706
.
THE JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
Vol. 14, No. 8
Jell Strength and Viscosity of Gelatin Solutions’*2 By Earle T. Oakes and Clarke E. Davis RBSEARCH LABORATORY, NATIONAL BISCUIT Co., NEWPORK, N.
This investigation shows that the solutions of d.ifferent gelatins bear the same qualitatioe variations for jell strength with pH. The form of the jell strength-pH curve is the same for different concentrations. The maximum for jell strength coincides in p H with the maximum increase in viscosity for aging of solution. Lack of agreement between viscosity and j e l l strength may result from differences in the ash content of gelatin. Viscosity-jell strength measurements properly carried out parallel each other on di$erent gelatins. The mean molecular weight of gelatin oaries directly with the grade and is of the order of magnitude of 1000. Additional data are submitted in favor of the gelatin-acid combination theory.
CAREFUL investigation of the viscosities of gelatin solutions indicated that their jell strength would exhibit considerable variation with change in pH, other factors remaining constant. Accordingly such an investigation was undertaken.
A
Fischer and Coffmana have investigated the effects of different acids, bases, and salts on gelatin solutions. Although no pH values are given, we may conclude from their work that gelatin solutions such as were employed by them are liquid in strong acid and strong basic solutions, while they are solid or semisolid in solutions near the neutral point. Patten and Johnson‘ have continued this work, measuring pH with Clark and Lubs indicators. Although no exact measurements were made of the rigidity of their solutions the following summary indicates their results: PH 2.8
3.6 4.0
7.9 8.2
State of Solution a t 18’ C. Liquid Semisolid Solid Semisolid Liquid
Sheppard, Sweet and Scott5 investigated the jell strength of gelatins and glues and described a very accurate form of testing machine. They did not, however, investigate the effect of pH on jell strength.
The method employed by the present authors is essentially one that has been in yse for several years in a large gelatin manufacturing concern.6 As employed by them it has given accurate results when all the conditions have been strictly adhered to. Particular attention must be given to the temperature control, because the jell strength changes enormously with change in temperature. The only difference between the method employed in this investigation and that described below are differences in concentration.
METHOD OF DETERMINING JELL STRENGTH Weigh into an ordinary glass of about 200-cc. capacity 30 g. of finely ground gelatin. Add 180 cc. of cold water, mix, and let stand 30 min. Heat on a water bath to 60” C., allow Received March 18, 1922. Published as Contribution No. 6 from the Research Laboratory of the National Biscuit Company. 8 . 7 . A m . Chem. SOC.,40 (1918),303. 4 J. Bid. Chem., 88 (1919), 179. 6 THISIOURNAL, 12 (1920), 1007. The United Chemical & Organic Products Co., of Chicago, Ill., designed and built this jell strength testing machine and materially assisted us by supplying their methods of measurements. Published through the courtesy of Dr. A. Schweizer. 1
9
Y
to cool to 25” to 30” C., and place in the thermostat a t 21 O * 0.1’ for 16 hrs. Test the jelly in the testing machine (Fig. 1). This consists essentially of a balance, with a plunger on the right arm, and a platform for supporting a beaker. Suspended from the left arm is a small bucket in which shot are poured t o balance the needle at zero when the beaker is placed on the right-hand scale pan. When the glass of jelly is placed on the adjustable platform below the plunger, this is adjusted until the surface of the jelly just touches the plunger when the pointer registers zero. - Water is then slowlv added to the beaker and the p l u n g e r forced into the gelatin until the pointer registers a certain arbitrary value. The number of cc. of water required to bring the pointer to a definite place on the scale (deflection 6) is taken as the jell strength of the jelly. pH measurements were made by means of the hydrogen electrode. Lipowitz’ first proposed testing glues ” U with instruments of FIQ. 1 this type in which the relative consistency of the jelly measured by its capacity for bearing a weight is the underlying principle. Numerous instruments differing in form but not in principle have since been used, not only by manufacturers and buyers but also by scientific investigators. Smith* has described an interesting instrument for measuring jell strength. It consists of a pressure chamber, one side of which is closed with a thin elastic rubber diaphragm, a rubber bulb to produce pressure, and a manometer to measure the pressure produced. Increasing pressure causes the diaphragm to displace the jelly and the pressure required to produce a certain displacement is taken as the jelly strength. Alexander0 points out that all methods which depend upon the breaking or compression of the jelly in open glasses are subject to two sources of error. The glasses usually vary in diameter, thus forming a surface of variable area, and there is always a “skin” of greater or less thickness which interferes with the accuracy of the test. To obviate these difficulties Alexander’o designed a machine to test the resiliency of jelly blocks free from containing walls.
