August, 1923
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
839
T h e Gelatin-Tannin Reaction’ By Arthur W. Thomas and Alexander Frieden COLUMBIA UNIVERSITY, NEWYORK,N. Y.
UA4NTITATIVE data on the mutual precipitation of gelatin and tannin are of timely value in the proper manipiilation of the gelatin test for tannin, so important in the leather industry, of the tannin test for proteins, of interest l o the biochemists, and also to aid in the elucidation of the question concerning the nature of the combinai ion between these substances of great chemical complexity.
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Gelatin is completely precipitated by gallotannic acid when the ratio of tannin to gelatin is not less than 2 :I and the gelatin-tannin precipitate is not soluble in excess of tannic acid. There is a definite hydrogen-ion concentration for each sort of vegetable tannin for best precipitation. If the solution is not adjusted to or near this CH+.the precipitate may f a i l to form. The presence of a neutral electrolyte broadens the CH+range for precipitation. At the optimum CH+ tannin can be detected in dilutions as high as I part tannin in IOD,ODO to 200,000 parts of water. depending upon the source of the tannin. Aging of gelatin solutions has no effect on the sensitivity in testing for tannin prooided bacterial action is srevented.
PREVIOUS WORK Seguin I * is credited with the discovery of the precipitation of gelatin by those vegetable extractives which have the power of tanning skin, although Seymour-Jones2 claims that in 1762 Lewis found that galls contained a n astringent substance which would precipitate gelatin from solution. Proust3 attempted to show that the gelatin-precipitating property was identical with the ability t o tan skins, and a year later Humphrey Davy4 pointed out that (1) different vegetable tannins required different amounts of gelatin for saturation, ( 2 ) the composition of the coagulum, which in the average was 100 gelatin t o 82.5 tannic acid, depended upon the concentrations of gelatin and tannin used, and (3) the coagulum was soluble in a n excess of gelatin. Following Davy, a number of investigators,& all criticizing each other, suggested composition ratios of the coagulum varying from 59 to 136 parts of tannin acid t o 100 parts of gelatin. Graeger6 suggested the quantitative use of the reaction for determination of the purity and amount of gelatin in a given solution, and Fehling’ devised a method whereby tannin could be determined by titration with a gelatin solution. These quantitative methods were based, of course, upon an accepted combining ratio which pleased the inventor of the method, but which unfortunately never agreed with that found by others. The importance of the presence of electrolyte for sharp coagulation was first noted by Risler-Beurat,a who recommended the addition of alum, and this innovation was demonstrated by Schneiderg to be an improvement in the quantitative determination of tannin. Later, Schultzlo recommended saturation with ammonium chloride, finding this salt more efficient than either sodium sulfate, acetate, or borate. Later workers offered evidence for and against the quantitative value of the tannin-gelatin reacti0n.l’ Lowenthal’s12 method, based on the titration of excess tannin by permanganate in the filtrate after mutual gelatin-tannin precipitation in the presence of sodium chloride and hydrochloric or sulfuric acid, won the approval of Procterls and Katheriner,14 although later SimandL6claimed the addition of acid to be a source of error, since “gelatin tannate” was dissolved thereby. Jeanla proposed the addition of sodium bicarbonate and sodium chloride, while Dreaper and Wilson17 demonstrated that the amount of acid or alkali is of deep significance, since an excess of either results in dissolution of the coagulum or prevention of formation of any precipitate whatsoever. Wood’s showed that borax and ammonium oxalate inhibited the reaction and that the lower the ash content of the gelatin, the less tannin is precipitated by it, which merely means that a certain amount and kind of neutral electrolyte is essential for best results. Trunkellg found that while the coagulum is peptized by an excess of gelatin, excess of tannin has no effect, and that under condition:, of complete mutual precipitation, the coagulum is 1 Presented before the Division of Leather Chemistry a t the 64th Meeting of the American Chemical Society, Pittsburgh, Pa., September 4 t o 8, 1922. Contribution No. 413 from t h e Chemical Laboratories, Columbia IJniversity. Numbers in the text refer to the bibliography a t the end of the article.
absolutely insoluble in water, although completely soluble in alcohol provided it is treated with alcohol before it has had a chance to dry! He also demonstrated that the combining capacity of gelatin is a function of its purity and claimed that aged gelatin solutions were not so efficient as freshly prepared ones.
