ice crystallisation.

(c) The Rigidity of Opaque and Clear Gels.—In order to compare the rigidity of opaque and clear gels, the following rough experiment was carried o u t:—Three series of gels containing 3 per cent, of gelatin were made up ; the first contained no added electrolytes and in consequence consisted of opaque gels, the second contained enough BaCl2, and the third enough HC1 to give clear gels. Some gels of each series were used to determine the velocities of ice crystallisation. Through the other gels a fine steel knitting needle was allowed to fall under its own weight, and the time taken was noted. The results show that 3 per cent, opaque gels offer less resistance to a falling body, that is they are less rigid, than 3 per cent, clear gels. It is evident that the rigidity of the 3 per cent, gels cannot be correlated with the velocity of ice crystallisation.

Table III.

ElectrolyteConcentration. Turbidity. Velocity of Ice

Crystallisation.Behaviour of Knitting

Needle.

NilM./100 BaCl2 M./250 HC1

5NilNil

3-1 cm./hr.3- 8 „ 14- 0 „ J

Sank rapidly through the gel. Penetrated slowly and came to

rest about half-way down the test-tube.

Y. The Effect of Prolonged Boiling of the Gelatin.■ It is well known that the prolonged boiling of gelatin solutions results in a product of little or no gelling power. This form of gelatin is usually referred to as (3-gelatin. Experiments were carried out to find whether the alteration of gelatin to (3-gelatin had any effect on the velocity of ice crystallisation.



316 E. H. Callow.

About 500 c.c. of a 6 per cent, solution of gelatin was made up in the usual way ; 100 c.c. was kept for controls and the remainder was heated, on a boiling water bath for six hours, in a Jena flask fitted with a reflux condenser. Both normal and boiled solutions gave gels, but the gels obtained from the boiled gelatin solution were much less rigid than the normal gels. The velocities of ice crystallisation at —3° C. were determined in the usual way, with the following results :—

r

.

r6 per cent, normal 0-4 cm./hr.6 per cent, boiled 5*3 cm./hr.

wThese results show that the velocity of ice crystallisation for a 6 per cent, gel I j is greatly increased by previously boiling the gelatin and thus increasing the : concentration of [3-gelatin at the expense of the normal gelatin.

A similar experiment was carried out with 1 per cent, gelatin. When boiled 11,, and normal solutions were cooled to room temperature, a striking difference flk was observed in their appearance. The normal 1 per cent, solution had set (] to a very turbid gel, while the boiled gelatin solution had not set and only I f, showed faint opalescence. At — 3° C., however, both types of gel wereequally turbid. The following results were obtained for the velocity of icecrystallisation :—

Normal 1 per cent, gelatin 770 cm./hr.Boiled 1 per cent, gelatin 900 cm./hr.Water . . 1,800 cm. /hr.

VI. Miscellaneous Experiments.(a) Starch, Agar-Agar and Di-Benzoyl Cystin Gels.—In order to obtain infor

mation about the velocity of ice crystallisation through supercooled gelsmade from substances other than gelatin, a few preliminary experiments havebeen carried out. Di-benzoyl cystin gels at —3° C. gave a velocity of ice crystallisation of 1,400 cm. /hr. as against 1,500 cm. /hr. for that of distilled water. Gels of starch and agar-agar were also found to give a velocity of ice crystallisation of the same order as that of distilled water at — 3° C. It was also observed that the addition of small quantities of acid or alkali to agar- agar gels had no effect on the velocity of ice crystallisation.

(b) The Destruction of Gel Structure by the Separation of Ice.—Ice was allowed

I 1'1

to separate out through a gel at —3° C., the velocity of ice crystallisation being3*0 cm./hr. The test-tube containing the gel was then placed at room temperature for eight hours. After this the test-tube was put back into the chamber Sat —3° C. and left for the usual time. On seeding, the ice crystals immediately- Ili




^spread to the bottom of the test-tube. Instead of the whole gel being inter- & penetrated with ice crystals, as is usual with such high velocities of ice crystal- a lisation, the advancing ice crystals kept to well-defined paths and formed p opaque white tracks, which appeared to occupy the spaces that had been taken pup by the ice in the original separation. This definitely proves that the I separation of ice irreversibly ruptures the structure of the gel.

