Analysis Of Glue And Gelatine
( Originally Published 1906 )
In discussing the methods applicable to the assay of the constituent elements of glue and gelatine, as well as of the impurities they may contain, it must be reaffirmed at the outset that analysis supplies no data as to the strength of the product. For the de-termination of this, the glue test is essential, sufficing for all practical purposes to acquaint the consumer with the relative merits of his purchase. As his experience in testing increases, he will be able to account properly for all undue characteristics.
Glue is in the main employed as a binding or adhesive medium; and its relative value for such use is comprehensively determined by the test. In the more extended applications of glue and gelatine, however, where, for example, the latter is used for alimentary purposes, or the former for finishing delicate fabrics or for sizing sensitive colors, it may not suffice merely to know the relative economy of the product in actual work. Both glue and gelatine enter frequently into the manufacture of complex products in which they are compounded with chemicals. In such instances the question of purity has largely to be considered, the manufacturer having often to purchase the glue and gelatine upon specification. Excesses of grease or soaps, acid or alkali, mucin or foreign mineral matter may all operate to the disadvantage of his processes. It is for chemical analysis to determine these limits; and the results of analysis often serve to amplify and confirm the qualitative observations of the test. Hence, where time permits, analytical data should be collected for such comparison.
The analysis of glue and gelatine comprehends the determination of moisture, ash, acidity, or alkalinity, fat, foreign matter and available glue present. It includes also the qualitative recognition of mucins.
1. Moisture. — A good glue contains not less than 8 nor more than 16 per cent of moisture. Moisture in excess renders the finished product more or less pliable and soon causes putrefaction. If too dry, on the other hand, the glue soon becomes brittle and gradually crumbles away. In this condition, it has lost much of its original strength.
To determine the content of moisture, about three grams of the sample, preferably in the ground or powdered state, are weighed out upon a tared watch-glass and dried in the oven at 110° C. to constant weight. The loss in weight represents the moisture which may be calculated to per cent based upon the weight of the sample taken.
2. Ash. — The dried sample from the moisture de-termination is transferred to a weighed platinum crucible. This should be capacious, so that the sample may be distributed over the bottom in - a fairly thin layer. Neglect of this precaution will retard incineration.
A small Bunsen flame is now placed underneath the crucible and gradually raised until the bulk of the carbon has been burned off. During incineration the glue swells, the contents of the crucible suddenly bursting into flame. At such times, the Bunsen flame should be withdrawn and the contents be permitted to burn quietly of their own accord. Incineration may be completed over the blast-lamp, although, if proper precautions have been observed, a pure white ash may be obtained with the heat of an ordinary Bunsen flame. Too much heat at the outset produces a form of carbon which is extremely difficult to burn off completely.
After the glue has been reduced to an ash, it should be observed if this has fused or not. This will serve to identify the glue as bone or hide, as the case may be. The ash of bone glue fuses and its aqueous solution is neutral, containing traces of phosphates and chlorine. The ash of hide glue does not fuse, owing to the presence of traces of lime which render its aqueous solution slightly alkaline. The solution is free from phosphates and chlorides.
In the ash will be found traces of media added to the glue to produce "color," which may be identified by applying the usual qualitative tests. At times such salts will be found, as the carbonate and the sulphate of lead, chromates and the salts of tungsten which have been added with a view to increasing the adhesiveness of the glue or to facilitate the drying of the solution. The presence of such salts as sulphate of barium, carbonate of calcium (chalk), carbonate of magnesia, or zinc oxide or sulphate, must not be construed as adulteration; other salts may properly be so regarded.
The ash will vary from 2 to 8 per cent, according to the quality of the glue. Its examination is of interest as confirmatory of certain test factors. Thus, it has already been pointed out that the viscosities of acid-treated bone glues, or of glues from mixed bone and hide stocks, are disproportionate to their jelly strengths, the latter being higher than the viscosity would indicate. Accordingly, if a given glue display this disrelation between viscosity and jelly strength, and yet the jelly be not sufficiently clear to warrant the assumption that the glue is from acid-treated stock, the examination of the ash will doubtless prove it to be a mixture of bone and hide, the ash partially fusing and its solution showing lime, phosphates, and chlorides.
A very clear glue may have a viscosity much higher than normal and its ash may contain alum. This is proof of the fact that the glue-liquor was clarified by means of alum, or that the alum was added with the deliberate intent of imparting to the solution of the finished glue a fictitious "body." Alum precipitates much of the viscous element (chondrin) of glue, but, if used in excess, the precipitate redissolves and thus the glue solution becomes very "stringy." The use of an excess of alum in clarification invariably produces a . foamy glue. These two phenomena are to be observed in rabbit glues, some of which test A Extra, having viscosities of 70 seconds. The ash of these glues is rich in alum.
