CHEMICAL PROPERTIES.
314. Solubility in Alcohol.—As has already been noted, the glycerids are freely soluble in ether, chloroform, carbon bisulfid, acetone, carbon tetrachlorid, and some other less commonly used solvents. Their solubility in absolute alcohol is variable and the determination of its degree may often be useful in analytical work.
The method used by Milliau for determining the degree of solubility is as follows:[267] The fatty matter is deprived of its free acids by shaking for half an hour with twice its volume of ninety-five per cent alcohol. After standing until the liquids are separated, the oil or fat is drawn off and washed three times with distilled water. The sample is deprived of water by filtering through a hot jacket filter and a given weight of the dry sample is well shaken with twice its weight of absolute alcohol. A weighed portion of the alcoholic solution obtained is evaporated to remove the alcohol and the weight of the residual fat determined. From the data obtained the percentage of solubility is calculated. Olive oils, when treated as described above, show a solubility of about forty-three parts per thousand of absolute alcohol, cotton oil sixty-two parts, sesamé forty-one parts, peanut sixty-six parts, colza twenty parts, and flaxseed seventy parts per thousand.
315. Coloration Produced by Oxidants.—When oils and fats are mixed with oxidizing reagents, such as sulfuric and nitric acids, the glycerids are partly decomposed with the production of colors which have some analytical significance. The most simple method of applying these tests is by the use of a thick porcelain plate provided with small cup-shaped depressions for holding the few drops of material required. Two or three drops of the oil under examination are placed in each of the cups, a like quantity of the oxidizing reagent added, and the mixture stirred with a small glass rod. The colors produced are carefully noted and the mixture is allowed to remain at room temperature for at least twelve hours in order that the final tint may be observed. The sulfuric acid used for this reaction should have a specific gravity of one and seven-tenths and the nitric acid should have the usual commercial strength of the strongest acid. Pure lard, when treated with sulfuric acid, as above described, shows but little change of color while the vegetable oils mostly turn brown or black. In addition to the reagents mentioned many others, including sulfuric and nitric acids, sulfuric acid and potassium bichromate, chlorin, ammonia, hydrogen peroxid, sodium hydroxid and aqua regia are used. Only a few of these tests seem to have sufficient analytical importance to merit any detailed description.[268]
316. Coloration in Large Masses.—Instead of applying the color test in the small way just described, larger quantities of the fat may be used, either in the natural state or after solution in petroleum or other solvent. For this purpose about ten cubic centimeters of the oil are shaken with a few drops of sulfuric acid or sulfuric and nitric acids. Lard, when thus treated (five drops of sulfuric acid to ten cubic centimeters of lard) shows practically no coloration. When treated with an equal volume of sulfuric acid and shaken, the lard on separating has a brown-red tint.[269]
Olive oil, with a few drops of sulfuric acid, gives a green color, while cottonseed, peanut and other vegetable oils, when thus treated with sulfuric and nitric acids, show brown to black coloration. The delicacy of the reaction may be increased by first dissolving the fat or oil in petroleum ether.
In the use of the coloration test with solvents, a convenient method is to dissolve about one cubic centimeter of the fat in a test tube in petroleum ether, add one drop of strong sulfuric acid and shake.
In the case of lard, the color does not change or becomes yellow or red. Cottonseed oil, similarly treated, shows a brown or black color.[270]
317. Special Nitric Acid Test.—A special nitric acid test for cottonseed oil is made with nitric acid of exactly 1.375 specific gravity at 15°. This test is especially valuable in detecting cottonseed in olive oil. The operation is conveniently conducted by shaking together equal volumes of the oil and acid in a test tube until an intimate mixture or emulsion is secured. When any considerable quantity of cottonseed oil is present an immediate brown coloration is produced, from the intensity of which the relative proportion of cottonseed oil in the case of a mixture may be roughly approximated. When only a little cottonseed oil is present in the mixture, the test tube containing the reagents should be set aside for several hours before the final observation is made.
318. Coloration with Phosphomolybdic Acid.—Among the color tests, one which we have found of use is the coloration produced in certain oils, mostly of a vegetable origin, by phosphomolybdic acid.[271]
The method of applying the test is extremely simple. A few cubic centimeters of the oil or melted lard are dissolved in an equal volume of chloroform, and a third volume of ten per cent phosphomolybdic acid added. The mouth of the test tube is closed with the thumb, and the whole is violently shaken. On being left in repose, the phosphomolybdic acid gathers at the top, and the coloration produced therein is easily observed. Cottonseed oil and peanut oil both give a beautiful green when treated in this way, which is turned to a blue on the addition of ammonia. Linseed oil gives a green color, but forms a kind of emulsion which obscures the color to some extent. The pure lards rendered in the laboratory give no coloration whatever to the reagent, but it retains its beautiful amber color in every case. Mixtures containing as little as ten per cent cottonseed oil and ninety per cent lard, show a distinct greenish tint, while twenty per cent cottonseed oil gives a distinct green. This reaction, therefore, may be considered of great value, and on account of its easy application it should come into wide use. But it is probable that different samples of cottonseed oil, refined to different degrees or in different ways, vary in their deportment with phosphomolybdic acid as they do with silver nitrate. In other words, there may be some samples of cottonseed oil which will not give the green color when treated as above, or so faintly as to have no diagnostic value in mixtures.
This reaction shows itself with nearly all vegetable oils but those which have been chemically treated either for the purpose of bleaching, or for the removal of the acidity, do not respond to the test at all, or else in a feeble manner, and that only after standing some time. Lard, goose fat, tallow, deer fat, butter fat, etc., show no change in color on being treated with this reagent, either with or without the addition of alkali. The presence of a small quantity of vegetable oil betrays itself by the appearance of the above mentioned coloration, the intensity of which forms an approximate measure of the amount of vegetable oil present in the sample. In experiments with suspected lards, which deviated in their iodin absorption numbers from those of genuine lard, the results were concordant, the color deepening as the iodin figure rose. The mineral fats (paraffin, vaselin) are without action on this reagent, and the only animal fat which reduces it is codliver oil.
In like manner some samples of lard may be found which exhibit a deportment with this reagent similar to that shown with vegetable oils, and tallow and lard oil have been shown to give more distinct reactions than some of the vegetable oils.[272]
The phosphomolybdic acid may be prepared by precipitating a solution of ammonium molybdate with sodium phosphate and dissolving the washed precipitate in a warm solution of sodium carbonate. The solution is evaporated to dryness and the dry residue subjected to heat. If a blue coloration be produced it may be discharged by adding a little nitric acid and reheating. The residue is dissolved in water, acidified with nitric and made of such a strength as to contain about ten per cent of the substance.
319. Coloration with Picric Acid.—If to ten cubic centimeters of oil a cold saturated solution of picric acid in ether be added and the latter be allowed to evaporate slowly, the acid remains dissolved in the oil, to which it communicates a brown color.
Pure lard, after the evaporation of the ether, appears of a citron-yellow color; if cottonseed oil be present, however, the mixture assumes a brown-red color.[273]
320. Coloration with Silver Nitrate.—A modification of Bechi’s method of reducing silver nitrate, given further on, has been proposed by Brullé.[274] The reagent employed consists of twenty-five parts of silver nitrate in 1,000 parts of alcohol of ninety-five per cent strength. Twelve cubic centimeters of the oil to be examined and five of the reagent are placed in a test tube, held in a vessel containing boiling water, and the ebullition continued for about twenty minutes. At the end of this time an olive oil, even if it be an impure one, will show a beautiful green tint. With seed oils the results are quite different. Cotton oil submitted to this treatment becomes completely black. Peanut oil shows at first a brown-red coloration and finally a somewhat green tint, losing its transparency. Sesamé oil is distinguished by a red-brown tint very pronounced and remaining red. Colza oil takes on a yellowish green coloration, becomes turbid and is easily distinguished in its reaction from olive oil. In mixtures of olive oil with the other oils, any notable proportion of the seed oils can be easily determined by the above reactions. Natural butter treated with this reagent retains its primitive color. That containing margarin becomes a brick-red and as little as five per cent of margarin in butter can be detected by this test. With ten per cent the tint is very pronounced.
321. Coloration with Stannic Bromid.—This reagent is prepared by adding dry bromin, drop by drop, to powdered or granulated tin held in a flask immersed in ice water, until a persistent red color indicates that the bromin is in excess. In the application of this reagent three or four drops of it are added successively to a little less than that quantity of the oil, the mixture well stirred and set aside for a few minutes. The unsaponifiable matters of castor oil give a green color when thus treated, sandal wood oil a blood-red color and cedar oil a purplish color.[275]
322. Coloration with Auric Chlorid.—The use of auric chlorid for producing colorations in oils and fats was first proposed by Hirschsohn.[276] One gram of auric chlorid is dissolved in 200 cubic centimeters of chloroform and about six drops of this reagent added to five cubic centimeters of the oil to be tested. In the case of cottonseed oil a beautiful red color is produced.
I have found that even pure lards give a trace of color sometimes with this reagent, and therefore the production of a slight red tint cannot in all cases be regarded as conclusive of the presence of cottonseed oil.[277]
In general, it may be said that the color reactions with fats and oils have a certain qualitive and sorting value, and in any doubtful case they should not be omitted. Their value can only be established by comparison under identical conditions with a large number of fats and oils of known purity. The analyst must not depend too confidingly on the data found in books, but must patiently work out these reactions for himself.
323. Thermal Reactions.—The measurement of the heat produced by mixing glycerids with reagents which decompose them or excite other speedy chemical reactions, gives valuable analytical data. These measurements may be made in any convenient form of calorimeter. The containing vessel for the reagents should be made of platinum or some other good conducting metal not affected by them.
The heat produced is measured in the usual way by the increment in temperature noted in the mass of water surrounding the containing vessel. The determination of the heat produced in chemical reactions is a tedious and delicate operation requiring special forms of apparatus for different substances. The time element in these operations is a matter of importance, since it is necessary to work in rooms subject to slight changes of temperature and to leave the apparatus for some time at rest, in order to bring it and its contents to a uniform temperature. For these reasons the more elaborate methods of calorimetric examination are not well suited to ordinary analytical work, and the reader is referred to standard works on thermal chemistry for the details of such operations.[278] For our purpose here a description of two simple thermal processes, easily and quickly conducted, will be sufficient, while a description of the method of determining the heat of combustion of foods will be given in another place.
324. Heat of Sulfuric Saponification.—Maumené was the first to utilize the production of heat caused by mixing sulfuric acid with a fat as an analytical process.[279] In conducting the process a sulfuric acid of constant strength should be employed inasmuch as the rise of temperature produced by a strong acid is much greater than when a weaker acid is employed. The process is at best only comparative and it is evident that the total rise of temperature in any given case depends on the strength of the acid, the character, and purity of the fat or oil, the nature of the apparatus and its degree of insulation, the method of mixing and the initial temperature. For this reason the data given by different analysts vary greatly.[280] For some of the methods of conducting the operation the reader may consult the work of Allen, cited above, or other authorities.[281]
In this laboratory the process is conducted as follows:[282] The initial temperature of the reagents should be a constant one. For oils 20° is a convenient starting point and for fats about 35°, at which temperature most of them are soft enough to be easily mixed with the reagent. The acid employed should be the pure monohydrated form, specific gravity at 20°, 1.845.
The apparatus used is shown in [Fig. 98].
Fig. 98.—Apparatus for Determining Rise
of Temperature with Sulfuric Acid.
