ESTIMATION OF FAT.

450. Form of Fat in Milk.—The fat in milk occurs in the form of globules suspended in the liquid, in other words in the form of an emulsion. Many authorities have asserted that each globule of fat is contained in a haptogenic membrane composed presumably of nitrogenous matter, but there is no convincing evidence of the truth of this opinion. The weight of experimental evidence is in the opposite direction. The supposed action of the membrane and the phenomena produced thereby are more easily explained by the surface tension existing between the fat globules and the menstruum in which they are suspended.

Babcock affirms that the spontaneous coagulation of the fibrin present in milk tends to draw the fat globules into clusters, and this tendency can be arrested by adding a little soda or potash lye to the milk as soon as it is drawn.[432]

The diameter of the fat globules is extremely variable, extending in some cases from two to twenty micromillimeters. In cow milk, the usual diameters are from three to five micromillimeters.

451. Number of Fat Globules in Milk.—The number of fat globules in milk depends on their size and the percentage of fat. It is evident that no definite statement of the number can be made. There is a tendency, on the part of the globules, to diminish in size and increase in number as the period of lactation is prolonged. To avoid large numbers, it is convenient to give the number of globules in 0.0001 cubic millimeter. This number may be found within wide limits depending on the individual, race, food and other local conditions to which the animal or herd is subjected. In general, in whole milk this number will be found between 140 and 250.

452. Method of Counting Globules.—The number of globules in milk is computed with the aid of the microscope. The most convenient method is the one devised by Babcock.[433] In carrying out this computation, capillary tubes, from two to three centimeters long and about one-tenth millimeter in internal diameter, are provided. The exact diameter of each tube, in at least three points, is determined by the micrometer attachment of the microscope, and from these measurements the mean diameter of the tube is calculated. This known, its cubic content for any given length is easily computed. Ten cubic centimeters of the milk are diluted with distilled water to half a liter and one end of a capillary tube dipped therein. The tube is quickly filled with diluted milk and each end is closed with a little wax to prevent evaporation. Several of these tubes being thus prepared, they are placed in a horizontal position on the stage of the microscope and covered with glycerol and a cover glass. The tubes are left at rest for some time until all the fat globules have attached themselves to the upper surfaces, in which position they are easily counted. The micrometer is so placed as to lie parallel with the tubes, and the number of globules, corresponding to each division of its scale, counted. The mean number of globules corresponding to each division of the micrometer scale is thus determined.

To compare the data obtained with each tube they are reduced to a common basis of the number of globules found in a length of fifty divisions of the micrometer scale in a tube having a diameter of 100 divisions, using the formula

N = 10000n
d²

in which n = the number of globules found in the standard length of tube measured and d = the diameter of the tube. It is not difficult to actually count all the globules in a length of fifty divisions of the scale, but the computation may also be made from the mean numbers found in a few divisions. The usual number of globules found in a length of 0.1 millimeter in a tube 0.1 millimeter in diameter, varies from fifty to one hundred.

Example.—The length of one division of the micrometer scale is 0.002 millimeter, and the internal diameter of the tube 0.1 millimeter. The content of a tube, of a length of 0.002 × 50 = 0.1 millimeter, is therefore 0.0007854 cubic millimeter.

The cubic content of a tube 100 scale divisions in diameter and fifty in length is 0.0031416 cubic millimeter. The number of globules found in fifty divisions of the tubes used is 40. Then the number which would be contained in a tube of a diameter of 100 divisions of the micrometer scale and a length of fifty divisions thereof is

N = 10000n = 400000 = 160.
d²2500

Since the milk is diluted fifty times, the actual number of globules corresponding to the volume given is 8000. It is convenient to reduce the observations to some definite volume, exempla gratia, 0.0001 cubic centimeter. The equation for this in the above instance is 0.0031416: 0.0001 = 8000: x, whence x = 223, = number of fat globules in 0.0001 cubic millimeter.

In one cubic millimeter of milk there are therefore 2,230,000 fat globules, and in one cubic centimeter 2,230,000,000 globules. In a single drop of milk there are from one to two hundred million fat globules.[434]

453. Classification of Methods of Determining Fat in Milk.—The fat, being the most valuable of the constituents of milk, is the subject of a number of analytical processes. An effort will be made here to classify these various methods and to illustrate each class with one or more typical processes. In general the methods may be divided into analytical and commercial, those of the first class being used for scientific and of the other for trade purposes. For normal milk, some of the trade methods have proved to be quite as accurate as the more chronokleptic analytical processes to which, in disputed cases, a final appeal must be taken. When the analyst is called upon to determine the fat in a large number of samples of milk some one of the trade methods may often be adopted with great advantage.

