AUTHORITIES CITED IN PART FOURTH.
[229] Benedikt and Lewkowitsch; Oils, Fats, Waxes, p. 1.
[230] Op. cit. supra, p. 46.
[231] Archiv für Physiologie, 1895, Band 61, S. 341: Chemiker-Zeitung Repertorium, Band 16, S. 338.
[232] Vid. op. cit. 1, p. 63.
[233] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 25.
[234] Journal of the Society of Chemical Industry, 1886, p. 508.
[235] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, p. 423.
[236] Vid. op. cit. supra, p. 435.
[237] Vid. op. cit. supra, p. 437.
[238] Vid. op. et loc. cit. supra.
[239] Benedikt and Lewkowitsch; Oils, Fats, and Waxes, pp. 96 et seq.: Zune; Analyse des Beurres, pp. 26 et seq.
[240] Journal of the Society of Chemical Industry, 1885, p. 535.
[241] Vid. op. cit. 1, p. 97.
[242] Vid. op. cit. 7, p. 443.
[243] Vid. op. cit. 1, pp. 97 and 98.
[244] Butter, its Analysis and Adulterations, p. 24.
[245] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 34.
[246] Vid. op. cit. 7, p. 447.
[247] Analyse des Beurres, pp. 33 et 63: Zeitschrift für Instrumentenkunde, 1887, Ss. 16, 55, 392, 444: Zeitschrift für physikalische Chemie, Band 18, S. 294. (Ou. pp. 328-9 and 334 read Amagat for Armagat.)
[248] Zeitschrift für physikalische Chemie, Band 18, S. 294.
[249] American Chemical Journal, Vol. 10, p. 392.
[250] Vid. op. cit. 7, pp. 473 et seq.
[251] Jean; Chimie Analytique des Matiéres Grasses, p. 26.
[252] Vid. op. cit. supra, p. 31.
[253] The Analyst, Vol. 20, p. 135.
[254] Schlussbericht über die Butteruntersuchungsfrage, Milchwirthschaftlicher Verein, Korrespondenzblatt, No. 39, 1891, S. 15.
[255] Vid. op. cit. 7, p. 75.
[256] Journal of the American Chemical Society, Vol. 15, p. 173.
[257] Communicated by Krug to author.
[258] Vid. op. cit. 7, pp. 449 et seq.
[259] Vid. op. cit. 28, Vol. 18, p. 189.
[260] Vid. op. cit. 7, Plates 32 and 35.
[261] Vid. op. cit. supra, p. 452.
[262] Vid. op. cit. supra., p. 93.
[263] Vogel; Practische Spectralanalyse, S. 279: Zune; Analyse des Beurres, Tome 2, p. 48: Benedikt and Lewkowitsch; Oils, Fats, Waxes, p. 83.
[264] Bulletin de l’Association Belge des Chimistes, Tome 9, p. 145.
[265] Journal of the Chemical Society, Abstracts, Vol. 46, p. 1078: Dingler’s Polytechnisches Journal, Band 252, S. 296.
[266] The Analyst, July 1894, p. 152.
[267] Rapport sur les Procédé pour reconnâitre les Falsifications des Huiles d’Olive, p. 37.
[268] Vid. op. cit. 7, p. 251.
[269] Taylor; Annual Report U. S. Department of Agriculture, 1877, p. 622: Milliau; Journal of the American Chemical Society, Vol. 15, p. 153.
[270] Gantter; Zeitschrift für analytische Chemie, 1893, Band 32, S. 303.
[271] Welmans; Journal of the Society of Chemical Industry, 1892, p. 548.
[272] Vid. op. cit. 1, p. 254.
[273] Pharmaceutische Zeitung, 1891, p. 798: The Analyst, Vol. 17, p. 59.
[274] Comptes rendus, Tome 112, p. 105.
[275] Pearmain and Moor; The Analyst, Vol. 20, p. 174.
[276] Pharmaceutische Zeitschrift für Russland, 1888, S. 721: American Journal of Pharmacy, 1889, p. 23.
[277] Vid. op. cit. 7, p. 502.
[278] Muir; Elements of Thermal Chemistry, p. 25 et seq.
[279] Comptes rendus, Tome 35 (1852), p. 572.
[280] Allen; Commercial Organic Analysis, Vol. 2, p. 56.
[281] Vid. op. cit. 1, p. 235; et op. cit. 23, p. 217.
[282] Vid. op. cit. 7, p. 44.
[283] Vid. op. cit. supra, p. 445: Proceedings American Public Health Association, Vol. 10.
[284] Vid. op. cit. 23, p. 61.
[285] Journal of the Society of Chemical Industry, 1891, p. 234.
[286] Vid. op. cit. 1, p. 240.
[287] The Analyst, Vol. 22, p. 58.
[288] Vid. op. cit. supra, Vol. 20, p. 146.
[289] Journal of the American Chemical Society, Vol. 17, p. 378.
[290] Dingler’s Polytechnisches Journal, 1884, Ss. 253-281: Journal of the Society of Chemical Industry, 1884, p. 641.
[291] Bulletin No. 46, Division of Chemistry, U. S. Department of Agriculture, p. 32.
[292] Liebermann; Berichte der deutschen chemischen Gessellschaft, Band 24, S. 4117.
[293] Vid. op. cit. 1, p. 136.
[294] Vid. op. cit. 57, 1895, pp. 130 and 1030.
[295] Zeitschrift für analytische Chemie, Band 32, Ss. 181 et seq.
[296] Vid. op. cit. 61, Vol. 16, p. 372.
[297] Zeitschrift für angewandte Chemie, 1895, S. 254.
[298] Chemiker-Zeitung, Band 19, Ss. 1786 and 1831.
[299] Vid. op. cit. 61, Vol. 16, p. 277.
[300] Pharmaceutical Journal, Sept. 25, 1880.
[301] Vid. op. cit. 59, Vol. 20, p. 50.
[302] Williams; vid. op. cit. supra, Vol. 20, p. 277.
[303] Vid. op. cit. 59, 1889, p. 61.
[304] Vid. op. cit. 61, Vol. 15, p. 110.
[305] Zeitschrift für physiologische Chemie, Band 14, S. 599; Band 12, S. 321; Band 16, S. 152.
[306] Vid. op. cit. 1, p. 60.
[307] Vid. op. cit. supra, p. 557.
[308] Vid. op. cit. 7, p. 459; vid. op. cit. 63, p. 27.
[309] Vid. op. cit. 69, 1895, S. 721.
[310] Vid. op. cit. 67, Band 18, S. 199: vid. op. cit. 7, pp. 58-461: vid. op. cit. 63, p. 30.
[311] Vid. op. cit. 52, p. 40.
[312] Vid. op. cit. 1, p. 119.
[313] Vid. op. cit. supra, p. 127.
[314] Monatshefte für Chemie und verwandte Theile anderer Wissenschaften, Band 8, S. 40.
[315] Vid. op. cit. 57, 1890, p. 846.
[316] Reichert; vid. op. cit. 67, Band 18, S. 68.
[317] Meissl; vid. op. cit. 62, Band 233, S. 229.
[318] Vid. op. cit. 1, p. 121.
[319] Vid. op. cit. 63, p. 28.
[320] Vid. op. cit. 67, Band 16, S. 145; Band 18, S. 68: vid. op. cit. 7, p. 53: vid. op. cit. 59, 1877, p. 147.
[321] Vid. op. cit. 57, 1888, pp. 526 and 697: American Chemical Journal, Vol. 10, p. 326: vid. op. cit. 1, pp. 123-127.
[322] Vid. op. cit. 1, p. 143.
[323] Vid. op. ch. 7, p. 143.
[324] Vid. op. cit. 67, 1890, S. 4.
[325] Allen; Commercial Organic Analysis, Vol. 2, pp. 224-236.
[326] Analyse Chimique des Matiéres Grasses, p. 13.
[327] Chemiker-Zeitung, Band 19, S. 451.
[328] Annali del Laboratorio Chimico, 1891-92, p. 197: Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, p. 465: Journal of Analytical and Applied Chemistry, Vol. 1, p. 449; Vol. 2, pp. 119 and 275; vid. op. cit. 311.
[329] Rapport presenté a l’Academie Sciences le 20 fevrier, 1883: Analyse des Matiéres Grasses, p. 17: Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, p. 446.
[330] Analyse des Matiéres Grasses, p. 15.
[331] Chemical News, 1888, p. 113: Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, p. 468.
[332] Vid. op. cit. 69, 1895, S. 535.
[333] Justus Liebig’s Annalen der Chemie, Band 192, S. 178: vid. op. cit. 67, Band 26, S. 575: vid. op. cit. 7, p. 514.
[334] Journal de Pharmacie et de Chimie, 1889, p. 447.
[335] Moniteur Scientifique, Tome 13, p. 263: vid. op. cit. 69, 1884, S. 262.
[336] Vid. op. cit. 7, p. 515.
PART FIFTH.
SEPARATION AND ESTIMATION OF
BODIES CONTAINING NITROGEN.
370. Nature of Nitrogenous Bodies.—The nitrogenous bodies, valuable as foods, belong to the general class of proteids and albuminoids. They are composed chiefly of carbon, hydrogen, oxygen, sulfur and nitrogen. Some of them, as lecithin and nuclein, contain phosphorus instead of sulfur, but these resemble the fats rather than the proteids.
Nitrogenous organic bodies of the class mentioned above are designated by the general name proteids. The term albumin is restricted in a physiological sense to a certain class of proteids. The term albuminoid is often used synonymously, as above, for proteids, but, more strictly speaking, it should be reserved for that class of bodies such as gelatin, mucin, keratin and the like, not really proteids, but, nevertheless, closely resembling them.[337] In chemical composition the proteids are characterized by the relative constancy of their nitrogen content, the mean percentage of this element being about sixteen, but varying in some instances more than two units from that number.
371. Classification of Proteids.—Many classifications of the proteids have been given based on physical, chemical and physiological characteristics. In respect of origin, they are divided into two great classes, viz., vegetable and animal. In respect of their physical and chemical properties the following classification of the proteids may be made.[338]
Albumins.—These are proteids soluble in water and not precipitated from their aqueous solutions by sodium chlorid or magnesium sulfate. They are easily coagulated by heat and are represented by three great classes, viz., egg-, serum-, and lactalbumin.
Egg albumin occurs in the white of egg; serum albumin is found in the serum of the blood. Vegetable albumins have been prepared from wheat, rye, potatoes, and papaws. (Carica Papaya). These vegetable albumins are coagulated by heat at about 70° and are not precipitated by the salt solutions named above, nor by acetic acid. The myrosin of mustard seeds also resembles vegetable albumin.
Globulins.—These bodies are insoluble in water, soluble in dilute solutions of neutral salts, but precipitated therefrom by saturation with sodium chlorid or magnesium sulfate. They are coagulated by heat. Among others belonging to this group are serum globulin, fibrinogen, myosin, crystalin, and globin.
Serum globulin is found in the serum of blood; cell globulin is found in lymph cells; fibrinogen occurs in the blood plasma; plasmin, in blood plasma; myosin, in dead muscles; vitellin, in the yolk of eggs; crystalin, in the lens of the eye; haemoglobin, in the red pigment of the blood; haemocyanin, in the blood of certain low grade animals.
Vegetable globulins are found in the cereals, leguminous plants, papaws and other vegetables, and are divided into two groups, myosins and paraglobulins. The vegetable myosins coagulate at from 55° to 60° and are precipitated from a saline solution by removing the salt by dialysis. In this form, however, they lose their true nature as globulins, becoming insoluble in weak saline solutions.