The test tube which holds the reagents is twenty-four centimeters in length and five in diameter. It is provided with a stopper having three perforations, one for a delicate thermometer, one for a bulb funnel for delivering the sulfuric acid, and one to guide a stirring rod bent into a spiral as shown. The thermometer is read with a magnifying glass. Fifty cubic centimeters of the fat are placed in the test tube and ten of sulfuric acid in the funnel and the apparatus is exposed at the temperature desired until all parts of it, together with the reagents, have reached the same degree. The test tube holding the oil should be placed in a vacuum-jacket tube, such as will be described in paragraph [316]. The oil is allowed to run in as rapidly as possible from the funnel and the stirring rod is moved up and down two or three times until the oil and acid are well mixed. Care must be exercised to stir no more than is necessary for good mixing. The mercury is observed as it ascends in the tube of the thermometer and its maximum height is noted. With the glass it is easy to read to tenths, when the thermometer is graduated in fifths of a degree. When oils are tested which produce a rise of temperature approaching 100°, in the above circumstances, (cottonseed, linseed and some others) either smaller quantities should be used or the oil diluted with some inert substance or dissolved in some inert solvent of high boiling point. For a study of the variations produced in the rise of temperature when varying proportions of oil and acid are used, the work of Munroe may be consulted.[283]
The thermélaeometer described by Jean is a somewhat complicated piece of apparatus and does not possess any advantage over the simple form described above.[284]
Instead of expressing the data obtained in thermal degrees showing the rise of temperature, Thompson and Ballentyne refer them to the heat produced in mixing sulfuric acid and water.[285]
The observed thermal degree of the oil and acid divided by that of the water and acid is termed the specific temperature reaction. For convenience in writing, this quotient is multiplied by 100. The respective quantities of acid and water are ten and fifty cubic centimeters. This method of calculation has the advantage of eliminating to a certain degree the variations which arise in the use of sulfuric acid of differing specific gravities. In the following table are given the comparative data obtained for some common oils.[286]
| Acid of 95.4 per cent. | Acid of 96.8 per cent. | Acid of 99 per cent. | ||||
|---|---|---|---|---|---|---|
| Kind of oil. | Rise of temp. with the oil. | Specific temp. reaction. | Rise of temp. with the oil. | Specific temp. reaction. | Rise of temp. with the oil. | Specific temp. reaction. |
| 0° | 0° | 0° | 0° | 0° | 0° | |
| Olive oil | 36.5 | 95 | 39.4 | 85 | 44.8 | 96 |
| Rapeseed oil | 49.0 | 127 | 37.0 | 89 | 58.0 | 124 |
| Castor oil | 34.0 | 88 | ||||
| Linseed oil | 104.5 | 270 | 125.2 | 269 | ||
325. Method of Richmond.—The rise of temperature produced by mixing an oil and sulfuric acid is determined by Richmond in a simple calorimeter, which is constructed by fitting a small deep beaker inside a larger one with a packing of cotton. The heat capacity of the system is determined by adding to ten grams of water, in the inner beaker, at room temperature, twenty-five grams of water of a noted higher temperature and observing the temperature of the mixture. The cooling of the system, during the time required for one determination of heat of sulfuric saponification, does not exceed one per cent of the whole number of calories produced.[287] Between the limits of ninety-two per cent and one hundred per cent the rise of temperature observed is directly proportional to the strength of the acid.
Relative Maumené Figure.—The total heat evolved per mean molecule is called by Richmond the relative maumené figure. It is calculated as follows:
- Let x = percentage of sulfuric acid in the acid employed;
- h = heat capacity of calorimeter in grams of water;
- R = observed rise of temperature (twenty-five grams of
- oil, five cubic centimeters sulfuric acid);
- K = potash absorbed for saponification (19.5 per cent of
- potassium hydroxid, standard of comparison);
- M = relative maumené figure:
| ThenM = R × | 21.5 | × | 20 + h | × | 19.5 |
| x - 78.5 | 20 | K |
326. Heat of Bromination.—The rise of temperature caused by mixing fats with sulfuric acid has long been used to discriminate between different fats and oils. Hehner and Mitchell propose a similar reaction based upon the rise of temperature produced by mixing bromin with the sample.[288] The action of bromin on unsaturated fatty bodies is instantaneous and is attended with a considerable evolution of heat. Since the action of bromin on many of the oils is very violent it is necessary to dilute the reagent with chloroform or glacial acetic acid. Owing to its high boiling point the acetic acid has some advantage over chloroform for this purpose. The tests are conveniently made in a vacuum-jacket tube. In such a tube there is no loss of heat by radiation. The bromin is measured in a pipette having at its upper end a tube filled with caustic lime held between plugs of asbestos. The bromin sample to be tested and the diluent employed are kept at the same temperature before beginning the trial. They are quickly mixed and the rise of temperature noted. The oil is first dissolved in the chloroform and the bromin then added.
A somewhat constant relation is noticed between the rise of temperature and the iodin number when one gram of oil, ten cubic centimeters of chloroform and one cubic centimeter of bromin are used.
If the rise in temperature in degrees be multiplied by 5.5 the product is approximately the iodin number of the sample. Thus a sample of lard gave a rise in temperature of 10°.6 and an iodin number of 57.15. The number obtained by multiplying 10.6 by 5.5 is 58.3.
In like manner the numbers obtained for some common oils are as follows:
| Material. | Rise of temperature with bromin. | Iodin No. | Calculated Iodin No. |
|---|---|---|---|
| Butter fat | 6.6 | 37.1 | 36.3 |
| Olive oil | 15.0 | 80.8 | 82.5 |
| Maize ” | 21.5 | 122.0 | 118.2 |
| Cotton ” | 19.4 | 107.1 | 106.7 |
| Castor ” | 15.0 | 83.8 | 82.5 |
| Linseed oil | 30.4 | 160.7 | 167.2 |
| Codliver ” | 28.0 | 144.0 | 140.0 |
327. Modification of the Heat of Bromination Method.—The method described above by Hehner and Mitchell presents many grave difficulties in manipulation, on account of the inconvenience of handling liquid bromin. The process is made practicable by dissolving both the oil or fat and the bromin in chloroform, or better in carbon tetrachlorid, in which condition the bromin solution is easily handled by means of a special pipette.[289]
In order to make a number of analyses of the same sample ten grams of the fat may be dissolved in chloroform or carbon tetrachlorid and the volume completed with the same solvent to fifty cubic centimeters. In like manner twenty cubic centimeters of the bromin are dissolved in one of the solvents named and the volume completed to 100 cubic centimeters therewith.
For convenience of manipulation the solutions are thus made of such a strength that five cubic centimeters of each represent one gram of the fat and one cubic centimeter of the liquid bromin respectively.
Fig. 99. Apparatus for Determining Heat of Bromination.
The apparatus used for the work is shown in the accompanying [figure]. The pipette for handling the bromin solution is so arranged as to be filled by the pressure of a rubber bulb, thus avoiding the danger of sucking the bromin vapor into the mouth. The filling is secured by keeping the bromin solution in a heavy erlenmeyer with a side tubulure such as is used for filtering under pressure. The solutions are mixed in a long tube, held in a larger vessel, from which the air is exhausted to secure a minimum radiation of heat. A delicate thermometer graduated in tenths serves to register the rise of temperature. The fat solution is first placed in the test tube, with care not to pour it down the sides of the tube but to add it by means of a pipette reaching nearly to the bottom. The whole apparatus having been allowed to come to a standard temperature the bromin solution is allowed to run in quickly from the pipette. No stirring is required as the liquids are sufficiently mixed by the addition of the bromin solution. The mercury in the thermometer rapidly rises and is read at its maximum point by means of a magnifying glass. With a thermometer graduated in tenths, it is easy to read to twentieths of a degree.
It is evident that the rise of temperature obtained depends on similar conditions to those mentioned in connection with sulfuric saponification. Each system of apparatus must be carefully calibrated under standard conditions and when this is done the comparative rise of temperature obtained with various oils and fats will prove of great analytical use. It is evident that the ratio of this rise of temperature to the iodin number must be determined for every system of apparatus and for every method of manipulation employed, and no fixed factor can be given that will apply in every case.
With the apparatus above described and with the method of manipulation given the following data were obtained for the oils mentioned:
| Rise of temperature. | |
| Olive oil | 20°.5 |
| Refined cottonseed oil | 25°.7 |
| Sunflowerseed oil | 28°.4 |
| Calycanthusseed oil | 29°.0 |
Bromin and chloroform, when mixed together, give off heat, due to the chemical reaction resulting from the substitution of bromin for hydrogen in the chloroform molecule and the formation of hydrobromic acid. For this reason the data obtained, when chloroform is used as a solvent, are slightly higher than with carbon tetrachlorid. The use of the latter reagent is therefore to be preferred.
328. Haloid Addition Numbers.—Many of the glycerids possess the property of combining directly with the haloids and forming thereby compounds in which the haloid, by simple addition, has become a part of the molecule. Olein is a type of this class of unsaturated glycerids. The process may take place promptly as in the case of bromin or move slowly as with iodin. The quantity of the haloid absorbed is best determined in the residual matter and not by an examination of the fat compound. By reason of the ease with which the amount of free iodin in solution can be determined, this substance is the one which is commonly employed in analytical operation on fats.
In general, the principle of the operation depends on bringing the fat and haloid together in a proper solution and allowing the addition to take place by simple contact. The quantity of the haloid in the original solution being known, the amount which remains in solution after the absorption is complete, deducted from that originally present, will give the quantity which has entered into combination with the glycerid.
329. Hübl’s Process.—In determining the quantity of iodin which will combine with a fat, the method first proposed by Hübl, or some modification thereof, is universally employed.[290] In the determination of the iodin number of a glycerid it is important that it be accomplished under set conditions and that iodid be always present in large excess. It is only when data are obtained in the way noted that they can be regarded as useful for comparison and determination. Many modifications of Hübl’s process have been proposed, but it is manifestly impracticable to give even a summary of them here. As practiced in the chemical laboratory of the Agricultural Department and by the Association of Official Agricultural Chemists, it is carried out as follows:[291]
(1) PREPARATION OF REAGENTS.
(a). Iodin Solution.—Dissolve twenty-five grams of pure iodin in 500 cubic centimeters of ninety-five per cent alcohol. Dissolve thirty grams of mercuric chlorid in 500 cubic centimeters of ninety-five per cent alcohol. The latter solution, if necessary, is filtered, and then the two solutions mixed. The mixed solution should be allowed to stand twelve hours before using.
(b). Decinormal Sodium Thiosulfate Solution.—Dissolve 24.8 grams of chemically pure sodium thiosulfate, freshly pulverized as finely as possible and dried between filter or blotting paper, and dilute with water to one liter, at the temperature at which the titrations are to be made.
(c). Starch Paste.—One gram of starch is boiled in 200 cubic centimeters of distilled water for ten minutes and cooled to room temperature.
(d). Solution of Potassium Iodid.—One hundred and fifty grams of potassium iodid are dissolved in water and the volume made up to one liter.
(e). Solution of Potassium Bichromate.—Dissolve 3.874 grams of chemically pure potassium bichromate in distilled water and make the volume up to one liter at the temperature at which the titrations are to be made.
(2). DETERMINATION.
(a). Standardizing the Sodium Thiosulfate Solution.—Place twenty cubic centimeters of the potassium bichromate solution, to which have been added ten cubic centimeters of the solution of potassium iodid, in a glass stopper flask. Add to this mixture five cubic centimeters of strong hydrochloric acid. Allow the solution of sodium thiosulfate to flow slowly into the flask until the yellow color of the liquid has almost disappeared. Add a few drops of the starch paste, and with constant shaking continue to add the sodium thiosulfate solution until the blue color just disappears. The number of cubic centimeters of thiosulfate solution used multiplied by five is equivalent to one gram of iodin.
Example.—Twenty cubic centimeters of potassium bichromate solution required 16.2 sodium thiosulfate; then 16.2 × 5 = 81 = number cubic centimeters of thiosulfate solution equivalent to one gram of iodin. Then one cubic centimeter thiosulfate solution = 0.0124 gram of iodin: (Theory for decinormal solution of sodium thiosulfate, one cubic centimeter = 0.0127 gram of iodin.)
(b). Weighing the Sample.—About one gram of butter fat is placed in a glass stopper flask, holding about 300 cubic centimeters, with the precautions to be mentioned for weighing the fat for determining volatile acids.