454. Dry Extraction Methods.—Among the oldest and most reliable methods of determining fat in milk, are included those processes based on the principle of drying the milk and extracting the fat from the residue by an appropriate solvent. The solvents generally employed are ether and petroleum spirit of low boiling point. The methods of drying are legion.

In extracting with ether, it must not be forgotten that other bodies than fat may pass into solution on the one hand and on the other any substituted glycerid, such as lecithin or nuclein, which may be present may escape solution, at least in part. Perhaps petroleum spirit, boiling at from 45° to 60°, is the best solvent for fat, but it is almost the universal custom in this and other countries to use ether.

455. The Official Methods.—In the methods adopted by the Association of Official Agricultural Chemists two processes are recommended.

(1) The Asbestos Process: In this process it is directed to extract the residue from the determination of water by the asbestos method ([447]) with anhydrous pure ether until the fat is removed, evaporate the ether, dry the fat at 100° and weigh. The fat may also be determined by difference, after drying the extracted cylinders at 100°.

(2) Paper Coil Method: This is essentially the method proposed by Adams as modified by the author.[435] Coils made of thick filter paper are cut into strips 6.25 by 62.5 centimeters, thoroughly extracted with ether and alcohol, or the weight of the extract corrected by a constant obtained for the paper. If this latter method be used, a small amount of anhydrous sodium carbonate should be added. Paper free of matters soluble in ether is also to be had for this purpose. From a weighing bottle about five grams of milk are transferred to the coil by a pipette, taking care to keep dry the lower end of the coil. The coil, dry end down, is placed on a piece of glass, and dried at a temperature of boiling water for one hour, or better, dried in hydrogen at a temperature of boiling water, transferred to an extraction apparatus and extracted with absolute ether or petroleum spirit boiling at about 45°. The extracted fat is dried in hydrogen and weighed. Experience has shown that drying in hydrogen is not necessary. The fat may be conveniently dried in partial vacuo.

456. Variations of Extraction Method.—The method of preparing the milk for fat extraction is capable of many variations. Some of the most important follow:

(1) Evaporation on Sand: The sand should be pure, dry and of uniform size of grain. It may be held in a dish or tube. The dish may be made of tin foil, so that it can be introduced with its contents into the extraction apparatus after the desiccation is complete. For this purpose, it is cut into fragments of convenient size after its contents have been poured into the extractor. The scissors used are washed with the solvent.

(2) Evaporation on Kieselguhr: Dry kieselguhr (infusorial earth, tripoli) may take the place of the sand as above noted. The manipulation is the same as with sand.

(3) Evaporation on Plaster of Paris,[436] (Soxhlet Method),[437] (4) On Pumice Stone, (5) On Powdered Glass, (6) On Chrysolite:[438] The manipulation in these cases is conducted as with sand and no detailed description is required.

(7) Evaporation on Organic Substances: These variations would fall under the general heading of drying on paper. The following materials have been used; viz., sponge,[439] lint,[440] and wood pulp.[441] In these variations the principal precautions to be observed are to secure the organic material in a dry state and free of any matter soluble in the solvent used.

(8) Dehydration with Anhydrous Copper Sulfate: In this process the water of the sample is absorbed by powdered anhydrous copper sulfate, the residual mass extracted and the butter fat obtained determined by saponification and titration.[442] In the manipulation about twenty grams of the anhydrous copper sulfate are placed in a mortar, a depression made therein in such a manner that ten cubic centimeters of milk can be poured into it without wetting it through to the mortar. The water is soon absorbed when the mass is ground with a little dry sand and transferred to the extractor.

Petroleum spirit of low boiling point is used as a solvent, successive portions of about fifteen cubic centimeters each being forced through the powdered mass under pressure. Two or three treatments with the petroleum are required. The residual butter fat, after the evaporation of the petroleum, is saponified with a measured portion, about twenty-five cubic centimeters, of seminormal alcoholic potash lye. The residual alkali is determined by titration with seminormal hydrochloric acid in the usual manner. From the data obtained is calculated the quantity of alkali employed in the saponification. The weight of butter fat extracted is then calculated on the assumption that 230 milligrams of potash are required to saponify one gram of the fat.

