Chapter XVI: Preface: To Volume Third (16)

In this laboratory the heating is accomplished in a small porcelain dish on which is often deposited a brilliant mirror of metallic silver. The white color of the porcelain also serves as a background for the observation of the coloration produced. In most instances a green color has been noticed after the reduction of the silver is practically complete. Unless cottonseed oil has been boiled or refined in some unusual way, the test, as applied above, is rarely negative. The reduction of the silver is doubtless due to some aldehydic principle, present in extremely minute quantities, and which may be removed by some methods of technical treatment. The silver nitrate test therefore is reliable when the reduction takes place, but the absence of a distinct reaction may not in all cases prove the absence of cottonseed oil.

=363. Milliau’s Process.=—Milliau has proposed the application of the silver salt directly to the free fat acids of the oil instead of to the oil itself.[329] About fifteen cubic centimeters of the oil are saponified with alcoholic potash in the usual manner, 150 cubic centimeters of water added to the dish and the mixture boiled until the alcohol is evaporated. The fat acids are freed by the addition of decinormal sulfuric acid and as they rise to the surface in a pasty condition are removed with a spoon. The free acids are washed with distilled water. The water is drained off and the free acids dissolved in fifteen cubic centimeters of ninety-two per cent alcohol and two cubic centimeters of a three per cent solution of silver nitrate. The test tube containing the mixture is well shaken and placed in a water-bath, out of contact with light, and left until about one-third of the alcohol is evaporated. Ten cubic centimeters of water are added, the heating continued for a few minutes and the color of the supernatant fat acids observed. The presence of cottonseed oil is revealed by the production of a lustrous precipitate which colors the fat acids black. In some cases the process of Milliau gives better results than the original method of Bechi, but this is not always the case. It does away with the use of amyl alcohol and colza oil, but its manipulation is more difficult. In all doubtful cases the analyst should apply both methods.

=364. Detection of Sesame Oil.=—Milliau has pointed out a characteristic reaction of this oil which may be used with advantage in cases of doubtful identity.[330] The identification is based on the fact that the free acids of sesame oil, or some concomitant thereof, give a rose-red color when brought in contact with a solution of sugar in hydrochloric acid.

The analytical process is conducted as follows: About fifteen grams of the oil are saponified with alcoholic soda and when the reaction is complete treated with 200 cubic centimeters of hot water and boiled until the alcohol is removed. The fat acids are set free with decinormal sulfuric acid and removed with a spoon as they rise to the surface in a pasty state, in which condition they are washed by shaking with water in a large test tube. When washed, the acids are placed in an oven at 105° until the greater part of the water is evaporated and the acids begin to become fluid. At this point they are treated with half their volume of hydrochloric acid saturated with finely ground sugar. On shaking the mixture, a rose color is developed which is characteristic of the sesame oil. Other oils give either no coloration or at most a yellow tint.

=365. The Sulfur Chlorid Reaction.=—Some vegetable oils, when treated with sulfur chlorid, give a hard product similar to elaidin, while lard does not. This reaction is therefore helpful in discriminating between some vegetable and animal glycerids. The process which is described by Warren has been used with some satisfaction in this laboratory.[331]

Five grams of the oil or fat are placed in a tared porcelain dish and treated with two cubic centimeters of carbon bisulfid and the same quantity of sulfur chlorid. The dish is placed on a steam-bath and its contents stirred until the reaction is well under way. The heating is continued until all volatile products are evaporated, the hard mass being well rubbed up to facilitate the escape of imprisoned vapors. The powdered or pasty mass is transferred to a filter and washed with carbon bisulfid to remove all unaltered oil. The washing with carbon bisulfid is hastened by pressure and about 200 cubic centimeters of the solvent should be used. After drying, the weight of insoluble matter is obtained and deducted from the total weight of the sample used.

The color and tenacity of the hard, insoluble portion are characteristic. The quantitive part of the operation appears to have but little value, but applied qualitively in this laboratory it produces hard, leathery masses with cotton, olive and peanut oils, and but little change in lard and beef fats. Qualitively applied, the process is conducted as described above but without making the weighings. In this instance it is as easy of application as the process of Bechi and is deserving of greater attention than has been given it by analysts.

In the combination which takes place between the sulfur and the fat it is probable that only addition products are formed, since the quantity of alkali required for saponification is not diminished by previously treating the fat with sulfur chlorid.[332] The reactions which take place are probably well represented by the following equations, in which oleic acid is treated with sulfur chlorid:

C₁₈H₃₄O₂ + S = C₁₈H₃₄S.O₂.
C₁₈H₃₄S.O₂ + NaOH = C₁₈H₃₄SO₂Na + H₂O.

