VISCOSIMETRIC STUDY OF THE HARDENING OF GELATI" BY CHROME ALUM * BY J. POURAD~ER Research Laboratories, Kodak Path6 S.A.F., Vincennes, France Received 10th July, 1953 The reaction of gelatin with chrome alum is followed by the change in viscosity of solutions containing chrome alum and gelatin. The type of effect obtained depends on the concentration of gelatin and the pH. At relatively high gelatin concentrations there is an increase in viscosity which at highest chrome alum and gelatin concentrations may lead to the formation of a rigid gel. At low concentrations of gelatin and at the pH of the isoelectric point a fall in viscosity can take place. These effects are explained on the basis of variations in the relative amounts of inter- and intramolecular bonding. It is considered that intramolecular bonding is favoured by proximity to the isoelectric point when the gelatin molecule is most tightly coiled, and by low concentrations of gelatin.Although the reaction of chromium salts on gelatin has been somewhat neglected as a subject of research, it is less complex than the reaction of the same salts on hides or leather. In fact, as gelatin is soluble in water above a certain temperature, it can be tanned by chromium salts in the homogeneous phase, and there is no risk of the reaction being disturbed by difficulties in penetration, like those which often occur with hides. This solubility also has the advantage of allowing one to apply to the reaction products the semiquantitative relations which have recently been established between the physical properties of the dilute solutions and the molec- ular characteristics of the solute.It would seem, therefore, that research on the reaction of chromium salts and gelatin, in addition to the interest which it presents for its own sake, should be capable of providing information on the tanning of collagen. EXPERIMENTAL kfATERIALs.-Gelatin.-hReasurements were carried out on a de-ashed Eastman Kodak gelatin obtained from limed calf-skin. Its isoelectric point 1 was at pH 4.75 : its number average molecular weight 2 was 65,000 : and its average molecular weight determined viscometrically 3 was 102,000. Gelatin concentrations are expressed in dry weight of gelatin (dried for 48 h in thin layers at 310" C ) in 100 ml of solution. Chrome alum.-The chromium salt used in this study was pure crystallized potassium chrome alum.Solutions were prepared by dissolving a known weight of chrome alum in twice-distilled water, and checking by a chromium determination. Chrome alum concentrations are expressed in terms of K2S04, Cr2(SO&, 24 H20. PROCEDURE.-The gelatin solution adjusted to the required pH was kept in a thermostat to reach temperature equilibrium. The titrated solution of chromium salt was then added, and after thorough agitation the mixture was kept in the thermostat with re- adjustment of the pH when necessary. The viscosity of the solution was measured at regular intervals. The addition of a chromium salt to the gelatin solution generally gives rise to a change in viscosity, which, according to the experimental conditions, increases or decreases.If q is the viscosity of a solution at a temperature t and TO the viscosity of the solvent at the same temperature, then the specific viscosity is given by the ratio q SF r= (q - TO)/TO. In order to estimate the action of a chromium salt on a gelatin in solution we took the hardening $1 defined as the relative variation in the specific viscosity ; H = His positive if the chromium saIt has brought about an increase in viscosity and is negative in the contrary case. * Communication no. 1494V, from the Kodak Research Laboratories. 180J . POURADTER 181 RESULTS The reaction between chromium salts and gelatin is not instantaneous but sometimes proceeds for several days. It is therefore necessary to specify the duration of the reaction for each test.On comparing the results obtained by treating solutions of different concentrations, it is found that the concentration of gelatin in the solution during the reaction is one I 2 3 4 5 6 7 Hours FIG. 1.-Variation of hardening as a func- FIG. 2.Variation of hardening as a func- tion of the reaction time. tion of the reaction time. Concentration of gelatin solution 0.85 % ; Concentration of gelatin solution 1-70 % ; chrome alum concentrations are indicated chrome alum concentrations are indicated on the curves ; buffer : acetic acid + on the curves ; buffer : acetic acid + sodium acetate ; pH = 4.75, t = 38" C ; sodium acetate ; pH = 475, t = 38" C ; SO$- concentration 8 x 10-3 M. SO$- concentration 8 x 10-3 M. of the factors which determine the sign and the amplitude of the variations in viscosity.For example, fig. 1 and 2 show the results of two series of measurements carried out under identical conditions on solutions of gelatin with concentrations of 0-85 % and 1-70 %. Whatever the quantity of alum added, the effect of the reaction with chrome alum is to decrease the viscosity of the solution at 0.85 % and to increase it at 1-70 %. As a general rule there is a diminution in the viscosity when the chromium salt is added to a dilute solution, and an increase when it is added to a concentrated solution (fig. 3). 0.5 4-0 4, - 0.0. minutes ,25 ,50 75 Hardening [.25 I / / e'- FIG. 3.Variation of hardening as a func- FIG. 4.