457. Gypsum Method for Sour Milk.—In sour milk, extraction of the dry residue with ether is attended with danger of securing a part of the free lactic acid in the extract. This may be avoided, at least in part, by making the milk neutral or slightly alkaline before desiccation. This method is illustrated by a variation of Soxhlet’s method of drying on gypsum proposed by Kühn.[443] The curdled milk is treated with potash lye of forty per cent strength until the reaction is slightly alkaline. For absorbing the sample before drying, a mixture is employed consisting of twenty-five grams of plaster of paris, four of precipitated carbonate of lime and two of acid potassium sulfate. To this mixture ten grams of the milk, rendered alkaline as above noted, are added in a desiccating dish, the excess of moisture evaporated at 100°, the residual mass finely ground and extracted with ether for four hours. A little gypsum may be found in the solution, but in such small quantities as not to interfere seriously with the accuracy of the results obtained.

458. Estimation of Fat in Altered Milk.—In altered milk the lactose has usually undergone a fermentation affording considerable quantities of lactic acid. If such milks be treated by the extraction method for fat, the results will always be too high, because of the solubility of lactic acid in ether.

Vizern[444] has proposed to avoid this error by first warming the soured milk for a few minutes to 40°, at which temperature the clabber is easily divided by vigorous shaking. Of the milk thus prepared, thirty grams are diluted with two or three volumes of water and poured onto a smooth and moistened filter. The vessel and filter are washed several times until the filtrate presents no further acid reaction. The filter and its contents are next placed in a vessel containing some fine washed sand. A small quantity of water is added, sufficient to form a paste. With a stirring rod, the filter is entirely broken up and the whole mass thoroughly mixed. Dried on the water bath the material is subjected to extraction in the ordinary way. Several analyses made on fresh milk and on milk kept for several months show that almost identical results are obtained.

In respect of this process there would be danger, on long standing, of the formation of free acids from butter glycerids, and these acids would be removed by the process of washing prescribed. In this case the quantity of fat obtained would be less than in the original sample.

459. Comparison of Methods.—An immense amount of work has been done by analysts in comparing the various types of extraction methods outlined above.[445]

The consensus of opinion is that good results are obtained by all the methods when properly conducted, and preference is given to the two methods finally adopted by the Association of Official Chemists. As solvents, pure ether and petroleum spirit of low boiling point are preferred. The direct extraction gravimetric processes are important, since it is to these that all the other quicker and easier methods must appeal for the proof of their accuracy.

460. Wet Extraction Methods.—It has been found quite impracticable to extract the fat from milk by shaking it directly with the solvent. An emulsion is produced whereby the solvent itself becomes incorporated with the other constituents of the milk, and from which it is not separated easily even with the aid of whirling. The disturbing element which prevents the separation of the solvent is doubtless the colloid casein, since, when this is previously rendered soluble, the separation of the solvent holding the fat is easily accomplished.

The principle on which the methods of wet extraction are based is a simple one; viz., to secure a complete or partial solution of the casein and subsequently to extract the fat with a solvent immiscible with water. The methods may be divided into three great classes; viz., (1) those in which the solvent is evaporated from the whole of the extracted fat and the residual matters weighed; (2) processes in which an aliquot part of the fat solution is employed and the total fat calculated from the data secured; (3) the density of the fat solution is determined at a definite temperature and the percentage of fat corresponding thereto determined from tables or otherwise. Methods (1) and (2) are practically identical in principle and one or the other may be applied according to convenience or to local considerations. The methods may be further subdivided in respect of the reagents used to secure complete or partial solution of the casein, as, for instance, alkali or acid.[446]

461. Solution in an Acid.—A good type of these processes is the method of Schmid.[447] In this process ten cubic centimeters of milk are placed in a test tube of about five times that content, graduated to measure small volumes. An equal quantity of hydrochloric acid is added, the mixture shaken, boiled until it turns dark brown, and cooled quickly. The fat is extracted by shaking with thirty cubic centimeters of ether. After standing some time the ethereal solution separates and its volume is noted. An aliquot part of the solution is removed, the solvent evaporated, and the weight of fat in the whole determined by calculation.