The vegetable paraglobulins are coagulated at from 70° to 75°. Vegetable vitellin, which is not included in this classification, can be obtained in a crystalline form and of remarkable purity.[339]
Albuminates.—This name is given to the compounds of the proteids with metallic oxids or bases, and also to acid and alkali albumins. They are insoluble in water or dilute neutral salts, but easily soluble in strong acids or alkalies. Casein is a type of this group.
Acid albumin is made from egg albumin by treatment with hydrochloric acid; alkali albumin is formed in egg albumin by the action of a dilute alkali; trinitroalbumin is formed from dry albumin by treatment with nitric acid; casein or caseinogen is the chief proteid in milk.
The chief vegetable albuminates are legumin and conglutin. Legumin is a vegetable casein and occurs chiefly in peas, beans and other leguminous seeds. It is prepared by extracting the meal of the seeds mentioned with dilute alkali, filtering the extract, precipitating with acetic acid, washing the precipitate with alcohol, and drying over sulfuric acid. Treated with sulfuric acid it yields leucin, tyrosin and glutamic and aspartic acids. Conglutin is prepared in a similar manner from almonds.
It is probable that these bodies do not exist as such in the fresh seeds in question but are produced therein from the other proteids by the alkali used in extraction. A further description of vegetable proteids will be found in the special paragraphs devoted to the study of these bodies in the principal cereals.
Proteoses.—This name is applied to proteids which are not coagulated by heat, but most of them are precipitated by saturated solutions of neutral salts. They are also precipitated by nitric acid. They are formed from other proteids by the action of proteolytic ferments. The albumoses represent this group.
Protoalbumose is soluble in distilled water and weak saline solutions and is precipitated by mercuric chlorid and copper sulfate.
Heteroalbumose is insoluble in distilled water, but soluble in weak saline solutions, from which it separates when the salts are removed by dialysis. Deuteroalbumose is soluble in distilled water and saline solutions and is not precipitated on saturation with sodium chlorid. It is thrown out by mercuric chlorid but not by copper sulfate.
Vegetable proteoses are known as phytalbumoses, two of which have been found in the juice of the papaw mentioned above. They have also been found in cereals.
Peptones.—These bodies are very soluble in water but are not thrown out by heat, by saturation with neutral salts, nor by nitric acid. They are completely precipitated by tannin and by strong alcohol.
The peptones are the only soluble proteids which are not precipitated by saturation with ammonium sulfate. The principal animal varieties are hemi- and anti-peptones. These forms of proteids do not appear to exist as such in vegetable products but are produced in large quantities by treating other proteids with pepsin or pancreatin. In sprouting plants, there appears to be a widely diffused ferment capable of converting the proteids of the cotyledons into peptonoid bodies and thus fitting them for entering the tissues of the new plant.
Insoluble Proteids.—This class includes a miscellaneous collection of nitrogenous bodies not belonging to any of the definite groups already mentioned. Fibrin and gluten are types of these insoluble bodies. Fibrin is formed from the fibrinogen of fresh blood and causes coagulation. When washed free of red blood corpuscles it is a white elastic solid. It is insoluble in water and is converted into albumoses and peptones by trypsin and pepsin. It swells up when treated with a very weak one-tenth per cent solution of hydrochloric acid and dissolves to acid albumin when heated therewith.
Gluten is the most important of the insoluble vegetable proteids and forms the chief part of the nitrogenous constituents of wheat. It is readily prepared by washing wheat flour in cold water, as will be described further on. It is probably a composite body formed by the process of extraction from at least two proteid bodies existing in wheat. When dried it forms a horny elastic mass of a yellow-gray color. Gluten is composed of two bodies, one soluble the other insoluble in alcohol. The part insoluble in alcohol has been called vegetable fibrin, and the soluble part is subdivided into two portions, one unicedin or vegetable unicin, and the other glutin (gliadin) or vegetable gelatin. Gluten, according to some authorities, does not properly exist in wheat flour, but is formed therein by the action of water and certain ferments from free existing proteids. A better explanation of the composition of gluten is that of Osborne, which will be given further on.
372. Albuminoids.—In this paragraph the term albuminoids is not employed as synonymous with proteids but as characteristic of a class of bodies nearly resembling them, but, nevertheless, differing from them in many important particulars. Following is an abstract of their classification as given in Watt’s dictionary.[340]
Collagen.—The nitrogenous portions of connective tissues are largely composed of collagen. By boiling water it is converted into gelatin. It may be prepared from tendons as follows: The tendinous tissues are shredded as finely as possible and extracted with cold water to remove the soluble proteids. Thereafter they are subjected for several days to the action of lime water, which dissolves the cement holding the fibers together. The residual insoluble matter is washed with water, weak acetic acid, and again with water. The residue is chiefly collagen, mixed, however, with some elastin and nuclein. With dilute acids and alkalies collagen swells up after the manner of fibrin. The organic nitrogenous matter of bone consists largely of collagen, which is sometimes called ossein.
Gelatin.—When the white fibers of collagen, obtained as above, are subjected to the action of boiling water or of steam under pressure they dissolve and form gelatin. Isinglass is a gelatin made from the swimming bladder of the sturgeon or other fish. Glue is an impure gelatin obtained from hides and bones. Pure gelatin may be prepared from the commercial article by removing all soluble salts therefrom by treatment with cold water, dissolving in hot water and filtering into ninety per cent alcohol. The gelatin separates in the form of white filaments and these are removed and dried. Gelatin is insoluble in cold but soluble in hot water. It is insoluble in alcohol, ether and chloroform. Its hot aqueous solutions deflect the plane of polarized light to the left. Its gyrodynat varies with temperature and degree of dilution and is also influenced by acids and alkalies. At 30° it is [α]D³⁰° = -130.
Gelatin is not precipitated by acetic acid nor lead acetate solution, in which respect it differs from chondrin.
If boiled for a day, or in a short time if heated to 140° in a sealed tube, gelatin loses its power of setting and is split up into two peptonoid bodies, semi-glutin and hemi-collin. Gelatin is easily digested but cannot take the place of other proteids in nutrition.
Mucin.—This albuminoid, together with globulin, forms the principal part of connective tissue. It is also present in large quantities in mucus and is the chief lubricant of mucous membranes. It is extremely difficult to prepare mucin in a state of purity, and it is not certain that it has ever been accomplished. It is precipitated but not rendered subsequently insoluble by sodium chlorid, magnesium sulfate and alcohol. When boiled with sulfuric acid it yields leucin and tyrosin and, with caustic soda, pyrocatechin.
Met- and Paralbumin.—Metalbumin is a form of mucin and differs from paralbumin by giving no precipitate when boiled. Both bodies yield reducing sugars when boiled with dilute sulfuric acid.
Nuclein.—The nitrogenous matters which form the nuclei of the ultimate cells are called nuclein. Nuclein resembles mucin in many physical properties but contains phosphorus. It is also, like mucin, resistant to pepsin digestion. The nuclein of eggs and milk probably contains iron. Nuclein is found also in cells of vegetable origin and in yeast and mildew.
Nucleoproteids.—These are bodies which yield both nuclein and albumin when boiled with water or treated with dilute acids or alkalies. Many nucleoproteids have the physical properties of mucus and the sliminess of the bile and of the synovial liquid is due to them. They are the chief nitrogenous constituent of all protoplasm.
Chondrin.—Chondrin is obtained from cartilage by boiling with water. The solutions of chondrin set on cooling in the manner of gelatin. They are precipitated by the same reagents used for throwing out gelatin and mucin. Chondrin is also levorotatory. By some authorities chondrin is regarded as a mixture of gelatin and mucin.
Elastin.—The elastic fibers of connective tissue are composed of this material. It can be prepared from the neck muscles by boiling with ether and alcohol to remove fats and then for a day and a half with water to extract the collagens. The residue is boiled with strong acetic acid and thereafter with strong soda until the fibers begin to smell. It is then treated with weak acetic acid and for a day with dilute hydrochloric acid. The acid is removed by washing with water and the residue is elastin. There is no solvent which acts on elastin without decomposing it. It is digested by both pepsin and trypsin with the formation of peptones.
Keratin.—This nitrogenous substance is found chiefly in hairs, nails, and horns. It is essentially an alteration proteid product due to peripheral exposure. It is prepared by digesting the fine ground material successively with ether, alcohol, water and dilute acids. The residue is keratin. An imperfect aqueous solution may be secured by heating for a long time under pressure to 200°. It is also dissolved by boiling the materials mentioned above with alkalies, and when the solution thus obtained is treated with water, hydrogen sulfid is evolved, showing that the sulfur of the molecule is loosely combined.
Horn swells up when treated with dilute acetic acid and dissolves in the boiling glacial acid. When treated with hot dilute sulfuric acid it yields aspartic and volatile fat acids, leucin and tyrosin. Keratin, when burning, gives off a characteristic odor as is perceived in burning hair.
Other Albuminoids.—Among the albuminoids of less importance may be mentioned neurokeratin found in the medullary sheath of nerve fibers; chitin occurring in the tissues of certain invertebrates; conchiolin, found in the shells of mussels and snails; spongin, occurring in sponges; fibroin forming silk and spiders webs; and hyalin or hyalogen found in edible birds’ nests.
The nitrogenous bases in flesh which are soluble in cold water, viz., kreatin, kreatinin, carnin, sarkin and xanthin are not classed among the albuminoid bodies, since they have a much higher percentage of nitrogen than is found in true proteid bodies, and are further differentiated from them by the absence of sulfur.
373. Other Forms of Nitrogen.—In addition to the proteids and albuminoids mentioned above, agricultural products may contain nitrogen in the form of ammonia, amid nitrogen and nitric acid. The quantities of nitrogen thus combined are not large but often of sufficient magnitude to demand special study. In general, these bodies belong to transition products, representing stages in the transfer of nitrogen from the simple to complex forms of combination, or the reverse.
For instance, the nitrogen which finally appears in the proteids of a plant has entered its organism chiefly as nitric acid, and the nitric acid which is found in a vegetable product is therefore a representative of the quantity of unabsorbed nitrogen present in the tissues at the moment when the vital activity of the plant is arrested. In some instances, it is found that the absorption of nitrates by vegetable tissues takes place in far larger quantities than is necessary for their nutrition, and in these cases the excess of nitrates accumulates, sometimes to a remarkable extent. In a case cited in the reports of the Kansas Agricultural Experiment Station, where Indian corn was grown on ground which had been used for a hog pen, the quantity of potassium nitrate found in the dried stalks was somewhat remarkable. When one of the stalks was cut in two and tapped lightly upon a table, crystals of potassium nitrate were easily obtained in the form of fine powder. On splitting the cornstalk the crystals in the pith could be seen without the aid of a microscope. On igniting a piece of the dried stalk it burned rapidly with deflagration. The percentage of potassium nitrate in the dried material was 18.8. Cattle eating this fodder were poisoned.[341]
In preserved meat products large quantities of oxidized nitrogen are often found, and these come from the use of potassium nitrate as a preserving and coloring agent. Ammonia is rarely found in vegetable tissues in greater quantities than mere traces, but may often exist in weighable amounts in animal products.
Amid nitrogen is found rather constantly associated with proteid matters in vegetable products. Asparagin and glutamin are instances of amid bodies of frequent occurrence. Betain and cholin are found in cottonseed.
