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:

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]

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:

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:

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:

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.

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]

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]

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.

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]

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.

Likewise since M grams of fat acid require the quantity of potassium hydroxid mentioned above we have:

1 : K = M : 56100,

Substituting this value of M in (1) we have

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

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).

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.

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:

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

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.