The schmid process has been improved by Stokes,[448] Hill,[449] and Richmond.[450] The most important of these variations consists in weighing instead of measuring the milk employed, thus insuring greater accuracy. Dyer and Roberts affirm that the ether dissolves some of the caramel products formed on boiling condensed milk with hydrochloric acid, and that the data obtained in such cases by the process of Schmid are too high.[451]

Since lactic acid is also slightly soluble in ether, sour milk should not be extracted with that solvent. In these cases petroleum spirit, or a mixture of petroleum and ether, as suggested by Pinette, may be used.[452] Another variation consists in extracting the fat with several portions of the solvent and evaporating all the extracts thus obtained to get the total fat. This method is perhaps the best of those in which the fat is extracted from the residual liquid after the decomposition of the casein by an acid, and may be recommended as both reliable and typical within the limitations mentioned above.

462. Solution in an Alkali.—The casein of milk is not so readily dissolved in an alkali as in an acid, but the solution is sufficient to permit the extraction of the fat. Soda and potash lyes and ammonia are the alkaline bodies usually employed. To promote the separation of the emulsions, alcohol is added with advantage. The principle of the process rests on the observed power of an alkali to free the fat globules sufficiently to allow them to dissolve in ether or some other solvent. When the solvent has separated from the emulsion at first formed, the whole or a part of it is used for the determination of fat in a manner entirely analogous to that employed in the process with the acid solutions described above. There are many methods based on this principle, and some of the typical ones will be given below. Experience has shown that extraction from an alkaline solution is more troublesome and less perfect than from an acid and these alkaline methods are, therefore, not so much practiced now as they were formerly.

463. Method of Short.—Instead of measuring the volume of the separated fat, Short has proposed a method in which the casein is dissolved in an alkali and the fat at the same time saponified. The soap thus produced is decomposed by sulfuric acid and the volume of the separated fat acids noted. This volume represents eighty-seven per cent of the corresponding volume of fat.[453]

The solvent employed is a mixture of sodium and potassium hydroxids, containing in one liter 125 and 150 grams, respectively, of these alkalies. The sample of milk is mixed with half its volume of the reagent and placed in boiling water for two hours. By this treatment the casein is dissolved and the fat saponified. After cooling to about 60°, the soap is decomposed by the addition of equal parts of sulfuric and acetic acids. The tubes containing the mixture are again placed in boiling water for an hour and they are then filled with boiling water to within one inch of the top. The tubes may either be furnished with a graduation or the column of fat be measured by a scale.

464. Method of Thörner.—The process of Short is conducted by Thörner as follows:[454]

Ten cubic centimeters of milk measured at 15° are saponified, in tubes fitting a centrifugal, by the addition of one and a half cubic centimeters of an alcoholic potash lye, containing 160 grams of potassium hydroxid per liter, or one cubic centimeter of an aqueous fifty per cent soda lye. The saponification is hastened by setting the tubes in boiling water, where they remain for two minutes. The soap formed is decomposed with a strong acid, sulfuric preferred, the tubes placed in the centrifugal and whirled for four minutes, when the fat acids will be formed in the narrow graduated part of the tube and the volume occupied thereby is noted after immersion in boiling water. Thörner’s process is not followed in this country, but is used to a considerable extent in Germany.[455]

465. Liebermann’s Method.—In this method, fifty cubic centimeters of milk, at ordinary temperatures, are placed in a glass cylinder twenty-five centimeters high and about four and a half internal diameter; five cubic centimeters of potash lye of 1.27 specific gravity are added, the cylinder closed with a well fitting cork stopper and thoroughly shaken.[456] After shaking, fifty cubic centimeters of petroleum spirit, boiling point about 60°, are added. The cylinder is again stoppered and vigorously shaken until an emulsion is formed. To this emulsion fifty cubic centimeters of alcohol of ninety-five per cent strength are added, and the whole again thoroughly shaken. After four or five minutes the petroleum spirit, containing the fat, separates. In order to insure an absolute separation of the fat, however, the shaking may be repeated three or four times for about one-quarter minute, waiting each time between the shakings until the spirit separates.

Of the separated petroleum spirit twenty cubic centimeters are placed in a small weighed flask. The use of the flask is recommended on account of the ease with which the petroleum spirit can be evaporated without danger of loss of fat. Instead of the flask a weighed beaker or other weighed dish may be employed.

The petroleum spirit is carefully evaporated on a water-bath and the residue dried at 110° to 120° for one hour. The weight found multiplied by five gives the content of fat in 100 cubic centimeters of the milk. The percentage by weight can then be calculated by taking into consideration the specific gravity of the milk employed.

