Nitrogenous Composition of Ammoniated Peat and Related Products

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Nitrogenous Composition of Ammoniated Peat and Related Products

L. A. PINCK, L. B. HOWARD, AND G . E. HILBERT Fertilizer Investigations, Bureau of Chemistry and Soils, Washington, D. C.

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A chemical study was made of ammoniated peat, lignin, dextrose, and starch. These products were fractionated by various solvents, and the distribution of the nitrogen was determined. Urea was found to be the only common product present in large quantities. It, is apparently formed from carbon dioxide liberated during the ammoniation. The remaining organic nitrogenous compounds present a rather complex mixture in which no single individual occurs in appreciable amounts. A considerable amount of a complex, highly insoluble, nitrogenous polymerization product is formed in the ammoniation of these related materials. The results of this work indicate that both the carbohydrate and lignin units of peat are extensively ammoniated.

CHOLL and D a v i s (6) have shown that the ammoniation of peat yields a nitrogenous p r o d u c t t h a t h a s potentialities as an organic fertilizer. 4 t the suggestion of Kunsman and Davis of this laboratory, a chemical investigation of this material was made with the aim of elucidating the nature of the major products formed as well as determining, if possible, the ingredients of peat that have suffered ammoniation. Except for private information from Davis that crystals of ammonium carbamate had been r e p e a t e d l y observed a t the outlet of the reaction bomb, which suggested t h e possibility of the presence of urea, no knowledge concerning the nature of the products formed was available in the literature. Peat is a decomposition product of organic matter consisting essentially of carbohydrates and lignin. The extent to which these units have been modified in peat depends greatly upon the various natural processes to which the material has been subjected; invariably, however, the evolution of peat is accompanied by the formation of acid products (humic acid). Because peat is a complex material and because it is reasonable to expect that its constituents would behave differently toward ammonia, it was felt that desirable information concerning the nature of the ammoniation would be obtained by carrying out simultaneous investigations on the ammoniated parent substances-for example, ammoniated lignin and various types of ammoniated carbohydrates. It seemed that the isolation of pure products would be favored from these materials; moreover, such information would be useful in attempting to isolate similar products, if present, from the more complex ammoniated peat. A similar method of attack was used in the study of the different ammoniated products. I t consisted essentially of extracting them with various solvents such as water, alcohol, acids, alkali, etc. The various fractions were investigated and finally analyzed for different types of organically bound nitrogen by a modification of the well-known Van Slyke procedure as adapted by Morrow and Gortner (4) for soil analysis, and the results obtained on the different fractions and products were compared. On theoretical grounds, since some knowledge of the active groups in peat is available, one can anticipate to a certain extent the types of nitrogenous products which could possibly form on ammoniation. Although this

*** affords a useful working hypothesis, such a discussionwould hardly be appropriate here. All the ammoniated products studied were kindly prepared by W. Scholl in the manner described by Scholl and Davis, by treating the sample with liquid ammonia in the ratio of 1 to 1 a t 180" C. for 24 hours. The raw materials used in the ammoniation experiments were peat from Capac, Mich., Merck's anhydrous dextrose, Mallinckrodt's soluble starch (indicator grade), and cellulose in the form of nonabsorbent cotton. The lignin used in this work was a much appreciated gift of Max Phillips.

The cellulose after ammoniation was pale yellow and in texture was quite similar t o the original raw material; since the nitrogen content was very low (0.8 to 2.2 per cent), it was not further investigated. The ammoniated products from pt, lignin,l and starch were dark brown amorphous solids. hey were finely powdered and dried to constant weight in a vacuum desiccator over sulfuric acid or phosphorus pentoxide; after this treatment they were quite hygroscopic. With ammoniated dextrose this procedure was not possible since the reaction mixture was partially liquid. The materials were then extracted with various solvents until no further appreciable amount of soluble substance was removed. When practical, the insoluble matter as well as the residue resulting from concentrations of the extract was dried to constant weight at room temperature, except when otherwise noted, and the nitrogen content was determined by the Kjeldahl method. In certain cases the fractions resulting from the hydrochloric acid treatments were also analyzed for chlorine. The analyses were kindly carried out by Mrs. E. K. Rist.