Perusal of this mass of experimental contradictions shows that one or more controlling conditions were not known by the earlier workers. One condition that was not controlled and which may account for the wide discrepancies is the hydrogenion concentration, which has been demonstrated by modern research to be of paramount importance in any reaction in which protein plays a role. The first to take this into consideration were Michaelis and Davidsohn,20 to whom we shall refer later.
PURPOSE OF PRESENT WORK The purpose of this investigation is to show the best ratio of tannin to gelatin and the optimum hydrogen-ion concentration for the mutual precipitation of gelatin and tannin, and to demonstrate the delicacy of the test, under proper conditions, for tannin in solutions of various important vegetable tanning materials. %IAXIMUIvf
MUTUAL PRECIPITATION
Gallotannic acid containing 11.6 per cent water and 0.07 per cent ash and gelatin containing 16.6 per cent water and 1.16 per cent ash were used, except for the experiments recorded in Tables I and 11, where an edible gelatin was used. Ratio of Tannin t o Gelatin 20 10 8 6 4 2
1
Volume of Precipitate cc.
TABLEI Appearance of Supernatant Liquid
Gelatin
-
--
0.4
0.9 0.9 1.4 1.5 2.5 1.8
Test- of Liquid Supernatant for
Clear; coiorleks Milky
Tannin
++ ++ +
+- Almost-..
The method used for determining the maximum mutual precipitation of gelatin and tannin was as follows: The required amount of tannin solution was allowed to run into a 100-cc. graduated tube with a long, tapering end graduated to 0.01 cc. (oil tube). The gelatin solution was then added, and the mixture shaken and centrifuged for 30 minutes a t a force of 1000 “times gravity.” The volume of the precipitate a t the bottom of the tube gave an indication of the maximum precipitation, though it is to be noted that the comparison of the volumes of precipitate was not always extremely exact, owing to imperfect packing. The appearance of the supernatant solution in the tube and the presence or absence of gelatin and tannin therein were additional aids in determining the maximum mutual precipitation. The solutions used in these experiments mere made up so that the total reacting mixture contained 1 gram in 100 cc.
IhTDliSTRIALA N D ENGINEERING CHEMISTRY
840
of solution. The proportions of gelatin and tannin were varied, a t the same time varying the hydrogen-ion concentration in order to get the approximate limits for the optimum. The results obtained at a reaction of CH+ = 10-4.0 are shown in Table I.? The ratios noted above were tried at CH+ = and IO-*. At -log CH+ = 2, dense turbidities formed at all ratios, but did not settle out even upon centrifuging. At -log CH+ = 6, slight turbidities were obtained, whereas a t -log CH+ = 8, no effect was obtained, the solutions remaining clear. These results show that the best ratio of gelatin and tannin is 1:2, and unless otherwise stated this ratio was adhered to in subsequent experiments. Solutions were prepared at various CH+'S between and where the total concentration of gelatin and tannin was 1, 0.1, and 0.02 per cent, all of which showed that the optimum reaction lay between the limits of -log CH+ = 4 to 5 . The more exact determination of the optimum reaction, where the total concentration of gelatin and tannin was 0.1 per cent, is s h o r n in Table 11. TABLE I1 Appearance of Supernatant -Log c,+ Solution Opalescent 3.9 Opalescent 4.1 Clear 4.2 Clear 4.3 Clear 4.4 Opalescent 4.5 Opalescent 4.6 Milky 4.7 Opalescence 4.9 Slight opalescence 5.1
Volume of Precipitate cc. 0 0.20 0.35 0.36 0.40 0.25 0 0 0 0
All these supernatant solutions gave precipitates on addition of gelatin to the supernatant solution, the minimum, however, being obtained with the solution at CH+ = 10-4.4. From these results, it is concluded that the optimum CH+ for the mutual precipitation of gelatin and gallotannic acid is 10-4.4. A 1 per cent solution of tannin was treated with a 0.1 per cent solution of gelatin a t various CH+,S,as in Table 11. The results obtained show that the optimum CH+remains about the same. -Log c, + 3.9 4.1 4.3 4.6 4.7 4.9 6.1 5.3
TABLEI11 Appearance of Supernatant Solution Clear yellowish Clear: yellowish Clear, yellowish Clear, yellowish Cloudy Milky Opalescent Slightly opalescent
Volume of Precipitate cc. 0.90 1 10 1.15 1 20 0.90 0.80 0 0
Vol. 15, No. 8
These figures show a slight difference in the optimum point for the three gelatins.