The occurrence of nitrogen, in the form of alkaloids, is of interest to agricultural chemists in this country, chiefly from its presence as nicotin in tobacco and from a toxicological point of view, but in other localities the production of alkaloids, as for instance in opium, tea and coffee, is a staple agricultural industry. The methods of separating and determining these forms of nitrogen will be given further on. This description can evidently not include an extended compilation of the methods of separating and determining alkaloidal bodies, with the exception of those with which the agricultural analyst will be called upon frequently to deal, viz., nicotin and caffein and nitrogenous bases such as betain and cholin.
QUALITIVE TESTS FOR
NITROGENOUS BODIES.
374. Nitric Acid.—Any nitric acid or nitrate which an agricultural product may contain may be leached out by treating the fine-ground material with cold water. From vegetable matters this extract is evaporated to a small bulk, filtered, if necessary, and tested for nitric acid by the usual treatment with ferrous sulfate and sulfuric acid. In the case of vegetable substances there will not usually be enough of organic matter to interfere with the delicacy of the reaction, but in animal extracts this may occur. Colored extracts should be decolorized with animal char (bone-black) before they are subjected to examination. It is not well to attempt to remove the organic matters, but, since they are more insoluble in water than the nitrates, the solution containing both may be evaporated to dryness and treated with a quantity of cold water insufficient for complete solution. The nitrates will be found in the solution obtained in a larger proportionate quantity than before.
375. Amid Nitrogen.—One or more atoms of the hydrogen in ammonia may be replaced by acid or basic bodies (alcohol radicles). In the former cases amids, in the latter amins result. In the ratio of displacement there are formed primary, secondary, and tertiary bodies determined by the number of hydrogen atoms replaced. The primary amids are the only ones of these bodies that are of interest in this connection.
The amids are easily decomposed, even on heating with water and the more readily with acids and alkalies, the amido radicle being converted into ammonia. A type of these reactions is given below.
CH₃.CO.NH₂ + H₂O = CH₂.CO.OH + H₃N.
On boiling an amid with hydrochloric acid, the ammonia is procured as chlorid whence it is easily expelled by heating with an alkali. In a body free of ammonia, an amid is easily detected by subjecting the substance containing it to the action of hot hydrochloric acid, filtering, neutralizing the free acid with sodium hydroxid, adding an excess thereof and distilling into an acid.[342] In case the quantity of ammonia produced is very small it may be detected by the nessler reagent.[343] Amids are soluble in a fresh, well washed preparation of cupric hydrate suspended in water. The hydrate also passes into solution forming a liquid of a deep blue color.
If amids be added to a cold solution of potassium nitrate in sulfuric acid free nitrogen is evolved.
376. Ammoniacal Nitrogen.—This combination of nitrogen may be detected by distilling the sample, or an aqueous extract thereof, with magnesia or barium carbonate. The ammonia is collected in an acid and detected therein by the usual qualitive reactions.
377. Proteid Nitrogen.—There are a few general qualitive reactions for proteid nitrogen and some special ones for distinct forms thereof. Below will be given a few of those reactions which are of most importance to the agricultural analyst:
Conversion into Ammonia.—All proteid matters are converted into ammonia on boiling with strong sulfuric acid in presence of an oxygen carrier. Mercury is the substance usually selected to effect the transfer of the oxygen. Bodies which are found to be free of nitrates, ammonia and amids, are subjected directly to oxidation with sulfuric acid, and the ammonia produced thereby is distilled and detected in the manner already suggested. If nitrogen be present in the form of ammonia, amids and nitrates, the substance may be heated with an acid, hydrochloric or acetic, thrown on a filter, washed with hot dilute acid and the residue tested as above for proteid nitrogen.
Biuret Reaction.—When proteid matter is dissolved in sulfuric acid, the solution, made alkaline with potassium hydroxid and treated with a few drops of a solution of copper sulfate, gives a violet coloration. This is commonly known as the biuret reaction, because the substance C₂H₆N₃O₂, biuret, left on heating urea to 160° gives the coloration noted in the conditions mentioned.
It has been found by Bigelow, in this laboratory, that if a solution is to be examined containing a very small amount of a proteid or similar body, the copper sulfate solution should not contain more than four grams of CuSO₄.5H₂O in 100 cubic centimeters of water, and the test should first be made by adding to the solution one or two drops of this copper sulfate solution, and then a strong excess of potassium or sodium hydroxid. The test may be repeated, using from one-half to two cubic centimeters of the copper sulfate solution, according to the amount of proteid present. If too much of the copper sulfate solution be employed its color may conceal that of the reaction.
Heating to the boiling point sometimes makes the violet color more distinct.
If a solid is to be examined it is first suspended in water, and in this state treated in the same manner as a solution. If solution is not complete, the mixture should be filtered when the color produced may be observed in the filtrate.
Proteoses and peptones give a red to red-violet and other proteids a violet to violet-blue coloration.
Xanthoproteic Reaction.—Strong nitric acid produces a yellow coloration of proteid matter, which is intensified on warming. On treating the yellow mixture with ammonia in slight excess the color is changed to an orange or red tint.
378. Qualitive Tests for Albumin.—Albumin is one of the chief proteids and exists in both animal and vegetable substances. It is soluble in cold water and may therefore be separated from many of its nearly related bodies which are insoluble in that menstruum. In aqueous solutions its presence may be determined by the general reactions for proteid matters given above or by the following tests:
Precipitation by Heat.—Albumin is coagulated by heat. Vegetable albumins become solid at about 65° and those of animal origin at a somewhat higher temperature (75°). Some forms of animal albumin, however, as for instance that contained in the serum, coagulate at a lower temperature.
Precipitation by Acids.—Dilute acids also precipitate albumins especially with the aid of heat. Practically all the albumins are thrown out of solution by application of heat in the presence of dilute acids.
Mercuric Salts.—Acid mercuric nitrate and a mixture of mercuric chlorid, potassium iodid and acetic acid completely precipitate all albuminous matters.[344]
The yellow or red color produced on heating albumin with the mercuric nitrate is known as Million’s reaction.
379. Qualitive Test for Peptones and Albuminates.—When peptones and albuminates are dissolved in an excess of glacial acetic acid and the solution treated with sulfuric acid a violet color is produced and also a faint fluorescence.
Separation of Peptones and Albumoses.—In a solution of peptones and albumoses the latter may be precipitated by saturating the solution with finely powdered zinc or ammonium sulfate.
Action of Phosphotungstic Acid.—All proteid matters in aqueous, alkaline or acid solutions, are precipitated by sodium phosphotungstate in a strongly acid solution. Acetic, phosphoric, or sulfuric acid may be used for producing the required acidity, preference being given to the latter.
Action of Trichloracetic Acid.—In the precipitation of albumin by trichloracetic acid, there is formed a compound of the two bodies which to 100 parts of albumin has 26.8 parts of the trichloracetic acid.
The different albuminoid bodies obtained by precipitation behave in a similar manner. There are formed flocculent precipitates insoluble both in dilute and concentrated acids in the cold and also at a high temperature, with the exception of the hemialbumose compound.[345]
Albumin peptone, however, gives with the acid named in concentrated solution a precipitate easily soluble in an excess of the reagent. In the analysis of cow’s milk but not of human milk, this acid can be used for the estimation of the albuminoid substances. With both kinds of milk it can be used for the estimation of the albumin after the removal of the casein.
After precipitation of the albuminoid bodies, the milk sugar can be estimated by polarizing the filtrate and, volumetrically after removal of the excess of the acid by evaporation. By means of trichloracetic acid it is possible to separate albumin peptone from mucus and mucus peptone. A similar reaction is also produced by dichloracetic acid, but the reaction with this last agent is less delicate than with the other. Neither mucus nor albumin is precipitated by chloracetic acid.
380. Action of Albumins on Polarized Light.—Many of the albumins and albuminates, when in solution, strongly deflect the plane of polarized light to the left.[346]
The gyrodynats of some of the albumins and albuminates are given below:
| Serum albumin | [α]D = | -57°.3 to -64°.6. |
| Egg albumin | [α]D = | -35°.5 to -38°.1. |
| Serum globulin | [α]D = | -47°.8. |
| Milk albumin | [α]D = | -76°.0 to -91°.0. |
Our knowledge of the gyrodynatic numbers of the proteids and allied bodies is too fragmentary to be of any great help in analytical work. In practice, the rotatory power of these bodies becomes a disturbing force in the determination of milk sugar.[347] A further study of this property of certain proteids may lead to analytical processes for their detection and determination, but no reliable methods for this can now be recorded.
381. Alkaloidal Nitrogen.—Only a general statement can be made here in respect of the detection of alkaloidal nitrogen in vegetable or animal tissues. Alkaloids are not found in healthy animal tissues and the description of methods for isolating and detecting ptomaines is foreign to the purpose of this work. In vegetable tissues the presence of alkaloids may be established by the following methods of examination.
The fine-ground tissues are made to pass a sieve of half millimeter mesh and when suspended in water are acidified with sulfuric. The mixture is then thoroughly extracted by shaking in a separatory funnel with petroleum ether, benzene and chloroform, successively. Some resins, glucosids and a few alkaloidal bodies not important here are extracted by this treatment.
The residue is made distinctly alkaline with ammonia and treated as above with the same solvents. In the solution obtained as last mentioned nearly all the alkaloidal bodies found in plants are contained.
All the alkaloids in a plant may be obtained by digesting the finely divided material with dilute sulfuric acid. The acid solution thus obtained is made nearly neutral with ammonia or magnesia, concentrated to a sirup, and gums, mucilage, etc. thrown out by adding about three volumes of ninety-five per cent alcohol. The alkaloids are found in the filtrate. The alcohol is evaporated from the filtrate and the residue tested for alkaloids by group reagents.[348] Potassium mercuric iodid and phosphotungstic and molybdic acids are types of these reagents.
The same group reagents may also be applied to the extracts obtained with petroleum ether, benzene and chloroform, in all cases, after the removal of the solvents by evaporation.
ESTIMATION OF NITROGENOUS BODIES
IN AGRICULTURAL PRODUCTS.
382. Total Nitrogen.—Any one of the methods heretofore described for the estimation of total nitrogen in soils or fertilizers is applicable for the same purpose to agricultural products. One among these, however, is so superior in the matter of convenience and certainty, as to make it preferable to any other. The moist combustion of the sample with sulfuric acid with subsequent distillation of the ammonia produced is the process which is to be recommended.[349]
The usual precautions for securing a representative sample should be observed, but no further directions are needed. In all cases hereafter, where the estimation of nitrogen is enjoined, it is understood that the moist combustion process is to be used unless otherwise stated.
383. Estimation of Ammoniacal Nitrogen.—If the distillation of ammonia be accomplished with the aid of magnesia alba or barium carbonate it may be safely conducted on the finely ground materials or, in case of animal bodies, in as fine a state of subdivision as may be conveniently secured. Since the salts of ammonia are easily soluble in water they may be all obtained in aqueous solution, and the distillation of this solution with magnesia gives correct results. Experience has shown that the stronger alkalies, such as sodium and potassium hydroxids, cannot be safely used in the distillation of ammonia from mixtures containing organic nitrogenous materials because of the tendency of these bodies to decomposition, in the circumstances, yielding a portion of their nitrogen as ammonia. Barium carbonate acts with less vigor on non-ammoniacal nitrogenous matters than magnesia, and in some cases, as pointed out further on, may be substituted therefor with advantage. There is no danger of failing to obtain a part of the ammonia on distillation with magnesia provided the latter does not contain more than a trace of carbonate.[350]
When no easily decomposable organic nitrogenous matters are present, the distillation may be conducted with the stronger alkalies in the manner prescribed.[351] All the necessary details of conducting the distillation are found in the preceding volumes of this work.