The results obtained by this method agree well with those obtained by the paper coil method, when petroleum spirit instead of sulfuric ether is used as the solvent for the fat. Sulfuric ether, however, gives an apparently higher content of fat because of the solution of other bodies not fat present in the milk.

466. Densimetric Methods.—Instead of evaporating the separated fat solution and weighing the residue, its density may be determined and the percentage of fat dissolved therein obtained by calculation, or more conveniently from tables. The typical method of this kind is due to Soxhlet, and until the introduction of modern rapid volumetric processes, it was used perhaps more extensively than any other proceeding for the determination of fat in milk.[457] The reagents employed in the process are ether saturated with water and a potash lye containing 400 grams of potash in a liter. The principle of the process is based on the assumption that a milk made alkaline with potash will give up all its fat when shaken with ether and the quantity of fat in solution can be determined by ascertaining the specific gravity of the ethereal solution.

Fig. 108. Areometric Fat Apparatus.

The apparatus is arranged as shown in [Fig. 108], whereby it is easy to drive the ethereal fat solution into the measuring vessel by means of the bellows shown. In the bottle, seen at the right of the engraving are placed 200 cubic centimeters of milk, ten of the potash lye and sixty of the aqueous ether. The milk and potash are first added and well shaken, the ether then added, and the contents of the bottle are shaken until a homogeneous emulsion is formed. The bottle is then set aside for the separation of the ethereal solution, which is promoted by gently jarring it from time to time. When the chief part of the solution has separated, a sufficient quantity of it is driven over into the measuring apparatus, by means of the air bulbs, to float the hydrometer contained in the inner cylinder. After a few moments the scale of the oleometer is read and the percentage of fat calculated from the table. All the measurements are made at a temperature of 17°.5. The temperature is preserved constant by filling the outer cylinder of the apparatus with water. If the room be warmer than 17°.5, the water added should be at a temperature slightly below that and vice versa. The oleometer carries a thermometer which indicates the moment when the reading is to be made.

The scale of the oleometer is graduated arbitrarily from 43 to 66, corresponding to the specific gravities 0.743 and 0.766, respectively, or to corresponding fat contents of 2.07 and 5.12 per cent, in the milk, a range which covers most normal milks.

In the use of the table the per cents corresponding to parts of an oleometer division can be easily calculated.

Table for Calculating Per Cent of Fat in Milk
by Areometric Method of Soxhlet.

Reading of
oleometer.		 Per cent fat
in milk.		Reading of
oleometer.		 Per cent fat
in milk.
432.07553.49
442.18563.63
452.30573.75
462.40583.90
472.52594.03
482.64604.18
492.76614.32
502.88624.47
513.00634.63
523.12644.79
533.25654.95
543.37665.12

467. Application of the Areometric Method.—Soxhlet’s method, as outlined above, with many modifications, has been extensively used in Europe and to a limited degree in this country, and the results obtained are in general satisfactory, when the sample is a mixed one from a large number of cows and of average composition.

The author has shown that the process is not applicable to abnormal milk and often not to milk derived from one animal alone.[458]

The chief difficulty is found in securing a separation of the emulsion. This trouble can usually be readily overcome by whirling. Any centrifugal machine, which can receive the bottle in which the emulsion is made, may be employed for that purpose.

Since the introduction of more modern and convenient methods of fat determination, the areometric method has fallen into disuse and perhaps is no longer practiced in this country. It is valuable now chiefly from the fact that many of the recorded analyses of milk fat were made by it, and also for its typical character in representing all methods of analysis of fat in milk based on the density of ethereal solutions.

468. The Lactobutyrometer.—A typical instrument for measuring the volume of fat in a milk is known as Marchand’s lactobutyrometer. It is based on the observation that ether will dissolve the fat from milk when the casein is wholly or partly dissolved by an alkali, and further, that the fat in an impure form can be separated from its ethereal solution by the action of alcohol. Experience has shown that all the fat is not separated from the ethereal solution by this process, and also that the part separated is a saturated solution in ether. The method cannot be rigorously placed in the two classes given above, but being volumetric demands consideration here chiefly because of its historical interest.[459]

The instrument employed by Marchand is a tube about thirty centimeters long and twelve in diameter, closed at one end and marked in three portions of ten cubic centimeters each. The upper part is divided in tenths of a cubic centimeter. The superior divisions are subdivided so that the readings can be made to hundredths of a cubic centimeter.