(c). Absorption of Iodin.—The fat in the flask is dissolved in ten cubic centimeters of chloroform. After complete solution has taken place thirty cubic centimeters of the iodin solution (1) (a) are added. The flask is now placed in a dark place and allowed to stand, with occasional shaking, for three hours.
(d). Titration of the Unabsorbed Iodin.—One hundred cubic centimeters of distilled water are added to the contents of the flask, together with twenty cubic centimeters of the potassium iodid solution. Any iodin which may be noticed upon the stopper of the flask should be washed back into the flask with the potassium iodid solution. The excess of iodin is taken up with the sodium thiosulfate solution, which is run in gradually, with constant shaking, until the yellow color of the solution has almost disappeared. A few drops of starch paste are added, and the titration continued until the blue color has entirely disappeared. Toward the end of the reaction the flask should be stoppered and violently shaken, so that any iodin remaining in solution in the chloroform may be taken up by the potassium iodid solution in the water. A sufficient quantity of sodium thiosulfate solution should be added to prevent a reappearance of any blue color in the flask for five minutes.
(e). Setting the Value of the Iodin Solution by the Thiosulfate Solution.—At the time of adding the iodin solution to the fat, two flasks of the same size as those used for the determination should be employed for conducting the operation described above, but without the presence of any fat. In every other respect the performance of the blank experiments should be just as described. These blank experiments must be made each time the iodin solution is used.
Example of Blank Determinations.—Thirty cubic centimeters of iodin solution required 46.4 cubic centimeters of sodium thiosulfate solution: Thirty cubic centimeters of iodin solution required 46.8 cubic centimeters of sodium thiosulfate solution: Mean, 46.6 cubic centimeters.
| Weight of fat | 1.0479 | grams | |
| Quantity of iodin solution used | 30.0 | cubic | centimeters |
| Thiosulfate equivalent to iodin used | 46.6 | ” | ” |
| Thiosulfate equivalent to remaining iodin | 14.7 | ” | ” |
| Thiosulfate equivalent to iodin absorbed | 31.9 | ” | ” |
| Percent of iodin absorbed, 31.9 × 0.0124 × 100 ÷ 1.0479 = 37.75. | |||
330. Character of Chemical Reaction.—The exact nature of the chemical process which takes place in this reaction is not definitely known. Hübl supposed that the products formed were chloro-iodid-additive compounds, and he obtained a greasy product from oleic acid, to which he ascribed the formula C₁₈H₃₄IClO₂. By others it is thought that chlorin alone may be added to the molecule.[292]
In general, it may be said that none of the glycerids capable of absorbing halogens is able to take on a quantity equivalent to theory.[293] While the saturated fatty acids (stearic series) theoretically are not able to absorb iodin some of them are found to do so to a small degree. It is evident, therefore, that it is not possible to calculate the percentage of unsaturated glycerids in a fat from their iodin number alone. According to the data worked out by Schweitzer and Lungwitz both addition and substitution of iodin take place during the reaction.[294] This fact they determined by titration with potassium iodate and iodid according to the formula 5HI + HIO₃ = 6I + 6H₂O. The authors confess that whenever free hydriodic acid is found in the mixture that iodin substitution has taken place and that for each atom of hydrogen eliminated from the fat molecule two atoms of iodin disappear, one as the substitute and the other in the form of hydriodic acid. When carbon bisulfid or tetrachlorid is used as a solvent no substitution takes place and pure additive compounds are formed.
The following process is recommended to secure a pure iodin addition to a glycerid: About one gram or a little less of the oil or fat is placed in a glass stopper flask, to which are added about seven-tenths of a gram of powdered mercuric chlorid and twenty-five cubic centimeters of a solution of iodin in carbon bisulfid. The stopper is made tight by smearing it with powdered potassium iodid, tied down, and the mixture is heated for some time under pressure. By this method it is found that no hydriodic acid is formed, and hence all the iodin which disappears is added to the molecule of the glycerid. The additive numbers obtained for some oils are appended:
| Oil. | Time of heating. | Temperature. | Per cent iodin added. | Per cent hübl number. |
| Lard oil | 30 minutes. | 50°.0 | 73.0 | 78.4 |
| Cottonseed oil | 2 hours. | 50°.0 | 103.0 | 106.5 |
| Oleic acid | 2 ” | 65°.5 | 93.8 |
331. Solution in Carbon Tetrachlorid.—Gantter has called attention to the fact that the amount of iodin absorbed by fat does not depend alone upon the proportion of iodin present but also upon the amount of mercuric chlorid in the solution.[295] Increasing amounts of mercuric chlorid cause uniformly a much greater absorption of the iodin. For this reason he proposes to discard the use of mercuric chlorid altogether for the hübl test and to use a solvent which will at the same time dissolve both the iodin and the fat. For this purpose he uses carbon tetrachlorid. The solutions are prepared as follows:
Iodin Solution.—Ten grams of iodin are dissolved in one liter of carbon tetrachlorid.
In the preparation of this solution the iodin must not be thrown directly into the flask before the addition of the tetrachlorid. Iodin dissolves very slowly in carbon tetrachlorid and the solution is made by placing it in a sufficiently large weighing glass and adding a portion of the carbon tetrachlorid thereto. The solution is facilitated by stirring with a glass rod until the added tetrachlorid is apparently charged with the dissolved iodin. The dissolved portion is then poured into a liter flask, new portions added to the iodin and this process continued until the iodin is completely dissolved, and then sufficient additional quantities of the tetrachlorid are added to fill the flask up to the mark.
332. Sodium Thiosulfate Solution.—Dissolve 19.528 grams of pure sodium thiosulfate in 1000 cubic centimeters of water. For determining the strength of the solution by titration, the solution of iodin in carbon tetrachlorid and a solution of sodium thiosulfate in water are each placed in a burette. A given volume of the iodin solution is first run into a flask with a glass stopper and afterward the sodium thiosulfate added little by little until, after a vigorous shaking, the liquid has only a little color. Some solution of starch is then added and shaken until the mixture becomes deep blue. The sodium thiosulfate solution is added drop by drop, with vigorous shaking after each addition, until the solution is completely decolorized. If both solutions have been correctly made with pure materials they will be of equal strength; that is, ten cubic centimeters of the iodin solution will be exactly decolorized by ten cubic centimeters of the sodium thiosulfate solution.
333. Method of Conducting the Absorption.—The quantity of the fat or oil employed should range from 100 to 200 milligrams, according to the absorption equivalent. These quantities should be placed in flasks with glass stoppers in the ordinary way. In the flasks are placed exactly fifty cubic centimeters of the iodin solution equivalent to 500 milligrams of iodin, and the flask is then stoppered and shaken until the fat or oil is completely dissolved. In order to avoid the volatilization of the iodin finally, sufficient water is poured into the flask to form a layer about one millimeter in thickness over the solution containing the iodin and fat. The stopper should be carefully inserted and the flask allowed to stand at rest for fifty hours.
334. Estimation of the Iodin Number.—This is determined in the usual way by titration of the amount of iodin left in excess after the absorption as above described. The iodin number is to be expressed by the number of milligrams of iodin which are absorbed by each 100 milligrams of fat.
Example.—One hundred and one milligrams of flaxseed oil were dissolved in fifty cubic centimeters of the carbon tetrachlorid solution of iodin and allowed to stand as above described for fifty hours. At the end of this time, 42.3 cubic centimeters of the sodium thiosulfate solution were required to decolorize the excess of iodin remaining.
Statement of Results.—Fifty cubic centimeters of the sodium thiosulfate equal 500 milligrams of iodin; therefore, 42.3 cubic centimeters of the thiosulfate solution equal 423 milligrams of iodin. The difference equals seventy-seven milligrams of iodin absorbed by 101 milligrams of the flaxseed oil. Therefore, the iodin number equivalent and the milligrams of iodin absorbed by 100 milligrams of flaxseed oil equal 76.2.
It is evident from the above determination that the iodin number of the oil, when obtained in the manner described, is less than half that secured by the usual hübl process. Since the solvent employed, however, is more stable than chloroform when in contact with iodin or bromin, the proposed variation is one worthy of the careful attention of analysts.
McIlhiney has called especial attention to the low numbers given by the method of Gantter, and from a study of the data obtained concludes that iodin alone will not saturate glycerids, no matter what the solvents may be.[296]
It is clear, therefore, that the process of Gantter cannot give numbers which are comparable with those obtained by the usual iodin method. Any comparative value possessed by the data given by the process of Gantter must be derived by confining it to the numbers secured by the carbon tetrachlorid process alone.
335. Substitution of Iodin Monochlorid for Hübl’s Reagent.—Ephraim has shown that iodin monochlorid may be conveniently substituted for the hübl reagent with the advantage that it can be safely used at once, while the hübl reagent undergoes somewhat rapid changes when first prepared. The present disadvantage of the process is found in the fact that the iodin monochlorid of commerce is not quite pure and each new lot requires to be titrated for the determination of its purity.
The reagent is prepared of such a strength as to contain 16.25 grams of iodin monochlorid per liter. The solvent used is alcohol. The operation is carried out precisely as in the hübl method, substituting the alcoholic solution of iodin monochlorid for the iodin reagent proposed by Hübl.[297] If the iodin monochlorid solution, after acting on the oil, be titrated without previous addition of potassium iodid a new value is obtained, the chloriodin number. In titrating, the sodium thiosulfate is added until the liquid, which is made brown by the separated iodin, becomes yellow. At this point the solution is diluted, starch paste added, and the titration completed.
336. Preservation of the Hübl Reagent.—To avoid the trouble due to changes in the strength of Hübl’s reagent, Mahle adds hydrochloric acid to it at the time of its preparation.[298] The reagent is prepared as follows: Twenty-five grams of iodin dissolved in a quarter of a liter of ninety-five per cent alcohol are mixed with the same quantity of mercuric chlorid in 200 cubic centimeters of alcohol, the same weight of hydrochloric acid of 1.19 specific gravity added and the volume of the mixture completed to half a liter with alcohol. After five days such a solution gave, on titration, 49.18 instead of 49.31 grams per liter of iodin.
It will be observed that this solution is double the usual strength, but this does not influence the accuracy of the analytical data obtained. It appears that the hübl number is not, therefore, an iodin number, but expresses the total quantity of iodin, chlorin and oxygen absorbed by the fat during the progress of the reaction.
337. Bromin Addition Number.—In the process of Hübl and others an attempt is made to determine the quantity of a halogen, e.g., iodin, which the oil, fat or resin will absorb under certain conditions. The numbers obtained, however, represent this absorption only approximately, because the halogen may disappear through substitution as well as absorption. Whether or not a halogen is added, i. e., absorbed or substituted, may be determined experimentally.
The principle on which the determination depends rests on the fact that a halogen, e. g., bromin, forms a molecule of hydrobromic acid for every atom of bromin substituted, while in a simple absorption of the halogen no such action takes place. If, therefore, bromin be brought into contact with a fat, oil or resin, the determination of the quantity of hydrobromic acid formed will rigidly determine the quantity of bromin substituted during the reaction. If this quantity be deducted from the total bromin which has disappeared, the relative quantities of the halogen added and substituted are at once determined. In the method of McIlhiney[299] bromin is used instead of iodin because the addition figures of iodin are in general much too low.
The Reagents.—The following solutions are employed:
1. One-third normal bromin dissolved in carbon tetrachlorid:
2. One-tenth normal sodium thiosulfate:
3. One-tenth normal potassium hydroxid.
The Manipulation.—From a quarter to one gram of the fat, oil or resin, is dissolved in ten cubic centimeters of carbon tetrachlorid in a dry bottle of 500 cubic centimeters capacity, provided with a well-ground glass stopper. An excess of the bromin solution is added, the bottle tightly stoppered, well shaken and placed in the dark. At the end of eighteen hours the bottle is placed in a freezing mixture and cooled until a partial vacuum is formed. A piece of wide rubber tubing an inch and a half long is slipped over the lip of the bottle so as to form a well about the stopper. This well having been filled with water the stopper is lifted and the water is sucked into the bottle absorbing all the hydrobromic acid which has been formed. The well should be kept filled with water, as it is gradually taken in until in all twenty-five cubic centimeters have been added. The bottle is next well shaken and from ten to twenty cubic centimeters of a twenty per cent potassium iodid solution added.