The difficulty arising from employing glasses of varying diameters was overcome in the present case by selecting and employing glasses of the same diameters. As no data on the “skin effect” were given by Alexander, this point was investigated. Three series of jellies were prepared from Ossein gelatin No. 2. In Series A, the gelatin “Neue. Chem. Tech. Unters.,” Berlin, 1861, 37. J . SOC.Chem. I n d . , 28 (1909), 262. 0 Allen’s “Commercial Organic Analysis,” 4th edition, 8, 608. 10 J. SOC. Chem. Ind., 25’ (1908),459. 7 6
THE JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY
Aug., 1922
solution was left uncovered in the thermostat. I n Series B, the surface of the gelatin was covered with toluene after the solution had cooled enough for the jelly t o set slightly. I n Series C, the surface of the hot gelatin solution was coyered by carefully floating toluene over the surface of the solution while
707
maximum of jell strength and shows that where the viscosity is increasing most rapidly with age of solution the jell strength is also greatest.12 RELATION BETWEEN JELL STRENGTH AND VISCOSITY I n Fig. 3 the viscosity-pH curve and the jell strength-pH curve for No. 2 gelatin have been reproduced. These curves furnish one reason for the lack of agreement obtained when the same series of gelatins have been classified by viscosity measurements and again by jell strength determinations. Suppose, for example, we select two gelatins exactly alike in every respect, except in their degree of acidity, the pH of Sample A being 8 and the pH of Sample B being 3. A will then give a high jell strength and low viscosity, while B will give a high viscosity and low jell strength. When brought t o the same pH they will have the same viscosity and the same jell strength. It is only when a given series of gelatins or glues are tested a t the same pH that we can expect to have the classification by viscosity measurements coincide with that for jell strength measurements. In order to test this relation between jell strength and viscosity more thoroughly, seven samples of gelatin were selected by the manufacturer as samples not classified alike by jell strength and viscosity measurements. Since these samples were not submitted as representative of special kinds of gelatin they were classified as miscellaneous and as such bear the prefix M, so as not to confuse them with the numerals previously employed in designating the kind of gelatin. TABLEI1
PH-
it was still hot and before any skin was formed. (In each case the toluene was poured off immediately before the test was run.) The results are given in Table I, Solution 2 being a duplicate of Solution 1 in each case. TABLE I-JELL
STRENGTH O F
Ai 490 Az 480
15 PER
BI 500
Bz 510
CENT
SOLUTION, GELATIN 2 Ci 500 c2 486
Per cent GELATIN Moisture M 2 8.16 7.62 M 3 7.86 M 1 M 4 8.10 M 6 7.86 M 5 8.53 M 7 8.33 X 8.93
Per cent Ash 1.23 1.03 0.59 1.12 1.04 0.84 0.86 2.39
Jell Strength 21 C. Mfgr’s Authors’ 000 605 600 600 550 550 450 430 410 405 340 330 310 300 59 59
Viscosity in Sec 60°
d’
..