(c) The “ Ageing” of Gelatin Gels.—When a gel is formed from a solution, the I final equilibrium is not immediately attained. In order to find out whether this (factor had been sufficiently considered in the routine adopted for the making I up of gelatin gels, the following experiment was carried o u t :—

Two similar series of gels were made up ; the first series was treated in the I usual way (i.e. kept at room temperature for three hours and at —3° C. for I sixteen hours), and the average value for the velocity of ice crystallisation was I found to be 6-95 cm. /hr. The other series was stored at 1° C. for a week and I then placed at —3°C. for sixteen hours. In this case the average value obtained I for the velocity of ice crystallisation was 7-00 cm./hr. These results show that I gels made up by the usual procedure had reached equilibrium as regards the I velocity of ice crystallisation before any determinations were carried out.

D isc u ssio n .

1. Opaque Gels.Dhere and Gorgolewski (1) showed that gelatin could not be rendered ash-

I free by ordinary dialysis. They found however that, on subjecting dialysed I gelatin to electrolysis, a further quantity of inorganic impurity could be I extracted. In this way they obtained a gelatin containing less than 0-05 per I cent. ash. Gels containing less than 8 per cent, of this purified gelatin exhibited I a white opalescence, the 2 per cent, gel being markedly opaque. Dhere (2) I described the appearance of this gel as follows : “ La gelatine opalescente est I d’un blanc legerement bleuatre en lumiere incidente, et d’un jaune plus ou I moins orange en lumiere transmise.” In the present investigation it has been I shown that these turbid gels exhibit maximum opacity when the concentration I of gelatin is about 2 per cent, (see fig. 10). Dhere and Gorgolewski (1) also I showed that this opalescence was removed by the addition of electrolytes.I Traces of alkali had this effect, but in the case of acids or neutral salts larger I quantities were required to give a clear gel.

I t is extremely probable that opalescence of turbid gelatin gels is due to the presence of fibrils such as are described by Lloyd (3) in the following words :

I “ Miss Laing has very kindly examined the turbid water gels for me by means of



318 E. H. Callow.

the ultramicroscope and finds th a t they contain long fibres similar to the fibres in soap curds.” There is good evidence for supposing tha t gelatin in. this fibrillar form takes no part in the formation of the actual gel structure. Thus, Dhere and Gorgolewski (1) found tha t solutions of their pure gelatin in distilled water were incapable of forming gels if the concentration of gelatin was less than 1 per cent. On cooling, the gelatin separated out in the form of white opaque flocculi. More recently Knaggs and Schryver (4) have shown that even a 2 per cent, solution of gelatin could be made to flocculate in the same way by subjecting it to prolonged electrolysis. The flocculi thus obtained were found to contain 7*5 per cent, of gelatin.

Opaque gels, therefore, appear to contain gelatin in two forms, the “ gel- structure ” form, which is present in all types of gelatin gels, and the fibrillar form, which is present only in opaque gels and is responsible for their turbidity. If this view is correct, an opaque gel will contain less gelatin present as structure and would thus be expected to exhibit less gel strength than a clear gel containing the same concentration of gelatin. The present investigation has shown that this actually is the case, for an opaque 3 per cent, gel was found to offer less resistance to the penetration of a falling body than a 3 per cent, clear gel (see Table III).

2. General Discussion.There appears to be no reference in the literature to the velocity of ice

crystallisation through supercooled gelatin gels, except for Walton and Brann’s paper (5) on “ The Effect of Dissolved Substances on the Velocity of Crystallisation of Water,” which includes a few experiments on the effect of gelatin in concentrations up to 1*5 per cent. Since Walton and Brann supercooled the gels to — 7*1° C., the velocities of ice crystallisation they obtained were naturally much greater than those recorded in the present paper, where a temperature of — 3° C. was employed. The greater part of the work by Walton and his collaborators (5, 6, 7) was carried out with true solutions.