The excess of bromin liberates a corresponding amount of iodin, which is determined by the thiosulfate solution in the usual way, after adding about seventy-five cubic centimeters of water. The total bromin which has disappeared is then calculated from the data obtained, the strength of the original bromin solution having been previously determined. The contents of the bottle are next transferred to a separatory funnel, the aqueous portion separated, filtered through a linen filter, a few drops of thiosulfate solution added, if a blue color persist, and the free hydrobromic acid determined by titration with potassium hydroxid, using methyl orange as indicator. The end reaction is best observed by placing the solution in a porcelain dish, adding the alkali in slight excess, and titrating back with tenth-normal hydrochloric acid until the pink tint is perceived. From the number of cubic centimeters of alkali used the amount of bromin present as hydrobromic acid is calculated, and this expressed as percentage gives the bromin substitution figure. The bromin substitution figure multiplied by two and subtracted from the total absorption gives the addition figure.
Following are the data for some common substances:
| Substance. | Total bromin absorption in eighteen hours. | Bromin addition figure. | Bromin substitution figure. | ||
|---|---|---|---|---|---|
| Rosin | 212.70 | 0.00 | 106.35 | ||
| Raw | linseed | oil | 102.88 | 102.88 | 00.00 |
| Boiled | ” | ” | 103.92 | 103.92 | 00.00 |
| Salad | cotton | ” | 65.54 | 64.26 | 0.64 |
| Sperm | ” | 56.60 | 54.52 | 1.04 | |
By the process just described it is possible to detect mixtures of rosins and rosin oils with animal and vegetable oils. In this respect it possesses undoubted advantages over the older methods.
338. Method Of Hehner.—The absorption of bromin which takes place when unsaturated fats are brought into contact with that reagent was made the basis of an analytical process, proposed by Allen as long ago as 1880.[300] In the further study of the phenomena of bromin absorption, as indicated by McIlhiney, Hehner modified the method as indicated below.[301] From one to three grams of the sample are placed in a tared wide-mouthed flask and dissolved in a little chloroform. Bromin is added to the solution, drop by drop, until it is in decided excess. The flask is placed on a steam-bath and heated until the greater part of the bromin is evaporated, when some more chloroform is added and the heating continued until all the free bromin has escaped. The flask is put in a bath at 125° and dried to constant weight. A little acrolein and hydrobromic acid escape during the drying and the residue may be colored, or a heavy bromo oil be obtained. The gain in weight represents the bromin absorbed. The bromin number may be converted into the iodin number by multiplying by 1.5875.[302]
Fig. 100.—
Olein
Tube.
339. Halogen Absorption and Addition of Fat Acids.—Instead of employing the natural glycerids for determining the degree of action with the halogens the acids may be separated by some of the processes of saponification hereafter described and used as directed for the glycerids themselves. It is doubtful if any practical advantage arises from this variation of the process. If the fat acids be separated, however, it is possible to get some valuable data from the halogen absorption of the fractions. Theoretically the stearic series of acids would suffer no change in contact with halogens while the oleic series is capable of a maximum absorptive and additive action. On this fact is based a variation of the iodin process in which an attempt is made to separate the oleic acid from its congeners and to apply the halogen to the separated product.
The method of separation devised by Muter is carried out as follows:[303] The separatory or olein tube consists of a wide burette stem, provided with a lateral stopcock, and drawn out below to secure a clamp delivery tube, and at the top expanded into a bulb closed with a ground glass stopper, as shown in [Fig. 100]. Forty cubic centimeters of liquid are placed in the tube and the surface is marked 0. Above this the graduation is continued in cubic centimeters to 250, which figure is just below the bulb at the top.
The process of analysis is conducted as follows: About three grams of the oil or fat are placed in a flask, with fifty cubic centimeters of alcoholic potash lye, containing enough potassium hydroxid to ensure complete saponification. The flask is closed and heated on a water-bath until saponification is complete. The pressure flask to be described hereafter may be conveniently used. After cooling, the excess of alkali is neutralized with acetic acid in presence of phenolphthalien and then alcoholic potash added until a faint pink color is produced. In a large porcelain dish place 200 cubic centimeters of water and thirty of a ten per cent solution of lead acetate and boil. Pour slowly, with constant stirring, into the boiling liquid the soap solution prepared as above described, and allow to cool, meanwhile continuing the stirring. At the end, the liquid remaining is poured off and the solid residue washed with hot water by decantation.
The precipitate of lead salts is finally removed from the dish into a stoppered bottle, the dish washed with pure ether, the washings added to the bottle together with enough ether to make the total volume thereof 120 cubic centimeters. The closed bottle is allowed to stand for twelve hours with occasional shaking, by which time the lead oleate will have been completely dissolved. The insoluble lead salts are next separated by filtration, and the filtrate collected in the olein tube. The washing is accomplished by ether and, to avoid loss, the funnel is covered with a glass plate. The ethereal solution of lead oleate is decomposed by dilute hydrochloric acid, using about forty cubic centimeters of a mixture containing one part of strong acid to four of water. The olein tube is closed and shaken until the decomposition is complete, which will be indicated by the clearing of the ethereal solution. The tube is allowed to remain at rest until the liquids separate and the aqueous solution is run out from the pinch-cock at the lower end. The residue is washed with water by shaking, the water drawn off as just described, and the process continued until all acidity is removed.
Water is then added until the separating plane between the two liquids is at the zero of the graduation, and enough ether added to make the ethereal solution of a desired volume, say 200 cubic centimeters. After well mixing, the ethereal solution or an aliquot part thereof, e.g., fifty cubic centimeters, is removed by the side tubulure and nearly the whole of the ether removed from the portion by distillation. To the residue are added fifty cubic centimeters of pure alcohol and the solution is titrated for oleic acid with decinormal sodium hydroxid solution. Each cubic centimeter of the hydroxid solution used is equivalent to 0.0282 gram of oleic acid. The total quantity of oleic acid contained in the amount of fat used is readily calculated from the data obtained.
To determine the iodin absorption of the free acid another measured quantity of the ethereal solution containing as nearly as possible half a gram of oleic acid, is withdrawn from the olein tube, and the ether removed in an atmosphere of pure carbon dioxid. To the residue, without allowing it to come in contact with the air, fifty cubic centimeters of Hübl’s reagent are added and the flask put aside in the dark for twelve hours. At the end of this time thirty-five cubic centimeters of a ten per cent solution of potassium iodid are added, the contents of the flask made up to a quarter of a liter with water, fifteen cubic centimeters of chloroform added, and the excess of iodin titrated in the way already described. The percentage of iodin absorbed is calculated as already indicated.
Lane has proposed a more rapid process for the above determination.[304] The lead soaps are precipitated in a large erlenmeyer and cooled rapidly in water, giving the flask meanwhile a circular motion which causes the soaps to adhere to its walls. Wash with hot water, rinsing once with alcohol, add 120 cubic centimeters of ether, attach a reflux condenser, and boil until the lead oleate is dissolved, cool slowly, to allow any lead stearate which has passed into solution to separate, and filter into the olein tube. The rest of the operation is conducted as described above. The percentage of oleic acid and its iodin absorption in the following glycerids are given in the table below:
| Cottonseed oil. | Lard. | Peanut oil. | |
| Per cent oleic acid | 75.16 | 64.15 | 79.84 |
| Per cent iodin absorbed | 141.96 | 99.48 | 114.00 |
340. Saponification.—In many of the analytical operations which are conducted on the glycerids it is necessary to decompose them. When this is accomplished by the action of a base which displaces the glycerol from its combination with the fat acids, the resulting salts are known as soaps and the process is named saponification. In general use the term saponification is applied, not only strictly, as above defined, but also broadly, including the setting free of the glycerol either by the action of strong acids or by the application of superheated steam. In chemical processes the saponification of a glycerid is almost always accomplished by means of soda or potash lye. This may be in aqueous or alcoholic solution and the process is accomplished either hot or cold, in open vessels or under pressure. It is only important that the alkali and glycerid be brought into intimate contact. The rate of saponification is a function of the intimacy of contact, the nature of the solvent and the temperature. For chemical purposes, it is best that the decomposition of the glycerid be accomplished at a low temperature and for most samples this is secured by dissolving the alkali in alcohol.
In respect of solvents, that one would be most desirable, from theoretical considerations, which acts on both the glycerids and alkalies. In the next rank would be those which dissolve one or the other of the materials and are easily miscible, as, for instance, carbon tetrachlorid for the glycerid and alcohol for the alkali. As a rule, the glycerid is not brought into solution before the saponification process is commenced. Instead of using an alcoholic solution of sodium or potassium hydroxid the sodium or potassium alcoholate may be employed, made by dissolving metallic sodium or potassium in alcohol. It is probable, however, that a little water is always necessary to complete the process.
If a fat be dissolved in ether and treated with sodium alcoholate, a granular deposit of soap is soon formed and the saponification is completed in twenty-four hours. As much as 150 grams of fat can be saponified with ten grams of metallic sodium dissolved in 250 cubic centimeters of absolute alcohol.[305] For practical purposes the alcoholic solution of the hydroxid is sufficient.
The chemical changes which fats undergo on saponification are of a simple kind. When the process is accomplished by means of alkalies, the alkaline base takes the place of the glycerol as indicated in the following equation:
| Triolein 884. | Potassium hydroxid 168. | ||
| C₃H₅(O.C₁₈H₃₃O)₃ | + | 3KOH | = |
| Potassium oleate 960. | Glycerol 92. | ||
| (KO.C₁₈H₃₃O)₃ | + | C₃H₅(OH)₃. | |
The actual changes which take place in ordinary saponification are not so simple, however, since natural glycerids are mixtures of several widely differing fats, each of which has its own rate of decomposition. Palmitin and stearin, for instance, are saponified more readily than olein and some of the saponifiable constituents of resins and waxes are extremely resistant to the action of alkalies. The above equation may be regarded as typical for saponification in aqueous or alcoholic solutions in open dishes or under pressure. If the alkali used be prepared by dissolving metallic sodium or potassium in absolute alcohol (sodium alcoholate or ethoxid) the reaction which takes place is probably represented by the equation given below:
C₃H₅(O.C₁₈H₃₃O)₃ + 3C₂H₅.ONa = C₃H₅(ONa)₃ + 3C₁₈H₃₃O.O.C₂H₅,
in which it is seen that complete saponification cannot occur without the absorption of some water, by which the sodium glyceroxid is converted into glycerol and sodium hydroxid, the latter compound eventually uniting with the ethyl ether of the fat acid.[306]
Glycerids are decomposed when heated with water under a pressure of about sixteen atmospheres or when subjected to a current of superheated steam at 200°. The reaction consists in the addition of the elements of water, whereby the glyceryl radicle is converted into free glycerol and the fat acid is set free. The chemical change which ensues is shown below:
C₃H₅(O.C₁₈H₃₃O)₃ + 3H₂O = 3C₁₈H₃₄O₂ + C₃H₅(OH)₃.
The details of saponification with sulfuric acid are of no interest from an analytical point of view.[307]
341. Saponification in an Open Dish.—The simplest method of saponifying fats is to treat them with the alkaline reagent in an open dish. In all cases the process is accelerated by the application of heat. Vigorous stirring also aids the process by securing a more intimate mixture of the ingredients. This method of decomposing glycerids, however, is not applicable in cases where volatile ethers may be developed. These ethers may escape saponification and thus prevent the formation of the maximum quantity of soap. While not suited to exact quantitive work, the method is convenient in the preparation of fat acids which are to be the basis of subsequent analytical operations, as, for instance, in the preparation of fat acids for testing with silver nitrate. Large porcelain dishes are conveniently used and the heat is applied in any usual way, with care to avoid scorching the fat.