EFFECTOF SALT Since 1860 it has been known that the precipitation of tannin by gelatin is made more complete by the addition of an electrolyte. I n the official method for testing for tannin used by the American Leather Chemists Association, the gelatin solution is made up to contain 10 per cent of sodium chloride. It was desirable, therefore, to study the effects of electrolyte on the mutual precipitation of gelatin and tannin. Preliminary experiments have shown that the addition of sodium or potassium chloride widens the range of CEI+for precipitation, and that these electrolytes will cause further precipitation only at -log CH+below 4 or above 5 . Between 4 and 5 the salt apparently exert2 no influence. Tannin and gelatin solutions with final concentration 0.1 per cent and tannin-gelatin ratio 2 : 1 were mixed in the usual way at various CH+ values. The solutions were (I) centrifuged for 30 minutes and (2) sodium chloride was added to make the final concentration of the solution 0.01 M . The solutions were again centrifuged and (3) more sodium chloride was added to make the final concentration 0.1 M . The results obtained are given in Table IV. TABLE IV-EFFECT -Log c, + 3.0 N o ppt. 3.5 No. ppt. 4.0 Ppt. 4.5 Ppt. 6.0 No ppt. 5 . 5 N o ppt.
OF SALT
(2) No GLCl 0.01 NaCl Faint opalescence Milky Slight opalescence Opalescent Slight opalescence Clear Slight opalescence Clear Slight opalescence Turbid Opalescent Clear
(3) 0.1 NaCl Increased turbidity
...... * .
No further effect No further effect Clear
.,....* .
Solutions were made in sodium chloride so that the final concentration of salt was in one case 0.01 M and in another 0.1 M. Higher concentrations did not seem to affect the mutual precipitation, but rather to salt out the tannin from the tannin solution. This salting-out effect was very marked with concentration of 1 M and upward. Table V gives the results obtained with 0.01 M sodium chloride solution, and Table VI, when a 0.1 M solution was used. -Log C, 1 2 3 4 5 6 7 S
+
TABLE V
----EFFECTMilky Opalescent Opalescent Ppt. Opalescent Clear Clear Clear
N o ppt. No ppt. No ppt.
N d bpt. No ppt. No ppt. N o ppt.
EFFECTOF ADDITIONAL NaCl None apparent Milky Ppt. None apparent Ppt. Slight Opalescence ppt.
h-one hIichaelis and DavidsohnZ0report the optimum reaction to vary between CH+ = 10-3.8 and 10-5.7 depending upon TABLEVI the ratios of tannin to gelatin and the masses of the same in EFFECTOF ADDITIONAL TESTF O R the solution. One reason for the discrepancy between their NaCh GELATIN -Log C, + -----EFFECT-findings and those of the authors is the fact that in a number None Negative 1 Very slight Clear s o h . of the very meager experiments made by them there was an PPt. ?;one Negative 2 Slight ppt. Clear soln. excess of gelatin. None Negative 3 Ppt. Clear soln, None Negative 4 Ppt. Clear s o h . I n anticipation of a possible criticism of the restriction None t 5 Ppt. Clear s o h . Ppt. Positive 6 Very slight Milky of the data to one given sample of gelatin, it was deemed PPt. advisable to try other specimens of gelatin. I n Tables I Ppt. Positive 7 No ppt. Opalescent Ppt. .... 8 No ppt. Clear and I1 an edible gelatin of unknown history was used. Duplicating the mutual precipitations over a range of CH+of 10-3.0 It is thus seen that the effect of electrolyte is only to widen to 10-5.6, using two different brands of gelatin, showed about the same results at each CH+, except that the greatest the range of CH+ for precipitation.$ However, while widenprecipitation of tannin occurred a t CH+ = 10-4.4for the first ing the range, it does not produce maximum precipitation, nor does it have an effect on the mutual precipitation at or gelatin, while a t CH+ = 10-4.6 for the second specimen. near the optimum CH+.