Table I1 gives the jell strength and viscosity values submitted with the samples by the manufacturer as well as jell strength, moisture, and ash obtained by ourselves on the
These results not only indicate the reliability of the method when carefully carried out but also serve to show that the skin on the surface of the gelatin had no appreciable effect upon the jell strength. Where the jelly had set beneath the toluene surface the skin could be broken more easily than where no toluene was used, but as will be seen from the results this had no effect upon the jell strengths. Naturally these remarks do not hold for solutions covered with foam which is not removed before the test is run.
EFFECT OF p H
ON
JELLSTRENGTH
The jell strength of a 5 per cent solution of hide gelatin (No. 3) was investigated over a range of pH 1.3 to 13.0. The results are plotted in Fig. 2, along with those obtained on 10 per cent solutions of the same gelatin, and show that the jell strength is a t a maximum at a pH approximately 8. Five per cent solutions of No. 2, a low-grade gelatin, and 15 per cent solutions of No. 1, a very low-grade gelatin, gave results in accord with No. 3. No. 1 gelatin was of such low jell strength that solutions more dilute than 15 per cent could not be used because they were liquid over too great a range. I n Fig. 2 are also plotted the values for the increase in ‘viscosity during the second hour, as determined by the writersll for 1 per cent solutions of the same gelatin a t different pH values. The maximum of this curve coincides with the 11
Davis, Oakes and Browne, J . A m . Chem. SOL, 43 (1921), 1526.
18 I t must be remembered that it is only possible to use a sterile gelatin for this work. At the pH of maximum jell strength liquefying bacteria develop very rapidly and during the 16-hr. conditioning period may transform an otherwise solid jelly into a viscous liquid.
THE JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY
708
samples as received. X is a standard low-grade gelatin included for comparison. The gelatins are listed in the order of their jell strength.
The viscosity measurements in Table I1 were made by the manufacturer and represent the number of seconds required by the hot solution as made up for jelly tests to run through a standard arbitrary viscometer. The jell strengths were determined as heretofore described, and as a check on the manufacturer our jell strengths were determined on the samples without altering the pH, which ranged from 4.0 to 4.4 in the various samples. The results of the two laboratories agree very well on jell strength, but it will be seen that the viscosity values do not parallel the jell strengths. Since pH exerts such an influence on both jell strength and viscosity it was not expected that the two measurements would grade a set of gelatins alike unless they were made a t the same pH. Accordingly these measurements were repeated on all samples at pH 8.0. The jell strengths are shown in Fig. 4,where it is seen that the jellies obey Hooke's law and the lines do not cross. TABLE 111-VISCOSITY GELATIN M M M M M M M
x
AND JELL
Viscosity 1.6 Per cent Solution p M 8.0; Age 3 Hrs.; 25' C.
STRENGTH Jell Strength 16 Per cent Solution pH 8.0; 21° C.
2 3 I 4 6 5 7
In determining viscosity the same procedure was employed as previously described.'l Fig, 5 gives the results for 1.5 per cent solutions at 25" C.; the absolute viscosity in centipoises being plotted against the age of solution. From this figure it is clear that the less the difference between two gelatins the further the age-viscosity curves will have to be prolonged to differentiate between them. These curves are really graphical representations of the tendency of each gelatin to form a jelly, and the viscosity at any age represents a stage in the process ultimately resulting in a solid jelly. It is to be expected, then, that these viscosity measurements and jell strength measure-
Vol. 14, No. 8
ments will parallel each other. The viscosity and jell strength measurements a t pH 8.0 are given in Table 111, where i t is seen that the two determinations classify the gelatins in exactly the same order, Having demonstrated that a t 25" viscosity measurements parallel those for jell strength, it wasdecided to investigate the causes for the discrepancies in the results when the viscosity was determined at a higher temperature. Since at 40" the viscosity does not change with age and as the thermostat is more easily controlled at 40" than at 60",the former temperature was selected. The writers are perfectly well aware that the system gelatin-water is quite different at 21' (the temperature at which the jell strengths were