342. Saponification under Pressure.—The method of saponification which has given the best satisfaction in my work and which has been adopted by the Association of Official Agricultural Chemists is described below.[308]
Reagents.—The reagents employed are a solution of pure potash containing 100 grams of the hydroxid dissolved in fifty-eight grams of recently boiled distilled water, alcohol of approximately ninety-five per cent strength redistilled over caustic soda, and sodium hydroxid solution prepared as follows:
One hundred grams of sodium hydroxid are dissolved in 100 cubic centimeters of distilled water. The caustic soda should be as free as possible from carbonates, and be preserved from contact with the air.
Apparatus.—A saponification flask; it has a round bottom and a ring near the top, by means of which the stopper can be tied down. The flask is arranged for heating as shown in [Fig. 101]. A pipette graduated to deliver forty cubic centimeters is recommended as being more convenient than a burette for measuring the solutions: A pipette with a long stem graduated to deliver 5.75 cubic centimeters at 50°.
Manipulation.—The fat to be examined should be melted and kept in a dry warm place at about 60° for two or three hours, until the water has entirely separated. The clear supernatant fat is poured off and filtered through a dry filter paper in a jacket funnel containing boiling water. Should the filtered fat, in a fused state, not be perfectly clear, it must be filtered a second time. The final drying is accomplished at 100° in a thin layer in a flat bottom dish, in partial vacuum or an atmosphere of inert gas.
The saponification flasks are prepared by thoroughly washing with water, alcohol, and ether, wiping perfectly dry on the outside, and heating for one hour at the temperature of boiling water. The hard flasks used in moist combustions with sulfuric acid for the determination of nitrogen are well suited for this work. The flasks should be placed in a tray by the side of the balance and covered with a silk handkerchief until they are perfectly cool. They must not be wiped with a silk handkerchief within fifteen or twenty minutes of the time they are weighed or else the electricity developed will interfere with weighing. The weight of the flasks having been accurately determined, they are charged with the melted fat in the following way:
Fig. 101.—Apparatus for Saponifying under Pressure.
The pipette with a long stem, marked to deliver 5.75 cubic centimeters, is warmed to a temperature of about 50°. The fat, having been poured back and forth once or twice into a dry beaker in order to thoroughly mix it, is taken up in the pipette, the nozzle of the pipette having been previously wiped to remove any externally adhering fat, is carried to near the bottom of the flask and 5.75 cubic centimeters of fat allowed to flow into the flask. After the flasks have been charged in this way they should be re-covered with the silk handkerchief and allowed to stand for fifteen or twenty minutes, when they are again weighed.
343. Methods of Saponification.—In the Presence of Alcohol.—Ten cubic centimeters of ninety-five per cent alcohol are added to the fat in the flask, and then two cubic centimeters of the sodium hydroxid solution. A soft cork stopper is inserted and tied down with a piece of twine. The saponification is completed by placing the flask upon the water or steam-bath. The flask during the saponification, which should last one hour, should be gently rotated from time to time, being careful not to project the soap for any distance up its sides. At the end of an hour the flask, after having been cooled to near the room temperature, is opened.
Without the Use of Alcohol.—To avoid the danger of loss from the formation of ethers, and the trouble of removing the alcohol after saponification, the fat may be saponified with a solution of caustic potash in a closed flask without using alcohol. The operation is carried on exactly as indicated above for saponification in the presence of alcohol, using potassium instead of sodium hydroxid solution. For the saponification, use two cubic centimeters of the potassium hydroxid solution which are poured on the fat after it has solidified in the flask. Great care must be taken that none of the fat be allowed to rise on the sides of the saponifying flask to a point where it cannot be reached by the alkali. During the process of saponification the flask can only be very gently rotated in order to avoid the difficulty mentioned. This process is not recommended with any apparatus except a closed flask with round bottom. Potash is used instead of soda so as to form a softer soap and thus allow a more perfect saponification.
The saponification may also be conducted as follows: The alkali and fat in the melted state are shaken vigorously in the saponification flask until a complete emulsion is secured. The rest of the operation is then conducted as above.
344. Saponification in the Cold.—By reason of the danger of loss from volatile ethers in the hot alcoholic saponification, a method for successfully conducting the operation in the cold is desirable. Such a process has been worked out by Henriques.[309] It is based upon the previous solution of the fat in petroleum ether, in which condition it is so easily attacked by the alcoholic alkali as to make the use of heat during the saponification unnecessary. The process is conveniently conducted in a porcelain dish covered with a watch glass. Five grams of the fat are dissolved in twenty-five cubic centimeters of petroleum ether and treated with an equal quantity of four per cent alcoholic soda lye. The process of saponification begins at once and is often indicated by the separation of sodium salts. It is best to allow the action to continue over night and, with certain difficultly saponifiable bodies, such as wool fat and waxes, for twenty-four hours. In the case of butter fat an odor of butyric ether may be perceived at first but it soon disappears. After the saponification is complete, the excess of alkali is determined by titration in the usual way with set hydrochloric acid, using phenolphthalien as indicator. For the determination of volatile acids, the mixture, after saponification is complete, is evaporated rapidly to dryness, the solid matter being reduced to powder with a glass rod, after which it is transferred to a distilling flask and the volatile acids secured by the usual processes. In comparison with the saponification and reichert-meissl numbers obtained with hot alcoholic potash, the numbers given by the cold process are found to be slightly higher with those fats which give easily volatile ethers. On account of the simplicity of the process and the absence of danger of loss from ethers, it is to be recommended instead of the older methods in case a more extended trial of it should establish the points of excellence claimed above.
345. Saponification Value.—The number of milligrams of potassium hydroxid required to completely saturate one gram of a fat is known as the saponification value of the glycerid. The process of determining this value, as worked out by Koettstorfer and modified in the laboratory of the Department of Agriculture, is as follows:[310]
The saponification is accomplished with the aid of potassium hydroxid and in the flask and manner described in the preceding paragraph. About two grams of the fat will be found a convenient quantity. Great care must be exercised in measuring the alkaline solution, the same pipette being used in each case and the same time for draining being allowed in every instance. Blanks are always to be conducted with each series of examinations. As soon as the saponification is complete, the flask is removed from the bath, allowed to cool and its contents are titrated with seminormal hydrochloric acid and phenolphthalien as indicator. The number expressing the saponification value is obtained by subtracting the number of cubic centimeters of seminormal hydrochloric acid required to neutralize the alkali after saponification from that required to neutralize the alkali of the blank determinations, multiplying the result by 28.06 and dividing the product by the number of grams of fat employed.
Example.—Weight of sample of fat used 1.532 grams: Number of cubic centimeters half normal hydrochloric acid required to saturate blank, 22.5: Number of cubic centimeters of half normal hydrochloric acid required to saturate the alkali after saponification 12.0: Difference, 10.5 cubic centimeters:
Then 10.50 × 28.06 ÷ 1.532 = 192.3.
This latter number represents the saponification value of the sample.
346. Saponification Equivalent.—Allen defines the saponification equivalent as the number of grams of fat saponified by one equivalent, viz., 56.1 grams of potassium hydroxid.[311] The saponification equivalent is readily calculated from the saponification value using it as a divisor and 56100 as a dividend. Conversely the saponification value may be obtained by dividing 56100 by the saponification equivalent. No advantage is gained by the introduction of a new term so nearly related to saponification value.
347. Saponification Value of Pure Glycerids.—The theoretical saponification values of pure glycerids are given in the following table.[312]
| Name. | Symbol. | Molecular weight. | Saponification value. |
|---|---|---|---|
| Butyrin | C₃H₅(O.C₄H₇O)₃ | 302 | 557.3 |
| Valerin | C₃H₅(O.C₅H₉O)₃ | 344 | 489.2 |
| Caproin | C₃H₅(O.C₆H₁₁O)₃ | 386 | 438.3 |
| Caprin | C₃H₅(O.C₁₀H₁₉O)₃ | 554 | 305.0 |
| Laurin | C₃H₅(O.C₁₂H₂₃O)₃ | 638 | 263.8 |
| Myristin | C₃H₅(O.C₁₄H₂₇O)₃ | 722 | 233.1 |
| Palmitin | C₃H₃(O.C₁₆H₃₁O)₃ | 806 | 208.8 |
| Stearin | C₃H₅(O.C₁₈H₃₅O)₃ | 890 | 189.1 |
| Olein | C₃H₅(O.C₁₈H₃₃O)₃ | 884 | 190.4 |
| Linolein | C₃H₅(O.C₁₈H₃₁O)₃ | 878 | 191.7 |
| Ricinolein | C₃H₅(O.C₁₈H₃₃O₂)₃ | 932 | 180.6 |
| Euricin | C₃H₅(O.C₂₂H₁₄O)₃ | 1052 | 160.0 |
From the above table it is seen that in each series of glycerids the saponification equivalent falls as the molecular weight rises.
348. Acetyl Value.—Hydroxy acids and alcohols, when heated with glacial acetic acid, undergo a change which consists in substituting the radicle of acetic acid for the hydrogen atom of the alcoholic hydroxyl group. This change is illustrated by the equations below:[313]
For a Fat Acid:
| Ricinoleic acid. | Acetic anhydrid. | ||
| C₁₇H₃₂(OH).COOH | + | (C₂H₃O)₂O | = |
| Acetyl-ricinoleic acid. | Acetic acid. | ||
| C₁₇H₃₂(O.C₂H₃O)COOH | + | HC₂H₃O₂. | |
For an Alcohol:
| Cetyl alcohol. | Acetic anhydrid. | ||
| C₁₆H₃₃.OH | + | (C₂H₃O)₂O | = |
| Cetyl acetate. | Acetic acid. | ||
| C₁₆H₃₃.C₂H₃O | + | HC₂H₃O₂. | |
Determination.—The method of determining the acetyl value of a fat or alcohol has been described by Benedikt and Ulzer.[314] The operation is conducted on the fat acids and not on the glycerids containing them.
The insoluble fat acids are prepared as directed in paragraph [340].
From twenty to fifty grams of the fat acids are boiled with an equal volume of acetic anhydrid, in a flask with a reflux condenser, for two hours. The contents of the flask are transferred to a larger vessel of about one liter capacity, mixed with half a liter of water and boiled for half an hour. To prevent bumping, some bubbles of carbon dioxid are drawn through the liquid by means of a tube drawn out to a fine point and extending nearly to the bottom of the flask. The liquids are allowed to separate into two layers and the water is removed with a syphon. The oily matters are treated several times with boiling water until the acetic acid is all washed out. The acetylated fat acids are filtered through a dry hot jacket filter and an aliquot part, from three to five grams, is dissolved in absolute alcohol. After the addition of phenolphthalien the mixture is titrated as in the determination of the saponification value. The acid value thus obtained is designated as the acetyl acid value. A measured quantity of alcoholic potash, standardized by seminormal hydrochloric acid, is added, the mixture boiled and the excess of alkali determined by titration. The quantity of alkali consumed in this process measures the acetyl value. The sum of the acetyl acid and the acetyl values is the acetyl saponification value. The acetyl value is therefore equal to the difference of the saponification and acid values of the acetylated fat acids. In other words, the acetyl value indicates the number of milligrams of potassium hydroxid required to neutralize the acetic acid obtained by the saponification of one gram of the acetylated fat acids.
Example.—A portion of the fat acids acetylated as described, weighing 3.379 grams, is exactly neutralized by 17.2 cubic centimeters of seminormal potassium hydroxid solution, corresponding to 17.2 × 0.02805 = 0.4825 gram of the hydroxid, hence 0.4825 × 1000 ÷ 3.379 = 142.8, the acetyl acid value of the sample.
