ESTIMATION OF NITROGEN IN SOILS.

395. Introductory Considerations.—The great economic and biologic value of nitrogen as a plant food renders its estimation in soils of especial importance. It is necessary, first of all, to remember that the nitrogen present in soils may be found in three forms; viz., first, in organic compounds, second, as ammonia, and third, as nitric or nitrous acid. Further than this each of these classes of nitrogen may be subdivided. The organic nitrogen may be in a form easily nitrified and rendered available for plant food, or it may be inert and resistant to nitrification, as in humus, or exist in an amid state. The ammoniacal nitrogen may exist in small quantities as gaseous ammonia, or be combined with mineral or organic acids. As nitric or nitrous acid the nitrogen will be found combined with bases, or perhaps in minute quantities as free acid, in passing under the influence of the nitrifying ferment from the organic to the inorganic state. To the latter state it must finally come before it is suited to absorption by plants.

In general, far the largest part of soil nitrogen, excluding the atmosphere diffused in the pores of the soil, is found in the organic state and is derived from the débris of animal and vegetable life and from added fertilizers. As ammonia, the nitrogen can only be regarded as in a transition state, arising from the processes of decay, or incomplete nitrification. As nitric acid, it is found as a completed product of nitrification, or as the result of electrical action. The processes of nitrification and the isolation and determination of the nitrifying organisms will be considered in a special chapter of this manual. By reason of the great solubility of the nitrates, and the inability of the soil to retain them, there can never be a great accumulation of nitric acid in the soil save in localities deficient in rain-fall or in specially protected spots, such as caves. The nitric acid, therefore, produced in the soil passes at once into growing vegetation, or is found eventually in the drainage waters.

The formation of ammonia in soil containing much vegetable matter is thought by Berthelot and André^[260] to be due to the progressive decomposition of amid principles under the influence of dilute acids or alkalies, either in the cold or at an elevated temperature. Soils of the above description, of themselves, contain neither free ammonia nor ammoniacal salts, and the ammonia which is found in the analysis of these soils comes from the reaction above indicated. The ammonia which comes from these soils, in place of what is given off to the surrounding atmosphere, comes from the same class of decompositions, and these decompositions, in this case, are effected by the water itself, and by the alkaline carbonates of the soil. The amid principles which are thus decomposed belong either to the class of amids proper, derived by the displacement of hydrogen in ammonia by acids, or to the class of alkalamids derived from nitrogenous bases, both volatile and fixed. Among these alkalamids some are soluble in water and some insoluble, and the decomposition of these last by acids or by alkalies may furnish bodies which themselves are either soluble or insoluble in water.

To determine the nature of the nitrogenous principles in a soil rather rich in humus, Berthelot and André applied the following treatment:

A soil containing 19.1 grams of carbon and 1.67 grams of nitrogen per kilogram was first subjected to treatment, at room temperature, with a concentrated solution of potash. By this treatment 17.4 per cent of the nitrogen content was set free under the form of ammonia. One-quarter of this was obtained during the first three days; one-eighth during the next three days. Afterward the action became much more feeble and was continued during forty days longer, and the evolution of the gas was diminished almost proportionately to the time. It appears from the above observations that the amid principles of the soil, decomposable by potash, belong to two distinct groups, which are broken up with very unequal velocities. The soil, treated on the water-bath for three hours at 100° with strong potash, showed the following behavior in respect of its nitrogenous constituents: Nitrogen eliminated in the form of ammonia, sixteen per cent; nitrogen remaining in the part soluble in potash, ten per cent; nitrogen remaining in the part insoluble in potash, seventy-four per cent.

Treatment with Acid.—The nitrogenous compounds of the soil are also decomposed by dilute acids, and often more rapidly than by the alkalies. The method of treatment is substantially the same as that set forth above. The decompositions effected either by alkalies or by acids tend in general to lower the molecular weights of the resulting products. The prolonged action of alkalies at the temperature of boiling water rendered soluble, after twenty-four hours of treatment, 93.6 per cent of the organic nitrogen found in the vegetable mould. By treating the earth successively with alkalies and acids 95.5 per cent of the total nitrogen were decomposed. These experiments show how the insoluble nitrogen in humic compounds can be gradually rendered assimilable. The action of vegetables is not assuredly identical with those which acids and alkalies exercise. However, both present certain degrees of comparison from the point of view of the mechanisms set in play by the earthy carbonates and carbon dioxid, as well as by the acids formed by vegetation. The reactions which take place naturally, while they are not so violent as those produced in the laboratory, make up by their duration what they lack in intensity.

For a more detailed study of the nature of the nitrogenous elements in soil the following method of treatment, due to Berthelot and André, is recommended:

Treatment of the Soil with Alkalies.—1. Reaction with cold, dilute solution of potash. Take fifty grams of the sample, dried at 110°, and mix with a large excess of ten per cent potash solution and place under a bell-jar containing standard sulfuric acid. The mixture is left for a long time in order to secure as fully as possible the ammonia set free.

Example: Fifty grams of a soil contained 0.0814 gram of nitrogen. Treated as above it gave the following quantities of nitrogen as ammonia:

It is seen that the action still continued after forty days. In the space of forty days 17.4 per cent of the total nitrogen contained in the soil had been converted into ammonia by dilute potash. According to the above observations the amid principles transformed into ammonia under the influence of dilute potash, exist in groups which are acted on with very unequal rapidity.

2. Reaction with hot dilute solution of potash. Take 200 grams of the soil sample, mix with one and one-half liters of dilute potash solution containing fifty grams of potash. Place in a flask and heat on boiling water-bath for six hours. The flask is furnished with a stopper and tubes, and a current of pure hydrogen is made to pass through the liquid, having the double object of preventing any oxidizing effect from the air and of carrying away the ammonia which may be formed. The escaping hydrogen and ammonia are passed into a bulb apparatus containing titrated sulfuric acid.

The sample of soil employed contained in 200 grams, 0.3256 gram of nitrogen. There was obtained at the end of six hours’ heating, 0.0366 gram of nitrogen. In other words, 11.24 per cent of the total nitrogen in the sample appeared as ammonia.

Examination of Residue.—After the separation of the ammonia as above described, pour the residue in the flask on a filter, wash with hot water, and determine nitrogen in filtrate and in solid matter on the filter by combustion with soda-lime. The filtrate is, of course, first evaporated to dryness after being neutralized with sulfuric acid.