determined) from that at 40". Just what are the differences in the system a t these two temperatures is still an open question, but since the differences do exist some chemists have argued that no relation between properties is to be expected. However, it has been repeatedly _shown that jell strength and viscosity decrease progressively with the hydrolysis of the gelatin. The greater the mean molecular weight the greater the jell strength and viscosity. It is only in about 25 per cent of the cases that this relation does not hold true and these are precisely the cases that demand attention. Reference to Table I1 shows that Samples 2 and 3 and Samples 6 and 5 are the most conspicuous examples of nonconformity to the general rule. These two sets were selected for detailed investigation. Viscosity determinations were made on 1 per cent and 5 per cent solutions of these gelatins a t pH 8.0 and 40 " C. The results are given in Table IV, and it is seen that they classify the gelatins exactly as in Table 11. TABLE IV-VISCOSITIESO F GSLATINSAT 40' C., pH 8.0 GELATIN M 2
3;
M 5
1 Per cent Solution 0.900 0.910 0.871 0.879
5 Per cent Solution 2.817 2.884 2.465-2.466 2.530-2.624
These results establish very clearly the fact that viscosity results above gelation temperatures may not classify gelatins in the same order as do jell strength measurements. I n seeking an explanation for these results it was noticed that the gelatins with the abnormally low viscosities had the higher ash content. From the earliest investigations the effect of neutral salts on the physical properties of the proteins has been recognized. Quite recently Loeb*8 has repeated much of this work with various salts but keeping the pH of the solution constant. He has shown that each of the salts normally occurring in the ash of gelatin lowers the viscosity to a marked extent. This is not surprising in view of the fact that these same salts also lower the viscosity of pure water quite as much as they do gelatin solutions, as will be seen from a comparison of Loeb's values for gelatin solutions with those of any of the physical tables for the same salts in pure water. Undoubtedly, then, these abnormally low viscosities are due to the high ash content of the gelatins. To test this by bringing the gelatins to the same ash content it was necessary to ash a quantity of Samples 3 and 5 and to add this ash to solutions of these same gelatins, so that they would have the same ash content as Samples 2 and 6, respectively. The samples could not all be washed to the same ash content for this would change the composition of the sample by removing some of the more soluble, more hydrolyzed, fractions. Not all of the added ash could be dissolved in the gelatin solution, but what did dissolve lowered the viscosity to such an extent as to bring the viscosity measurements a t 40" into accord with the jell strength measurements at 21". The difference in ash content of gelatins is, then, the main cause for the lack 18
J . Gen. Physiol., 9 (1921). 391.
*
Aug., 1922
THE JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY
709
gelatins are more completely hydrolyzed and have more of the lower molecular weight fractions. The high-grade gelatins are less hydrolyzed and have a greater mean molecular weight. Among others, Bogue17 has touched upon this phase. I n seeking an explanation for the maximum in the viscosity-pH curve many considerations lead to the conclusion that a compound is formed at this point. The fact that the higher the ACID AND ALKALI-GELATIN COMBINATION AND THE MOLEC- mean molecular weight of the gelatin the less acid is required ULAR WEIGHTOF GELATIN to titrate over the range pH 4.7 t o pH 3.5 lent considerable support to this view. I n order to test this point more thorThe behavior of various proteins when subjected to the effects of added acids, bases, and salts has long since been the subject of many investigations.14 Ostwald and FischeP give an excellent summary of the situation up to that date (1915) in the following paragraph:
of agreement between classifying gelatins by viscosity and jell strength measurements, and for a given ash content viscosity measurements may be substituted for jelly strength measurements. Viscosity measurements may be expressed in absolute units and may be very accurately determined; this is not true of jell btrength measurements.
'
The most striking fact that the study of the influence of electrolytes on the viscosity of purified proteins has brought out is the enormous change in viscosity which is produced by traces of electrolytes. * * * * * It remains for future investigators t o give us a clear and comprehensive presentation of this subject.