After the addition of 32.8 cubic centimeters more of the seminormal potash solution, the mixture is boiled to saponify the acetylated fat acids. The residual potash requires 14.2 cubic centimeters of seminormal hydrochloric acid. The quantity of potash required for the acetic acid is therefore 32.8 - 14.3 = 18.5 cubic centimeters or 18.5 × 0.02805 = 0.5189 gram of potassium hydroxid. Then 0.5189 × 1000 ÷ 3.379 = 153.6 = acetyl value of sample. The sum of these two values, viz., 142.8 and 153.6 is 296.4, which is the acetyl saponification value of the sample. As with the iodin numbers, however, it is also found that acids of the oleic series give an acetyl value when treated as above, and it has been proposed by Lewkowitsch to determine, in lieu of the data obtained, the actual quantity of acetic acid absorbed by fats.[315] This is accomplished by saponifying the acetylated product with alcoholic potash and determining the free acetic acid by distillation, in a manner entirely analogous to that used for estimating volatile fat acids described further on.
The rôle which the acetyl value plays in analytical determinations is interesting, but the data it gives are not to be valued too highly.
349. Determination of Volatile Fat Acids.—The fat acids which are volatile at the temperature of boiling water, consist chiefly of butyric and its associated acids occurring in the secretions of the mammary glands. Among vegetable glycerids cocoanut oil is the only common one which has any notable content of volatile acids. The boiling points of the above acids, in a pure state, are much higher than the temperature of boiling water; for instance, butyric acid boils at about 162°. By the expression volatile acids, in analytical practice, is meant those which are carried over at 100°, or a little above, with the water vapor, whatever be their boiling point. The great difficulty of removing the volatile from the non-volatile fat acids has prevented the formulation of any method whereby a sharp and complete separation can be accomplished. The analyst, at the present time, must be content with some approximate process which, under like conditions, will give comparable results. Instead, therefore, of attempting a definite determination, he confines his work to securing a partial separation and in expressing the degree of volatile acidity in terms of a standard alkali. To this end, a definite weight of the fat is saponified, the resulting soap decomposed with an excess of fixed acid, and a definite volume of distillate collected and its acidity determined by titration with decinormal alkali. The weight of fat operated on is either two and a half[316] or five grams.[317]
Numerous minor variations have been proposed in the process, the most important of which is in the use of phosphoric instead of sulfuric acid in the distillation. An extended experience with both acids has shown that no danger is to be apprehended in the use of sulfuric acid and that on the whole it is to be preferred to phosphoric.[318]
The process as used in this laboratory and as adopted by the official agricultural chemists is conducted as follows:[319]
350. Removal of the Alcohol.—The saponification is accomplished in the manner already described, ([341-344]) and when alcoholic potash is used proceed as follows:
The stopper having been laid loosely in the mouth of the flask, the alcohol is removed by dipping the flask into a steam-bath. The steam should cover the whole of the flask except the neck. After the alcohol is nearly removed, frothing may be noticed in the soap, and to avoid any loss from this cause or any creeping of the soap up the sides of the flask, it should be removed from the bath and shaken to and fro until the frothing disappears. The last traces of alcohol vapor may be removed from the flask by waving it briskly, mouth down, to and fro.
Dissolving the Soap.—After the removal of the alcohol the soap should be dissolved by adding 100 cubic centimeters of recently boiled distilled water, or eighty cubic centimeters when aqueous potassium hydroxid has been used for saponification, and warming on the steam-bath, with occasional shaking, until the solution of the soap is complete.
Setting free the Fat Acids.—When the soap solution has cooled to about 60° or 70°, the fat acids are separated by adding forty cubic centimeters of dilute sulfuric acid solution containing twenty-five grams of acid in one liter, or sixty cubic centimeters when aqueous potassium hydroxid has been used for saponification.
Melting the Fat Acid Emulsion.—The flask is restoppered as in the first instance and the fat acid emulsion melted by replacing the flask on the steam-bath. According to the nature of the fat examined, the time required for the fusion of the fatty acid emulsions may vary from a few minutes to several hours.
The Distillation.—After the fat acids are completely melted, which can be determined by their forming a transparent, oily layer on the surface of the water, the flask is cooled to room temperature, and a few pieces of pumice stone added. The pumice stone is prepared by throwing it, at a white heat, into distilled water, and keeping it under water until used. The flask is connected with a glass condenser, [Fig. 102], slowly heated with a naked flame until ebullition begins, and then the distillation continued by regulating the flame in such a way as to collect 110 cubic centimeters of the distillate in, as nearly as possible, thirty minutes. The distillate should be received in a flask accurately marked at 110 cubic centimeters.
Fig. 102.—Apparatus for the Distillation of Volatile Acids.
Titration of the Volatile Acids.—The 110 cubic centimeters of distillate, after thorough mixing, are filtered through perfectly dry filter paper, 100 cubic centimeters of the filtered distillate poured into a beaker holding about a quarter of a liter, half a cubic centimeter of phenolphthalien solution added and decinormal barium hydroxid solution run in until a red color is produced. The contents of the beaker are then returned to the measuring flask to remove any acid remaining therein, poured again into the beaker, and the titration continued until the red color produced remains apparently unchanged for two or three minutes, The number of cubic centimeters of decinormal barium hydroxid solution required should be increased by one-tenth to represent the entire distillate.
The number thus obtained expresses, in cubic centimeters of decinormal alkali solution, the volatile acidity of the sample. In each case blank distillations of the reagents used should be conducted under identical conditions, especially when alcoholic alkali is used for saponification. It is difficult to secure alcohol which will not yield a trace of volatile acid in the conditions named. The quantity of decinormal alkali required to neutralize the blank distillate is to be deducted from that obtained with the sample of fat.
351. Determination of Soluble and Insoluble Fat Acids.—The volatile fat acids are more or less soluble in water, while those which are not distillable in a current of steam are quite insoluble. It is advisable, therefore, to separate these two classes of fat acids, and the results thus obtained are perhaps more decidedly quantitive than are given by the distillation process just described. The methods used for determining the percentage of insoluble acids are essentially those of Hehner.[320] Many variations of the process have been proposed, especially in respect of the soluble acids.[321]
The process, as conducted in this laboratory and approved by the Association of Official Agricultural Chemists, is as follows:
Preparation of Reagents.—Sodium Hydroxid Solution.—A decinormal solution of sodium hydroxid is used. Each cubic centimeter contains 0.0040 gram of sodium hydroxid and neutralizes 0.0088 gram of butyric acid (C₄H₈O₂).
Alcoholic Potash Solution.—Dissolve forty grams of good caustic potash in one liter of ninety-five per cent alcohol redistilled over caustic potash or soda. The solution must be clear and the potassium hydroxid free from carbonates.
Standard Acid Solution.—Prepare accurately a half normal solution of hydrochloric acid.
Indicator.—Dissolve one gram of phenolphthalien in 100 cubic centimeters of ninety-five per cent alcohol.
Determination.—Soluble Acids.—About five grams of the sample are placed in the saponification flask already described, fifty cubic centimeters of the alcoholic potash solution added, the flask stoppered and placed in the steam-bath until the fat is entirely saponified. The operation may be facilitated by occasional agitation. The alcoholic potash is always measured with the same pipette and uniformity further secured by allowing it to drain the same length of time (thirty seconds). Two or three blank experiments are conducted at the same time.
In from five to thirty minutes, according to the nature of the fat, the liquid will appear perfectly homogeneous and, when this is the case, the saponification is complete and the flask is removed and cooled. When sufficiently cool, the stopper is removed and the contents of the flask rinsed with a little ninety-five per cent alcohol into an erlenmeyer, of about 200 cubic centimeters capacity, which is placed on the steam-bath together with the blanks until the alcohol is evaporated.
The blanks are titrated with half normal hydrochloric acid, using phenolphthalien as indicator, and one cubic centimeter more of the half normal hydrochloric acid than is required to neutralize the potash in the blanks is run into each of the flasks containing the fat acids. The flask is connected with a reflux condenser and placed on the steam-bath until the separated fat acids form a clear stratum on the upper surface of the liquid. The flask and contents are cooled in ice-water.
The fat acids having quite solidified, the liquid contents of the flask are poured through a dry filter into a liter flask, taking care not to break the cake. Between 200 and 300 cubic centimeters of water are brought into the flask, the cork with the condenser reinserted and the flask placed on the steam-bath until the cake of acid is thoroughly melted. During the melting of the cake of fat acids, the flask should occasionally be agitated with a rotary motion in such a way that its contents are not made to touch the cork. When the fat acids have again separated into an oily layer, the flask and its contents are cooled in ice-water and the liquid filtered through the same filter into the same liter flask as before. This treatment with hot water, followed by cooling and filtration of the wash water, is repeated three times, the washings being added to the first filtrate. The mixed washings and filtrate are made up to one liter, and 100 cubic centimeters thereof in duplicate are titrated with decinormal sodium hydroxid. The number of cubic centimeters of sodium hydroxid required for each 100 cubic centimeters of the filtrate is multiplied by ten. The number so obtained represents the volume of decinormal sodium hydroxid neutralized by the soluble fat acids of the fat, plus that corresponding to the excess of the standard acid used, viz., one cubic centimeter. The number is therefore to be diminished by five, corresponding to the excess of one cubic centimeter of half normal acid. This corrected volume multiplied by 0.0088 gives the weight of soluble acids as butyric acid in the amount of fat saponified.
Insoluble Acids.—The flask containing the cake of insoluble fat acids from the above determination and the paper through which the soluble fat acids have been filtered are allowed to drain and dry for twelve hours, when the cake, together with as much of the fat acids as can be removed from the filter paper, is transferred to a weighed evaporating dish. The funnel, with the filter, is then placed in an erlenmeyer and the paper thoroughly washed with absolute alcohol. The flask is rinsed with the washings from the filter paper, then with pure alcohol, and the rinsings transferred to the evaporating dish. The dish is placed on the steam-bath until the alcohol is evaporated, dried for two hours at 100°, cooled in a desiccator and weighed. It is again placed in the air-bath for two hours, cooled as before and weighed. If there be any considerable decrease in weight, reheat two hours and weigh again. The final weighing gives the weight of insoluble fat acids in the sample, from which the percentage is easily calculated.
The quantity of non-volatile and insoluble acids in common glycerids is from ninety-five to ninety-seven parts in 100. The glycerids yield almost the same proportion of fat acids and glycerol when the acids are insoluble and have high molecular weights. When the acids are soluble and the molecular weight low the proportion of acids decreases and that of glycerol increases.
In the following table will be found the data secured by quantitive saponification and separation of soluble and insoluble acids found in the more common glycerids:[322]
| Molecular weight of | Yield per 100 parts of glycerid. | ||||
|---|---|---|---|---|---|
| Glycerid. | Fat acid. | Glycerid. | Fat acid. | Fat acid. | Glycerol. |
| Stearin | Stearic | 890 | 284 | 95.73 | 10.34 |
| Olein | Oleic | 884 | 282 | 95.70 | 10.41 |
| Palmitin | Palmitic | 806 | 256 | 95.28 | 11.42 |
| Myristin | Myristic | 722 | 228 | 94.47 | 12.74 |
| Laurin | Lauric | 638 | 200 | 94.95 | 14.42 |
| Caprin | Capric | 594 | 172 | 93.14 | 15.48 |
| Caproin | Caproic | 386 | 116 | 90.16 | 23.83 |
| Butyrin | Butyric | 302 | 88 | 87.41 | 30.46 |
The general expression for the saponification of a neutral fat is C₃H₅O₃.R₃ + 3H₂O = 3R.OH + C₃H₈O₃, in which R represents the acid radicle. It is evident from this that the yield of more than 100 parts of fat acids and glycerol given by glycerids is due to the absorption of water during the reaction.
352. Formulas for General Calculations.—For calculating the theoretical yields of fat acids and glycerol, the following general formulas may be used:
- Let M = the molecular weight of the fat acid:
- K = saponification value:
- F = the quantity of free fat acids in the glycerid:
- N = the quantity of neutral fat in the glycerid:
- A = the number of milligrams of potassium hydroxid required
- to saturate the free acid in one gram of the sample.
- The free acid is determined by the method given below.
M grams of a fat acid require 56100 milligrams of potassium hydroxid for complete neutralization while F grams corresponding to 100 grams of fat are saturated by 100 × A milligrams of the alkali.