The insoluble part contained 0.041 gram of nitrogen, i. e., 12.84 per cent of the entire amount.

The soluble part contained 0.2411 gram of nitrogen, i. e., 74.05 per cent of the whole.

Summary of Data.—In the sample analyzed the following data were obtained:

The same experiment in which the heating on the water-bath was continued for thirteen hours gave the following data:

Further Treatment of Matter Insoluble in Hot Dilute Potash.—A portion of the insoluble portion from the last experiment was treated for thirteen hours longer under the same conditions with dilute hot potash. The soluble and insoluble portions were determined as already described. Of the nitrogen insoluble after thirteen hours, 64.21 per cent remained insoluble after the second thirteen hours. This fact shows that slow and progressive decomposition of the alkalamids in the soil occurs under the influence of hot dilute potash.

Treatment of Matter Insoluble in Hot Dilute Potash with Hydrochloric Acid.—A part of the material insoluble in hot potash after thirteen hours is mixed with dilute hydrochloric acid, in such proportion as to have one-fifth the weight of pure hydrochloric acid to the dry solid matter. Heat in flask on a boiling water-bath for thirteen hours. Determine the nitrogen in the insoluble residue.

Example: In the case given it was found that 54.91 per cent of the nitrogen insoluble in dilute hot potash were dissolved by the hot hydrochloric acid.

This fact shows that insoluble nitrogen compounds contained in the soil are dissolved by dilute acids even more readily than by dilute alkalies at the temperature of boiling water.

Several reactions appear to take place simultaneously when potash is brought into contact with the nitrogenous principles of arable earth. Some of these principles, during the first period of the action become soluble and even form compounds which are not precipitable by acids. When, however, the action of the potash is prolonged, the dissolved bodies lose little by little a part of their nitrogen as ammonia or as soluble alkalamids. They become thus changed either to compounds no longer soluble in the potash, or to those insoluble in the solution when acidified. These compounds, it is true, contain nitrogen, but are poorer in this element and have a higher molecular weight, or, in other words, are condensation products. These last principles are not absolutely stable in the presence of potash, but are decomposed much more slowly than the original principles from which they were derived.

In general, it may be said that under the influence of alkalies on the nitrogenous principles of the soil there is a tendency to form two classes of bodies, the one more soluble with a lower molecular weight, the other less soluble with a higher molecular weight. The inverse relation between solubility and condensation is in agreement with what is observed in similar reactions with organic bodies in general. It certainly plays an important rôle in the transformations which an arable soil undergoes, either through the mild influences of the air and natural waters, or the more energetic action of vegetables themselves.

The methods of estimating nitric nitrogen will be made the theme of a special study in connection with the chapter on nitrification. There will be considered first, therefore, the methods of determining organic and ammoniacal nitrogen with only such incidental treatment of the methods for nitric nitrogen as the processes applicable to the other forms may contain.

396. Provisional Methods of the Association of Official Agricultural Chemists.^[261]—The nitrogen compounds in the soil are usually placed in three classes.

1. The nitrogen combined with oxygen as nitrates, or nitrites, existing as soluble salts in the soil.

2. The nitrogen combined with hydrogen as ammonia, or organic nitrogen easily convertible into ammonia. The ammonia may exist as salts, or may be occluded by hydrated ferric or aluminum oxids and organic matter in the soil.

3. The inert nitrogen of the soil or the humus nitrogen.

Active Soil Nitrogen.—The material proposed for reducing the nitrates to ammonia, and at the same time to bring ammonia salts and organic nitrogen into a condition for separation by distillation, is sodium amalgam. Liquid sodium amalgam may be readily prepared by placing 100 cubic centimeters of mercury in a flask of half a liter capacity, covering the warmed mercury with melted paraffin, and dropping into the flask at short intervals pieces of metallic sodium, the size of a large pea (taking care that the violence of the reaction does not project the contents from the flask), till 6.75 grams of sodium have combined with the mercury. The amalgam contains one-half per cent of sodium and may be preserved indefinitely under the covering of paraffin. To estimate the active soil nitrogen, weigh fifty grams of air-dried soil and place it in a clean mortar. Take 200 cubic centimeters of ammonia-free distilled water, rub up the soil with a part of the water to a smooth paste, transfer this to a flask of one liter capacity, washing the last traces of the soil into the flask with the rest of the water. Add twenty-five cubic centimeters of the liquid sodium amalgam and shake the flask so as to break the sodium amalgam into small globules distributed through the soil. Insert a stopper with a valve and set aside in a cool place for twenty-four hours. Pour into the flask fifty cubic centimeters of milk of lime, and distill, on a sand-bath, 100 cubic centimeters into a flask containing twenty cubic centimeters of decinormal sulfuric acid, and titrate with decinormal soda solution, using dimethyl-orange as indicator. Estimate the nitrogen of the ammonia found as active soil nitrogen.

If the ammonia produced is too small in amount to be readily estimated volumetrically, determine the ammonia by nesslerizing the distillate.

Estimation of Nitrates in the Soil.—When it is desired to estimate separately the nitrates in the soil the following method may be used: Evaporate 100 cubic centimeters of the soil extract to dryness on the water-bath, dissolve the soluble portion, of the residue in 100 cubic centimeters of ammonia-free distilled water, filtering out any insoluble residue, place the solution in a flask and add ten cubic centimeters of liquid sodium amalgam, insert stopper with valve, set it aside to digest in a cool place for twenty-four hours, add fifty cubic centimeters of milk of lime, distill and titrate as above, and estimate the nitrogen as N₂O₅.

Nesslerizing may be substituted for titration when the amount of nitrates is small.

An approximate estimation of the amount of nitrates will be of value in determining which method of estimation to use. This may be done by evaporating a measured quantity of the soil extract, say five cubic centimeters, on a porcelain crucible cover on a steam-bath or radiator, having first dissolved a minute fragment of pure brucin sulfate in the soil extract. When dry pour over the residue concentrated sulfuric acid, free from nitrates, and observe the color reactions produced.

If the nitrate (reckoned as KNO₃) left upon evaporating the quantity of water taken does not exceed the two-thousandth part of a milligram, only a pink color will be developed by adding the sulfuric acid; with the three-thousandth part of a milligram, a pink with faint reddish lines; with the four-thousandth part, a reddish color; with the five-thousandth part, a red color.