=366. Detection of Cholesterin and Phytosterin in Glycerids.=—Cholesterin is often found in animal glycerids and a corresponding body, phytosterin, is sometimes found in oils of a vegetable origin.[333] When one of these two bodies is present it may be useful in distinguishing between animal and vegetable glycerids. They are detected as follows: Fifty grams of the glycerids in each case are saponified with alcoholic alkali, preferably potash, in order to have a soft soap. After saponification is complete, the alcohol is evaporated and the residual soap dissolved in two liters of water. The mixture is shaken with ether and the ethereal solution evaporated to a small bulk. The residue, which may contain a small quantity of unsaponified fat, is again treated with alcoholic potash and subjected a second time to the action of ether, as indicated above, with the addition of a few drops of water and of alcohol if the emulsion separate slowly. The ethereal extract finally secured is allowed to evaporate slowly and the cholesterin (phytosterin) is obtained in a crystalline form. The melting point of the cholesterin crystals is 146° and that of the phytosterin 132°.

Cholesterin crystallizes in thin rhombic tables while phytosterin separates in stellar aggregates or in bundles of long needles.

When dissolved in chloroform the two products show different color reactions with sulfuric acid, cholesterin giving a cherry and phytosterin a blue-red tint. In a mixture of animal and vegetable glycerids the two products are obtained together and the melting point of the mixture may afford some idea of the relative quantities of each present. It is evident, however, that no reliable judgment can be formed from these data of the relative proportions of the two kinds of glycerids in the original sample.

=367. Cholesterin and Paraffin in Ether Extracts.=—In ethereal extracts of some bodies, especially of flowers of the chrysanthemum, paraffin is found combined with cholesterin. The two bodies may be separated as follows:[334]

The ether extract is treated with aqueous then with alcoholic potash several times; the residue soluble in ether is a solid body melting at from 70° to 100°.

If the ethereal solution be cooled in a mixture of snow and salt, a crystalline deposit is formed. This substance, purified by repeated precipitations, is obtained colorless in fine crystalline scales melting at 64°. It is very soluble in ether, benzene and chloroform, almost insoluble in cold alcohol, and somewhat soluble in hot.

Its percentage composition is:

Per cent.
Carbon 85.00
Hydrogen 14.95

It is therefore a paraffin.

The ethereal solution, freed by the above process from paraffin, leaves on evaporation a crystalline mass which is cholesterin, retaining still a small quantity of fat matters. In treating the crystals with alcoholic potash these fat bodies are saponified and the residue is taken up with ether. The cholesterin is obtained in fine needles melting at from 170° to 176°. It presents all the reactions of cholesterin, especially the characteristic reaction with chloroform and sulfuric acid.

=368. Absorption of Oxygen.=—Among oils a distinction is made between those which oxidize readily and those which are of a more stable composition. Linseed oil, for instance, in presence of certain metallic oxids, absorbs oxygen readily and is a type of the drying oils, while olive oil represents the opposite type.

The method of determining the quantity of oxygen absorbed is due to Livache and is carried out as follows:[335]

Precipitated metallic lead (by zinc) is mixed in a flat dish, with the oil to be tested, in the proportions of one gram of lead to three-quarters of a gram of oil, and exposed to the air and light of the workroom. The dish is weighed from time to time until there is no longer any increase in weight.

Instead of lead, finely divided copper has been used by Krug in this laboratory, but the percentage of absorption of oxygen is not so high with copper as with lead. Krug found the quantities of oxygen absorbed, after nine days, by the samples treated with copper and lead respectively to be the following:

Copper, per cent Lead, per
oxygen absorbed. cent oxygen
absorbed.

Olive oil 1.69 2.03
Cottonseed oil 4.25 5.30
Peanut oil 2.74 3.87
Linseed oil 5.55 7.32

Livache found that linseed oil absorbed about twice as much oxygen as indicated by the data just given.

=369. Elaidin Reactions.=—In discriminating between oils and fats having a preponderance of olein and others with a smaller proportion of that glycerid, the conversion of the olein into its isomer elaidin is of diagnostic value. The following will be found a convenient method of applying this test:[336]

About ten cubic centimeters of the oil are placed in a test tube together with half that quantity of nitric acid and one gram of mercury. The mixture is shaken until the mercury dissolves when the mass is allowed to remain at rest for twenty minutes. At the end of this time it is again shaken and placed aside. In from one to three hours the reaction is complete. Olive, peanut and lard oils give very hard elaidins. The depth to which a plunger of given weight and dimensions sinks into an elaidin mixture at a given temperature, has been used as a measure of the percentage of olein contained in the sample of oil, but it is evident that such a determination is only roughly approximate. Copper may be used instead of mercury for the generation of the oxids of nitrogen, but it is not so effective. The vapors of nitric oxids may also be conducted directly into the oil from a convenient generator. The reaction may also be accomplished by shaking the oil with nitric acid and adding, a drop at a time, a solution of potassium nitrite.

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]_{D}³⁰° = -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 Aspartic Ammonium
acid. acid. sulfate.
2C₄H₈N₂O₃ + 2H₂O + H₂SO₄ = 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.

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.