I t is well known that the velocity of ice crystallisation through supercooled solutions depends on the degree of supercooling, which in its turn is controlled by the temperature employed and the freezing point of the solution. Walton and his co-workers (5, 6, 7) found that there may also be a further effect on the velocity of ice crystallisation due to an alteration of the availability of the water. The nature of the dissolved substance determines the magnitude of this effect, and these workers discuss this in terms of the hydration theory.

Owing to its high molecular weight, gelatin can have little or no effect on the freezing point of water. Moreover, since in the present investigation the



temperature used was a constant, the degree of supercooling was practically the same for all experiments. With low concentrations of gelatin, the velocity of ice crystallisation was found to decrease slightly with increasing concentrations of gelatin. In all these cases, however, the velocity was still of the same order of magnitude as that for distilled water. It is here suggested that such decreases in the velocity of ice crystallisation are due to the decreasing avail-

t ability of the water. With concentrations of gelatin above 1 per cent. (2 per cent, in the region of the iso-electric point) even a slight increase in the concentration was found to cause an enormous decrease in the velocity of ice crystallisation. For example, at pH 1-50 the velocity of ice crystallisation

a- through a 1 per cent, gel was 960 cm./hr., whilst for a 1-| per cent, gel it was only 40 cm./hr. It is suggested that this is due to the retarding effect of the

ifc actual gel structure, a certain minimum strength being necessary to bring about e this result. This minimum strength is not attained in the case of low con

centrations of gelatin. The total concentration of gelatin necessary to give tel this minimum strength of structure is greater in the case of opaque gels than mil for clear ones, for in opaque gels some of the gelatin is present in the fibrillar jtl form, which appears to take no part in gel structure. In the case of clear gels,

this minimum of gel strength appears to be attained when the concentration of gelatin exceeds 1 per cent, (see fig. 2). However, with opaque gels at pH 4-75, more than 2 per cent, of gelatin is necessary to give this minimum strength of gel structure (see fig. 3).

When the velocity of ice crystallisation is very rapid, the force created by the advancing ice crystals is sufficient to shatter the gel structure completely, and


the ice formed interpenetrates the whole gel, the appearance resembling that obtained when ice is formed from distilled water. When, however, the gel strength exceeds this minimum, the ice formed can only advance along a few paths, and its velocity is correspondingly retarded ; the ice crystals formed are much larger and resemble rootlets in their manner of growth. When the size of ice crystals becomes very large, it exerts a splitting effect on the gel owing to its wedge-like growth, and this enables the ice to penetrate more rapidly. Thus two opposing factors are acting at the same time ; increases in the concentration of gelatin increase the time required for the crystallisation of the ice, but with very slow crystallisation the large crystals formed actually tend to decrease the time required. For the concentrations of gelatin used in this investigation, these very slow crystallisations were only obtained around the iso-electric point, and it is probable that the upper part of the curve at pH 4-75 owes its shape to this cause (see fig. 3).

y |



320 E. H. Callow.

The effect of alterations of hydrogen-ion concentration on the velocity of ice crystallisation was studied chiefly by varying the concentration of hydrochloric acid present in the gel. When the concentration of gelatin was above 3 per cent., all the curves obtained were similar in shape. In each case the lowest point on the curve was practically a t the iso-electric point, whilst the highest point was at pH 2*60. For concentrations of gelatin lower than 3 per cent., the curves were again very similar in shape, except for an anomalousf region around the iso-electric point, where the velocities observed were much higher ( see figs. 4 and 7). All the gels in this region exhibited turbidity. As! already explained, turbid gels contain some of the gelatin in the form of fibrils,) leaving less gelatin available for actual gel structure. Thus, as the turbidity! increases the gel strength decreases, and, unless the gel structure is strong! enough to exert its specific retarding effect, the velocity of ice crystallisation is correspondingly increased. For instance, in the case of 2 per cent, gels, as! the turbidity increases the velocity of ice crystallisation also increases, both attaining a maximum a t the iso-electric point. With higher concentrations of gelatin, however, this is not the case, for here, even with turbid gels, the gel structure is strong enough to exert ijs specific retarding effect on the velocity of ice crystallisation.