3. Acidity and Alkalinity. — For the determination of alkali, one gram of the sample is dissolved in 500 c. c. of distilled water, so as to form a practically colorless solution. A few drops of alcoholic solution of phenolphthalein are added and the whole titrated with decinormal hydrochloric acid until the pink coloration just disappears.
In the case of acidity, direct titration of the glue solution with standard alkali would supply figures representing the sum of mineral and free organic acids. Hence, for the determination of mineral acidity, 50 grams of the sample are suspended in a flask in 80 c. c. of cold distilled water for ten hours. The flask is then fitted to a condenser and the volatile acids driven over by means of a current of steam, the distillate being collected in a graduted cylinder. When 300 c. c. have come over, the distillation is interrupted and the distillate is titrated with standard alkali. In the presence of sulphurous acid, the cylinder should contain a known quantity of standard alkali added previous to distillation. The figures obtained represent acidity due to hydrochloric and sulphurous acids, which should not exceed 0.2 per cent.
For the determination of free organic acids in addition to sulphurous acid, Kalmann proposes the following method :
One gram of the coarsely powdered glue is dissolved in water over the water-bath and then titrated with standard alkali, using phenol-phthalein as an indicator. The liquid is now cooled and, after the addition of some starch solution, is titrated with decinormal iodine until a permanent blue color is established. The amount of iodine used is the equivalent of the sulphurous acid in the sample,
The blue color is now discharged by the addition of a drop of sodium acid-sulphite and again titrated with decinormal alkali and phenol-phthalein. The second volume of alkali indicates the amount of hydriodic acid formed, which should correspond with the result of the iodine titration, showing that this was in no wise affected by the organic matter of the glue.
(a) Kissling's Method. Twenty grams of the sample are dissolved in 150 c. c. of water containing 10 c. c. of hydrochloric acid, 1.20 specific gravity. The liquid is heated three or four hours under a reflux con-denser on the water-bath. The solution is cooled, 50 c. c. of petroleum ether added, and the whole well shaken, when, after standing until clear, an aliquot part of the solvent is withdrawn into a weighed dish, the solvent evaporated, and the residual grease weighed.
(b) Extraction Method. — This is by far more convenient of operation than the foregoing.
About five grams of the finely ground or powdered sample are covered with an equal weight of water, soaked up and melted over the water-bath. An absorbent material is now added to remove the water. S. Rideal recommends plaster of paris. The author finds that this seriously interferes with the drying of the mass and uses fine infusorial earth which has previously been well extracted with petroleum ether, and kept in a tightly stoppered bottle. The mass, when dry, is placed in a mortar and carefully reduced to powder. This must be done cautiously, as some is apt to spatter while grinding. It is best first to break the mass roughly on a large sheet of paper and transfer small portions at a time to the mortar, and so powder them.
The powder is now transferred to the capsule of the Soxhlet extraction apparatus, and extracted with petrolic ether for two hours. At the end of this time the flask containing extracted grease and solvent is connected with a condenser and the bulk of the solvent distilled off. The balance is transferred to a weighed dish, the flask rinsed well with the smallest possible quantity of fresh solvent, the washings added to the contents of the dish, and the whole cautiously evaporated over the water-bath. The residual grease or fat is then weighed.
5. Approximate Assay of Constituent Elements.
(a) Gelatin. Gelatin is distinguished from chondrin and mucin by certain characteristic reactions. Its assay in glue is of but little interest, but in ascertaining the purity of commercial gelatine, it is a factor of importance, a good gelatine consisting chiefly of gelatin and little else.
(aa) Preparation. For the purpose of confirming its qualitative reactions, pure gelatin may be obtained as follows:
1. Method of Davidowsky. Buckshorn is treated with dilute hydrochloric acid for the purpose of dissolving out the calcium phosphate, leaving the glue-yielding material. The latter is treated with milk of lime to free it from fat, washed, boiled and the result-ant solution permitted to jell. The jelly is now treated with successive cold waters which serve to extract all coloring matter. This accomplished, the jelly is dissolved, strained and mixed with an equal volume of 95 per cent alcohol, a precipitate of gelatin resulting which may contain phosphates. These may be re-moved by redissolving the precipitate, acidulating the solution, and bringing it into a dialyser, where the salts and acid diffuse in the water which must be renewed.
A jelly of pure gelatin remains behind. This is evaporated to driness.