By increasing or diminishing the amount of soil extract evaporated to secure a color reaction of a certain intensity, an approximate estimate may be made of the amount of nitrates present.

Blank experiments to test the acid and the brucin sulfate will be required before confidence can be placed in such estimations.

Total Nitrogen of Soils.—The total nitrogen of soils may be determined by the usual combustion with soda-lime, but this process is often unsatisfactory because of the large amount of material required when the organic matter or humus is small in amount.

A modification of the kjeldahl method is more easy to carry out and gives results equally satisfactory. Place twenty grams of soil in a kjeldahl flask, and add twenty cubic centimeters of sulfuric acid (free from ammonia) holding in solution one gram of salicylic acid. If the soil contain much lime or magnesia in the form of carbonate, enough more sulfuric acid must be added to secure a strongly acid condition of the contents of the flask. Add gradually two grams of zinc dust, shaking the contents of the flask to secure intimate mixture. Place the flask in a sand-bath and heat till the acid boils, and maintain the boiling for ten minutes. Add one gram of mercury and continue the boiling for one hour, adding ten cubic centimeters of sulfuric acid if the contents of the flask are likely to become solid. Cool the flask and wash out the soluble materials with 200 cubic centimeters of pure water, leaving the heavy earthy materials. Rinse the residue with 100 cubic centimeters of water, and add this to the first washings. Place this soluble acid extract in a liter digestion flask, add thirty-five cubic centimeters of a solution of potassium sulfid, and shake the flask to secure intimate mixture of the contents. Introduce a few fragments of granulated zinc, pour in seventy-five cubic centimeters of a saturated solution of caustic soda, connect the flask with a condenser and distill 150 cubic centimeters into a flask containing twenty cubic centimeters of acid, using the same acid and alkali for titration used in the kjeldahl method under fertilizers.

Enter the nitrogen found in this operation as total soil nitrogen.

The difference between the total soil nitrogen and the active soil nitrogen will express the inert nitrogen of the soil.

397. Hilgard’s Method.^[262]—The humus determination will, in the case of virgin soils, usually indicate approximately the store of nitrogen in the soil, which must be gradually made available by nitrification. Ordinarily (outside of the arid regions) the determination of ammonia and nitrates present in the soil is of little interest for general purposes, since these factors will vary with the season and from day to day. Kedzie proposes to estimate the active soil nitrogen (ammonia plus nitrates and nitrites) by treatment of the whole soil with sodium amalgam and distillation with lime. The objection to this process is that the formation of ammonia by the reaction of the alkali and lime upon the humus amids would greatly exaggerate the active nitrogen and lead to a serious overestimate of the soil’s immediate resources.

The usual content of nitrogen in black soil-humus is from six to eight per cent in the regions of summer rains. From late determinations it would seem that in the arid regions the usually small amount of humus (often less than two-tenths per cent) is materially compensated by a higher nitrogen percentage. It thus becomes necessary to determine the humus nitrogen directly; and this is easily done by substituting in the grandeau process of humus extraction potash or soda-lye for ammonia water, and determining the nitrogen by the kjeldahl method in the filtrate.

The lye used should have the strength of four per cent in the case of potassium hydroxid, three per cent in that of sodium hydroxid. The black humus filtrate is carefully neutralized with sulfuric acid, evaporated to a small bulk in a beaker or evaporating basin, and the reduced liquid finally evaporated to dryness in the kjeldahl flask itself by means of a current of air. The beaker or basin is washed either with some of the alkaline lye, or, after evaporation, with warm concentrated sulfuric acid, which is then used in the nitrogen determination in the usual way.

For the determination of nitrates in the soil it is, of course, usually necessary to use large amounts of material, say not less than 100 grams, and, according to circumstances, five or more times that amount. In the evaporated solution the nitric acid is best determined by the reduction method, as ammonia.

Usually the soil filtrate is clear and contains no appreciable amount of organic matter that would interfere with the determination; yet in the case of alkaline soils (impregnated with sodium carbonate) a very dark colored solution may be obtained. In that case the soil may advantageously be mixed with a few per cent of powdered gypsum before leaching; or the gypsum may be used in the filtrate to discolor it by the decomposition of sodium carbonate and the precipitation of calcium humate. The evaporated filtrate can then be used for the nitrate determination by either the kjeldahl, griess, or the nessler process, which will, of course, include such portions of the ammoniacal salts as may have been leached out.

For the separate determination of these and of the occluded ammonia, when desired, it is probably best to mix the wetted soil intimately with about ten per cent of magnesium oxid and distill into titrated hydrochloric acid. For general purposes, however, this determination is usually of little interest.

398. Müller’s Modified Kjeldahl Method.—Numerous difficulties, as stated by Müller,^[263] have attended the attempts to apply the kjeldahl method for the estimation of nitrogen to samples of soil, and he has modified the method to some extent and made comparisons of the quantity of nitrogen by this modified method and by the soda-lime method.

The principal difficulty encountered by him has been in the regular heating of the mixture of fuming sulfuric acid and soil. The particles of soil are deposited at the bottom of the flask and the result is that the bottom layers become overheated, and, being poor conductors of heat, fail to transmit a sufficient quantity of heat to penetrate to the upper layers of the liquid to complete the reaction. In order to avoid this difficulty Müller heats his flask in a small stove formed with a straight vertical cylinder of iron or copper, the upper end of which is covered with a sheet of iron pierced with a hole which allows the neck of the flask to pass through, while the lower end is closed with a piece of sheet iron furnished on its upper surface with a layer of asbestos. This cylinder of metal is surrounded with a second one, concentric with the first through which passes a current of heated gases furnished by an ordinary bunsen. By heating the flask in this stove or furnace an even distribution of the heat is secured to all parts of the mixture, but the little drops of sulfuric acid, which are condensed on the cold part of the neck, sometimes lead to the fracture of the glass as they run down the sides of the flask to the hot portions. To prevent the reflux of this condensed acid, which only needs to be done near the end of the reaction, when it is necessary to heat to a very high temperature, the neck of the flask is bent at the point immediately above its emergence at the upper surface of the furnace, and carried into a flask of about seventy-five cubic centimeters capacity, which will receive the drops of sulfuric acid condensed during the operation. The furnace has the following dimensions; height, twelve centimeters; diameter of interior cylinder, five and one-half centimeters; diameter of exterior cylinder, seven and one-half centimeters.