The tube is filled with milk to the first mark and two or three drops of a twenty-five per cent solution of soda lye added thereto. Ether is poured in to the second mark, the tube closed and vigorously shaken. Alcohol of about ninety per cent strength is added to the upper mark, the tube closed, shaken and allowed to stand in a vertical position, with occasional jolting, until the separation of the liquids is complete. In order to promote the separation the tube is placed in a cylinder containing water at 40°.

When the separation is complete the milk serum is found at the bottom, the mixture of alcohol and ether in the middle and the fat at the top. The mixture of ether and alcohol contains 0.126 gram of fat, and each cubic centimeter of the separated ether fat 0.233 gram of fat. The total volume of the separated fat, multiplied by 0.233, and the product increased by 0.126, will give the weight of fat in the ten cubic centimeters of milk employed.

Example.—Milk used, ten cubic centimeters of 1.032 specific gravity = 10.32 grams. The observed volume of the saturated ether fat solution is two cubic centimeters. Then the weight of fat is 2 × 0.233 + 0.126 = 0.592 gram. The percentage of fat in the sample is 0.592 × 100 ÷ 10.32 = 5.74.

In the apparatus used in this laboratory the upper division of the graduation is marked 12.6, because this represents the quantity of fat which remains in the ether-alcohol mixture for one liter of milk. From this point the graduation is extended downward to ninety-five, which, for ten cubic centimeters of milk, represents 0.95 gram. After the fat has separated, enough ninety-five per cent alcohol is added to bring the upper surface exactly to the graduation 12.6. The number of grams per liter of milk is then read directly from the scale.

In respect of applicability, the observation made regarding Soxhlet’s areometric method may be repeated.

In practical work in this country the lactobutyrometer is no longer used, but many of the recorded determinations of fat in milk have been made by this method.

469. Volumetric Methods.—For practical purposes, the volumetric methods of estimating fat in milk have entirely superseded all the other processes. It has been found that the fat readily separates in a pure state from the other constituents of milk whenever the casein is rendered completely soluble; whereas no process has yet been devised whereby the fat can be easily separated in a pure state from milk which has not been treated with some reagent capable of effecting a solution of the casein. The volumetric methods may be divided into two classes; viz., (1) Those in which the fat is separated by the simple action of gravity, and (2) those in which the natural action of gravity is supplanted by centrifugal motion. Each of these classes embraces a large number of variations and some of the typical ones will be described in the following paragraphs. As solvents for the casein a large number of reagents has been used, including alkalies and single and mixed acids. In practice, preference is given to the least complex and most easily prepared solvents.

470. Method Of Patrick.—A typical illustration of the method of collecting the fat after solution of the casein, without the aid of whirling, is found in the process devised by Patrick.[460]

The solvent employed is a mixture of acetic, sulfuric and hydrochloric acids, saturated with sodium sulfate, in the respective volumetric proportions of nine, five and two. The separation is accomplished in a large test tube drawn out near the top into a constricted neck which is graduated to measure the volume of the separated fat or to give direct percentage results.

The tube should have a content of about twenty-five cubic centimeters below the upper mark on the neck. In use 10.4 cubic centimeters of milk and a sufficient quantity of the mixed acids to fill it nearly to the upper mark are placed in the tube, together with a piece of pumice stone, and the mixture boiled. On cooling below 100°, the fat will separate and the volume thereof may be measured in the constricted portion of the tube. The volume of the fat may be converted into weight on multiplying by 0.88 at 60°, or more conveniently the percentage of fat be taken from a table. In practice, the tube is filled with the milk and acid mixture nearly up to the neck, its contents well mixed and additional acid mixture added until the liquid is raised in the tube above the neck. After mixing a second time, the contents are boiled for five minutes and the fat allowed to collect in the expanded part of the tube above the neck. When the fat has collected, the mixture is boiled gently a second time for a few minutes. By this treatment the fat is mixed with the upper portions of the acid liquid and clarified. The clearing of the fat may be hastened by sprinkling over it a little effloresced sodium sulfate. The fat is brought into the graduated neck by opening a small orifice in the belly of the tube, which is closed by means of a rubber band. When the temperature has reached 60°, the space occupied by the fat is noted and the numbers obtained express the percentage of fat in the sample.