384. Estimation of Amid Nitrogen.—In bodies containing no ammonia, or from which the ammonia has been removed by the method described in the preceding paragraph, the nitrogen in the amid bodies is converted into ammonia by boiling for about an hour with five per cent sulfuric or hydrochloric acid. The ammonia thus produced is estimated in the usual manner after distillation over magnesia free of carbonate. The free acid is exactly neutralized with sodium or potassium carbonate before the addition of the magnesia. The results are given in terms of asparagin. The reaction which takes place in the decomposition of the amid body is indicated by the following equation:
| Asparagin. | Sulfuric acid. | ||
| 2C₄H₈N₂O₃ + 2H₂O | + | H₂SO₄ | = |
| Aspartic acid. | Ammonium sulfate. | ||
| 2C₄H₇NO₄ | + | (H₄N)₂SO₄. | |
Half of the nitrogen contained in the amid body is thus obtained as ammonia.
It is advisable to calculate all the amid nitrogen in agricultural products as asparagin.
385. Sachsse’s Method.—A method for the determination of amid bodies by liberation of free nitrogen has been described by Sachsse and Kormann.[352] It is based on the reaction which takes place when amid bodies are brought into contact with nitrites in presence of an acid. The mixture of the reagents by which the gas is set free is accomplished in the apparatus shown in [Fig. 103]. The vessel A has a capacity of about fifty cubic centimeters and carries a stopper with three perforations for the arrangement shown.
Fig. 103.—Apparatus
for Amid Nitrogen.
Fig. 104.—Sachsse’s Eudiometer.
About six cubic centimeters of a concentrated aqueous solution of potassium nitrite are placed in A and the lower parts of the tubes a and b are filled with water to a little above e in order to exclude the air therefrom. Dilute sulfuric acid is placed in one of the funnels and an aqueous solution of the amid in the other. The air is displaced from the empty part of A by introducing the sulfuric acid, a little at a time, whereby nitrous acid and nitric oxid are evolved. This operation is continued until all the air has been driven out through c d, the open end of d being kept in the liquid in the dish shown in [Fig. 104]. The eudiometer in which the evolved nitrogen is measured is shown in [Fig. 104], and should have a capacity of about fifty cubic centimeters, and be graduated to fifths. It is filled with the solution of ferrous sulfate contained in B by sucking at g, after which the clamp h is replaced, the cock f closed, and the free end of d placed in the lower end of the eudiometer. The solution of the amid is run slowly into the generator A, [Fig. 103], together with small additional quantities of the sulfuric acid when the evolution of gas becomes slow. From time to time h is opened and fresh quantities of the ferrous solution allowed to flow into the eudiometer. Any trace of the amid remaining in the funnel is washed into A with pure water, with care to avoid the introduction of air. When the liquid in A assumes a permanent blue color the decomposition is complete. The residual gas is driven out of A by filling with water. The tubes d and h, after all the nitric oxid is absorbed, are removed from the eudiometer which is transferred to a cylinder containing water and immersed therein until the two liquid surfaces are at the same level and the volume of the nitrogen observed. After correction for temperature and pressure, the weight of the nitrogen is calculated. Twenty-eight parts by weight of nitrogen correspond to 150 of pure asparagin, 181 of tyrosin and 131 of leucin.[353] This method of procedure is difficult of manipulation and is apt to give results that are too high. It cannot be preferred to the more simple and accurate processes already described.
386. Preparation of Asparagin.—In case the analyst desires to prepare a quantity of asparagin for comparative purposes it may be easily accomplished in the following way: A sufficient quantity of pease or beans is sprouted in a dark place and allowed to grow until the reserve food of the seed is exhausted. The young sprouts are gathered, shredded and subjected to strong pressure. The juice thus obtained is boiled to coagulate the albumin, and thrown on a filter. The filtrate is evaporated to a thin sirup and set aside to allow the asparagin to separate in a crystallized form. If the crystals at first formed are colored they may be dissolved, decolorized with bone-black, and recrystallized. Instead of the above method the young shoots may be shredded, extracted with hot water and the extract treated as above. A larger yield of the asparagin is obtained by the latter process than by the one mentioned above.[354]
387. Detection and Estimation of Asparagin and Glutamin.—Of all the amid bodies asparagin is the most important from an agricultural standpoint, because of its wide distribution in vegetable products.[355] Asparagin is easily obtained from the aqueous extracts of plants by crystallization.[356] In addition to its crystalline characteristics asparagin may be identified by the following tests. Heated with alkalies, including barium hydroxid, asparagin yields ammonia. Boiled with dilute acids it forms ammonium salts. A warm aqueous solution dissolves freshly prepared copper hydroxid with the production of a deep blue color. Sometimes, on cooling, crystals of the copper compound formed are separated. Asparagin crystallizes with one molecule of water. Glutamin gives essentially the reactions characteristic of asparagin, but crystallizes without water in small white needles. Asparagin is easily detected with the aid of the microscope by placing sections of vegetable tissues containing it in alcohol. After some time microscopic crystals of asparagin are separated. The presence of large quantities of soluble carbohydrates seriously interferes with the separation of asparagin in crystalline form.
For the detection of glutamin the liquid containing it is boiled with dilute hydrochloric acid, by which ammonia and glutamic acid are formed. On the addition of lead acetate to the solution the glutamic acid is thrown out as a lead salt, in which, after its decomposition with hydrogen sulfid, the characteristic properties of glutamic acid can be established.
The above process is chronophagous and also uncertain where the quantity of glutamin is very small and that of other soluble organic matters very large. A much better process, both for the detection of glutamin and asparagin, is the following, based on the property possessed by mercuric nitrate of precipitating amids.
The aqueous extract containing the amid bodies is mixed with lead acetate until all precipitable matters are thrown out and the mixture poured into a filter. To the filtrate is added a moderately acid solution of mercuric nitrate. The precipitate produced is collected on a filter, washed, suspended in water, decomposed with hydrogen sulfid and again filtered. The amid bodies (glutamin, asparagin, etc.) are found in the filtrate and can be detected and estimated by the processes already described. A reaction showing the presence of an amid body is not a positive proof of the presence of asparagin or glutamin, since among other amids, allantoin may be present. This substance is found in the sprouts of young plants and also in certain cereals, as shown by researches in this laboratory.[357] Allantoin, glutamin, and asparagin, when obtained in solution by the above process, may be secured, by careful evaporation and recrystallization, in well defined crystalline forms. Asparagin gives lustrous, rhombic prisms, easily soluble in hot water, but insoluble in alcohol and ether.
Allantoin is regarded as a diureid of glyoxalic acid and has the composition represented by the formula C₄H₆N₄O₃. It crystallizes in lustrous prisms having practically the same solubility as asparagin.
Glutamin is the amid of amidoglutaric acid. It crystallizes in fine needles. Its structural formula is represented as
- CO.NH₂
- /
- C₃H₅(NH₂)
- \
- CO₂H.
387. Cholin and Betain.—Cholin is a nitrogenous base found in both animal and plant tissues. Its name is derived from the circumstance that it was first discovered in the bile. It is found in the brain, yolk of eggs, hops, beets, cottonseed and many other bodies. When united with glycerolphosphoric acid it forms lecithin, a compound of great physiological importance. From a chemical point of view, cholin is oxyethyltrimethyl-ammonium hydroxid,
- OH
- /
- C₂H₄; (C₅H₁₅NO₂).
- \
- N(CH₃)₃.OH
It is crystallized with difficulty and is deliquescent. Its most important compound, from an analytical point of view, is its platinum salt C₅H₁₄ONCl₂PtCl₄. This salt crystallizes in red-yellow plates and is insoluble in alcohol.
Betain, C₅H₁₁NO₂, is the product of the oxidation of cholin.
In this laboratory the bases are separated from cottonseed and from each other by the process described below.[358]
About five pounds of fine-ground cottonseed cake are extracted with seventy per cent alcohol. The material should not be previously treated with dilute mineral acids because of the danger of converting a part of the cholin into betain. The alcohol is removed from the filtered extract and the residue dissolved in water. The aqueous solution is treated with lead acetate until no further precipitation takes place, thrown on a filter, the lead removed from the filtrate with hydrogen sulfid and the liquid evaporated to a viscous syrup. The sirup is extracted with alcohol containing one per cent of hydrochloric acid. The solution thus obtained is placed in a deep beaker and the bases precipitated by means of an alcoholic solution of mercuric chlorid. The complete separation of the salts requires at least two weeks.
The double salts of the bases and mercury thus obtained, after freeing from the mother liquor, are recrystallized from a solution in water and from the pure product thus obtained the mercury is removed after solution in water, by hydrogen sulfid. The filtrate, after separating the mercury, contains the bases as chlorids (hydrochlorates). The solution of the chlorids is evaporated slowly in (pene) vacuo to a thick sirup and set over sulfuric acid to facilitate crystallization. The hydrochlorates are obtained in this way colorless and in well-shaped crystalline forms.
In a quantitive determination, a small amount of the fine meal is extracted at once with one per cent hydrochloric acid in seventy per cent alcohol, the salts obtained purified as above and weighed.
The following process serves to determine the relative proportions of cholin and betain in a mixture of the two bases.
A definite weight of the chlorids, prepared as directed above, is extracted by absolute alcohol. This treatment dissolves all the cholin chlorid and a little of the betain salt. The alcoholic solution is evaporated and again extracted with absolute alcohol. This process is repeated three times and at the end the cholin chlorid is obtained free of betain. In a sample of cottonseed cake examined in this laboratory the two bases were found present in the following relative proportions, viz., cholin 17.5 per cent, betain 82.5 per cent. Thus purified the cholin is finally precipitated by platinum chlorid. For a description of the special reaction, by means of which cholin and betain are differentiated, the paper cited above may be consulted.
These bodies have acquired an economic interest on account of their occurrence in cottonseed meal, which is so extensively used as a cattle food. It is evident from the relative proportions in which they occur that the less nocuous base, betain, is the more abundant. It is possible, however, that the base originally formed is cholin and that betain is a secondary product.
Experience has shown that it is not safe to feed cottonseed meal to very young animals, while moderate rations thereof may be given to full-grown animals without much expectation of deleterious results. In the case of toxic effects it is fair to presume that a meal has been fed in which the cholin is relatively more abundant than the betain.
389. Lecithin.—Lecithin is a nitrogenous body, allied both to the fats and proteids and containing glycerol and phosphoric acid. Its percentage composition is represented with some accuracy by the formula C₄₂H₈₆NPO₉, or according to Hoppe-Seyler, C₄₄H₉₀NPO₉. It appears to be a compound of cholin with glycerolphosphoric acid. It is widely distributed both in animal and vegetable organisms, in the latter especially in pease and beans.
From a physiological point of view, lecithin is highly important as the medium for the passage of phosphorus from the organic to the inorganic state, and the reverse. This function of lecithin has been thoroughly investigated in this laboratory by Maxwell.[359]
In the extraction of lecithin from seeds (pease, beans, etc.) it is not possible to secure the whole of the substance by treatment with ether alone.[360]
The extraction of the lecithin may, however, be entirely accomplished by successive treatments for periods of about fifteen hours with pure ether and alcohol. This is better than to mix the solvents, since, in this case, the ether having the lower boiling point is chiefly active in the extraction. When the extraction is accomplished by digestion and not in a continuous extracting apparatus the two solvents may be mixed together and thus used with advantage. After the evaporation of the solvents, the lecithin is ignited with mixed sodium and potassium carbonate whereby the organic phosphorus is secured without loss in an inorganic form. Where greater care is desired, the method described for organic phosphorus in soils may be used.[361] The inorganic phosphorus thus obtained is estimated in the usual way as magnesium pyrophosphate.