It is supported on a triangle of large iron wire and is heated by an ordinary bunsen, or by a concentric bunsen, according to the temperature which it is necessary to obtain. The proportions which should be observed between the amount of earth employed and the sulfuric acid are about as follows:

Of the dry earth, fifteen grams; of the fuming sulfuric acid, thirty cubic centimeters. There should also be added to the mixture about three-tenths of a gram of pure stearic acid, or better, benzoic acid. When the soil to be analyzed does not contain carbonate, the sulfuric acid should be added in two portions. At first add about twenty cubic centimeters of the acid, and after shaking it, the other ten cubic centimeters, running it in from a burette or a pipette in such a manner as to wash thoroughly the neck and sides of the flask. If the earth contain carbonate, however, it is necessary to add the fuming acid in small portions of about five cubic centimeters at a time, waiting each time until the disengagement of the gas caused by the previous addition has ceased. A soil which contains from thirty to forty per cent of calcium carbonate should be carefully treated in a porcelain capsule with a slight excess of sulfuric acid, pure and dilute. The mixture is afterward to be evaporated to dryness upon a sand-bath and the residue heated in a drying oven to 110°. The mass is then pulverized, introduced into the flask, treated with three-tenths of a gram of benzoic acid and thirty cubic centimeters of fuming sulfuric acid, and treated as indicated above. In all cases it is necessary to continue the heating until the contents of the flask are colorless.

With soils containing considerable quantities of iron, however, a slight red color will probably be observed which will not interfere with the accuracy of the tests.

The heating should at first be gentle and the temperature afterward elevated little by little, and finally the heat should be sufficiently great to distill about one and one-half cubic centimeters of sulfuric acid. The operation lasts from twelve to thirteen hours. As the reaction is terminated the cooled mass is taken up with water absolutely free from ammonia. It is filtered into a flask, and washed upon the filter until the volume of the filtered liquid is about 350 cubic centimeters. Afterward an excess of soda-lye, at 50° baumé is added, then a few pieces of quartz to facilitate boiling. The flask is then connected with a condenser, the liquid distilled and received in a conical flask closed by a cork having two holes, of which one permits the entrance of the end of the condenser, and the other a glass tube which is connected with a small flask containing water, the neck of the receiving flask being inclined toward the condenser to avoid the entrainment of any of the alkaline liquid which may be distilled. The receiving flask rests upon two or three pieces of sheet iron and is heated with an ordinary burner, and ebullition is perfectly regular. From 170 to 180 cubic centimeters of the liquid are distilled in from three and one-half to four hours. The distilled liquid, treated with a few drops of litmus, is titrated by a solution of sulfuric or hydrochloric acid, of which one cubic centimeter corresponds to 0.001 cubic centimeter of nitrogen.

399. Modification of the Kjeldahl Method by Arnold and Wedemeyer.^[264]—For the oxidizing liquid a mixture of three grams of benzoic acid with forty cubic centimeters of H₂SO₄ is employed. After placing in the digestion flask with the nitrogenous body the whole is gently shaken for a few minutes to prevent clotting. The temperature is then raised until acid vapors begin to come off, when one gram of copper sulfate and one gram of mercuric oxid are added; and after ten to fifteen minutes, to avoid foaming, ten to twenty grams of potassium sulfate. The sublimate noticed on the walls of the flask is benzoic acid and does not interfere with the accuracy of the determination.

This method has given good results with the alkaline nitrates, the nitrates of barium, mercury, silver, lead, and with strychnia, ammonia, pyridin, azobenzol, dinitrobenzol, and picric acid.

400. Prevention of Bumping During Distillation.—Daffert has employed the modified kjeldahl method, but found considerable difficulty in using the same owing to the violent bumping of the liquid in the distillation. This was especially the case where the sample contained a large proportion of sand. To overcome this annoyance and danger he devised the following process:^[265]

Fit into the mouth of a large-mouthed distillation flask a stopper having two perforations. Through one of the perforations pass the usual distillation tube, through the other a similar tube connected with a supply of steam. Bring the contents to a brisk boil, after which a small current of steam is turned on, allowing the same to pass in a small stream throughout the distillation. By this means, not only is all danger from bumping avoided, but the time required for the distillation shortened. By the old method it usually requires from fifteen to twenty minutes, whereas the former requires from six to ten minutes.

It is advisable to filter all samples of soils having a large proportion of sand.

401. Determination of Organic Nitrogen by the Soda-Lime Method.—In the description of the method following, the directions of the French Commission of Agricultural Chemists have been taken as the basis of the analytical process.^[266] This method is, in this country, almost superseded by the moist combustion process with sulfuric acid. By reason of its long use, however, and because it is still regarded as the best method by the agricultural chemists of France, Italy, and England, it merits a full description. It is recommended also by Berthelot and André,^[267] by the International Congress of Chemists, held in Paris in 1889, by the Italian chemists, and by the official Belgian method,^[268] in all cases where nitrates are not present in notable quantities.

The nitrogen which is found in soils in the organic state is transformed into ammonia when it is heated with soda-lime. This reaction is the base of the process of analysis which has so long been used for this class of bodies. The analytical process is conducted as follows:

A well-cleaned glass combustion tube, closed at one end, is used. The length of the tube is from thirty-five to forty centimeters. It is filled first to a depth of two centimeters with calcium oxalate; afterwards to a depth of five centimeters with soda-lime in small fragments; afterwards with the mixture to be analyzed; viz., of ten grams of the sample of soil, or twenty grams if poor in nitrogen and organic matters, with soda-lime reduced to a coarse powder. This mixture should occupy a length of about twenty centimeters in the tube. The soil and soda-lime are mixed in a mortar. Afterwards the mortar is rubbed with small quantities of soda-lime, and this, together with the copper boat which has been used in introducing the mixture, is thoroughly washed with the soda-lime, which is poured into the tube until it is filled to within four centimeters of its open extremity. The open end of the tube is then closed with a wad of asbestos packed sufficiently tight to prevent the carrying off of the soda-lime by the gas which may be generated during the combustion. The combustion should be commenced by heating the tube near the open extremity until it is red and carrying the heat progressively towards the part containing the soil mixed with the soda-lime. An ordinary gas combustion furnace should be used and the heat graduated in such a way that the bubbles of gas pass off regularly and not too rapidly. The gas is conducted into a bulb tube containing a decinormal standard sulfuric acid colored with litmus. The combustion is continued until the whole of the organic material is decomposed, care being taken not to raise the combustion tube above a low redness in order to avoid its softening. At the end, however, the temperature of the combustion tube should be raised to a bright red, and the part containing the calcium oxalate should be heated little by little for the purpose of evolving hydrogen, which is used to drive out the last traces of ammonia. After the combustion is completed, and the last traces of ammonia driven out, the standard acid which has received the evolved ammonia is removed, the tube leading to it washed, the wash-water collected with the rest of the liquid and titrated with a standard solution of lime-water, the strength of which has previously been determined against standard sulfuric acid.

