Part 93

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. 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. 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. 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.

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.

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.

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.

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. 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.

PROTEID BODIES IN MILK.

=476. Kinds of Proteid Bodies in Milk.=—The proteid bodies in milk are all found in at least partial solution. Some authorities state that a portion of the casein is present in the form of fine particles suspended after the manner of the fat globules. The number and kind of proteid bodies are not known with definiteness. Among those which are known with certainty are casein, albumin, peptone and fibrin. The latter body was discovered in milk by Babcock. Lactoglobulin and lactoprotein are also names given to imperfectly known proteid bodies in milk. Lactoprotein is not precipitated either by acids or by heat and is therefore probably a peptone. By far the greater part of the proteid matters in milk is casein. Casein has been called caseinogen by Halliburton, and paracasein by Schulze and Röse. Casein has intimate relations to the mineral matters in milk, and is probably itself made up of several proteid bodies of slightly differing properties. In general all that class of proteid matter contained in milk which is precipitated by rennet or a weak acid, or spontaneously on the development of lactic acid, is designated by the term casein, while the albumins and peptones in similar conditions remain in solution. Casein contains phosphorus, presumably as nuclein. Fibrin is recognized in milk by the reactions it gives with hydrogen peroxid or gum guiacum. The decomposition of hydrogen peroxid is not a certain test for fibrin, inasmuch as pus and many other bodies will produce the same effect. If the milk decompose hydrogen peroxid, however, before and not after boiling, an additional proof of the presence of fibrin is obtained, since boiled fibrin does not act on the reagent. The gum guiacum test is applied by dipping a strip of filter paper into the milk and drying. The solution of gum guiacum is applied to the dried paper and the presence of fibrin is recognized by the blue color which is produced. The fibrin is probably changed into some other proteid during the ripening of cream in which the fibrin is chiefly found. The albumin in milk is coagulated by boiling, while the casein remains practically unaffected when subjected to that temperature.

=477. Estimation of Total Proteid Matter.=—The total proteid matter in milk is determined by any of the general methods applicable to the estimation of total nitrogen, but the moist combustion method is by far the most convenient. From the total nitrogen, that which represents ammonia or other nonproteid nitrogenous bodies, is to be deducted and the remainder multiplied by an appropriate factor. Practically all the nitrogen obtained is derived from the proteid matters and, as a rule, no correction is necessary. The factors employed for calculating the weight of proteid matter from the nitrogen obtained vary from 6.25 to 7.04. It is desirable that additional investigations be made to determine the magnitude of this factor. It is suggested that provisionally the factor 6.40 be used. In the method adopted by the Association of Official Agricultural Chemists it is directed that about five grams of milk be placed in the oxidizing flask and treated without previous evaporation exactly as described for the estimation of total nitrogen in the absence of nitrates. The nitrogen obtained is multiplied by 6.25 to get the total proteid matter. In order to prevent the too great dilution of the sulfuric acid, the milk may be evaporated to dryness or nearly so before oxidation. In this laboratory it is conveniently done by placing the milk first in the oxidizing flask, connecting this with the vacuum service and placing the flask in hot water. The aqueous contents of the milk are quickly given off at a temperature not exceeding 85°, and the time required is only a few minutes.

The milk may also be dried in dishes made of thin glass or tin foil and, after desiccation, introduced with the fragments of the dishes into the oxidizing flask.

The preliminary drying in the oxidizing flask is recommended as the best.

Söldner oxidizes the nitrogen in human milk by boiling ten cubic centimeters thereof for three hours with twenty-five of sulfuric acid, fifty milligrams of copper oxid and three drops of a four per cent platinic chlorid solution, and, after distilling the ammonia, uses the factor 6.39 for calculating the proteid matter. According to this author human milk is much less rich in nitrogenous constituents than is generally supposed, containing not more than one and a half per cent thereof in average samples collected at least a month after parturition.

=478. Precipitation of Total Proteids with Copper Sulfate.=—This method of throwing out the total proteids of milk is due to Ritthausen. The proteids and fat are precipitated together by the addition of a measured volume of copper sulfate solution, containing 63.92 grams of the crystallized salt in one liter. The process, as modified by Pfeiffer, is conducted as follows:

Ten grams of milk are diluted with ten times that much water, five cubic centimeters of the copper sulfate solution added and then soda lye solution drop by drop until the copper is just precipitated. This is determined by testing a few drops of the filtrate with soda lye, which, when the copper is precipitated, will give neither a turbidity nor a blue color.

The mixture is poured into a dry tared filter, the precipitate washed with hot water, dried to constant weight and weighed. The fat is removed from the dry pulverized mass by extraction with ether and the residue dried and weighed.

The quantity of copper oxyhydrate contained in the precipitate is calculated from the quantity of the copper solution used and amounts to 0.2026 gram. The casein thus prepared contains not only the copper compound named, but also some of the sodium sulfate formed on the addition of the soda lye and other mineral salts present in the milk and from which it is quite impossible to completely free it. There are also many other objections to the process, and the product is of such a nature as to render the data obtained by the method very doubtful.

This method is chiefly valuable on account of its historical interest. Not only are the drying and weighing of the precipitate rendered unnecessary by the modern methods of determining nitrogen, but there are numerous sources of error which seem to throw doubt on the accuracy of the results. The copper hydroxid does not lose all its water even on drying at 125°. The method therefore can only be recommended for practical purposes when all the tedious processes of drying, extracting and calculating the quantity of copper oxid are abandoned and the moist washed precipitate used directly for the determination of nitrogen.