If the proteid matters contain salts of ammonium it is probable that a difficultly soluble double sulfate of zinc and ammonium, (NH₄)₂SO₄.ZnSO₄.6H₂O, will be found in the precipitate. Ammonium salts, if present, should therefore be removed by distillation with magnesia. It is better, however, to throw down the ammonia with the first zinc precipitate, distil this with magnesia and determine the amount of nitrogen derived from the ammonia compounds. In a second sample, the total nitrogen is determined by moist combustion and the difference between the two results gives that due to albumoses.
419. Examination for Muscular Tissue.—Some samples of meat extracts contain small quantities of finely ground muscular tissue. For detecting this the extract is treated with cold water and the insoluble residue examined with a microscope. If muscular tissue be found, about eight grams of the extract or twenty-five of the fluid preparation, are treated with cold water, the insoluble matter collected upon a filter, washed with cold water, and the nitrogen determined in the residue. The percentage of nitrogen multiplied by 6.25 gives the quantity of muscle fiber proteids present. The filtrate from the above determination is acidified with acetic, boiled, any precipitate which is formed collected and the nitrogen therein determined. The nitrogen obtained multiplied by 6.25 gives the quantity of coagulable albumin present. An aliquot portion of the filtrate is used for the determination of nitrogen and the percentage therein found, deducted from the total nitrogen of the sample, gives a remainder which may be used as a representative of the whole of the nitrogen present in the form of albumin and muscular tissue.
420. Estimation of Gelatin.—The tin foil dish and its contents used for the determination of water, as above described, are cut into small pieces, placed in a beaker and extracted four times with absolute alcohol. After the removal of the alcohol, the residue is extracted with ice water containing ten per cent of alcohol, in which a small piece of ice is kept to avoid a rise of temperature. The beaker should be shaken during the extraction, which should last for about two minutes. Where large numbers of samples are treated at once, any convenient form of shaking machine may be employed. At least two extractions with ice water must be made. The residue is then collected upon a filter and washed with ice water until the washings are completely colorless. The residue on the filter is replaced in the beaker, boiled with water, well washed on the filter with boiling water, the filtrate and washings concentrated and the nitrogen therein determined.
The principle of this determination is based on the fact that gelatin is almost completely insoluble in ice water while serum peptones and albumin peptones are almost completely soluble in that reagent. On the other hand, the flesh bases and the proteids present are almost completely removed by the preliminary treatment with alcohol and ice water or are left undissolved by the hot water. The solution in boiling water, therefore, contains practically nothing but gelatin.[397]
In a later article, Stutzer modifies the method given above as follows:[398]
Of dry and moist extracts from five to seven grams and of liquid extracts from twenty to twenty-five grams are used for the determination and placed in tin foil dishes, as described above. In case of solid extracts, a sufficient quantity of warm water is added to completely dissolve them, the solution being facilitated by stirring. In case the solution is too thin it should be concentrated before going further. It is treated with a sufficient amount of dust-free ignited sand to completely absorb it, and the dish and its contents are then dried to a constant weight. The dried contents of the dish are rubbed up in a mortar, the dish cut into fine bits, and all placed in a beaker. The solid syrphete[399] is extracted four times with 100 cubic centimeters of absolute alcohol, the alcohol in each case being poured through an asbestos filter for the purpose of collecting any matters suspended therein. In a large flask are placed 100 grams of alcohol, 300 grams of ice and 600 grams of cold water, and the flask is placed in a large vessel and packed with finely divided ice. Four beakers marked b, c, d, e are also placed in ice and the beaker containing the syrphete, left after extraction with absolute alcohol as above mentioned, is marked a and also placed in pounded ice. The extraction with cold alcoholic water proceeds as follows:
In beaker a are poured 100 cubic centimeters of the mixture in the large flask, its contents are stirred for two minutes and then the liquid portion poured off into beaker b to which, at the same time, a piece of ice is added. In beaker a are poured again 100 cubic centimeters from the large flask, treated as above described, and the liquid extract poured into beaker c. In like manner the extraction in beaker a is continued until each of the beakers has received its portion of the extract. By this time the liquid over the sand in beaker a should be completely colorless. The filtration of the liquid extract is accomplished as follows:
In a funnel of about seven centimeters diameter is placed a perforated porcelain plate about four centimeters in diameter which is covered with asbestos felt with long fiber. Three filters are prepared in this way. On the first filter are poured the contents of beaker b. After the liquid has passed through, the sand and other residue in beaker a are transferred to the filter and the beaker and residue washed with the alcoholic ice water from the large flask. The filtration should be accomplished under pressure. On the second filter are poured the contents of beaker c. On the third filter the contents of beakers d and e. The washing with alcoholic ice water from the large flask is continued in each instance until the filtrate is colorless. At the same time the asbestos filter, which was used in the first instance for filtering the absolute alcohol extract, is washed with the alcoholic ice water mixture from the large flask. At the end the sand remaining in beaker a together with all the asbestos filters are brought together into a porcelain dish, boiled two or three times with water, the aqueous solution filtered and the filtrate concentrated and used for the estimation of the nitrogen. The quantity of nitrogen found multiplied by 6.25 represents the proteid matter in the gelatin of the sample.
The object of the multiple filters, described above, is to accelerate the process, and they are required because the gelatin quickly occludes the filter pores. For this reason the asbestos filters are found to operate better than those made of paper. It should be mentioned that the residue of the peptones insoluble in alcohol may contain, in addition to gelatin, also small quantities of albumoses. From the quantity of albumose nitrogen found, it is understood that the nitrogen in the form of coagulable albumin, determined as described in the first process mentioned above, is to be deducted, since these coagulable albumins are insoluble in alcohol.
421. Estimation of Nitrogen in the Flesh Bases Soluble in Alcohol.—About five grams of the dry extract, ten grams of the extract containing water or twenty-five grams of the liquid extract are placed in a beaker and enough water added in each case to make about twenty-five cubic centimeters in all. Usually no water need be added to the liquid extracts. Very thin peptone solutions should be evaporated until the content of water is reduced to seventy-five per cent. The solution, prepared as above indicated, is treated slowly with constant stirring with 250 cubic centimeters of absolute alcohol, the stirring continued for some minutes and the vessel set aside for twelve hours, at the end of which time the precipitate is separated by filtration and washed repeatedly with strong alcohol. Leucin, tyrosin and a part of the flesh bases are dissolved by alcohol. The alcohol is removed by distillation and the residue dissolved in water. Any flocky residue which remains on solution with water is removed by filtration, the nitrogen determined therein and the quantity thereof added to the albumose nitrogen found, as hereafter described.