When the strength of gel structure is sufficient to cause a specific retarding! effect, increases in the strength of gel structure, due to the presence of electro-! lytes, no longer cause a decrease in the velocity of ice crystallisation. Thus, the velocity of ice crystallisation through 3 per cent, opaque gels was invariably slower than through 3 per cent, clear gels, although the former offered far less resistance to the penetration of a falling body. Since in these cases the strength of gel structure cannot account for the change in velocity of ice crystallisation,! some other factor must be sought. I t is suggested, tentatively, that this! factor may be an increase or decrease in the amount of water readily available; for the formation of ice. Assuming this to be the case, the results obtained by] varying the concentration of electrolytes, may be interpreted as follows :—At the iso-electric point where the velocity of ice crystallisation is at a minimum,] the amount of readily available water will also be a minimum. Increases in the concentration of hydrochloric acid will cause increases in the amount of 1 readily available water, until pH 2-60 is reached, after which the amount j decreases with further increases of acid. The occurrence of this maximum at 1 pH 2-60 is of great interest, as pH 2*5 (approx.) was given, by both Lloyd (8) j and Loeb (9), as the hydrogen-ion concentration at which all the gelatin in a | hydrochloric-acid solution exists in the form of gelatin chloride. Moreover, j




Lloyd (8) has shown that gelatin swells to its maximum extent when immersed in HC1 solution at jpH 2*5. Experiments in which the hydrogen-ion concentration was varied by addition of sulphuric acid showed that this acid has less effect than hydrochloric acid, the maximum velocity of ice crystallisation attained in the case of gelatin sulphate being only half that attained by gelatin chloride gels. Thus it would appear that sulphuric acid is not able to increase the amount of water readily available for ice formation to the same extent as hydrochloric acid. The addition of sodium hydroxide also increases the velocity of ice crystallisation, and hence the amount of readily available water, but not to the same extent as hydrochloric acid {see fig. 4).

I t has already been mentioned that there is a general resemblance between the curves obtained, during the present investigation, for 4 per cent, gelatin gels at varying hydrogen-ion concentrations and Loeb’s curves (10) for the swelling of gelatin under the same conditions of hydrogen-ion concentration ( figs. 4 and 5). Further, the results obtained by Loeb, for the effect of different concentrations of sodium chloride on the relative viscosity of a 0 • 8 per cent, solution of gelatin chloride at 24° C., resemble the results recorded in the present investigation for the effect of different concentrations of sodium chloride on the velocity of ice crystallisation of 3 per cent, gels of gelatin chloride {see figs. 8 and 9). Loeb considered that a solution of gelatin at 24° C. contains aggregates of gelatin molecules suspended in a true solution of gelatin in water. He attributed changes in the viscosity, obtained by varying the hydrogen-ion concentration of the gelatin solution, to changes in the volume occupied by these aggregates, due to their swelling. Loeb showed further that both the viscosity and swelling of gelatin at different hydrogen-ion concentrations are dependent on the potential difference between the gel or gelatin aggregates and the surrounding fluid. Since the results obtained in the present investigation resemble those of Loeb, it is probable that in some way the velocity of ice crystallisation is affected by the potential difference developed between the solid and liquid phases of the gel. The magnitude of this potential difference will be rigidly controlled by the amount and type of electrolytes present and will be a minimum at the iso-electric point. I t is tentatively suggested that at the iso-electric point, where the solid gel structure is practically uncharged, the gelatin is combined with the maximum amount of water and hence the amount of water readily available for ice formation is at a minimum. In the presence of electrolytes the solid gelatin structure becomes charged and is unable to combine effectively with as much water as uncharged gelatin. Thus more water is rendered available for the formation of ice, and the velocity of ice crystallisation is correspondingly increased.