402. Preparation of the Standard Sulfuric Acid.—The sulfuric acid to be used in making the standard solutions should be previously boiled for half an hour in a platinum dish and allowed to cool in a desiccator. It should contain 61.25 grams of sulfuric acid in one liter. It is recommended that the flask which holds the sulfuric acid should be one which has been used for a long time for holding concentrated sulfuric acid, in order to avoid any action of the alkali in the glass upon the acid after its strength has been determined. The solution before described is of such strength as to have each cubic centimeter equivalent to one milligram of nitrogen.

For the estimation of the nitrogen in the soil a tenth normal solution should be used, which is prepared by taking 100 cubic centimeters of the normal solution, described above, and diluting to one liter.

Preparation of the Lime-Water.—From 200 to 300 grams of slaked lime are placed in a closed flask of about five liters capacity. This is filled with water and shaken frequently, and left to deposit the matter in suspension. The water which contains the saline particles which may have been present in the lime is then poured off. Fresh water is then poured on and the flask shaken from time to time. To use this lime-water the clear part of it is decanted into a flask, avoiding, as much as possible, access to the air. The flask is closed with a cork carrying two tubes drawn out and bent at a right angle. One of these serves for pouring off the water and the other serves for the entrance of the air. These two tubes are themselves closed by means of a rubber tube carrying a pinch-cock. The strength of the lime-water is fixed by titration with the decinormal standard sulfuric acid.

Preparation of the Soda-Lime.—Six hundred grams of slaked lime in fine powder are saturated with 300 grams of caustic soda dissolved in 300 cubic centimeters of water. The whole is rubbed into a paste and introduced into a crucible which is heated to redness. The contents of the crucible, still hot, are poured out, and rapidly reduced to fragments in a copper mortar in such a manner as to have the pieces about the size of a pea, and without having too much finely powdered soda-lime mixed with it. While the matter is still hot it is placed in a flask and well-stoppered. In order that this reagent should contain no nitrogen it is indispensable to use in its preparation materials which contain no trace of nitrates.

Preparation of the Calcium Oxalate.—In a small copper vessel place 100 grams of oxalic acid and add gradually, bringing it to boiling, enough water to dissolve it. Afterwards place in the solution small portions of slaked lime in a state of powder, constantly testing it until turmeric paper indicates that there is a little lime in excess. It is then evaporated, stirring vigorously on the open fire, and the evaporation is finally finished on a steam-bath. The dried material is placed in a flask and well-stoppered. The oxalic acid which is used in this preparation should be free from every trace of nitrogen.

Preparation of the Litmus Solution.—Five grams of litmus are placed in a flask with a flat bottom. Afterwards a few cubic centimeters of ammonia are added, twenty five grams of crystallized sodium carbonate, and ten cubic centimeters of water. This mixture is left to digest for sometime, with frequent stirring, at a temperature of from 60°–80°. The digestion is finished in about four or five days, during which time, at intervals, a few drops of ammonia are added, sufficient to maintain always the ammoniacal odor. At the end of this time 200 cubic centimeters of water are added and the digestion allowed to continue several days more, still maintaining the solution alkaline with ammonia. A slight excess of hydrochloric acid is added, and the matter which is precipitated is received upon a filter where it is washed several times with cold water and allowed to dry at a low temperature.

For use, from one to two grams of this dry precipitate are dissolved in 100 cubic centimeters of alcohol, and there is thus obtained a litmus solution of extreme sensibility.

403. Treatment of Soil Containing Nitrates.—Nitrates exist in small quantities in all arable soils. When treated for nitrogen by the soda-lime method above described, a part of the nitric nitrogen is changed to the state of ammonia, while another part escapes estimation altogether, causing an error which it is important to point out. When the soils contain only small quantities of nitrates this error is insignificant and does not affect sensibly the results, but in the case of earths rich in nitrates it is necessary first to eliminate them before the determination of the nitrogen by the soda-lime method. The operation is carried on as follows:

Twenty grams of the soil are washed on a small funnel, furnished with a plug of asbestos, with small quantities of pure water, in such a way as to cause thirty to forty cubic centimeters of water to pass through. The whole of the nitrate is thus removed. The soil is now dried and submitted to analysis by the soda-lime method as just described. There are removed with the nitrate only small traces of organic nitrogen, too small to influence the results of the analysis. If, however, it is desired to remove altogether this slight cause of error, evaporate the wash-waters, above described, to two or three cubic centimeters; add a few drops of a concentrated solution of ferrous chlorid and as much hydrochloric acid, and boil some minutes in order to drive off, in the state of nitrogen dioxid, all the nitric acid. The residue is evaporated to dryness and contains the traces of organic nitrogen. This is added to the soil which is to be treated by the soda-lime method.

404. Müller’s Method.—The determination of nitrogen in the soil by soda-lime is carried on as follows by Müller:^[269]

Fifteen grams of fine earth, dried and mixed with a little sugar, are mixed with thirty grams of soda-lime in powder. The bottom of the combustion tube contains a little moist soda-lime, which is heated at the end of the operation at the same time that a current of pure hydrogen is made to pass through it, and the temperature of the tube is raised, little by little, to a distinct redness. The contents of the receiving bulbs are distilled, after the addition of water and soda, in the same apparatus which served in the estimation of nitrogen, by the kjeldahl method; the determinations and titrations are made also under the same conditions.