Then M : 56100 = F : 100A.
| Whence F = | AM | (1). |
| 561 |
Likewise since M grams of fat acid require the quantity of potassium hydroxid mentioned above we have:
1 : K = M : 56100,
| Whence M = | 56100 | (2). |
| K |
Substituting this value of M in (1) we have
| F = | A × 56100 | = | 100A | (3). |
| 561 × K | 561 × K |
It is evident that it is not necessary to calculate the acid value (A) of the sample and the saponification value (K) of the free fat acids, the ratio A/K alone being required. It will be sufficient therefore to substitute for A and K the number of cubic centimeters of alkali solutions required for one gram of the fat and one gram of the fat acids, respectively. If a and b represent these numbers the formula may be written
| F = | 100a | (4); |
| b |
| and N = 100 - F = | 100a | (5). |
| b |
To simplify the determinations, it may be assumed that the free fat acids have the same molecular weight as those still in combination with the glycerol in any given sample. On this assumption, the process may be carried on by determining the acid value A and the saponification value K for the total fat acids. The mean molecular weight M, the percentage of free fat acids F, and the proportion of neutral fat N, may then be calculated from the formulas (2), (3), (4), and (5).
Further, let G = the quantity of glycerol and L that of fat acids obtainable from one gram of neutral fat, that is, ¹/₁₀₀ of H the percentage of total fat acids.
The molecular weight of the neutral fat in each case is 3M + 38. Therefore, 3M + 38 parts of neutral fat yield 3M parts of fat acids and ninety-two parts of glycerol (C₃H₈O₃ = 92).
| Then L = | H | = | 3M | (6); |
| 100 | 3M + 38 |
| and G = | 92 | (7). |
| 3M + 38 |
N per cent of neutral fat yields, therefore, on saponification, the following theoretical quantities of fat acids F, and glycerol G expressed as parts per hundred.
| F = N × | 3M | (8); |
| 3M + 38 |
| and G = N × | 92 | (9). |
| 3M + 38 |
Formula (9) expresses also the total yield of glycerol from any given sample. For a further discussion of this part of the subject a work of a more technical character may be consulted.[323]
353. Determination of a Free Fat Acid in a Fat.—The principle of the method rests upon the comparative accuracy with which a free fat acid can be titrated with a set alkali solution when phenolphthalien is used as an indicator. Among the many methods of manipulation which the analyst has at his command there is probably none more simple and accurate than that depending on the solution of the sample in alcohol, ether, chloroform, or carbon tetrachlorid. Any acidity of the solvent is determined by separate titration and the proper correction made. Either an aqueous or alcoholic solution of the alkali may be used, preferably the latter. The alkaline solution may be approximately or exactly decinormal, but it is easier to make it approximately so and to determine its real value before each operation by titration against a standard decinormal solution of acid. About ten grams of the sample and fifty cubic centimeters of the solvent will be found convenient quantities.
Example.—Ten grams of rancid olive oil dissolved in alcohol ether require three and eight-tenths cubic centimeters of a solution of alcoholic potash to saturate the free acid present. When titrated with decinormal acid the potash solution is found to contain 25.7 milligrams of potassium hydroxid in each cubic centimeter. The specific gravity of the oil is 0.917 and the weight used therefore 9.17 grams. Then the total quantity of potassium hydroxid required for the neutralization of the acid is 25.7 × 3.8 = 97.7 milligrams.
The acid value A is therefore:
| A = | 3.8 × 25.7 | = 10.6 |
| 9.17 |
It is customary to regard free acid as oleic, molecular weight 282. On this assumption the percentage of free acids in the above case is found by the formula
| A (per cent) = | 3.8 × 25.7 × 282 | = 5.35 |
| 561 × 9.17 |
354. Identification of Oils and Fats.—Properly, the methods of identifying and isolating the different oils and fats should be looked for in works on food adulteration. There are, however, many characteristics of these glycerids which can be advantageously discussed in a work of this kind. Many cases arise in which the analyst is called upon to determine the nature of a fat and discover whether it be admixed with other glycerids. It is important often to know in a given case whether an oil be of animal or vegetable origin. Many of the methods of analysis already described are found useful in such discriminations. For instance, a large amount of soluble or volatile acids in the sample under examination, would indicate the presence of a fat derived from milk while the form of the crystals in a solid fat would give a clue to whether it were the product of the ox or the swine. In the succeeding paragraphs will be briefly outlined some of the more important additional methods of determining the nature and origin of fats and oils of which the history is unknown.
The data obtained by means of the methods which have been described, both physical and chemical, are all useful in judging the character and nature of a glycerid of unknown origin. The colorations produced by oxidizing agents, in the manner already set forth will be found useful, especially when joined to those obtained with cottonseed and sesame oils yet to be described. For instance, the red coloration produced by nitric acid of 1.37 specific gravity is regarded by some authorities as characteristic of cottonseed oil as well as the reduction by it of silver nitrate. The coloration tests with silver nitrate (paragraph [320]) and with phosphomolydic acid (paragraph [318]) are also helpful in classifying oils in respect of their animal or vegetable origin. The careful consideration of these tests, together with a study of the numbers obtained by treating the samples with iodin, and the heat of bromination and sulfuric saponification, is commended to all who are interested in classifying oils. In addition to these reactions a few specific tests are added for more detailed work.
355. Consistence.—It has already been said that oils are mostly of vegetable origin and the solid fats of animal derivation. In the animal economy it would be a source of disturbance to have in the tissues a large body of fat which would remain in a liquid state at the normal temperature of the body. Nearly all the animal fats are found to have a higher melting point than the body containing them. An exception is found in the case of butter fat, but it should be remembered that this fat is an excretion and not intended for tissue building until it has undergone subsequent digestion. Fish oils are another notable exception to the rule, but in this case these oils can hardly be regarded as true glycerids in the ordinary sense of that term.
In general, it may be said that a sample of a glycerid, which in its natural state remains liquid at usual room temperatures, is probably an oil of vegetable origin. Fish oils have also an odor and taste which prevent them from being confounded with vegetable oils. In oils which are manufactured from animal glycerids such as lard oil, the discrimination is more difficult but peculiarities of taste and color are generally perceptible.
356. Nature of the Fat Acid.—When it is not possible to discriminate between samples by the sensible physical properties just described, much light can be thrown on their origin by the determination of their other physical properties, such as specific gravity, refractive index, melting point, etc., in the manner already fully described. Further light may be furnished by saponification and separation of the fat acids. The relative quantities of oleic, stearic, palmitic, and other acids will help to a correct judgment in respect of the nature of the sample. The vegetable oils and lard oils, for instance, consist chiefly of olein; lard and tallow contain a large proportion of stearin; palm oil and butter fat contain considerable portions of palmitin, and the latter is distinguished moreover by the presence of soluble and volatile acids combined as butyrin and its associated glycerids.
Oleic acid can be rather readily separated from stearic and palmitic by reason of the solubility of its lead salts in ether. One method of accomplishing this separation has already been described (paragraph [339]).
357. Separation with Lime.—A quicker, though perhaps not as accurate a separation of the oleic from the palmitic and stearic acids, is accomplished by means of lime according to the method developed by Bondzyuski and Rufi.[324] This process is used chiefly, however, to separate the free fat acids (palmitic, stearic) from the neutral fat and the free oleic acid. It probably has no point of superiority over the lead process.
358. Separation of the Glycerids.—The fact that olein is liquid at temperatures allowing palmitin and stearin to remain solid, permits of a rough separation of these two classes of bodies by mechanical means. The mixed fats are first melted and allowed to cool very slowly. In these conditions the stearin and palmitin separate from the olein in a crystalline form and the olein is removed by pressure through bags. In this way lard is separated into lard oil, consisting chiefly of olein, and lard stearin, consisting largely of stearin. Beef (caul) fat is in a similar manner separated into a liquid (oleo-oil) and a solid (oleo-stearin) portion. It is evident that these separations are only approximate, but by repeated fractionations a moderately pure olein or stearin may be obtained.
359. Separation as Lead Salts.—Muter’s process, with a special piece of apparatus, has already been described ([339]). For general analytical work the special tube may be omitted. In a mixture of insoluble free fat acids all are precipitated by lead acetate, and the resulting soap may be extracted with ether, either with successive shakings or in a continuous extraction apparatus. In this latter case a little of the lead stearate or palmitate may pass into solution in the hot ether and afterwards separate on cooling. When the operation is conducted on from two to three grams of the dry mixed acids, the percentage proportions of the soluble and insoluble acids (in ether) can be determined. The lead salt which passes into solution can be decomposed and the oleic acid removed, dried and weighed. Dilute hydrochloric acid is a suitable reagent for decomposing the lead soap. The difference between the weight of the oleic acid and that of the mixed acids before conversion into lead soap furnishes the basis for the calculation. For further details in respect of the fat acids the reader may consult special analytical works.[325]
360. Separation of Arachidic Acid.—Peanut oil is easily distinguished from other vegetable glycerids by the presence of arachidic acid.
The method used in this laboratory for separating arachidic acid is a modification of the usual methods based on the process as carried out by Milliau.[326] About twenty grams of the oil are saponified with alcoholic soda, using twenty cubic centimeters of 36° baumé soda solution diluted with 100 cubic centimeters of ninety per cent alcohol. When the saponification is complete, the soda is converted into the lead soap by treatment with a slight excess of a saturated alcoholic solution of lead acetate. Good results are also obtained by using dilute alcohol, viz., fifty per cent, instead of ninety per cent, in preparing the lead acetate solution.
While still warm the supernatant liquid is decanted, the precipitate washed by decantation with warm ninety per cent alcohol and triturated with ether in a mortar four times, decanting the ethereal solution in each instance. By this treatment all of the lead oleate and hypogaeate are removed and are found in the ethereal solution, from which they can be recovered and the acids set free by hydrochloric acid and determined in the usual way.
The residue is transferred to a large dish containing two or three liters of pure water and decomposed by the addition of about fifty cubic centimeters of strong hydrochloric acid. The lead chlorid formed is soluble in the large quantity of water present, which should be warm enough to keep the free acids in a liquid state in which form they float as a clear oily liquid on the surface. The free acids are decanted and washed with warm water to remove the last traces of lead chlorid and hydrochloric acid. The last traces of water are removed by drying in a thin layer in vacuo. Practically all of the acids, originally present in the sample except oleic and hypogaeic, are thus obtained in a free state and their weight is determined.
The arachidic acid may be separated almost quantitively by dissolving the mixed acids in forty cubic centimeters of ninety per cent alcohol, adding a drop of hydrochloric acid, cooling to 16° and allowing to stand until the arachidic acid has crystallized. The crystals are purified by washing twice with twenty cubic centimeters of ninety per cent and three times with the same quantity of seventy per cent alcohol. The residual impure arachidic acid is dissolved in boiling absolute alcohol, poured through a filter and washed with pure hot alcohol. The filtrate is evaporated to dryness and heated to 100° until a constant weight is obtained. From the above data, the percentages of oleic, hypogaeic, arachidic and other acids in the sample examined are calculated.
In the above process, owing to the pasty state of the lead soaps, the trituration in a mortar with ether is found troublesome. The extraction of the lead oleate and hypogaeate is facilitated by throwing the pasty ethereal mass on a filter and washing it thoroughly with successive portions of about fifty cubic centimeters of ether. By this variation, it was found by Krug in this laboratory, that less ether was required and a more complete removal of the lead oleate effected. The solution of the lead oleate is completed by about half a dozen washings with ether as above described. The extraction may also be secured by placing the lead soaps in a large extracting apparatus and proceeding as directed in paragraph [40]. The residue is washed from the filter paper into a large porcelain dish and decomposed as already described with hydrochloric acid. After the separation is complete, the mixture is cooled until the acids are solid. The solid acids are then transferred to a smaller dish, freed of water and dissolved in ether. The ethereal solution is washed with water to remove any traces of lead salt or of hydrochloric acid. After the removal of the ether, the arachidic acid is separated as has already been described.