?All hydrogen-ion concentrations reported in this paper were determined electrometrlcally. Solutions were adjusted to the hydrogen-ion concentrations throughout by means of sodium hydroxide or hydrochloric acid, except for t h e data recorded in Tables VI11 and I X where phosphate buffers were b e d for the dilutlons up to C H + = lo-'.
$ In accordance with Weiske, Z. physiol. Chem., 7, 460 (1882), who found t h a t gelatin and tannin wpuld not precipitate each other from all solutions, while the addition of a neutral salt to such solutions effected a positive result.
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I S D C S T R I A L A N D ENGINEERIIVG CHE,MISTRY
August, 1923
APPLICATION
VEGETABLE TAKNING EXTRACTS
54 1
a turbidity, but due to the darkening of the solutions at higher alkalinities such end point became impossible t o of tannin by the “gelatin-salt” test. In order to establish detect and it was necessary to look for the appearance of , was the delicacy of the test with several important tanning a definite coagulum. At CH+ = 1 0 ~ ~ 0precipitation found to take place-at higher concentrations of tannin than materials, it was required first to determine the optimum hydrog en-ion concentration for each extract. While this a t 10-9, to be sure-but the values were too indefinite to be was found to be a t CH+ = 1 0 - ~to . ~~ o - ~forJ gallotannic reported, on account of the very dark color of the solutions. The figures strikingly demonstrate the significance of acid, it is not necessarily true for all tannins, as the figures hydrogen-ion concentration. in the gelatin-tannin reaction. in Table VI1 show. Tests on. the acid side of the optimum were not performed TABLE VII-OPTIMUM C, RANGEFOR TANNIN-GELATIN PRBCIPITATIONbecause the tannin particles are precipitated in acid solutions. EXTRACT -Log c, t It is to be emphasized that the delicacies tabulated above Larch 4 0 t o 3 5a Oak 3 5to4 0 are extreme figures representing the faintest turbidities Hemlock 4 Oto4 5 and precipitates visible upon careful scrutiny only by one Quebracho 4 Oto4 5 Wattle 4 5to4 0 very familiar with the technic. In practical and hurried Gambier 3.5 to 4 0 use the delicacies noted above would not be obtained. a T h e optimum reaction is given last in each case. In testing spent tan liquors it would be necessary, owing The optimum for gallotannic acid-gelatin precipitation to the color of such liquors, to set up a blank for comparison suggested that the combination took place a t or very close with the tube to which the gelatin-salt reagent is added. to the isoelectric point of gelatin (CH+ = 10-4.6 to Since the standard gelat,in-salt reagent is readily putresbut the data offered above indicate that the hydrogen-ion cible and its efficiency thereby rapidly declines, it is necesconcentration is of significance to the components of the sary to prepare it fresh daily. A series of gelatin solutions tanning extract as well as to the gelatin. was prepared and allowed to stand a t room temperature t’o The delicacy of the test for tannin at the optimum CH+ see whether this troublesome feature could be eliminat’ed. for each material is given below (Table TIII). The gelatin Four mixt,ures were prepared, all 1 per cent gelatin, as follows: solution consisted of 1 gram gelatin and 10 grams sodium (a)in distilled water at -log CH+ = 7 , plus 1 per cent sodium chloride, made up to 100 cc. in distilled water. Two drops fluoride, ( b ) same as (a) except use of toluene instead of of this solution were let fall into 10 cc. of the tannin solution sodium fluoride, (c) in distilled water adjusted to -log CH+= to be tested. Since so little gelatin is used it is unnecessary 4.6 plus 1 per cent sodium fluoride, and (d) same as (c) except to adjust the gelatin to the optimum CH+. To demonstrate that toluene was used instead of sodium fluoride. the necessity of adjusting the tannin solutions to optimum ’ The oldest solutions, prepared