Ammoniated Peat ETHEREXTRACT. A 200-gram specimen of ammoniated peat was extracted with three I-liter portions of ether over a total period of 12 days. The ether extract obtained after filtration was distilled under diminished pressure and yielded a dark brown gummy residue. After air-drying, the ether-insoluble WATER EXTRACT. portion was heated in an oven a t 70" C. for 2 days, and then thoroughly extracted with three 1.5-liter portions of water for 20-hour periods a t the boiling point, using a reflux condenser. The combined filtrates and washings were concentrated to a definite volume and aliquots removed for analysis. Further concentration of the extract yielded a dark brown sirup. 1 One hundred gram8 of either peat or lignin yield 90 t o 100 grama of ammoniated product.

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ALCOHOLEXTRACT.The water-insoluble fraction was dried a t 70' C. for 3 days and extracted with 1250 cc. of 95 per cent alcohol for 20 hours under reflux. This extraction was repeated. Removal of the alcohol yielded a black gummy residue. DILUTEHYDROCHLORIC ACIDEXTRACT. After drying the fraction insoluble in alcohol, it was extracted twice with 1 liter of 2 per cent hydrochloric acid. The residue was washed well with about 1200 cc. of water, when it was practically free from acid. The washings and extracts were combined and concentrated under diminished pressure to dryness; the residue was freed from acid by repeated addition of water and subsequent removal by distillation. The soluble residue was dried over sodium hydroxide before sampling for analysis. STRONGHYDROCHLORIC ACID EXTRACT. The insoluble fraction was extracted twice by boiling for 20 hours with 1 liter of 20 per cent hydrochloric acid. After washing the insoluble residue thoroughly with about 1500 cc. of water, the extracts and washings were combined, concentrated, and dried, in the same manner as that described for the extraction with 2 per cent hydrochloric acid. The insoluble fraction was powdered until it passed a 100-mesh sieve and then digested with boiling water for 20 hours in order t o remove chloride ion more thoroughly. After filtration the insoluble product was washed with hot water and dried to constant weight a t 85" C. These latter washings were combined and concentrated; as the residue was small, it was discarded. SODIUMHYDROXIDE EXTRACT.The fraction insoluble in 20 per cent hydrochloric acid was then digested with 10 per cent sodium hydroxide, and a determination was made of the volatile nitrogen as well as that extracted. A 1-gram sample, together with 400 cc. of 10 per cent sodium hydroxide, mas placed in a Kjeldahl flask, and the mixture was cautiously distilled over a period of 10 hours; water was added from

Grams Combined N Extd. from Ammoniated Peat Containing Percentage of 24.5 g. N Total N 0.7 3.0 8.10 33.2 0.9 3.8 1.6 6.5 1.3 5.2 2.8b 11.3 15.4 63.0 8.8 35.9

a Includes an estimate of the 1088 of volatile nitrogenous compounds during the extraction. b Includes the volatile nitrogen.

TABLE11. DISTRIBUTION OF NITROQEN IN EXTRACTS OF AMMONIATED PEAT (In per cent) -Consecutive Types of N Ether Water Humin I 0.7 0.6 Water-sol. 2.3 32.4 Amide 1.4 24.9b Humin I1 0.0 0.5 Total basic" 0.2 1.6 "Total nonbasic" 0.6 5.3 "Primary amino nonbasic" 0.1 1.2 "Nonamino" 0.5 4.1

Extracts 2 3 H I 1.1 0.6 2.6 5.9 1.2 2.9 0.1 0.6 0.7 1.8 0.5 0.8

;',"p-

0.1 0.4

0.2 0.6

203 H I 0.4 4.8 2.5 0.2 1.2 0.8 0.2 0.6

20% HC1

Ext. of AmmoniTotal ated Peat 3.4 48.0 46.4 33.0 32.9 1.4 2.3 3.7 5.5 8.0 7.9 1.8 6.2