For analytical purposes, the extraction of lecithin from vegetable substances is conducted in this laboratory as follows:[362] The fine-ground pea or bean meal is placed in an extraction apparatus and treated continuously with anhydrous ether for fifteen hours. The ether in the apparatus is replaced with absolute alcohol and the extraction continued for six hours longer. The alcoholic extract is evaporated to dryness and treated with ether. The part of the lecithin at first insoluble in ether becomes soluble therein after it has been removed from the vegetable tissues by alcohol. Moreover, any trace of inorganic phosphorus which may have been removed by the alcohol, is left undissolved on subsequent treatment with ether. The ether extract from the alcohol residue is added to that obtained directly, the ether removed by evaporation, and the total lecithin oxidized and the residue used for the estimation of phosphorus as already described.
In determining the lecithin in eggs, the procedure employed for vegetable tissues is slightly changed.[363] The whole egg, excluding the shell, is placed in a flask with a reflux condenser and boiled for six hours with absolute alcohol. The alcohol is then removed from the flask by evaporation and the residue treated in like manner with ether for ten hours. The ether is removed and the dry residue rubbed to a fine powder, placed in an extractor and treated with pure ether for ten hours. The extract thus secured is oxidized after the removal of the ether by fusion with mixed alkaline carbonates and the phosphorus determined in the usual way.
390. Factor for Calculating Results.—The percentage of lecithin is calculated from the weight of magnesium pyrophosphate obtained by multiplying it by the factor, 7.2703.[364] This factor is calculated from the second formula for lecithin given above, in which the percentage of phosphorus pentoxid, P₂O₅, is 8.789.
Example.—In fifty-four grams of egg, exclusive of the shell, is found an amount of organic phosphorus yielding 0.0848 gram of magnesium pyrophosphate. Then 0.0848 × 7.2703 = 0.61652 and 0.61652 × 100 ÷ 54 = 1.14. Therefore the percentage of lecithin in the egg is 1.14.
391. Estimation of Alkaloidal Nitrogen.—The alkaloids contain nitrogen in a form more difficult of oxidation than that contained in proteid or albuminoid forms. It is doubtful whether any of the nitrogen in alkaloids becomes available for plant nutrition by any of the usual processes of fermentation and decay to which nitrogenous bodies are submitted in the soil. Likewise, it is true that it is not attacked by the digestive processes in any way preparatory to its assimilation as food by the animal tissues. Alkaloidal nitrogen is therefore not to be regarded as a food either for the animal or plant.
For the general methods of estimating alkaloids the reader is referred to standard works on plant chemistry and toxicology. The alkaloids of interest in this manual are those which are found in tobacco, tea, coffee and a few other products of agricultural importance. The best methods of isolating and estimating these bodies will be given in the part of the volume devoted to the special consideration of the articles mentioned.
SEPARATION OF PROTEID BODIES
IN VEGETABLE PRODUCTS.
392. Preliminary Treatment.—The chief disturbing components of vegetable tissues, in respect of their influence on the separation and estimation of the proteid constituents, are fats and oils and coloring matters. In many cases these bodies are present in such small quantities as to be negligible, as, for instance, in rice. In other cases they exist in such large proportions as to present almost insuperable difficulties to analytical operations, as is the case with oily seeds. In all instances, however, it is best to remove these bodies, even when present in small proportions, provided it can be done without altering the character of the proteid bodies. This is secured by extracting the fine-ground vegetable material first with petroleum ether, and afterwards with strong alcohol and ether. Practically, all of the fatty bodies and the greater part of the most objectionable coloring matters are removed by this treatment. The extraction should in all cases be made at low temperatures, not exceeding 35°, to avoid the coagulating effect of higher temperatures upon the albuminous bodies which may be present.
In this laboratory, fatty seeds, as for instance peanuts, are first ground into coarse meal, then extracted with petroleum ether, ground to a fine meal and the fat extraction completed with petroleum ether, ninety-five per cent alcohol and pure sulfuric ether. The residue of the last solvent may be removed by aspirating air through the extracted meal. In some cases, it is advisable to extract with ethyl ether before as well as after the alcoholic extraction. This treatment removes at least a part of the water and prevents the dilution of the first part of alcohol added to such an extent as to make it dissolve some of the proteid matters. In each case, a portion of the alcoholic extract should be tested qualitively for proteid matter. If any be found, stronger alcohol should be used for, at least, the first extraction. A portion of the meal, prepared as above directed, is extracted with a ten per cent solution of sodium chlorid, as described further on, and a measured portion of the filtered extract diluted with water until the proteid matter in solution begins to be precipitated. By this treatment the proper strength of the salt solution, to be used for the subsequent extraction, is determined. To save time in dialyzing, the solution of salt employed as a solvent should be as dilute as possible.
The mixture of meal and solvent sometimes filters with difficulty. In these cases, it is advisable to first pour it into a linen bag from which the liquid portion can be removed by gentle pressure and subsequently filtered through paper. As a last resort, the liquid secured from the linen filter can be saturated with ammonium, zinc or magnesium sulfate, whereby all the proteid matters are thrown out. After filtering, the residue is again dissolved in salt solution and can then be readily filtered through paper.
The clear filtrate should be tested by fractional precipitation by heat and the final filtrate by acetic acid, as will be described further on.
The proteid matter may be further freed from amid compounds by treatment with copper sulfate.[365] This treatment is not advisable, however, except for the purpose of determining the total proteid nitrogen in the sample. The action of the water, heat and cupric sulfate combined is capable of inducing grave changes in the character of the residual matter which would seriously interfere with the results of subsequent studies of the nature of the proteid bodies.
In many instances, as with cereal grains, the separation of the proteid bodies is accomplished by no further preliminary treatment than is necessary to reduce them to the proper degree of fineness.
393. Diversity of Character.—The proteids which occur in vegetable products are found in all parts of the tissues of the plants, but in cereals especially in the seeds. In grass crops and in some of the legumes, such as clover, the nitrogenous matters are chiefly found in the straw and leaves. The general classification of these bodies has already been given, but each kind of plant presents marked variations, not only in the relative proportions of the different classes, but also in variations in the nature of each class. For this reason the study of vegetable proteids is, in some respects, a new research for each kind of plant examined. There are, however, some general principles which the analyst must follow in his work, and an attempt will be made here to establish these and to construct thereon a rational method of conducting the investigation. In the separation and estimation of complex bodies so nearly related to each other, it is difficult not only to secure satisfactory results, but also to prevent the transformation of some forms of proteid matter into others nearly related thereto by the action of the solvents used for separation and precipitation.
394. Separation of Gluten from Wheat Flour.—The most important proteid in wheat is the body known as gluten, a commercial name given to the nitrogenous matters insoluble in cold water. The gluten thus obtained does not represent a single chemical compound, but is a complex consisting of at least two proteid bodies, which together form an elastic, pasty mass, insoluble in cold water containing a trace of mineral salts. This mass has the property of holding mechanically entangled among its particles bubbles of gas, which, expanding under the action of heat during cooking, give to bread made of glutenous flours its porous property.
In respect of proteids, the American wheats, as a rule, are quite equal to those of foreign origin. This is an important characteristic when it is remembered that both the milling and food values of a wheat depend largely on the nitrogenous matter which is present. It must not be forgotten, however, that merely a high percentage of proteids is not always a sure indication of the milling value of a wheat. The percentage of gluten to the other proteid constituents of a wheat is not always constant, and it is the gluten content of a flour on which its bread making qualities chiefly depend. The percentage of moist gluten gives, in a rough way, the property of the glutenous matter of absorbing and holding water under conditions as nearly constant as can be obtained. In general, it may be said that the ratio between the moist gluten and the dry gluten in a given sample is an index for comparison with other substances in the same sample. Upon the whole, however, the percentage of dry gluten must be regarded as the safer index of quality. In respect of the content of glutenous matter, our domestic wheats are distinctly superior to those of foreign origin. They are even better than the Canadian wheats in this respect. It may be fairly inferred, therefore, that while our domestic wheats give a flour slightly inferior in nutritive properties to that derived from foreign samples, it is nevertheless better adapted for baking purposes, and this quality more than compensates for its slight deficiency in respect of nutrition, a deficiency, which, however, is so minute as to be hardly worth considering.[366]
The gluten is separated in this laboratory from the other constituents of a flour by the following process:
Ten grams of the fine-ground flour are placed in a porcelain dish, well wet with nearly an equal weight of water at a temperature of not to exceed 15°, and the mass worked into a ball with a spatula, taking care that none of it adheres to the walls of the dish. The ball of dough is allowed to stand for an hour, at the end of which time it is held in the hand and kneaded in a stream of cold water until the starch and soluble matter are removed. The ball of gluten thus obtained is placed in cold water and allowed to remain for an hour when it is removed, pressed as dry as possible between the hands, rolled into a ball, placed in a flat bottom dish and weighed. The weight obtained is entered as moist gluten. The dish containing the ball of gluten is dried for twenty hours in a steam-bath, again weighed, and the weight of material obtained entered as dry gluten. The determination of dry and moist gluten cannot in any sense be regarded as an isolation and estimation of a definite chemical compound. For millers’ and bakers’ purposes, however, the numbers thus obtained have a high practical value. A typical wheat grown in this country will contain about 26.50 per cent of moist and 10.25 per cent of dry gluten.
The gluten of wheat is composed of two proteid bodies, gliadin and glutenin.[367] Gliadin contains 17.66 per cent, and glutenin 17.49 per cent of nitrogen. Gliadin forms a sticky mass when mixed with water and is prevented from passing into solution by the small content of mineral salts present in the flour. It serves to bind together the other ingredients of the flour, thus rendering the dough tough and coherent. Glutenin serves to fix the gliadin and thus to make it firm and solid. Glutenin alone cannot yield gluten in the absence of gliadin, nor gliadin without the help of glutenin. Soluble metallic salts are also necessary to the formation of gluten, and act as suggested above, by preventing the solution of the gliadin in water, during the process of washing out the starch. No fermentation takes place in the formation of gluten from the ingredients named.
The gluten, which is obtained in an impure state by the process above described, is, therefore, not to be regarded as existing as such in the wheat kernel or flour made therefrom, but to arise by a union of its elements by the action of water.
395. Extraction with Water.—It is quite impossible to get an extract from fine-ground vegetable matter in pure water because the soluble salts of the sample pass at once into solution and then a pure water solvent becomes an extremely dilute saline solution. The aqueous extract may, however, be subjected to dialysis, whereby the saline matter is removed and the proteid matter, not precipitated during the dialytic process, may be regarded as that part of it in the original sample soluble in pure water. Nevertheless, in many instances, it is important to obtain an extract with cold water. In oatmeal the aqueous extract is obtained by Osborne as follows:[368] Five pounds of fine-ground meal are shaken occasionally with six liters of cold water for twenty-four hours, the liquid removed by filtration and pressure and the extraction continued with another equal portion of water in the manner noted. The two liquid extracts are united and saturated with commercial ammonium sulfate which precipitates all the dissolved proteid matter. The filtrate obtained is collected on a filter, washed with a saturated solution of ammonium sulfate and removed as completely as possible from the filter paper by means of a spatula. Any residual precipitate remaining on the paper is washed into the vessel containing the removed precipitate and the undissolved precipitate well beaten up in the liquid, which is placed in a dialyzer with a little thymol, to prevent fermentation, and subjected to dialysis for about two weeks. At the end of that time, the contents of the dialyzer are practically free of sulfates. The contents of the dialyzers are then thrown on a filter and in the filtrate are found those proteids first extracted with water, precipitated with ammonium sulfate and redissolved from this precipitated state by pure water. In the case of oatmeal, this proteid matter is not coagulated by heat, and may be obtained in the dry state by the evaporation of the filtrate last mentioned at a low temperature in vacuo. It is evident that the character of the proteid matter thus obtained will vary with the nature of the substance examined. In the case of oats, it appears to be a proteose and not an albumin.