This summary of the situation in 1915 is excellent, concise, and accurate. Thanks to Loeb, attention has been directed to activity relationships, and he has studied the effects of different hydrogen-ion concentrations in addition to the effects of different quantities of electrolytep. Although many of Loeb's results are open to different interpEtations and therefore many of his conclusions may be unacceptable t o other investigators in this field, the fact remains that many of the confusing data have been classified and correlated by considering the effects of different hydrogen-ion concentrations. The implied hope of Ostwald and Fischer is rapidly being attained. Previous investigators had found maxima in the viscosityacidity curves and in the viscosity-alkali curves, but since these maxima were a t different points for different acids and alkalies no general conclusions could be arrived at. Loeb has shown the existence of a maximum at pH 3.5, regardless of the acid employed. This has been verified by various other investigators, including the authors. I n seeking the cause of this maximum the older investigators suggested hydrolysis as the cause of the decline in viscosity after reaching the maximum. Northrupla has shown that between the limits pH 2.0 and pH 10.0 the rate of hydrolysis is practically constant. The same thing was found by Davis, Oakes and Br0wne.l' Loeb seeks the explanation for these maxima in the Donnan equilibrium relationship, but his arguments are not entirely convincing. I n experimenting with a number of different grades of gelatin, the authors discovered that the higher grades required less acid to bring them to the point of maximum viscosity at pH 3.5. I n fact it was found that a definite relationship exists between the grade of a gelatin and the acid required to titrate it over the range pH 4.7 t o pH 3.5. Since there is probably no gelatin that is not made up of a series of the products of hydrolysis of the original tissue, no gelatin can have what may he called a molecular weight. What is determined is the mean molecular weight of all the various fractions making up the gelatin sample. The low-grade 14 Von Schroeder, Z . 9hysik. Chem., 46 (1903),75;Lewites, Kolloid-Z., 2 (1908),208;Gokun, Ibid., 3 (1908),8 4 ; Laqueur and Sackur, Hofmeister's Be&., 3 (1903), 193; Hardy, J . Physiol., 83 (1905), 251; Pauli and coworkers, Kolloid-Z., S (1908). 2; '7 (1910). 241; Biochem. Z., 2'7 (1910), 296; Zoja, Koltoid-Z., 3 (1908),249;Procter, Ibid., S (1908), 307;J.Chem. SOC.,106 (19141,313; Procter and Wilson, Ibid., 109 (1916), 307;Tolman, J . Am. Chem. SOC.,36 (1913),307,317;Tolman andstearn, I b i d . , 4 0 (1918). 264;Robertson, Van Slyke, Ostwald, and Fischer in their texts and journal articles. 15 "Handbook of Colloid Chemistry," 170. 16 J. Gen. Physiol., 3 (1921), 715.
AGE IN HOURS oughly a number of gelatins of varying grades, and therefore different mean molecular weights, were titrated over this range. I n calculating the mean molecular weights from these titration results, the quantity of acid required to titrate pure water over the same range was subtracted from that required by the gelatin and two assumptions were made. One assumption was that the gelatin molecules were monoacidic bases in their combining with acid. The second assumption was that the hydrolysis of the gelatin acid compound was negligible. Neither of these assumptions is justified on the basis of experimental facts, but the results are undoubtedly valuable in showing the variation of mean molecular weight with physical properties. Also these results serve to fix the order of the mean molecular weight of gelatin since neither assumption could account for the differences between 800 and 100,000 as have been obtained by previous investigators. Paall8 and Procterls report a molecular weight of around 840, while Smith20obtains a molecular weight around 96,000. By dissolving 1g. of gelatin in 99 cc. of water and titrating over the range pH 4.7 to pH 3.5 with 0.2 M HC1 and subtracting the amount of acid necessary to titrate the same amount of water over this range, the molecular weights were calculated as described above. These values are given in Table V along with the jelly strength of each gelatin. Chem. Met. Eng., 23 (1920), 5 , 61, 105, 154, 197. Em., 26 (18921, 1202. 10 J . Chem. SOC.,106 (1914), 313. 1.J. A m . Chem. SOC.,4 3 (1921), 1350. 17