Blank determinations are also made under the same conditions to determine the amount of correction to be made by the two methods. Soda-lime, heated with pure sugar, gave 0.0002 gram of nitrogen for a total weight of fifty-five grams of the soda-lime contained in the tube. The fuming sulfuric acid gave 0.0011 cubic centimeters of ammoniacal nitrogen for the volume of thirty cubic centimeters.

The numbers obtained by the kjeldahl method in general, are lower than those obtained by the soda-lime method when no stearic or benzoic acid is used. The numbers obtained when stearic acid alone was used were sometimes inferior to those obtained by the soda-lime method. The numbers obtained when benzoic acid is used are, in general, about the same as those obtained by the soda-lime method.

It would seem that the double distillation, outlined above, for the kjeldahl method, would not be necessary if due care were exercised in the first distillation. This variation, therefore, seems to be unnecessary.

In the soda-lime method, time would be saved by the reception of the ammonia in standard acid, and its titration in the usual way, unless a further purification of the nitrogenous products of the combustion by the final distillation be desired.

405. Volumetric Determination of the Nitrogen.—Instead of separating the nitrates, the total nitrogen in the soil can be determined directly by the classic method of Dumas, which consists in bringing the whole of the nitrogen into a gaseous state and afterwards measuring its volume.

The following method illustrates the general principles of the determination:

A glass combustion tube closed at one end, about one meter in length, is selected. In the bottom of this tube is placed some potassium bicarbonate in a crystalline form, in small pieces, filling the tube to a distance of about twenty centimeters. Afterwards copper oxid is placed to the depth of ten centimeters and finally a mixture of from twenty to thirty grams of the earth with thirty to forty grams of copper oxid in a fine state of subdivision, and about ten grams of metallic copper obtained by reducing the copper oxid by hydrogen. Next the tube is filled with copper oxid to a depth of from twenty to twenty-five centimeters, and afterwards with reduced copper to the depth of at least twenty-five centimeters, and after this another layer of copper oxid of about five centimeters, and finally a plug of asbestos. The combustion tube is closed with a stopper carrying a glass tube of about ninety centimeters in length, of which the extremity, bent into the form of a ᥩ, extends to a mercury trough. The glass combustion tube is surrounded with brass gauze, except that part which contains the potassium bicarbonate. The beginning of the operation consists in heating the tube to decompose a part of the potassium bicarbonate, until the whole of the apparatus is filled with carbon dioxid. In order to determine that the whole of the air has been expelled and that the apparatus is entirely filled with carbon dioxid, a part of the gas which is disengaged, is received into a jar filled with mercury, in which a little potash-lye has been placed. If the gas is entirely absorbed by the potash, so that there remain only unappreciable particles, the tube can be regarded as completely free of air. When assurance is given that the air is all out of the apparatus, a jar of about 300 cubic centimeters capacity, filled with mercury and containing from thirty to forty cubic centimeters of a solution of potash of a density of 42° baumé, is placed over the outlet tube. The combustion is commenced by heating the anterior part of the tube, avoiding the heating of the part containing the earth. When the first part of the tube has reached the red stage the part containing the earth is gradually heated in order to obtain a gentle evolution of gas. The temperature of the tube is carried to redness and the heating gradually carried back toward the closed extremity, but avoiding raising the temperature of the part containing the potassium bicarbonate. The red heat is continued as long as bubbles of gas are discharged into the reservoir. When the evolution of gas has ceased the apparatus is again filled with carbon dioxid for the purpose of driving out the last traces of nitrogen, by heating again the part of the tube containing the potassium bicarbonate. The evolution of the carbon dioxid should be maintained for about fifteen minutes. At the end of this time all the nitrogen will be found in the receiving jar. Sometimes a small quantity of nitrogen dioxid is formed incidentally in the operation. After waiting for a quarter of an hour, in order to permit all the carbon dioxid which may have escaped into the reservoir to be completely absorbed, the receiving jar is carried to a water-basin and the mercury allowed gradually to escape; its place being taken by the water. The gas is then transferred into an azotometer where its volume and temperature are read in the usual way.

In order to absorb any nitrogen dioxid which may be admixed with the nitrogen itself, a little crystal of ferrous sulfate is introduced. The reservoir containing the nitrogen is carried to the mercury trough, and the water which it contains is nearly all run out in such a way as to be replaced with mercury, great care being exercised to avoid any escape of gas. Afterwards there is introduced over the mercury a crystal of ferrous sulfate and the azotometer is shaken until this crystal is dissolved by the water which it still contains. It is then allowed to remain for twenty hours. At the end of this time the nitrogen dioxid is absorbed and the volume of the gas is again read as before. One-half only of the total loss should be subtracted, since the volume of the nitrogen dioxid is twice the volume of the nitrogen itself. For the practice of this method, in connection with the use of a mercury pump, the directions which will be given under fertilizers may be consulted.

406. Estimation of Ammonia.—Ammonia exists ordinarily only in very small quantities in the soil, since it is incessantly transformed into nitrate or diffused in the air. Nevertheless, it is sometimes interesting to determine its quantity.

The method of determining the ammonia in soils is one of extreme delicacy on account of the small proportion therein, and the difficulty of expelling it without at the same time converting some of the organic nitrogen into ammoniacal compounds. The various methods employed for this purpose may be classified as follows:

1. Treatment of the soil with soda-lye in the cold, and the absorption of the ammonia given off by standard sulfuric acid.

2. The method of Boussingault, which consists in replacing the soda-lye with magnesia and distilling the ammonia at a boiling temperature, absorbing the distillate in a standard acid.

3. A modification of the above method, due to Schloesing, which consists first in extracting the ammonia by hydrochloric acid and subjecting the extract to distillation with magnesia.

4. The method of Knop consists in treating the soil in a closed cylinder with soda-lye containing bromin. The ammonia set free by the lye is decomposed in the presence of bromin into free nitrogen and hydrochloric acid. The nitrogen is collected and measured in an azotometer. The brom-soda-lye is prepared by dissolving 100 grams of sodium hydroxid in 1,200 cubic centimeters of water and adding twenty-five cubic centimeters of bromin.

