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°.[481] 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.
479. Proteid Bodies by Ammonium Sulfate.—All the proteid bodies except peptones are precipitated from milk on saturation with ammonium sulfate. This method has little analytical value because of the presence of nitrogenous salt in the precipitate. Zinc sulfate may be substituted for the ammonium salt and thus a determination of proteid matter other than peptone be obtained. This result subtracted from the total proteid nitrogen gives that due to peptone.
480. Total Proteid Matter by Tannic Acid.—For the determination of the total proteid matter in milk Sebelien uses the following process.[482] From three to five grams are diluted with three or four volumes of water, a few drops of a saline solution added (sodium phosphate, sodium chlorid, magnesium sulfate, et similia), and the proteid bodies thrown out with an excess of tannic acid solution. The precipitate is washed with an excess of the precipitant and the nitrogen therein determined and multiplied by 6.37.
481. Separation of Casein from Albumin.—Sebelien prefers magnesium sulfate or sodium chlorid to acetic acid as the best reagent for separating casein from lactalbumin. Of the two saline reagents mentioned, the former is the better. The milk is first diluted with a double volume of the saturated saline solution and then the fine powdered salt added until saturation is secured. The casein is completely thrown out by this treatment, collected on a filter, washed with the saturated saline solution, and the nitrogen therein determined. The difference between the total and casein nitrogen gives the quantity due to the albumin plus the almost negligible quantity due to globulin.[483]
482. Van Slyke’s Method of Estimating Casein.—The casein may be separated from the other albuminoids in milk by the procedure proposed by Van Slyke.[484] Ten grams of the fresh milk are diluted with ninety cubic centimeters of water and the temperature raised to 40°. The casein is thrown down with a ten per cent solution of acetic acid, of which about one and a half cubic centimeters are required. The mixture is well stirred and the precipitate allowed to subside. The whey is decanted onto a filter, and the precipitate washed two or three times with cold water, brought finally onto the filter and washed once or twice with cold water. The filter paper and its contents are used for the determination of nitrogen in the usual way. The casein is calculated from the nitrogen found by multiplication by the factor 6.25. Milk may be preserved for this method of determination by adding to it one part of finely powdered mercuric chlorid for each two thousand parts of the sample. The method is not applicable to curdled milk.
483. Theory of Precipitation.—Most authorities now agree in supposing that the state of semisolution in which the casein is held in milk is secured by the presence of mineral matters in the milk, in some intimate combination with the casein. Among these bodies lime is of the most importance. The action of the dilute acid is chiefly on these mineral bodies, releasing them from combination with the casein, which, being insoluble in the milk serum, is precipitated.
484. Factors for Calculation.—Most analysts still use the common proteid factor, 6.25, in calculating the quantity of proteids from the nitrogen determined by analysis. For casein many different factors have been proposed. According to Makeris the factor varies from 6.83 to 7.04.[485] Munk gives 6.34 for human and 6.37 for cow milk.[486] Sebelien adopts the latter factor, and Hammersten nearly the same; viz., 6.39. The weight of authority, at the present time, favors a factor considerably above 6.25 for calculating the casein and, in fact, the total proteids of milk from the weight of nitrogen obtained.
485. Béchamp’s Method of Preparing Pure Casein.—The casein in about one liter of milk is precipitated by adding gradually about three grams of glacial acetic acid diluted with water. The addition of the acid is arrested at the moment when litmus paper shows a slightly acid reaction. The precipitate thus produced, containing all the casein, the milk globules and the microzymes, is separated by filtration, being washed by decantation before collecting it on the filter. On the filter it is washed with distilled water and the fat removed by shaking with ether. The residue is suspended in water, dissolved in the least possible quantity of ammonium carbonate, any insoluble residue (microzymes, globules) separated by filtration and the pure casein thrown out of the filtrate by the addition of acetic acid. The washing with distilled water, solution in ammonium carbonate, filtration and reprecipitation are repeated three or four times in order to obtain the casein entirely free of other substances. Casein thus prepared is burned to a carbon free ash with difficulty and contains but little over one-tenth per cent of mineral matter.[487]
486. Separation of Casein with Carbon Dioxid.—The supersaturation of the lime compounds of casein with carbon dioxid diminishes the solvent action of the lime and thus helps to throw out the proteid matter. For this reason Hoppe-Seyler recommends the use of carbon dioxid to promote the precipitation of the casein.[488] The milk is diluted with about twenty volumes of water and treated, drop by drop, with very dilute acetic acid as long as a precipitate is formed. A stream of pure carbon dioxid is conducted through the mixture for half an hour, and it is allowed to remain at rest for twelve hours, when the casein will have all gone down and the supernatant liquid will be clear. Albumins and peptones are not thrown out by this treatment.
The method of precipitation is advantageously modified by saturating the diluted milk with carbon dioxid before adding the acetic acid, less of the latter being required when used in the order just noted.[489]