on June 24 and tested on CH+,the limits of delicacy are also given for the cases where November 25, all proved to be as efficient as a freshly prethe tannin solutions were diluted with distilled water jast as pared gelat’in-salt’reagent. The reaction of - log CH+ = 4.6 they were without paying any attention to the acidity. as well as 7 was selected, because gelatin is readily putresTABLE VI11 cible at CH+ = 10-7, while it is exceedingly resistant t’o Limit of Detection Parts Tannin in Parts bacteria at 4.6, not entirely because of its isoelectric condia t Optimum C,+ Water in Distilled Water EXTRACT 1 P a r t in 1 Part in tion at the latt’er, but also because the activity of liquefying Larch 150,000 20,000 bacteria is apparently inhibited or prevepted at CH+= 10-4.4. Oak 150,000 7500 Hemlock 130,000 6500 It is therefore unnecessary to prepare the gelatin-salt Quebracho 130,000 17,000 Wattle 200,000 20,000 reagent fresh daily, since it has been shown that such soluGambier 110,000 30,000 tions kept for five months at least, provided a suitable preIt is evident that when extreme delicacy is required, the servative such as toluene or sodium fluoride is present, reaction of the tannin solution must be adjusted, since, as are just as efficient as freshly prepared solutions. shown above, there is a marked falling off in limits of detection A4CKKOWLEDGXIENT when an extract is diluted with distilled water without regard The authors are indebted to A. F. Gallun h Sons Co., of to the CH+. In this connection it is interesting to note that Wilson and Kernz1reported the delicacy of the “gelatin-salt Milwaukee, Wis., for grants in aid of this investigation. test” (dilution at which test was negatire) as follows: queBIBLIOGRAPHY bracho, 1: 11,300; hemlock bark, 1: 5600; oak bark, 1: 5700; 1-Ann. chim., 20, 15 (1796). larch bark, 1: 6300; and gambier, 1: 6500. In their experi2-J. SOC.Leacher Trades Chem., 4 , 119 (1920). ments the extracts, as found, mere diluted with distilled water. 3-Ann. chim., 41, 332 (1802). , 233 (1803). 4-Phil. T ~ a n s .93, Owing to the confliction in the definition of tannin, it should Ann., 31, 124 ( 1 8 3 9 ) ;Lipowitz, Jahresber. chem. Tech., be emphasized that all the figures in this paper are based on 1861,5--Mulder, 624; Mdller, 2. angew. Chem., 15, 482, 1237 ( 1 9 0 2 ) ;Bottinger, A n n . , the Wilson and Kern21,z2 method, which defines tannin as those 244, 227 (1888), Schroder, Gerberei-Chem., 396, 1898. substances which irreversibly combine with hide substance. 6-Polytech. J . , 126, 124 (1852). 7-Ibid., 130, 53 (1853). The extracts were then made up at -log CH+ = 6, 7 , 5, 9, 8-Bull. soc. i n d . Mzdhowse, 3 0 , 263 (1860). and 10, and the limits of detection of tannin were determined 9-Jahresber. chem. Tech., 537, 1860. with results as tabulated below. In each case the gelatin 10-Polylech. J . , 182, 155 (1866). solution was adjusted to the same CH+as the tannin solution. Ibid., 205, 137 (1872j; Estcourt, Chem. News, 29, 109 11-Wagner, The abrupt change in delicacy between CH+ = 1 0 - 6 and (1874). 12--2. anal. Chem., 16, 33 (1877). 10-7 is, due to a difference in determination of end point. 13-Chem. News, 36, 58 (1877). At 10-0 the limit of detection was based upon appearance of 14-Polytech. J . , 227, 4 8 1 ( 1 8 7 8 ) ;228, 53 (1878). TO
Xo data are available concerning the limits of detection
TABLE IX
-- Log c,+ Wattle Quebracho 1,arch Hemlock Gambier Oak
Limits of Detection P a r t Tannin in Parts Water--6 7 8 9 30,000 8000 4000 1500 7500 2800 30,000 20,000 1000 500 15,000 1750 2400 1500 10,000 4800 760 400 10,000 1000 750 500 7000 1750
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Ij-Ibzd., 227, 490 (1878); 244, 391 (1882). 16-Z. angew. Chem., 11, 265 (1898). 17-J. S O L .Chem. Ind., 25, 515 (1906). lS-Ibid., 27, 384, 1164 (1908). 19-Biochem. Z., 26, 458 (1910). aO-Ibid., 54, 323 (1914). 21-J. I n d . Eng. Chem., 12, 465 (1920). 22--lbid., 13, 772 (1921).
