Quantities used for Analysis.—In conducting the separations above noted, it will be found convenient to use in each case about five grams of the solid or twenty of the liquid extract. In the nitrogen determinations, the weight of the sample should be inversely proportional to its content of nitrogen.
417. Preparation of the Phosphotungstic Reagent.—The phosphotungstic reagent is conveniently prepared as follows:
Dissolve 120 grams of sodium phosphate and 200 of sodium tungstate in one liter of water and add to the solution 100 cubic centimeters of strong sulfuric acid. When the reagent is prepared for general purposes it is customary to acidify with nitric, but in the present instance, inasmuch as the precipitate is used for the determination of nitrogen, it is evident that sulfuric should be substituted for nitric acid. In all cases the analyst must be assured of the strong acidity of the reagent, and in addition to this the solutions of proteid matter to which the reagent is added must first be made strongly acid with sulfuric.
418. Zinc Sulfate as Reagent for Separating Albumoses from Peptones.—When the albumoses are separated from the peptones, by precipitation with ammonium sulfate, there may be danger of some of this reagent adhering to the albumose, and in this way the quantity of nitrogen obtained on analysis may be increased. To avoid an accident of this kind Bömer replaces the ammonium by zinc sulfate.[395]
Since the precipitation of the albumoses by saturated saline solutions depends on their hydrolytic power, the substitution of another salt for ammonium sulfate capable of strongly attracting water, may be made if that salt does not possess any objectionable property. Crystallized zinc sulfate will dissolve in less than its own weight of cold water and is therefore well suited for the purpose in view.
In the case of a meat extract, the precipitation is accomplished as follows: Fifty cubic centimeters of the extract, freed from all solid matter by filtration and containing about two grams of the soluble proteids, are saturated in the cold with finely powdered zinc sulfate. The separated albumoses collect on the surface and are skimmed off, poured on a filter and washed with cold saturated zinc sulfate solution. The filter and its contents are used for the determination of nitrogen by moist combustion.[396]
The filtrate from the precipitated albumoses gives no biuret reaction, and, therefore, as in the use of ammonium sulfate, is free of albumin.
The biuret reaction is applied to the zinc sulfate filtrate as follows: The filtrate is greatly diluted with water and freed of zinc by means of a saturated solution of sodium carbonate. The filtrate free of zinc is evaporated on the steam-bath, made strongly alkaline with sodium hydroxid and treated with a few drops of a two per cent copper sulfate solution, added successively.
Another advantage possessed by the zinc sulfate is found in the fact that in the filtrate from the separated albumoses the peptones and other flesh bases can be thrown out by phosphotungstic acid. Before the application of the reagent, the filtrate should be made strongly acid by adding about an equal volume of dilute sulfuric acid (one part of acid to four of water.)
The nitrogen in the precipitate thus obtained is determined by moist combustion in the manner already suggested.