322 E. H. Callow.

loss of the ability to form a gel. Thus, Blasel and Matula (11) prepared de- aminated gelatin and found th a t it was incapable of forming a gel. It is also well known that no gel can be formed by the breakdown products (referred to as (3-gelatin and gelatoses in the literature) th a t are obtained by the prolonged boiling of gelatin solutions. Walton and Brann (5) claim th a t the velocity of ice crystallisation at — 7 • 1° C. through (3-gelatin is exactly the same as through normal gelatin gels of the same concentration. The present investigation however, definitely shows that, a t a tem perature of —3° C., gels in which some of the gelatin has been changed to (3-gelatin exhibit a greater velocity of ice crystallisation than normal gels. Owing to the methods employed in the manufacture of gelatin, it is highly probable th a t different samples of gelatin contain varying amounts of (3-gelatin. This would explain the fact that two different samples of “ ash-free ” gelatin gave different results for the velocity of ice crystallisation. Consequently for each series of curves the same sample of gelatin w7as used throughout, so th a t the results were strictly comparable.

1 • A method is described for measuring the velocity of ice crystallisation

through supercooled gelatin gels.this method, experiments carried out a t — 3° C. showed that increases

in the concentration of gelatin cause decreases in the velocity of ice crystal lisation. Such decreases are considerable for concentrations of gelatin abo 1 per cent, (above 2 per cent, a t pH 4-75), e.g. a t pH 1*50 the velocity ° ee crystallisation through a 1 per cent, gel was 960 cm./hr. (about half t ve ocity through distilled water) and th a t through a 1-5 per cent, gel xvaS on y 40 eras./hr. For lower concentrations of gelatin increases in the con

els he’1011 ^aVe muck k®8 e&©ct, the velocity of ice crystallisation through su g6** \vug °f,the 8ame order °1 magnitude as through distilled water.

S u m m a r y .

f * When tbe hydrogen-ion concentration was varied by means of H a * rjoiflf0



4 The presence of sufficient neutral salt caused a slight increase in the velocity of ice crystallisation through gelatin-water gels. But when NaCl was added to gelatin-chloride gels there was a marked decrease in the velocity of ice crystallisation. This antagonistic effect of NaCl resembles that obtained by Loeb (10) for the swelling, etc., of gelatin.

5. Opaque gels were shown to offer less resistance to the penetration of a falling body than clear ones containing the same concentration of gelatin. It is suggested that opaque gels owe their turbidity to the presence of fibrils such as described by Lloyd (3). These fibrils appear to take no part in the formation of gel structure.

6. Abnormally high results were obtained for the velocity of ice crystallisation through gels containing less than 2 • 5 per cent, gelatin in the region of the isoelectric point. These anomalies were correlated with the degree of turbidity.

7. The velocities of ice crystallisation through gels obtained from a gelatin solution which had previously been boiled for six hours were more rapid than through unboiled controls, thus showing that (3-gelatin does not retard the velocity of ice crystallisation to the same extent as normal gelatin.

In conclusion I gladly take this opportunity of thanking Mr. 6. S. Adair, Fellow of King’s College, Cambridge, for the interest he has shown and for much helpful criticism and advice offered during the course of this investigation. My thanks are also due to Mr. Pique for constructing the curves.


REFERENCES.

(b Dher6 and Gorgolewski, ‘ Comptes Rendus,’ vol. 150, p. 934 (1910).(2) Dhdr6, * J. Physiol et Path. Gen.,’ vol. 13, p. 157 (1911).J U°yd’ ‘ Biochemical J.,’ vol. 16, p. 530 (1922).* Knaggs and Schryver, ‘ Biochemical J.,’ vol. 18, p. 1079 (1924).

Walton and Brann, ‘ J. Am. Chem. Soc.,’ vol. 38, p. 317 (1916).? alton and Brann, ‘ J. Am. Chem. Soc.,’ vol. 38, p. 1161 (1916).

8 n ‘ J- Am- Chem- Soc.,’ vol. 40, p. 1168 (1918).Lloyd, ‘ Biochemical J.,’ vol. 14, p. 147 (1920).

1 ' te b > ^ticle in ‘ Colloidal Behaviour,’ Pt. I, Edited by Bogue,(1924).

u!™ eb' ‘ S t e in s and the Theory of Colloidal Behaviour,’ p. 78, New York(1 « ) • 1 and Matula, ‘ Biochem. Z.,’ vol. 58, p. 417 (1914).

p. 33, New York



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