396. Action of Water on Composition of Proteids.—When a body, such as oatmeal, containing many proteids of nearly related character, is exposed to the action of a large excess of water, the proteid bodies may undergo important changes whereby their relations to solvents are changed. After oatmeal has been extracted with water, as described above, the proteid matter originally soluble in dilute alcohol undergoes an alteration and assumes different properties. The same remark is applicable to the proteid body soluble in dilute potash. Nearly all the proteid matter of oatmeal is soluble in dilute potash, if this solvent be applied directly, but if the sample be previously treated with water or a ten per cent salt solution the subsequent proportion of proteid matter soluble in dilute potash is greatly diminished.[369] Water applied directly to the oatmeal apparently dissolves an acid albumin, a globulin or globulins, and a proteose. The bodies, however, soluble in water, exist only in small quantities in oatmeal. Experience has shown that in most instances, it is safer to begin the extraction of a cereal for proteid matter with a dilute salt solution rather than with water, and to determine the matters soluble in water alone by subsequent dialysis.
397. Extraction with Dilute Salt Solution.—In general, it is advisable to begin the work of separating vegetable proteids by extracting the sample with a dilute brine usually of ten per cent strength. As conducted by Osborne and Voorhees, on wheat flour, the manipulation is carried on as follows:[370]
The fine-ground whole wheat flour, about four kilograms, is shaken with twice that weight of a ten per cent sodium chlorid solution, strained through a sieve, to break up lumps, and allowed to settle for sixteen hours. At the end of this time, about half of the supernatant liquid is removed by a siphon or by decantation and filtered. Two liters more of the salt solution are added, the mixture well stirred and the whole brought onto the filter used above. The filtrate is collected in successive convenient portions and each portion, as soon as it is obtained, is saturated with ammonium sulfate. All the proteid matter is precipitated by this reagent. The precipitate is collected on a filter, redissolved in a convenient quantity of the salt solution and dialyzed for fourteen days or until all sulfates and chlorids are removed. The proteid matter, which is separated on dialysis, in this instance, is a globulin.
The proteid matter not precipitated on dialysis is assumed to be that part of the original substance soluble in water.
A part of the water soluble proteid matter obtained as above is coagulated by heat at from 50° to 80°. The part not separated by heat gives a precipitate on saturation with sodium chlorid.
In wheat there are found soluble in water two albumins and a proteose.[371]
In separating the albumin coagulating at a low boiling point from the dialyzed solution mentioned above, it is heated to 60° for an hour, the precipitate collected on a gooch, washed with hot water (60°), and then successively with ninety-five per cent alcohol, water-free alcohol and ether. On drying the residual voluminous matter on the filter over sulfuric acid, it becomes dense and horny, having in an ash free state, according to Osborne, the following composition:
| Per cent. | |
| Carbon | 53.06 |
| Hydrogen | 6.82 |
| Nitrogen | 17.01 |
| Sulfur | 1.30 |
| Oxygen | 21.81 |
398. Treatment without Precipitation with Ammonium Sulfate.—Where abundant means are at hand for dialyzing large volumes of solution, the preliminary treatment of the solution made with ten per cent sodium chlorid with ammonium sulfate may be omitted.
When the precipitated proteids are to be used for the estimation of the nitrogen therein contained, it has been proposed to substitute the corresponding zinc salt for the ammonium sulfate.[372] This reagent has given satisfactory results in this laboratory and while a larger experience is desirable before commending it as an acceptable substitute in all cases, yet its obvious advantage, in being free of nitrogen for the use mentioned, entitles it to careful consideration.
The manipulation, with the exception of the precipitation with ammonium sulfate, is the same as that described in the preceding paragraph. The globulins are completely precipitated when the dialysis is complete and may be separated from the soluble albumins and proteoses by filtration.
399. Separation of the Bodies Soluble in Water.—Albumins.—By the methods of treatment just described, the proteid matters soluble in ten per cent sodium chlorid solution are separated into two classes, viz., globulins insoluble in pure water and albumins and proteoses soluble in pure water. The aqueous solution will also contain any amids or nitrogenous bases soluble in the dilute saline solution and in water. Osborne and Voorhees have found that the best way of separating the albumins in the pure aqueous solution is by the application of heat.[373] By means of a fractional coagulation the albumins are divided into classes, viz., those separating at from 60° to 65° and those remaining in solution at that temperature but separating up to 85°. The respective quantities of these albumins are determined by collecting them in a filter and estimating the nitrogen therein by moist combustion in the usual way. Even a larger number of albumins may be secured, as in the maize kernel, by such a fractional precipitation by means of heat. Chittenden and Osborne find in this instance that the precipitation begins at about 40°.[374]
Proteose.—After the separation of the albumins by heat the filtrate may still contain proteid matter. This matter belongs to the proteose class. It may be partially secured by concentrating the filtrate, after the removal of the albumins, to a small bulk when a part of the proteose body will separate. It may be thrown out entirely by treating the filtrate above mentioned with fine-ground salt until it is saturated or by adding salt until the solution contains about twenty per cent thereof and precipitating the proteose by acetic acid.[375]
400. Separation of the Globulins.—The globulins which are extracted with ten per cent solution of sodium chlorid and precipitated on dialysis may be separated by fractional solution into several bodies of nearly related properties. This solution is conveniently accomplished by saline solvents of increasing strength. In the case of the maize globulins, Chittenden and Osborne employ dilute solutions of common salt for effecting the separation, beginning with a quarter of a per cent and ending with a two per cent mixture.[376]
401. Proteids Soluble in Dilute Alcohol.—Some of the proteid bodies which are soluble in dilute salt solution and in water are also soluble in alcohol. Since these bodies are more easily identified by the processes already described, attention will be given in this paragraph solely to those proteid bodies which are insoluble in water or dilute salt solution and are soluble in dilute alcohol.
For the extraction of these bodies, the residue, left after extraction with a ten per cent solution of sodium chlorid or with water, is mixed with enough strong alcohol to secure by the admixture with the water present in the sample an alcohol of about seventy-five per cent strength. The mixture is well shaken and digested for some time, at a temperature of about 46°, and thrown on a filter which is kept at about the same temperature. The residue is again mixed with alcohol of the same strength (seventy-five per cent) using about four liters for two and a half kilos of the original material. During the second digestion the temperature is kept at about 60°. The latter operation is repeated three times and in each case the filtrate obtained is evaporated separately.[377] This process is especially applicable to the meal from maize kernels, which contains a high relative percentage of an alcohol soluble proteid, zein.
The chief part of the zein is found in the first two extracts, obtained as described above. On evaporation, the zein separates as a tough, leathery, yellow-colored mass on the walls of the containing vessel. It is cut into small pieces and digested for several days in cold, pure alcohol. This is followed by digestion with a mixture of ether and pure alcohol, and finally with pure ether. By this treatment a part of the zein becomes insoluble in seventy-five per cent alcohol. The part soluble in dilute alcohol is precipitated by pouring it into water.
Another method of preparing zein is to extract the meal with seventy-five per cent alcohol after it has been treated with a ten per cent salt solution.
In this case the extraction is continued with seventy-five per cent alcohol in successive portions until no more proteid matter passes into solution. The several extracts are united and the alcohol removed by distillation, by which process the zein is separated. It is washed with distilled water, until the sodium chlorid is removed, dissolved in warm alcohol of about eighty per cent strength and any insoluble matter removed by filtration. On evaporating the filtrate nearly to dryness, the zein is separated and pressed as free of water as possible, yielding a yellow, elastic substance resembling molasses candy. This preparation is purified by digestion with pure alcohol and ether in the manner described. The two zeins which are secured by the treatment, one soluble and the other insoluble in alcohol, are practically identical in composition.[378]
Zein freshly precipitated by pouring its alcoholic solution in water is wholly insoluble in water, and, on boiling therewith, is changed into the variety insoluble in dilute alcohol. Boiled with dilute sulfuric acid, six in 300 cubic centimeters of water, it melts, forming a gummy mass, which is very slowly attacked by the acid yielding proteoses and peptones. Heated with stronger sulfuric acid it undergoes decomposition, yielding leucin, tyrosin, and glutamic acid.
402. Solvent Action of Acids and Alkalies.—In the preceding paragraphs, a synopsis has been given of the methods of separating proteid matters in such a manner as to secure them in a pure state in the same conditions as they exist in the natural substances. A very large percentage of the proteid matter is still left undissolved after extraction with the solvents already mentioned.
Often important information may be gained concerning the nature of the residual proteid matters by fractional extraction with dilute acids and alkalies. When the strength of these solutions is such that they contain about one per cent of the acid or alkali, the whole of the proteid matter may be dissolved by boiling successively with acid and alkali for half an hour. The proteid matter passing into solution in these cases is usually changed in character, assuming the nature of proteoses or allied bodies, when treated with an acid, and becoming albuminates when boiled with an alkali. Easily soluble carbohydrate matter is also removed by this treatment so that the residue obtained consists largely of cellulose and is known as crude or insoluble fiber. The removal of all the bodies soluble in dilute boiling acid and alkali is accomplished by the method described in paragraph [272].
For research purposes, the solvent action of dilute alkali is of chief importance to the analyst, and the extraction of the proteid matter, after all that is soluble in water, common salt solution and alcohol has been removed, should commence with a solution of potassium or sodium hydroxid containing not over two-tenths per cent of the alkali.
It has been shown by Osborne that the solvent action of very dilute alkali, in the cold, may be exerted without changing the character of the dissolved proteid.[379]
403. Method of Extraction.—The solvent employed is usually a two-tenths per cent solution of potassium hydroxid. It may be added directly to the substance or may follow extraction with water, salt solution or alcohol. In the former case, the manipulation is illustrated by the following description of the treatment of oatmeal:[380]
One hundred grams of oatmeal are mixed with half a liter of a two-tenths per cent potassium hydroxid solution and allowed to stand for some time at room temperature. The mixture is strained through a cloth to remove the chaff and the residue is stirred with another small portion of the solvent, again strained in the same cloth and the residue squeezed dry. The strained liquids are united and enough more of the solvent added to make the volume 700 cubic centimeters. After standing for some time, the insoluble matter settles to the bottom of the vessel and the supernatant liquid is decanted. More solvent is added to the residue, well mixed therewith and treated as above. It is advisable to make a third extraction in the same way. The extracts are united, passed through a filter, the proteid matter in solution thrown out by acetic acid, washed with water, alcohol and ether and dried over sulfuric acid.