This process is illustrative of the principle of analysis, but is no longer used in analytical determinations.

471. The Lactocrite.—One of the earliest methods for fat estimation in milk, depending on the solution of the casein and the collection of the fat by means of whirling, is based on the use of a centrifugal machine known as the lactocrite. This apparatus is modeled very like the machine usually employed for creamery work,[461] and at one time was extensively used, but it has now given place to less troublesome and expensive machines. The acid mixture for freeing the fat of casein is composed of glacial acetic acid carrying five per cent of sulfuric. The samples of milk are heated with the acid mixture in test tubes provided with stoppers and short glass tubes to return the condensation products. The hot mixture is poured into a small metallic cylindrical cup holding about three cubic centimeters. This cup fits by means of an accurately ground shoulder on a metal casing, carrying inside a heavy glass graduated tube of small internal diameter. The excess of the milk mixture escapes through a small aperture in the metallic screw cap of the metal holder. The metal holder is cut away on both sides in order to expose the graduations on the glass tube. The glass tube is held water-tight by means of perforated elastic washers. Thus prepared the tubes are inserted in the radial holes of a revolving steel disk previously heated to a temperature of 60°. The whirling is accomplished in a few minutes by imparting to the steel disk a speed of about 6,000 revolutions per minute. At the end of this operation the fat is found in a clear column in the small glass tube and the number of the divisions it occupies in this tube is noted. Each division of the scale represents one-tenth per cent of fat.

This apparatus is capable of giving accurate results when all its parts are in good working order. In this laboratory the chief difficulty which its use has presented is in keeping the joints in the glass metal tube tight.

This description of the apparatus is given to secure an illustration of the principle involved, a principle which has been worked out in later times into some of the most rapid and practical processes of estimating fat in milk.

472. Modification of Lindström.—Many modifications have been proposed for conducting the determination of fat by means of the lactocrite, but they do not involve any new principle and are of doubtful merit. In the modification suggested by Lindström, which has attained quite an extended practical application, the solvent mixture is composed of lactic and sulfuric acids and the butyrometer tubes are so changed as to permit the collection of the fat in the graduated neck after whirling, by means of adding water. The apparatus is also adjusted to secure the congelation of the fat column before its volume is noted.[462] The analyst can read the fat volume at his leisure when it is in the solid state and is not confused by changes of volume during the observation. The best acid mixture has been found to be composed of 100 volumes of lactic, an equal amount of acetic and fifteen volumes of sulfuric acids.[463]

Fig. 109.
Babcock’s Butyrometer
and Acid Measure.

473. The Babcock Method.—Among the many quick volumetric methods which have been proposed for the determination of fat in milk, none has secured so wide an application as that suggested by Babcock.[464]

The chief point of advantage in the use of this method is found in effecting the solution of the casein by means of sulfuric acid of about 1.83 specific gravity. By this reagent the casein is dissolved in a few moments without the aid of any other heat than that generated by mixing the milk with the reagent. The bottle in which the separation is made is shown in [Fig. 109]. The graduations on the neck are based on the use of eighteen grams of milk. To avoid the trouble of weighing, the milk is measured from a pipette graduated to deliver eighteen grams of milk of the usual specific gravity. While it is true that normal milk may vary somewhat in its density, it has been found that a pipette marked at 17.6 cubic centimeters delivers a weight which can be safely assumed to vary but slightly from the one desired. The graduated bottle holds easily thirty-five cubic centimeters of liquid in its expanded portion and the volume of milk just noted is mixed with an equal volume of sulfuric acid, conveniently measured from the lip cylinder shown in the [figure]. The complete mixture of the milk and acid is effected by gently rotating the bottle until its contents are homogeneous. The final color of the mixture varies from dark brown to black.

While still hot, the bottles are placed in a centrifugal machine and whirled for at least five minutes. The most convenient machine, where it is available, is the one driven by a jet of steam. The revolutions of the centrifugal should be at least 700 per minute for a twenty inch and 1,200 for a twelve inch wheel. After five minutes the bottles are removed and filled to the upper mark or nearly so with hot water, replaced in the machine and whirled for at least one minute. The fat will then be found in a clearly defined column in the graduated neck of the bottle. In reading the scale, the extreme limits between the lowest point marked by the lower meniscus and the highest point marked by the edge of the upper meniscus are to be regarded as the termini of the fat column.