5. The process described under 4, as shown by Baumann,^[270] does not give accurate results and it has been modified by him as follows: Two hundred grams of soil are treated with 100 cubic centimeters of dilute hydrochloric acid (one part acid and four of water) free of ammonia; 300 cubic centimeters of ammonia-free distilled water are added and the whole digested for two hours with frequent stirring. If a soil contain much calcium carbonate larger quantities of acid must be used. Two hundred cubic centimeters of the filtrate are placed in an evolution flask, connected with an azotometer, with five grams of freshly burned magnesia. The mixture is then oxidized as follows: Ozone is generated by adding three parts by weight of sulfuric acid to one part of dry and powdered potassium permanganate. A stream of air is drawn through the ozone generator by an aspirator, and the ozone is conducted into a flask containing the hydrochloric acid extract of the soil and magnesia. The oxidation is completed in about ten minutes. The mixture is then brought into the evolution flask of the azotometer and the nitrogen set free and measured in the usual way.

It has been shown that if asparagin or glutamin be present in the soil they are decomposed by the soda-lye and the results obtained are too high. It has been further proved that soils which contain a notable quantity of humus give, with soda-lye in the cold, a practically continuous evolution of ammonia. Moreover, soils which are rich in humus and which have been treated by distillation with magnesia give, on subsequent treatment with soda-lye, considerable additional quantities of ammonia.

Comparison of Methods of Estimating Ammonia.—Baumann has determined the ammonia-nitrogen in various soils by the soda-lime method; distillation of the hydrochloric acid extract with magnesia, and the azotometric method modified as indicated above. These methods will be designated as 1, 2, 3, respectively in the following table.

From the above figures it is seen that the method usually attributed to Schloesing gives uniformly higher numbers than either of the other processes, while the third gives slightly higher values than the second.

407. The Magnesia Distillation Process.—If a sample of soil be distilled directly with magnesia and water, there is danger on the one side of not extracting all the ammonia, by reason of the absorbing power of these bodies, and on the other, of transforming into ammonia the nitrogen of the organic matters. It is therefore preferable to separate the ammonia from the soil in the form of chlorid, and to subject this extract to distillation.

In fifty grams of the soil the humidity is determined by drying at 100° until there is no further loss of weight. The quantity of moisture being known, 200 grams of soil are taken and moistened with water, and then there is added, in small portions, some dilute hydrochloric acid, shaking frequently until the whole of the calcium carbonate present is decomposed. The liquor should remain acid at the end of the operation, but without containing a notable excess of acidity. Knowing beforehand the quantity of moisture contained in the 200 grams, water is added until the total quantity shall be equal to 500 cubic centimeters. The whole is then shaken and allowed to repose, and filtered rapidly, covering the funnel with a glass vessel and receiving the liquid which runs through in a flask with a narrow opening. Two hundred and fifty cubic centimeters of this liquor, or mixture, represent 100 grams of earth of known humidity. This quantity is introduced into a flask for determining the ammonia and five grams of calcined magnesia added. Before commencing the distillation, assurance should be had that the magnesia has completely saturated the acid in excess, and that the liquor is alkaline.

If, by chance, the liquor should be still acid it would be necessary to add sufficient magnesia in order that the reaction should be manifestly alkaline. Afterwards the distillation is begun and the ammonia is received in an appropriate vessel containing one-tenth normal sulfuric acid and titrated in the usual way, or nesslerized.

Inasmuch as the quantities of ammonia contained in the earth are generally very small it is necessary to be very particular in order to avoid errors. The distilled water which is employed should be deprived of all traces of ammonia by prolonged ebullition, and the hydrochloric acid should be distilled in the presence of a little sulfuric acid. The treatment with hydrochloric acid is for the purpose of destroying the absorbing properties of the soil for ammonia, and to permit this last to enter into solution as chlorid. When there is need of very great precision it is convenient to make a blank operation with the hydrochloric acid and water which are employed, in order to make a correction for the traces of ammonia which these reagents may contain.

408. Estimation of Ammoniacal and Amid Nitrogen by the Method of Berthelot and André.^[271]—Heat one hundred grams of earth for thirty hours on a steam-bath with about .500 cubic centimeters of dilute hydrochloric acid (fifteen grams of hydrochloric acid to 500 cubic centimeters of water). At the end of this time throw the contents of the flask on a filter and wash with hot water until acid reaction has ceased. Determine both the ammoniacal and amid nitrogen in the soluble, and the total nitrogen in the insoluble portion, the ammoniacal by distillation with magnesia, and the amid and total with soda-lime.

Example. A soil contained 0.1669 per cent total nitrogen. Of this there were obtained:

Treatment of the Insoluble Portion.—Treat the part insoluble in hydrochloric acid with a three per cent solution of potash on a steam-bath for thirty hours. Estimate the nitrogen remaining insoluble, from which the part dissolved can be determined by difference. The potash will dissolve usually about two-thirds of the remaining nitrogen.

About ninety per cent of the total nitrogen present in an arable soil will be rendered soluble by successive treatment with acid and alkali. The reverse treatment will give practically the same result. It is therefore immaterial, from an analytical standpoint, whether the acid or alkali be used first.

409. Estimation of Volatile Nitrogenous Compounds Emitted by Arable Soil.—The following method, due to Berthelot and André,^[272] may be practiced:

Porcelain pots, containing one kilogram of soil, are placed under bell-jars of fifty liters capacity adjusted to glass dishes designed to receive the waters of condensation.

During the first period the pots are to be sprinkled from time to time, during the duration of the experiment, through the upper tubulature, so as to prevent the soil from becoming dry. The water is partly condensed on the sides of the bell-jar. It is removed each week through the inferior tubulature, treated with a little dilute sulfuric acid, and preserved for further study. A small vessel containing dilute sulfuric acid is placed near the porcelain pot for the purpose of collecting, as far as possible, the evolved ammonia.

During the second period the pots are not sprinkled, the soil becomes dry and there is no longer any condensation of water on the walls of the bell-jar. The two periods should include about five months, from May to October.

At the end of the second period the following determinations are to be made:

1. The ammonia absorbed by the dilute sulfuric acid.

2. The ammonia set free by distillation with magnesia, such as may have accumulated in the condensed water.

3. The organic nitrogen contained in the latter after elimination of the ammonia. This is determined by adding a slight excess of acid, evaporation to dryness, and combustion with soda-lime, or by moist combustion with sulfuric acid.

Example:

Earth Employed.—One kilogram of sandy clay containing total nitrogen, 0.09 gram. Nitrogen in sprinkling water, 0.000048 gram.