The methods of procedure, when the sample has been previously extracted with water, salt solution or alcohol, are essentially the same as that just described and the reader may consult the paper of Osborne for details.[381]
404. Methods of Drying Separated Proteids.—In the preceding paragraphs, the analyst has been directed, in most instances, to dry the proteid matter, after it is secured in as pure a form as possible, at room temperature, over sulfuric acid. By this treatment the preparation may be obtained in a form suited to the study of its physical properties, since its solubility has not been affected by subjecting it to a high temperature. When it is desired to use the sample only for chemical analysis it is not necessary to wait on the slow process above mentioned. In this case the sample may be dried in an inert atmosphere at the temperature of a steam-bath or even at 110°. It is better, however, to avoid so high a temperature and to conduct the desiccation in vacuo at a heat not above that of boiling water. The sample, before drying, should be reduced to the finest possible state of comminution, otherwise particles of aqueous vapor may be retained with great tenacity.
In many cases it is advisable to dry the sample pretty thoroughly, then grind to a fine powder and finish the desiccation with the pulverulent mass. This treatment can be followed when the quantity of the material is considerably in excess of that required for the analytical operations.
405. Determination of Ash.—No method of treatment is known by means of which vegetable proteid matters may be obtained entirely free of mineral matters. The mineral bases may be naturally present in the proteid matter as organic and inorganic salts, or they may be mechanically entangled therewith, having been derived either from the other tissues of the plant or from the solvents employed. It is necessary in calculating the analytical data to base the computation on the ash free substance. The percentage of ash is determined by any of the standard processes or by heating the sample in a combustion tube, to very low redness, in a current of oxygen. The total residue obtained is used in calculating the percentage of ash, and the weights of material subsequently used for the determination of carbon, hydrogen, nitrogen and sulfur are corrected for the calculations by deducting the quantity of mineral matter contained therein.
By reason of the highly hygroscopic nature of the dry proteid bodies, they must be kept over a desiccating material and weighed quickly on a balance, in an atmosphere which is kept free of moisture by the usual methods.
406. Carbon and Hydrogen.—Carbon and hydrogen are estimated in proteid matters by combustion with copper oxid. Osborne prefers to burn the sample in a platinum boat in a current of air or of oxygen free of moisture and carbon dioxid.[382] It is advisable to use also a layer of lead chromate in addition to the copper oxid and metallic copper. The method of conducting the combustion has already been described.[383] The analyst should have at his disposal a quantity of pure sugar, which may be used from time to time in testing the accuracy of the work. In beginning a series of combustions this precaution should never be omitted. The addition of the lead chromate is to make more certain the absorption of oxidized sulfur produced during the combustion.
407. Estimation of Nitrogen.—In most cases it is found convenient, during the progress of separating vegetable proteids, to determine the quantity of each kind by estimating the nitrogen by moist combustion and computing the quantity of proteid matter by multiplying the nitrogen by 6.25. The estimation of the nitrogen is made either on an aliquot part of the extract or by direct treatment of the residue.
In the pure extracted proteid matter the nitrogen is most conveniently determined by moist combustion, but it may also be obtained either by combustion with soda-lime or with copper oxid, or by other reliable methods.[384]
The percentages of nitrogen found in the principal proteid bodies, together with the factors for computing the weights of the proteid bodies from the weights of nitrogen found, are given below:
| Name of body. | Percentage of nitrogen. | Factor. |
|---|---|---|
| Mucin | 13.80 to 14.13 | 7.25 to 7.08 |
| Chondrin | 14.20 to 14.65 | 7.04 to 6.83 |
| Albuminates | 13.87 | 7.21 |
| Oat proteids | 15.85 | 6.31 |
| Serum globulin | 15.63 | 6.40 |
| Egg albumin | 15.71 to 17.85 | 6.37 to 5.60 |
| Maize proteids | 16.06 | 6.22 |
| Casein | 15.41 to 16.29 | 6.49 to 6.13 |
| Serum albumin | 15.96 | 6.27 |
| Syntonin | 16.10 | 6.21 |
| Keratin | 16.20 to 17.70 | 6.17 to 5.65 |
| Fibrinogen | 16.65 | 6.01 |
| Peptones | 16.66 to 17.13 | 6.00 to 5.84 |
| Elastin | 16.75 | 5.97 |
| Wheat proteids | 16.80 to 18.39 | 5.95 to 5.44 |
| Fibrin | 16.91 | 5.91 |
| Flax seed proteids | 17.70 to 18.78 | 5.65 to 5.33 |
408. Determination of Sulfur.—Sulfur is a characteristic constituent of the proteid bodies, existing in quantities approximating one per cent of their weight.
In the estimation of sulfur, it is first converted into sulfuric acid, which is thrown out by a soluble barium salt and the sulfur finally weighed as barium sulfate.
All the sulfur existing in the organic state in a proteid may be obtained by burning in a current of oxygen and conducting the gaseous products of combustion through solid sodium or potassium carbonate at or near a red heat.[385] The organic sulfur may also be converted into sulfuric acid by fusing the proteid body with a mixture of sodium hydroxid and potassium nitrate. The fused mass, after cooling, is dissolved in water, the solution acidified with hydrochloric, evaporated to dryness to decompose nitrates and remove excess of hydrochloric acid and dissolved in a large excess of water. After standing for a day, the solution is filtered and the sulfuric acid thrown out of the hot filtrate with a slight excess of barium chlorid solution. The usual precautions in precipitating, filtering and igniting the barium sulfate are to be observed.[386]
409. General Observations.—In the preceding paragraphs have been stated the general principles upon which the separation of vegetable proteid matters depends, and a description has been given of the several processes by which this separation is accomplished. In each case, however, special conditions exist which require special modifications of the general processes, and these can only be successfully secured by the skill, judgment and patient labor of the investigator. Many of these cases have been already worked out, and the valuable data secured by Chittenden, Osborne and others, are accessible to the analyst in the papers already cited. In the case of the proteids in the peanut, a similar work has been done in this laboratory by Bigelow, the data of which have not yet been published. It is only by a careful study of the work already done as outlined here and as published in full in the cited papers, that the analyst will be able to secure trustworthy guidance for future investigations.
410. Dialysis.—One of the most important of the operations connected with the separation and analysis of proteids is the removal of the salts whereby their solutions are secured. This is accomplished by subjecting the solutions of the proteid matters to dialysis. The solution is placed in bags made of parchment dialysis paper. These bags are tied about a glass tube, whereby access may be had to their contents during the progress of the work. Since the volume of the liquid increases during the process, the bags should not be filled too full in the beginning.
Fig. 105. Dialyzing Apparatus.
In this laboratory the dialysis is carried out by Bigelow with the city water from the Potomac, which is first passed through a battery of porous porcelain filtering tubes to remove any suspended silt or micro-organisms. If unfiltered water be used, the germs therein cause a fermentation in the proteid matter, which seriously interferes with the value of the data obtained, and which can only be avoided by the use of an antiseptic, such as an alcoholic solution of thymol. Even with filtered water, the use of a few drops of the solution mentioned is often necessary. To avoid the use of too great quantities of the filtered water, the dialyzers are arranged en batterie, as shown in the [figure]. The filtered water enters the first vessel and thence passes through all. The parchment bags are frequently changed from vessel to vessel, each being brought successively into the first vessel in contact with the fresh water. In some cases the final steps in the dialysis may be accomplished in distilled water.
It is advisable to conduct a fractional preliminary dialysis of the salt solution containing proteids in such a way as to secure the globulins precipitated in each interval of twenty-four hours. Each portion thus secured may be examined with the microscope. Usually a period of two weeks is required to entirely remove the mineral salts from solution. If prepared parchment tubes be used for the dialysis, they should be first tested for leaks, and should not be more than half filled. By the use of a large number of these tubes a greater surface is exposed to dialytic action, and the time required to complete the operation is correspondingly decreased.
SEPARATION AND ESTIMATION OF
NITROGENOUS BODIES IN ANIMAL PRODUCTS.
411. Preparation of the Sample.—Animal products present many difficulties in respect of the reduction thereof to a sufficiently comminuted condition for analytical examination. In the case of bones, the choppers used for preparing them for feeding to fowls are the most efficient apparatus for reducing them to fragments. In this condition they may be ground to a finer state in a sausage machine. The flesh of animals may be reduced by this machine, with two or three grindings, to a fairly homogeneous mass. Subsequent grinding in a mortar with powdered glass or sharp sand may serve to reduce the sample to a finer pulp, but is not usually necessary and should be avoided when possible. The sample thus prepared serves for the estimation of water, ash and fat by methods already described. The sample should be prepared in quantities of considerable magnitude, the whole of any organ or separate portion of the body being used when possible. In examining the whole body the relative weights of blood, bones, viscera, muscle, hide and other parts should first of all be ascertained and noted.
412. Treatment of Muscular Tissues for Nitrogenous Bodies.—For the present purpose a brief sketch of the method of separating the nitrogenous bodies in the muscular tissues of the body is all that can be attempted. For methods of examining the different organs and parts of the body in greater detail, standard works on physiological chemistry may be consulted.[387]
Extraction with Cold Water.—A noted quantity of the finely divided tissues is mixed with several volumes of ice cold water and well rubbed occasionally for several hours, the temperature meanwhile being kept low. The mixture is poured into a linen bag and the liquid portion removed by gentle pressure. The residue in like manner is treated with fresh portions of cold water until it gives up no further soluble matters. An aliquot portion of the extract is concentrated to a small bulk and serves for the determination of total nitrogen. The methods of separating and estimating nitrogenous bodies in flesh soluble in water will be given in considerable detail further on.
Extraction with Ammonium Chlorid and Hydrochloric Acid.—The residue, after exhaustion with cold water, is extracted with a solution of ammonium chlorid containing 150 grams of the salt in a liter. This method of extraction is entirely similar to that with water just described. Globulins and myosin pass into solution by this treatment. The residual mass is washed as free as possible of the solvent and is then further extracted with dilute hydrochloric acid containing four cubic centimeters of the fuming acid in a liter. The treatment with dilute acid is continued until no further substance passes into solution. This is determined by neutralizing a portion of the extract with sodium carbonate, or by the direct addition of potassium ferrocyanid. In either case absence of a precipitate indicates that no nitrogenous matters are present in the solution.
Extraction with Alkali.—The residue from the acid extraction is washed with water until the acid is removed and then extracted in a similar manner with a dilute solution of sodium or potassium hydroxid containing not to exceed two grams of the caustic to the liter. When this residue is finally washed with water and a little acetic acid, it will be found that practically all the purely albuminous bodies contained in the tissues have been extracted with the exception of any fibrin, which the blood, present in the tissues at the commencement of the extraction, may have contained. The extract should be acidified with acetic as soon as obtained.
Extraction with Boiling Water.—The residual matter boiled for some time with water will part with its collagen, which, when transformed by the heat into glutin, passes into solution.
The sarcolemma, membranes, elastic fibers and keratin remain undissolved.
413. Contents of the Several Extracts.—By the systematic treatment of muscular tissues in the manner just described, the nitrogenous bodies they contain are separated into five classes, viz.:
Cold Water Extract.—This contains serum albumin, serum globulin, muscle albumin, myosin, mucin and peptone.
Ammonium Chlorid Extract.—This solution contains the globulins and also in many cases some myosin and serum globulin.
Hydrochloric Acid Extract.—When the extractive matter removed by hydrochloric acid, thrown out by sodium carbonate and well washed with water, has a neutral reaction, it consists of syntonin, when acid, of an albuminate.
Alkali Extract.—The acid albumin of the animal tissue is found in the alkaline solution and may be thrown out by making the solution slightly acid.
Insoluble Residue.—The fifth class contains the insoluble nitrogenous bodies mentioned above.
