487. Separation of Albumin.—In the filtrate from the casein precipitate the albumin may be separated by heating to 80°. It may also be precipitated by tannic acid, in which case it may contain a little globulin. It may also be thrown out by saturation with ammonium or zinc sulfates. The latter reagent is to be preferred when the nitrogen is to be determined in the precipitate. The quantities of albumin and globulin, especially the latter, present in milk are small compared with its content of casein.
488. Separation of Globulin.—The presence of globulin in milk is demonstrated by Sebelien in the following manner:[490] The milk is saturated with finely powdered common salt and the precipitate produced is separated by filtration. This filtrate in turn is saturated with magnesium sulfate. The precipitate produced by this reagent is collected on a filter, dissolved in water and precipitated by saturation with sodium chlorid. This process is repeated several times. The final precipitate is proved to be globulin by the following reactions: When a solution of it is dialyzed the proteid body separates as a flocculent precipitate, which is easily dissolved in a weak solution of common salt. The clear solution thus obtained becomes turbid on adding water, and more so after the addition of a little acetic acid. A neutral solution of the body is also completely precipitated by saturation with sodium chlorid. These reactions serve to identify the body as a globulin and not an albumin. All the globulin in milk is not obtained by the process, since a part of it is separated with the casein in the first precipitation.
489. Other Precipitants of Milk Proteids.—Many other reagents besides those mentioned have been used for precipitating milk proteids, wholly or in part. Among these may be mentioned the dilute mineral acids, lactic acid, rennet, mercuric iodid in acetic acid, phosphotungstic acid, acid mercuric nitrate, lead acetate and many others.
It has been shown by the author that many of these precipitants do not remove all the nitrogen but that among others the mercury salts are effective.[491] When nitrogen is to be subsequently determined the acid mercuric nitrate cannot be employed.
490. Precipitation by Dialysis.—Since the casein is supposed to be held in solution by the action of salts it is probable that it may be precipitated by removing these salts by dialysis.
491. Carbohydrates in Milk.—The methods of determining lactose in milk, both by the copper reduction and optical processes, have been fully set forth in foregoing paragraphs ([243], [244], [259], [262]). In general, the optical method by double dilution is to be preferred as practically exact and capable of application with the minimum consumption of time.[492] For normal milks a single polarization is entirely sufficient, making an arbitrary correction for the volume occupied by the precipitated proteids and fat. This correction is conveniently placed at six and a half per cent of the volume of milk employed.
The polarimetric estimation of lactose in human milk is likely to give erroneous results by reason of the existence in the serum of polarizing bodies not precipitable by the reagents commonly employed for the removal of proteids.[493] The same statement may be made in respect of ass and mare milk. The use of acetopicric acid for removing disturbing bodies, as proposed by Thibonet[494] does not insure results free from error. With the milks above mentioned, it is safer to rely on the data obtained by the alkaline copper reagents.
492. Dextrinoid Body in Milk.—In treating the precipitate, produced in milk by copper sulfate, with alcohol and ether for the purpose of removing the fat, Ritthausen isolated a dextrin like body quite different from lactose in its properties.[495] The alcohol ether extract evaporated to dryness leaves a mass not wholly soluble in ether, and therefore not composed of fat. This residue extracted with ether, presents flocky particles, soluble in water and mostly precipitated therefrom by alcohol. This body has a slight reducing effect on alkaline copper salts and produces a gray color with bismuth nitrate. The quantity of this material is so minute as to lead Ritthausen to observe that it does not sensibly affect the fat determinations when not separated. It is not clearly demonstrated that it is a dextrinoid body and the analyst need not fear that the optical determination of milk sugar will be sensibly affected thereby.
Raumer and Späth assume that certain discrepancies, observed by them in the data obtained for lactose by the copper and optical methods, are due to the presence of this dextrinoid body, but no positive proof thereof is adduced.[496]
493. Amyloid Bodies in Milk.—Herz has observed in milk a body having some of the characteristics of starch.[497] Observed by the microscope, these particles have some of the characteristics of the starch grains of vegetables, with a diameter of from ten to thirty-five micromillimeters. They are colored blue by iodin. When boiled with water, however, these particles differ from starch in not forming a paste. The particles are most abundant in the turbid layer found immediately beneath the ether fat solution in the areometric process of Soxhlet.