-Variation of hardening as a func- tion of the reaction time. tion of the chrome alum concentration.Gelatin concentrations are indicated on the Gelatin concentrations are indicated on the curves ; chrome alum concentration 1.5 % ; curves ; reaction time : 1 h ; pH = 4.75, pH = 4.75, t = 39.8" C. t = 39.8" C.182 HARDENING OF GELATIN The limits of concentration depend essentially on the experimental conditions (pH, con- centration and nature of the salts present, etc.). A complete series of measurements was made on isoelectric gelatin solutions (the pH of which had been readjusted regularly during the reaction) containing variable quan- tities of chrome alum. The hardening values obtained at the end of 1 h at 39.5" C are set out in fig. 4. Consideration of these curves leads to four cases : (i) gelatin concentrations equal to or greater than 4 % : the hardening increases with the concentration of chrome alum and above a certain value of this concentration the solution becomes rigid ; (ii) gelatin concentration of 2 % : the hardening increases with the chrome alum con- centration and tends towards a limiting value ; (iii) gelatin concentration of 1 %: the hardening increases with the concentration of chrome alum, passes through a maximum and then decreases ; (iv) gelatin concentrations equal to or lower than 0.5 %: the addition of chrome alum brings about a diminution in the viscosity of the solution.The strange behaviour of the tests in (iii) was found once more when, not the variations in viscosity, but the Harde niny FIG. 5.-Hardening as a function of the FIG. 6.-Hardening as a function of the PH.PH. Concentration of gelatin solution 4 %; Concentration of gelatin solution 4 %; chrome alum concentration 3 % ; reaction chrome alum concentration 1.5 % ; reaction times are indicated on the curves; t = times are indicated on the curves; t = 39.8" c. 39.8" C. variations in melting point of gelatin gels treated with different chromium salts were considered. The classification of (i), (ii), (iii) and (iv) is not absolute since measurements carried out under different experimental conditions have shown that if the nature of the phenomena is unchanged, the gelatin concentrations corresponding to the different kinds of behaviour vary with the experimental conditions. Among these the most important seem to be pH and the nature of any salts present.In order to study the influence of pH let us consider first the simple case of a gelatin solution of a concentration sufficient for the chromium salt always to bring about an in- crease in the viscosity, and containing only the hydrochloric acid or sodium hydroxide necessary to adjust the pH. Two series of measurements have been carried out on 4 % gelatin solutions maintained at 39.8" C and containing respectively 1.5 and 3.0 % chrome alum with respect to the weight of dry gelatin. The results corresponding to reaction times specified in regard to the curves are shown in fig. 5 and 6. Consideration of these curves leads to the three following observations. (i) Whatever the reaction time, the hardening, which is zero below a limiting pH of about 3, increases with pH to pass through a maximum, and then decreases again.J .POURADIER 183 These results are in agreement with those published earlier by different authors 4-6. It is very difficult to study the process at pH values above 6 since the chromium hydroxide precipitates and interferes with the measurement of viscosity. (ii) Contrary to what is observed in the study of most of the properties of gelatin there is no discontinuity in the curves at the isoelectric pM. (iii) The optimum pH depends on the reaction time and decreases as the latter in- creases. This observation should be compared with the results obtained by Houck and Dittmar 6 with more concentrated solutions. The phenomena are more complicated when the gelatin solutions are sufficiently dilute for the addition of chromium salt to bring about a diminution in viscosity at certain pH values.These experimental conditions have never been studied to our knowledge. However, they are very interesting from the point of view of explaining the mechanism of tanning, since at low concentrations the molecular interactions are so small that the phenomena may be analyzed into their components. The most characteristic measurements were made on 0.85 % gelatin solutions, the pH of which was buffered by a mixture of acetic acid and sodium acetate in suitable FIG. 7.-Variation of hardening as a function of the reaction time. Concentration of the gelatin solution 0.85 % ; chrome alum concentrations are indicated on the curves ; buffer : acetic acid + sodium acetate ; pH = 4.00, t = 38" C ; SO$- concentration 8 x 10-3 M.proportions. The results of measurements made at pH = 4.7 are aIready set out in fig. 1. According to these experiments, whatever the quantity of chrome alum added and what- ever the reaction time, the addition of the chromium salt brings about a diminution in viscosity. On the other hand, in studying the same solutions at more acid pH values, it is seen that the addition of chrome alum produces an increase in viscosity. The results of measurements made at pH 4.0 are brought together in fig. 7. It should be noted, moreover, that analogous results to those corresponding to a pH of 4.0 were obtained by treating the gelatin at a high pH in the presence of salts capable of forming chromium complexes and preventing the precipitation of chromium hydroxide.DISCUSSION In order to explain the variations in mechanical properties of chrome-tanned collagens, Spiers 7 in 1934 suggested that the chromium atoms would link together two adjacent polypeptide chains, one being attached to the chromium by a car- boxyl group and the other by an amino group. This hypothesis was taken up by Gustavson,* who suggested that the chromium complexes form bridges by184 HARDENING OF GELATIN attaching themselves with covalent forces to the acid groups of one chain and with co-ordination valencies to the basic groups of the other chain. We shall show that this hypothesis of bridge formation between the polypeptide chains can be applied to gelatin provided that factors inherent in the structure of that protein are taken into account.If the chromium can join together two neighbouring molecdes by attaching itself to a group R1 of the one, and to a group Rz of the other, it should be just as capable of linking two