414. General Observations.—Only a brief résumé of the methods of treating animal tissues for nitrogenous bases is given above, since a more elaborate discussion of these principles and methods would lead too far away from the main purpose of this manual. For practical purposes, the most important of these bodies are those soluble in water and the methods of treating these will be handled at some length. Unfortunately, the methods of determining the exact qualities of these bodies are not as satisfactory in case of animal as in vegetable nitrogenous bodies. The flesh bases, soluble in water, contain a much larger percentage of nitrogen than is found in true proteid bodies, and therefore the multiplication of the weight of nitrogen found therein by 6.25 does not give even a near approximation of the actual quantities of the nitrogenous bodies present in the sample.
Some of the flesh bases contain more than twice as much nitrogen as is found in proteids, and in such cases 3.12, and not 6.25, would be the more correct factor to use in the computation. When possible, therefore, these bodies should be precipitated and weighed after drying, but this is not practicable in many instances. The sole resource of the chemist in such cases is to determine the nature of the body as nearly as possible by qualitive reactions, then to determine the total nitrogen therein and multiply its weight by the corresponding factor. The principal flesh bases have the following percentages of nitrogen and the approximate factors for calculating analytical data are also given:
| Name of base. | Formula. | Per cent nitrogen. | Factor. |
|---|---|---|---|
| Glutin | C₁₃H₂₀N₄O₅ | 17.95 | 5.57 |
| Carnin | C₇H₈N₄O₂ | 31.11 | 3.21 |
| Kreatin | C₄H₁₉N₃O₂ | 32.06 | 3.12 |
| Kreatinin | C₄H₇N₃O₂ | 37.17 | 2.69 |
| Sarkin | C₅H₄N₄O | 41.18 | 2.43 |
415. Composition of Meat Extracts.—The meat extracts of commerce contain all the constituents of meat that are soluble in warm water. The parts which are soluble in warm water and not in cold are found in the cold aqueous solution as suspended or sedimentary matters. Among the nitrogenous bodies present are included albumin, albumose and peptone among the proteids, carnin, kreatin, kreatinin, sarkin and xanthin among the non-proteids, and inosinic and uric acids and urea among other nitrogenous bodies. Among the non-nitrogenous bodies are found lactic and butyric acids, inosit and glycogen. Among mineral bodies occurs the phosphates and chlorids of the common bases. In addition to these bodies, meat extracts may also contain gelatin and other decomposition products of proteid matter. Since meat extract is supposed to be prepared by the digestion of the meat free of bones and put in cold water or in warm water not above 75°, the presence of gelatin would indicate a different method of preparation, viz., either by boiling water or water heated above the boiling point under pressure. In a properly prepared extract, the percentage of gelatin is very small.
Approximately one-tenth of the whole nitrogen present is in the form of albumoses and only a trace as peptones. By far the greater part of the nitrogen exists as flesh bases (kreatin, etc.). The composition of three meat extracts, numbers one and two solid and number three liquid, is given in the subjoined table.[388]
| No. 1. Per cent. | No. 2. Per cent. | No. 3. Per cent. | |||
|---|---|---|---|---|---|
| Total nitrogen | 9.28 | 9.14 | 2.77 | ||
| Nitrogen | as | albumin | trace | 0.08 | trace |
| ” | ” | albumose | 0.96 | 1.21 | 0.70 |
| ” | ” | peptone | trace | trace | none |
| ” | ” | flesh bases | 6.81 | 5.97 | 1.56 |
| ” | ” | ammonia | 0.47 | 0.41 | 0.09 |
| ” | in | compounds insoluble in | |||
| sixty-six per cent alcohol | 0.21 | 0.33 | 0.25 | ||
| ” | ” | other bodies | 0.83 | 1.14 | 0.17 |
417. Analysis of Meat Extracts.—The analysis of a meat extract should include the determination of the water, ash and total nitrogen. After multiplying the nitrogen which exists as proteids by 6.25 and adding together the percentages of all the ingredients, ash, water, etc., including ammonia, the sum is to be subtracted from 100 and the difference entered as non-nitrogenous organic matter. The nature of this conglomerate has already been explained.
Water.—It is advisable to determine the water in a partial vacuum ([20]) or in an atmosphere of hydrogen ([23-25]).
The water may also be determined in solid extracts by placing about five grams of the material in a flat bottom tin foil dish about fifty-five millimeters in diameter and twenty millimeters deep. The material is dissolved in enough warm water to fill the dish a little over one-half and the liquid is then absorbed by adding a weighed quantity of fibrous asbestos or of dry fragments of pumice stone. The asbestos is to be preferred because of the fact that it may be subsequently cut into small bits for the determination of the gelatin. The dish thus prepared is dried to constant weight in a steam-bath or vacuum oven. The weight of the dish and of the added absorbent, together with that of the material employed and of the dried dish and its contents, give the data for calculating the percentage of water. The contents of the dish are used as described further on for the determination of gelatin. In liquid extracts the water is determined in an entirely analogous manner, using about twenty grams of the material and omitting the solution in water.
In solid extracts, the part insoluble in cold water is determined separately.
Ash.—The ash is determined by ignition at the lowest possible temperature, best in a muffle ([28-32]). The ash should be examined qualitively. Where a quantitive analysis is desired, larger quantities of the extract are incinerated and the constituents of the ash determined in the usual way.[389]
Total Nitrogen.—Since nitrates are not present unless added in the manufacture, the total nitrogen is best determined by moist combustion.[390]
Nitric Nitrogen.—The extract should be tested for nitrates and if present they are determined in the manner already described.[391]
Ammoniacal Nitrogen.—When ammonia is present it is determined by distillation with magnesia.[392]
Since boiling with magnesia may cause the distillation of more ammonia than is present as ammonium salts, the plus being due to the decomposition of some other nitrogenous compounds, Stutzer replaces the magnesia with barium carbonate.[393]
Proteid Nitrogen Insoluble in Sixty-Two Per Cent Alcohol.—The aqueous solution is treated with strong alcohol until the mixture contains about sixty-two per cent of the reagent. The precipitate produced is separated by filtration, washed with sixty-two per cent alcohol and the nitrogen therein determined.
Albumose Nitrogen.—This is secured by saturating the aqueous solution with zinc or ammonium sulfate. The separated albumoses are skimmed from the surface, thrown in a filter, washed with a saturated solution of zinc sulfate and the nitrogen determined therein by moist combustion. In the filtrate from the above separation, peptone is detected qualitively by adding a few drops of dilute solution of copper sulfate (biuret reaction).
Kreatin, Kreatinin and Other Flesh Bases.—The clear, aqueous solution of the extract is acidified with sulfuric, mixed with a solution of sodium phosphotungstate and allowed to stand for about six days. The precipitate is collected, washed with a solution of the precipitant, and the nitrogen therein determined. The nitrogen found, less that due to ammonia, represents the total nitrogenous matter precipitated by the phosphotungstic acid. From this quantity is deducted the nitrogen in the proteids, precipitated by sixty-two per cent alcohol and by ammonium or zinc sulfate, and the remainder represents the nitrogen in flesh bases.
The nitrogen thrown out by the phosphotungstic acid is deducted from the total nitrogen, and the remainder represents the nitrogenous bodies not precipitable by the reagent named.
This method of separating the nitrogenous matters in meat extracts is based on the observation that these bodies contain at most only a small quantity of peptones, so small as to be safely negligible.[394]
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.
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.
The volume of the aqueous solution is completed with water to half a liter. One hundred cubic centimeters of this solution are used for the determination of total nitrogen, and another 100 cubic centimeters for the determination of ammoniacal nitrogen by distillation with barium carbonate. A part of the ammonia may have escaped during the preliminary distillation of the alcohol and therefore the amount found may not represent the whole amount originally present. The use of the above determination is principally to ascertain the correction to be made in the amount of total nitrogen found in the first 100 cubic centimeters of the solution.
422. Treatment of the Residue Insoluble in Alcohol.—The residue insoluble in alcohol is washed from the filter into the beaker in which the first solution was made. The aqueous mixture is warmed on a water-bath until the alcohol adhering to the precipitate is completely evaporated, when the contents of the beaker are poured upon a filter free of nitrogen. A small part of the albumose, by reason of the treatment with alcohol, tends to remain undissolved, and it is advisable to collect this albumose upon a filter, wash it well with hot water and estimate the nitrogen therein. The quantity of nitrogen thus found is to be added to the albumose nitrogen determined as described later on.
The total filtrate obtained from the last filtration is made up to a volume of half a liter, of which fifty cubic centimeters are used for the determination of total nitrogen, fifty cubic centimeters for the determination of gelatin, albumose and peptone, and 100 cubic centimeters for the residual peptones. The albumose, together with the gelatin and peptones carried down with it, is precipitated with zinc or ammonium sulfate solution, and its per cent calculated from the amount of nitrogen found in the precipitate. The true peptone is determined by subtracting the quantity of nitrogen determined as albumose from the total nitrogen in solution.
The rest of the liquid, viz., 300 cubic centimeters, is evaporated to a small volume and tested qualitively for true peptones as follows:
To separate the albumose and gelatin a concentrated liquor is treated with an excess of finely divided ammonium sulfate so that a part of the salt remains undissolved. The separated albumose, gelatin and undissolved ammonium salts are collected on a filter, the filtrate mixed with a few drops of dilute copper sulfate solution and a considerable quantity of concentrated soda or potash lye added. Care should be taken that the quantity of copper is not too great, otherwise the peculiar red coloration will be obscured by the blue color of the copper solution.
423. Pancreas Peptone.—The filtrate obtained as described above, by treating the portion of the material insoluble in alcohol with warm water, contains in addition to the albumose and gelatin the whole of the pancreas peptone which may be present. To separate this peptone, 100 cubic centimeters of the aqueous solution are evaporated in a porcelain dish until the volume does not exceed ten cubic centimeters. When cool, at least 100 cubic centimeters of a saturated cooled solution of ammonium sulfate solution are added, the mixture thoroughly stirred, the precipitate collected upon a filter and washed with a cold saturated solution of ammonium sulfate. The contents of the filter are dissolved in boiling water, the filter thoroughly washed and the filtrate and washings evaporated in a porcelain dish with the addition of barium carbonate until, on the addition of new quantities of barium carbonate, no further trace of ammonia can be discovered. The residue is extracted with water, the barium sulfate and carbonate present separated by filtration, well washed and the nitrogen determined in the evaporated filtrate and washings in the usual way and multiplied by 6.25 to determine the quantity of pancreas peptone.
424. Albumose Peptone.—A part of the albumose peptone which may be present is determined in conjunction with the other bodies mentioned above. The chief quantity is found in the solution of the residue insoluble in alcohol in the following manner:
Fifty cubic centimeters of the solution of this residue in hot water are mixed with an equal volume of dilute sulfuric acid, one volume of acid to three of water, in the cold, and a solution of sodium phosphotungstate added until it produces no further precipitate. The precipitate is washed with dilute sulfuric acid and the nitrogen determined therein. The nitrogen thus found is derived from the albumose, pancreas peptone and gelatin. The quantity of nitrogen in the pancreas peptone and gelatin, as above described, is subtracted from the total quantity found in the phosphotungstic acid precipitated, and the remainder represents the nitrogen due to the albumose.
425. Nitrogen in the Form of Flesh Bases Insoluble in Alcohol.—This is determined by subtracting the quantity of nitrogen, determined by the phosphotungstic acid method already described, from the total quantity of nitrogen found in the precipitate insoluble in alcohol and soluble in water.