2. Method of Allen. — High-grade gelatine is soaked in successive quantities of cold water for several days. This removes salts and coloring matter. It is then dissolved in boiling water and filtered into 00 per cent alcohol, which precipitates the gelatin in white, stringy masses. These are collected, redissolved in hot water and reprecipitated by alcohol. The ash of the product is about 0.6 per cent.
Pure gelatin, in the dry state, is fairly transparent and glassy in appearance. It is somewhat yellowish and free from odor. It is not affected by exposure to the air. When heated, it softens without melting, swells considerably and decomposes with an odor resembling that of burned hair. The ash is blackish and combustible only with great difficulty.
Gelatin is insoluble in cold water, in which it swells and loses its transparency; but in hot water it dissolves with subsequent formation of a strong jelly. This may be re-melted and re-jelled a number of times, but pro-longed treatment destroys the gelatinizing property, although this is said to be restored by precipitating the gelatin by alcohol from the final solution. (Allen.)
Gelatin is precipitated from aqueous solution by chlorine, alcohol, platinic chloride, tannin, mercuric chloride, picric, phospho-molybdic, and phospho-tungstic acids and acid solution of chromic acid.
When a current of chlorine is passed through a solution of gelatine about one per cent in strength, the liquid remains clear for a time, but after a while froths strongly, each particle of froth becoming encased in a white pellicle. The frothing subsides as soon as the chlorine is in excess, which is noted from the yellow color of the solution, the liquid becoming clear and the gelatin is thrown down in the form of a white, granular precipitate. This, when washed and thoroughly dried, is a fairly white powder insoluble in water or alcohol, but soluble in alkalies.
Addition of a saturated aqueous solution of picric acid to a cold aqueous solution of gelatine produces a precipitate which first dissolves upon shaking, but which becomes permanent upon the addition of an excess of the precipitant. Upon heating, this precipitate is dissolved, but is again formed as a yellowish, sticky mass, as the solution cools. Platinic chloride and sulphate produce much the same precipitate..
Gallotannic acid precipitates gelatin as a fine white powder, which, upon exposure to the air, becomes buff-colored. Once thoroughly dried, this is soluble only in strong alkalies.
(b) Chondrin. The distinction between chondrin and gelatin was first pointed out by Muhlder. These have several reactions in common, but chondrin is precipitated by a number of substances which fail to react with gelatin.
(bb) Preparation. For the preparation of pure chondrin, Davidowsky recommends the selection of the cartilages of the ribs, of the larynx -- with the exception of those of the epiglottis, and of the windpipe and bronchi. These are boiled from 24 to 48 hours, the chondrin precipitated from solution by alcohol, re-dissolved in warm water, evaporated, and dried. The product so obtained closely resembles gelatin.
Like gelatin, chondrin is precipitated from aqueous solution by alcohol, tannin, and mercuric chloride. Unlike gelatin, it is precipitated by mineral acids organic acids, alum, sulphate of alumina, acetate and subacetate of lead, and sulphate of iron. All of these precipitates are soluble in excess of precipitant.
Of the precipitates produced by acids, those from phosphoric, hydrochloric, nitric, and sulphuric are soluble in excess; whereas, those from pyrophosphoric, sulphurous, hydrofluoric, carbonic, arsenic, tartaric, citric, oxalic, lactic, and succinic are insoluble in excess of precipitant.
(c) Mucin. While this is in no sense a constituent element of glue, it is properly discussed in connection with gelatin and chondrin, since, like them, it emanates from the glue-yielding tissues. Its presence in glue is due to defective liming or faulty washing after liming.
Mucins are slimy substances which mix with water in all proportions without undergoing real solution. They may be precipitated by alcohol or by saturation with common salt, ammonium sulphate and other neutral salts. They are soluble in alkalies and a 10 per cent solution of common salt.
Estimation of Glue Content. — This is at best only approximate, indirect methods of calculation supplying more reliable results than direct. The latter are based upon the precipitation of the gelatin and chondrin by tannic acid, and either weighing the precipitate of "tannate of gelatin," which is assumed to have the composition tannin 57.26 per cent, gelatin 42.74 per cent; or estimating the nitrogen content of the precipitate and calculating this to gelatin. An optional method is to determine the total nitrogen content of the glue, calculating the result to gelatin.
Rideal has pointed out the complexity of the organic matters of glue. Besides albumoses and peptones, several varieties of gelatin and chondrin may be present, as is evidenced by differences of elementary composition revealed by analysis. Each variety exerts a variable precipitating influence upon the tannin, which, in addition, throws down the peptones and other non-gelatinous matter. Through co-estimation of these with the gelatin and chondrin, the figures obtained are extremely inaccurate. Equally unsatisfactory is the estimation of the nitrogen content of the glue, this being that of all the nitrogenous matters present whether gelatinous or not.