The melting point of pure arachidic acid varies from 73° to 75°.
361. Detection of Arachis (Peanut) Oil.—Kreis has modified the usual process of Renard for the detection of arachis oil, by precipitating the solution of the fat acid with an alcoholic instead of an aqueous solution of lead acetate, in a manner quite similar to that described above.[327] The fat acids are obtained in the usual manner, washed with hot water and the acids from twenty grams of the oil dissolved in 100 cubic centimeters of ninety per cent alcohol. The solution is cooled in ice-water and the fat acids precipitated by the addition of fifteen grams of lead acetate dissolved in 150 cubic centimeters of ninety per cent alcohol. The precipitate, after standing for two hours, is separated by filtration through cotton wool and is extracted for six hours with ether. The residue is boiled with 250 cubic centimeters of five per cent hydrochloric acid until the fat acids appear as a clear oily layer upon the surface. The acids thus obtained are washed with hot water to remove lead chlorid, dried by pressing between blotting paper, dissolved in 100 cubic centimeters of ninety per cent alcohol, cooled to 15° and allowed to stand for several hours, after which time any arachidic acid present is separated by crystallization and identified in the usual manner.
When it is not important to obtain all of the acid present, the process may be simplified in the following manner:
The fat acids obtained from twenty grams of oil are dissolved in 300 cubic centimeters of ether and treated at the temperature of ice-water with a quantity of the alcoholic lead acetate solution mentioned above. Lead oleate remains in solution and the precipitate which forms after a few hours consists almost wholly of the lead salts of the solid fat acids. The precipitate is collected, washed with ether and identified in the usual manner.
362. Cottonseed Oil, Bechi’s Test.—Crude, fresh cottonseed oil, when not too highly colored, and generally the refined article, may be distinguished from other oils by the property of reducing silver salts in certain conditions. The reaction was first noticed by Bechi and has been the subject of extensive discussions.[328]
The process as proposed by Bechi has been modified in many ways but apparently without improving it. It is conducted as follows: One gram of silver nitrate is dissolved in 200 cubic centimeters of ninety-eight per cent alcohol and forty cubic centimeters of ether and one drop of nitric acid added to the mixture. Ten cubic centimeters of the oil are shaken in a test tube with one cubic centimeter of this reagent, and then with ten cubic centimeters of a mixture containing 100 cubic centimeters of amyl alcohol and ten of colza oil. The mixture is divided into two portions, one of which is put aside for future comparison and the other plunged into boiling water for fifteen minutes. A deep brown or black color, due to the reduction of silver, reveals the presence of cottonseed oil.
In this laboratory the heating is accomplished in a small porcelain dish on which is often deposited a brilliant mirror of metallic silver. The white color of the porcelain also serves as a background for the observation of the coloration produced. In most instances a green color has been noticed after the reduction of the silver is practically complete. Unless cottonseed oil has been boiled or refined in some unusual way, the test, as applied above, is rarely negative. The reduction of the silver is doubtless due to some aldehydic principle, present in extremely minute quantities, and which may be removed by some methods of technical treatment. The silver nitrate test therefore is reliable when the reduction takes place, but the absence of a distinct reaction may not in all cases prove the absence of cottonseed oil.
363. Milliau’s Process.—Milliau has proposed the application of the silver salt directly to the free fat acids of the oil instead of to the oil itself.[329] About fifteen cubic centimeters of the oil are saponified with alcoholic potash in the usual manner, 150 cubic centimeters of water added to the dish and the mixture boiled until the alcohol is evaporated. The fat acids are freed by the addition of decinormal sulfuric acid and as they rise to the surface in a pasty condition are removed with a spoon. The free acids are washed with distilled water. The water is drained off and the free acids dissolved in fifteen cubic centimeters of ninety-two per cent alcohol and two cubic centimeters of a three per cent solution of silver nitrate. The test tube containing the mixture is well shaken and placed in a water-bath, out of contact with light, and left until about one-third of the alcohol is evaporated. Ten cubic centimeters of water are added, the heating continued for a few minutes and the color of the supernatant fat acids observed. The presence of cottonseed oil is revealed by the production of a lustrous precipitate which colors the fat acids black. In some cases the process of Milliau gives better results than the original method of Bechi, but this is not always the case. It does away with the use of amyl alcohol and colza oil, but its manipulation is more difficult. In all doubtful cases the analyst should apply both methods.
364. Detection of Sesame Oil.—Milliau has pointed out a characteristic reaction of this oil which may be used with advantage in cases of doubtful identity.[330] The identification is based on the fact that the free acids of sesame oil, or some concomitant thereof, give a rose-red color when brought in contact with a solution of sugar in hydrochloric acid.
The analytical process is conducted as follows: About fifteen grams of the oil are saponified with alcoholic soda and when the reaction is complete treated with 200 cubic centimeters of hot water and boiled until the alcohol is removed. The fat acids are set free with decinormal sulfuric acid and removed with a spoon as they rise to the surface in a pasty state, in which condition they are washed by shaking with water in a large test tube. When washed, the acids are placed in an oven at 105° until the greater part of the water is evaporated and the acids begin to become fluid. At this point they are treated with half their volume of hydrochloric acid saturated with finely ground sugar. On shaking the mixture, a rose color is developed which is characteristic of the sesame oil. Other oils give either no coloration or at most a yellow tint.
365. The Sulfur Chlorid Reaction.—Some vegetable oils, when treated with sulfur chlorid, give a hard product similar to elaidin, while lard does not. This reaction is therefore helpful in discriminating between some vegetable and animal glycerids. The process which is described by Warren has been used with some satisfaction in this laboratory.[331]
Five grams of the oil or fat are placed in a tared porcelain dish and treated with two cubic centimeters of carbon bisulfid and the same quantity of sulfur chlorid. The dish is placed on a steam-bath and its contents stirred until the reaction is well under way. The heating is continued until all volatile products are evaporated, the hard mass being well rubbed up to facilitate the escape of imprisoned vapors. The powdered or pasty mass is transferred to a filter and washed with carbon bisulfid to remove all unaltered oil. The washing with carbon bisulfid is hastened by pressure and about 200 cubic centimeters of the solvent should be used. After drying, the weight of insoluble matter is obtained and deducted from the total weight of the sample used.
The color and tenacity of the hard, insoluble portion are characteristic. The quantitive part of the operation appears to have but little value, but applied qualitively in this laboratory it produces hard, leathery masses with cotton, olive and peanut oils, and but little change in lard and beef fats. Qualitively applied, the process is conducted as described above but without making the weighings. In this instance it is as easy of application as the process of Bechi and is deserving of greater attention than has been given it by analysts.
In the combination which takes place between the sulfur and the fat it is probable that only addition products are formed, since the quantity of alkali required for saponification is not diminished by previously treating the fat with sulfur chlorid.[332] The reactions which take place are probably well represented by the following equations, in which oleic acid is treated with sulfur chlorid:
C₁₈H₃₄O₂ + S = C₁₈H₃₄S.O₂.
C₁₈H₃₄S.O₂ + NaOH = C₁₈H₃₄SO₂Na + H₂O.
366. Detection of Cholesterin and Phytosterin in Glycerids.—Cholesterin is often found in animal glycerids and a corresponding body, phytosterin, is sometimes found in oils of a vegetable origin.[333] When one of these two bodies is present it may be useful in distinguishing between animal and vegetable glycerids. They are detected as follows: Fifty grams of the glycerids in each case are saponified with alcoholic alkali, preferably potash, in order to have a soft soap. After saponification is complete, the alcohol is evaporated and the residual soap dissolved in two liters of water. The mixture is shaken with ether and the ethereal solution evaporated to a small bulk. The residue, which may contain a small quantity of unsaponified fat, is again treated with alcoholic potash and subjected a second time to the action of ether, as indicated above, with the addition of a few drops of water and of alcohol if the emulsion separate slowly. The ethereal extract finally secured is allowed to evaporate slowly and the cholesterin (phytosterin) is obtained in a crystalline form. The melting point of the cholesterin crystals is 146° and that of the phytosterin 132°.
Cholesterin crystallizes in thin rhombic tables while phytosterin separates in stellar aggregates or in bundles of long needles.
When dissolved in chloroform the two products show different color reactions with sulfuric acid, cholesterin giving a cherry and phytosterin a blue-red tint. In a mixture of animal and vegetable glycerids the two products are obtained together and the melting point of the mixture may afford some idea of the relative quantities of each present. It is evident, however, that no reliable judgment can be formed from these data of the relative proportions of the two kinds of glycerids in the original sample.
367. Cholesterin and Paraffin in Ether Extracts.—In ethereal extracts of some bodies, especially of flowers of the chrysanthemum, paraffin is found combined with cholesterin. The two bodies may be separated as follows:[334]
The ether extract is treated with aqueous then with alcoholic potash several times; the residue soluble in ether is a solid body melting at from 70° to 100°.
If the ethereal solution be cooled in a mixture of snow and salt, a crystalline deposit is formed. This substance, purified by repeated precipitations, is obtained colorless in fine crystalline scales melting at 64°. It is very soluble in ether, benzene and chloroform, almost insoluble in cold alcohol, and somewhat soluble in hot.
Its percentage composition is:
| Per cent. | |
| Carbon | 85.00 |
| Hydrogen | 14.95 |
It is therefore a paraffin.
The ethereal solution, freed by the above process from paraffin, leaves on evaporation a crystalline mass which is cholesterin, retaining still a small quantity of fat matters. In treating the crystals with alcoholic potash these fat bodies are saponified and the residue is taken up with ether. The cholesterin is obtained in fine needles melting at from 170° to 176°. It presents all the reactions of cholesterin, especially the characteristic reaction with chloroform and sulfuric acid.
368. Absorption of Oxygen.—Among oils a distinction is made between those which oxidize readily and those which are of a more stable composition. Linseed oil, for instance, in presence of certain metallic oxids, absorbs oxygen readily and is a type of the drying oils, while olive oil represents the opposite type.
The method of determining the quantity of oxygen absorbed is due to Livache and is carried out as follows:[335]
Precipitated metallic lead (by zinc) is mixed in a flat dish, with the oil to be tested, in the proportions of one gram of lead to three-quarters of a gram of oil, and exposed to the air and light of the workroom. The dish is weighed from time to time until there is no longer any increase in weight.
Instead of lead, finely divided copper has been used by Krug in this laboratory, but the percentage of absorption of oxygen is not so high with copper as with lead. Krug found the quantities of oxygen absorbed, after nine days, by the samples treated with copper and lead respectively to be the following:
| Copper, per cent oxygen absorbed. | Lead, per cent oxygen absorbed. | |
| Olive oil | 1.69 | 2.03 |
| Cottonseed oil | 4.25 | 5.30 |
| Peanut oil | 2.74 | 3.87 |
| Linseed oil | 5.55 | 7.32 |
Livache found that linseed oil absorbed about twice as much oxygen as indicated by the data just given.
369. Elaidin Reactions.—In discriminating between oils and fats having a preponderance of olein and others with a smaller proportion of that glycerid, the conversion of the olein into its isomer elaidin is of diagnostic value. The following will be found a convenient method of applying this test:[336]
About ten cubic centimeters of the oil are placed in a test tube together with half that quantity of nitric acid and one gram of mercury. The mixture is shaken until the mercury dissolves when the mass is allowed to remain at rest for twenty minutes. At the end of this time it is again shaken and placed aside. In from one to three hours the reaction is complete. Olive, peanut and lard oils give very hard elaidins. The depth to which a plunger of given weight and dimensions sinks into an elaidin mixture at a given temperature, has been used as a measure of the percentage of olein contained in the sample of oil, but it is evident that such a determination is only roughly approximate. Copper may be used instead of mercury for the generation of the oxids of nitrogen, but it is not so effective. The vapors of nitric oxids may also be conducted directly into the oil from a convenient generator. The reaction may also be accomplished by shaking the oil with nitric acid and adding, a drop at a time, a solution of potassium nitrite.