In testing cream by the babcock process, it may either be diluted until the column of fat secured is contained in the graduated part of the neck or specially graduated bottles may be used.

Condensed Milk: In applying the babcock test to condensed milk, it is necessary to weigh the sample and to use only about eight grams.[465] This quantity is placed in the bottle and dissolved in ten cubic centimeters of water and the analysis completed as above. The reading noted is multiplied by eighteen and divided by the weight of the sample taken.

Skim Milk: In determining the fat in skim milk and whey, it is desirable to use a bottle of double the usual capacity, but with the same graduation on the neck. The percentage of fat noted is divided by two.

Cheese: Five grams are a convenient quantity of cheese to employ. To this quantity in the bottle are added fifteen cubic centimeters of hot water and the flask gently shaken and warmed until the cheese is softened. The treatment with acid and whirling are the same as described above. The noted reading is multiplied by eighteen and divided by five.

474. Solution in Amyl Alcohol and Hydrochloric and Sulfuric Acids.—Leffmann and Beam have proposed to aid the solution of the casein in sulfuric acid by the previous addition to the milk of a mixture of equal volumes of amyl alcohol and hydrochloric acid.[466] In this process the same graduated flasks may be used as in the babcock process, or a special flask may be employed. In this case the graduation of the neck is for fifteen cubic centimeters of milk, and each one and a half cubic centimeters is divided into eighty-six parts. The quantity of milk noted is placed in the flask, together with three cubic centimeters of the mixture of amyl alcohol and hydrochloric acid, and well shaken. To the mixture, sulfuric acid of 1.83 specific gravity is added until the belly of the flask is nearly full and the contents well mixed by shaking. When the casein is dissolved, the addition of the sulfuric acid is continued until the flask is filled to the upper mark and again the contents mixed. It is well to close the mouth of the flask with a stopper while shaking. The bottle is placed in a centrifugal and whirled for a few moments, when the fat is collected in the graduated neck and its volume noted.

The process is also known in this country as the beimling method.[467] The fat separated in the above process is probably mixed with a little fusel oil, and therefore it is advisable to use the specially graduated bottle instead of one marked in absolute volumes.[468]

The method, when conducted according to the details found in the papers cited, gives accurate results, but is somewhat more complicated than the babcock process and is not now used to any great extent in analytical work.

475. Method of Gerber.—The method proposed by Gerber for estimating fat in milk is based on the processes of Babcock, Beimling and Beam already described. The tubes in which the decomposition of the milk and the measurement of the fat are accomplished are of two kinds, one open at only one end for milk and the other open at both ends for cheese. They are closed during the separation by rubber stoppers.[469]

Fig. 110. Gerber’s Butyrometers.

The apparatus have been greatly improved and simplified since the first description of them was published and have come into extensive use in Europe and to a limited extent in this country.[470]

The butyrometer tubes are made of various sizes and shapes, but the most convenient are those noted above as shown in [Fig. 110].

Before adding the strong sulfuric acid, one cubic centimeter of amyl alcohol is mixed with the milk in the butyrometer. This admixture serves to clarify the fat and render the reading more easy.

The centrifugal is run by hand, and the required speed of rotation is given it by means of a cord wrapped spirally about its axis, as shown in [Fig. 111]. The cord in the new machines is replaced by a leather strap working on a ratchet.

Fig. 111. Gerber’s Centrifugal.

The process is more speedy than that of Babcock, and the results have been shown by a large experience to be reliable and accurate.

The sulfuric acid employed is of 1.825 to 1.830 specific gravity. There is no danger of loss by the formation of volatile ethers where the quantity of amyl alcohol used does not exceed one cubic centimeter. In a comparison of the respective merits of the methods of Babcock, Thörner and Gerber, made by Hausamann, the first place is awarded to the Gerber process.[471] In the [figure 110], the butyrometers marked 2, 5 and 8 are for milk, and those numbered 1, 3 and 7 are for cream and cheese. In conducting the analysis, ten cubic centimeters of the sulfuric acid are placed in the butyrometer with one cubic centimeter of the amyl alcohol. When mixed, eleven cubic centimeters of the milk are added and the contents of the tube well mixed, the tube stoppered and placed in the centrifugal. The larger tubes, open at both ends, require double the quantities of the reagents mentioned. The measurements are made at about 15°.

Minute directions for conducting the analyses with milk, skim milk, buttermilk, cream, condensed milk, cheese and butter accompany each apparatus.