For technical purposes, it suffices to estimate the non-glue, comprising fats, soaps, and extraneous mineral matter, and calculating the available glue by difference. This method fails to take into account such non-gelatinous substances as albumoses and peptones.
Stelling's Method. Fifteen grams of the sample are placed in a 250 c. c. flask, covered with 60 e. c. water and soaked over night. The following morning, the mass is melted and any loss through evaporation made good. The flask is then made up to the mark with 96 per cent alcohol and the contents thoroughly shaken. After standing for about six hours, from 25 to 50 c. c. of the supernatant liquid are withdrawn, evaporated to driness, and the residual non-glue dried and weighed.
In criticism of the above method, Kissling points out that the non-glue consists of fat as well as decomposition products of gelatin. He also observes that gelatin is not totally insoluble in alcohol of 72 per cent strength. Nevertheless, he considers the method reliable as partially indicating the value of a glue. He found that the non-gelatin varied from 7.6 per cent in the case of a hide glue, to 23.2 per cent in a very inferior bone product.
By Stelling's method, any mucin present is thrown down by the alcohol and hence is not estimated with the non-glue, thus rendering the figure for available glue, obtained by difference, high. This is not a very serious objection inasmuch as, when present in glue, the amount of mucin is quite small, the presence of even this rendering the glue unfit for many uses. Hence its co-estimation indirectly with the true glue occasions no serious error. On the other hand, the substances precipitable by alcohol carry down with them much, if not all, of the media added to the glue to produce color. Hence, in the case of opaque or colored glues, the coloring material must be estimated separately and added to the figure for non-glue. The author does not regard as pertinent Kissling's objection to the co-estimation of fat with the non-glue. True, if much grease be present in the residue of non-gelatinous material it will interfere with accurate weighing because of its hygroscopic nature. For this reason, it is well to extract the grease before estimating the non-glue. In all instances, however, the figure for grease must be added to that for non-glue. Again, the glue should be dissolved in not more than twice its weight of water that the subsequent dilution of the alcohol may not be so great as to permit the return of any of the precipitate to solution. With these modifications, the method yields reliable results; and it will be found that the figures for true glue, calculated by difference, are well in accord with the grading assigned by test.
Foreign Matter. — This includes insoluble organic as well as mineral matter. For the determination, about two grams of the glue are soaked and dissolved in a liter flask with the aid of 200 c. c. of water. The flask is then made up to the mark, care being taken to mix the contents thoroughly. The flask is then kept in a warm place from 24 to 48 hours, during which the insoluble material settles to the bottom. The bulk of the supernatant solution is decanted off, the balance being poured through a tared filter and the residue washed with hot water until free from all glue. The filter, with its contents, is then dried at a temperature not exceeding 100° C. and weighed. The figures so obtained serve to correct the result for available glue.
Both glue and gelatine exhibit characteristic behavior toward certain reagents, the reactions serving to identify them when associated with other substances, and frequently to separate them quantitatively.
Gallotannic acid produces a buff-colored precipitate, known as tanno-gelatin. This is supposed to be the chemical base of leather. The reaction takes place in solutions containing only 0.005 per cent of glue or gelatine.
Added to a neutral or slightly alkaline solution of gelatine, formaldehyde produces a white, stringy precipitate of formo-gelatin. The purer the product, the more readily the precipitate forms, low-grade glues requiring some time to respond to the test.
The bichromates of potassium and ammonium, added to solutions of glue and gelatine, render the product evaporated therefrom insoluble after exposure to sun-light.
When compounded at high temperature with dilute mineral acids, glue loses its power of gelatination. This is restored by the addition of common salt.
A glue solution boiled with slaked lime loses its gelatinizing power; and if the solution be subsequently evaporated, the product is a colorless, gummy mass, soluble in cold water and a solution of common salt.
Potassium or sodium carbonate, neutral potassium tartrate, magnesium sulphate, and rochelle salts coagulate glue solutions by removing the water from them.
Gelatination may be destroyed by the addition of the chlorides of ammonium and barium, nitrate of potassium, and saturation with common salt.
Glue and gelatine are distinguished from albumins in that they yield no precipitate with potassium ferrocyanide or ferricyanide.
Purity of Commercial Gelatine. — For this, Vogel em-ploys a 10 per cent solution of silver nitrate to which has been added enough ammonia to re-dissolve the precipitate which first forms. This reagent is then mixed with an equal volume of the solution under examination. Any impurity is indicated by a coloration varying from yellow to deep brown.