Nitrogen in Exhaled Products.—

Conclusions.—The exhalation of nitrogenous compounds takes place with a certain relative activity, about two milligrams in two months and a half, as long as the soil is kept moist by sprinkling.

In the second period, without sprinkling, the exhalation is reduced to a mere trace.

The vessel containing the dilute sulfuric acid placed near the porcelain pot absorbs only about one-half of the ammoniacal nitrogen set free. The nitrogen emitted under other forms than ammonia is, in every instance, greatly superior in quantity, and this is the most important of the observed phenomena. This is true at least with the kind of soil with which the experiment was made. With arable soil containing twenty times as much nitrogen as the soil described above this order is reversed,^[273] the ammoniacal prevailing over the non-ammoniacal nitrogen volatilized.

These phenomena are doubtless greatly influenced in soil under culture by microbes, and the lowest orders of vegetation to which are doubtless due the traces of non-ammoniacal volatile nitrogenous compounds, a sort of vegetable ptomaines.

410. General Conclusions.—In the light of our present knowledge concerning the methods of nitrogen determination in the soil in the form of organic compounds and ammonia, moist combustion with sulfuric acid is to be preferred to the older soda-lime process. For the nitrogen combined as ammonia, the extraction of the sample with hydrochloric acid and subsequent distillation with an excess of freshly calcined magnesia, are recommended. For the study of the progressive decomposition of the nitrogenous compounds, the various processes devised by Berthelot and André are the best.

The origin of the nitric acid in the soil, the methods of studying the various nitrifying organisms, and of estimating the nitric acid produced, will form the subject of the next part.

Note.—At the Eleventh Annual Convention of the Association of Official Agricultural Chemists, held in Washington, August 23, 24 and 25, 1894, the following process of soil extraction was adopted as the official method:

Preparation of the Sample.—500 grams or more, of the air-dried soil, which may be either the original soil or that which has been passed through a sieve of coarser mesh, are sifted upon a sieve with circular openings one-half millimeter in diameter, rubbing, if necessary, with a rubber pestle in a mortar, until the fine earth has been separated as completely as possible from the particles that are too coarse to pass through the sieve. The fine earths thoroughly mixed and preserved in a tightly stoppered bottle from which the portions for analysis are weighed out.

The coarse part is weighed and may be subjected to further examination, (as in Bulletin 38, Div. of Chem., pp. 65, 75 and 200.) It may sometimes be necessary to wash the soil through the one-half millimeter sieve with water, in which case proceed as directed on pp. 65 and 75 of the above Bulletin. The use of water is to be avoided whenever possible.

Determination of Moisture.—Heat two to five grams of the air-dried soil in a flat-bottomed, tared platinum dish; heat for five hours in a water-oven kept briskly boiling; cover the dish, cool in a desiccator, and weigh.

Repeat the heating, cooling, and weighing at intervals of two hours till constant weight is found, and estimate the moisture by the loss of weight. Weigh rapidly to avoid absorption of moisture from the air. An air-bath must not be used in this determination.

Determination of Volatile Matter.—The platinum dish and soil used to determine moisture are used also to determine volatile matter. Heat the dish and dried soil to full redness until all organic matter is burned away. If the soil contain appreciable quantities of carbonates, the contents of the dish, after cooling, are to be moistened with a few drops of a saturated solution of ammonium carbonate, dried and heated to dull redness to expel ammonium salts, cooled in the desiccator and weighed.

The loss in weight represents the organic matter, water of combination, ammonium salts, etc.

Extraction of Acid-Soluble Materials.—In the following scheme for soil analysis it is intended to use the air-dried soil from the sample bottle for each separate investigation. The determination of moisture, made once for all on a separate portion of air-dried soil, will afford the datum for calculating the results of analysis upon the soil dried at the temperature of boiling water. It is not desirable to ignite the soil before analysis or to heat it so as to change its chemical properties.

The acid digestion is to be performed in a flask so arranged that the evaporation of acid shall be reduced to a minimum, but to take place under atmospheric pressure and at the temperature of boiling water. Any flask resistant to acids is suitable, but it is not necessary to use a condenser, as a simple bohemian glass tube eighteen inches in length will answer the purpose of preventing loss of acid. Where it is not desired to determine sulfur trioxid, an erlenmeyer fitted with a rubber stopper and hard glass tube will answer. The flask must be immersed in the water-bath up to the neck or at least to the level of the acid and the water must be kept boiling continuously during the digestion.

In the following scheme, ten grams of soil are taken, this being a convenient quantity in most soils, in which the insoluble matter is about eighty per cent. If desired, a larger quantity of such soil may be taken, using a proportionately larger quantity of acid and making up the soil solution to a proportionately larger volume. In very sandy soils, where the proportion of insoluble matter is ninety per cent or more, twenty grams of soil are to be digested with 100 cubic centimeters of acid and the solution made up to 500 cubic centimeters or a larger quantity may be used, preserving the same proportions. It is very important that the analyst assure himself of the purity of all the reagents to be used in the analysis of soils before beginning the work.

Acid Digestion of the Soil.—Place ten grams of the air-dried soil in a 150 to 200 cubic centimeter bohemian flask, add 100 cubic centimeters of pure hydrochloric acid of specific gravity 1.115, insert the stopper with condensing tube, place in a water or steam-bath and digest for ten hours continuously at the temperature of boiling water, shaking once each hour. Pour the clear liquid from the flask into a small beaker, wash the residue out of the flask with distilled water on a filter adding the washings to the contents of the beaker. The residue after washing until free of acid, is to be dried and ignited as directed below. Add one or two cubic centimeters of nitric acid to the filtrate, and evaporate to dryness on the water-bath, finishing on a sand or air-bath to complete dryness; take up with hot water and a few cubic centimeters of hydrochloric acid, and again evaporate to complete dryness. Take up as before, filter and wash thoroughly with cold water or with hot water slightly acidified at first with hydrochloric acid. Cool and make up to 500 cubic centimeters. This is solution “A.” The residue is to be added to the main residue and the whole ignited and weighed, giving the insoluble matter.

The determination of the various components of the solution remains essentially as described in the provisional methods of the Association which have already been given.

It is directed that all results of soil analysis be calculated on the basis of the sample dried to constant weight at the temperature of boiling water.