groups, Rl and R2, of the same molecule, thus forming an intramolecular bridge. Together with intermolecular bridges, intramolecular bridges may also be formed, and the relative proportions of the two kinds of bridge must depend on experimental conditions. It is probable that an intramolec- ular bridge is formed when two groups R1 and R2 of the same molecule are neighbours, and that, on the other hand, the chromium forms an intermolecular bridge when two groups belong to different chains which are close to one another. This observation is valid for any kind of process of fixation of chromium and in particular when that reagent is attached simultaneously or successively with the two groups, R1 and R2.A complete theory of the action of chromium salts on gelatin must therefore take account of the existence of these two kinds of bridges which modify the physical and mechanical properties of gelatin in different ways. The intermolec- ular bridges by linking together the molecules increase the apparent molecular weight of the treated gelatin and bring about an increase in cohesion. When the number of these bridges is sufficiently large, all the molecules are united into a rigid three-dimensional lattice. On the other hand, the intramolecular bridges by bringing together different parts of the same molecule do not contribute to the cohesion of the system. Moreover, it should not be forgotten that when it is attached to the polar groups of the gelatin the chromium modifies the interactions between the gelatin and water and between the molecules of gelatin.Consequently, every time the chromium, in order to form an intramolecular bridge, blocks groups which entered into the linking of the molecules among themselves, it diminishes the cohesion of the gelatin. If intramolecular bridges only were formed, the treated gelatin could be represented diagrammatically by a structure like grains of rice. In fact, intermolecular and intramolecular bridges are formed sim~iltaneously and the properties of the hardened gelatin depend on the relative proportion of each type of bridge. Finally, it is possible that in the presence of a substance capable of actively forming complexes with the chromium, the latter is attached to a single group of the gelatin and that its action is limited in this way to blocking the groups under consideration.Since the probability of intra- or intermolecular bridge formation depends on the proximity of two groups, R1 and R2, of one and the same chain or of different chains, it is logical to conclude that the concentration of the gelatin solution at the time of tanning should have an effect on the properties of the hardened gelatin. If the solution is very dilute, the gelatin molecules in solution are dispersed at a distance from one another, and, in the main, intramolecular bridges should be formed. These diminish the attractive forces which unite the different molecules and consequently bring about a lowering of the viscosity and similarly a lowering of melting point of the gels.On the other hand, in concentrated solutions there should be a relatively high proportion of intermolecular bridges tending to form a rigid three-dimensional lattice. The influence of pH on hardening may be explained by taking into account at the same time the possibilities of reaction of the chromium salt with the different groups of the gelatin, and of the degree of unfolding or of coiling of the polypeptide chains. It has been shown earlier 299 that the gelatin molecule, which has the form of a more or less tight ball at the isoelectric point, unfolds when the pH is varied and is more spread out as the pH becomes farther from the isoelectric point.J .POURADIER 185 Since the maximum coiling into a ball occurs at the isoelectric point, it is at that pH that one group R1 of a molecule of gelatin is most likely to be next to a group IR2 of the same molecule, and consequently it is at this pH where there is the greatest likelihood of finding intramolecular bridges. If the pH is made to vary, for example, by acidifying the solution, an unfolding of the gelatin molecule is brought about and at the same time the formation of intermolecular bridges is encouraged. This analysis of the phenomena makes it possible to explain the difference in behaviour of 0.85 % gelatin solution treated with chrome alum at pH 4.75 and pH 4.0. In an isoelectric solution at this dilution, the ball- like molecules are at a distance from one another and intramolecular bridges are formed almost exclusively, bringing about a decrease in viscosity, whereas at pH 4.0 the molecules are sufficiently expanded for them to form intermolecular bridges in sufficient number to increase the cohesion appreciably.These few considerations show the interest offered by the present hypothesis of the formation of inter- and intramolecular bridges, a hypothesis which makes it possible to explain qualitatively most of the results obtained." * The reaction of chrome alum on gelatin causes variations in the melting point of the gels and modifies the molecular weight of the treated gelatin. The results of our measurements on this subject have already been published.10 1 Pouradier and Roman, Compt. rend., 1949, 229, 1325. 2 Pouradier and Venet, J. Chim. Phys., 1950, 47, 11. 3 Pouradier and Venet, J. Chim. Phys., 1950,47, 391. 4 Crabtree and Russell, Sci. Znd. Phot., 1930, (2nd series), 1, 271, 309, 352, 393, 437, 5 Rousselot, Sci. Znd. Phot., 1936 (2nd series), 7, 193. 6 Houck and Dittmar quoted in Mees' The Theory of the Photographic Process (The 7 Spiers, J. Int. SOC. Leather Trades Chem., 1934, 18, 114. 8 Gustavson, J. Int. SOC. Leather Trades Chem., 1936, 20, 398. 9 Abribat, Pouradier and Venet, J. Polymer Sci., 1949, 4, 523. 463. Macmillan Company, New York, 1942). 10 Pouradier, Roman, Venet, Chateau and Accary, Bull. SOC. Chim., 1952, 19, 928.