LthaF

EItmotion

-

mtmotion

I

A

soluble I 1 5 n W N

I

Soluble 8 b x 10.8% A

(1) TU. d u e i i tha d i t h n n c e b e t n a u the e@$ HC1 and 10%N ~ O Binsoluble material. FIGURE1. F R A C T I O N A T I O N OF AMXONIATED PEAT

TABLEI. FRACTIONATION OF AMMONIATED PEAT

Solvent Ether Water Alcohol 2% HCl 2 0 7 HC1 10% NaOH Total soluble Insoluble

Watar

IluolUblc IS-%

441

1.7 6.2

a The sum of the ercentage of nitrogen in humin I and in the fraction insoluble in 20% H& is 53.6% of the total. b Includes 15.5y0 urea nitrogen and 2.4Y0 ammonium nitrogen.

time t o time to keep the mixture a t about its original volume. The distillate was collected in a measured quantity of standardized acid and the liberated base was determined by backtitration as ammonia; 6.1 nig. volatile nitrogen were found.

The data obtained in this study have been corrected to account for samples removed for analytical purposes and are presented in Figure 1 and Table I. ISOLATION OF UREA. Because of the partial hydrolysis of urea in the water-soluble fraction, the nitrogen content varied with the conditions of heating; and on long periods of digestion a deposit of colorless crystals (ammonium bicarbonate and/or carbamate) was formed in the condenser tube. As one might expect, the distillate obtained from concentration contained appreciable quantities of ammonia. The sirupy residue obtained from the aqueous extract, after standing for several days, deposited a mass of colorless needles contaminated with a dark brown amorphous substance. This material was taken up in ethyl alcohol, filtered, and concentrated by evaporation. The residue was recrystallized several times from butyl alcohol until free from colored impurities. The crystals melted a t 132' C. and, when mixed with pure urea, melted a t 132.5" C.; a test with urease was positive. The urea and ammonia (probably present in the form of salts) content of the dried ammoniated peat was kindly determined by Mr. Yee by a modification of the urease method of Fox and Geldard (3). The samples for analysis were prepared by extracting about 5.0 to 7.5 grams of the finely pulverized ammoniated peat with 50 cc. of cold water by vigorous mechanical shaking for approximately one hour. The liquid was clarified, and aliquots of 5 cc. were taken for analysis. The urea and ammonium nitrogen were found t o be 15.5 and 2.4 per cent, respectively, of total original nitrogen. DISTRIBUTION OF NITROGEN IN VARIOUS EXTRACTS. In general, the distribution of the different types of nitrogen in

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the various extracts of ammoniated peat was studied by using a modification of the method of Van Slyke as adapted by Morrow and Gortner for the study of the nature of the organic nitrogen in soil. As the nitrogen content of the extracts of the substances used in this work is much higher than that obtained in soil extracts, it was found convenient to alter the quantities of materials used in their procedure. The classification of the various types of nitrogen has also been somewhat changed to suit better this particular problem.

Ether -

gltraotion

Water -

amotion

bloohol -

Brtraotion

(11 N i t r o e n value Calculated by differenoe.

FIGURE 2. FRACTIONATION OF AMMONIATED LIGNIN

The details of the method for determining the distribution of the nitrogen in the ether, alcohol, and 2 and 20 per cent hydrochloric acid extracts in which urea is absent are as follows : A sample containing 0.15 to 0.25 gram nitrogen was digested with about 100 cc. of boiling 20 per cent hydrochloric acid for 48 hours under reflux. The acid was removed under diminished pressure; a t the final stages of the distillation, water was added to facilitate more effective removal of the acid. The residue was taken up in distilled water and filtered into a 250-cc. volumetric flask. The insoluble residue was analyzed for nitrogen and classified as “humin I.” After anal zing 10- or 25-cc. aliquots of the aqueous filtrate for “water-solutle” nitrogen, duplicate 100cc. aliquots were treated with 1 gram of magnesium oxide and 60 cc. of ethyl alcohol, and distilled a t reduced pressure at 50” to 60’ C . for 1.5 to 2 hours into measured quantities of standard acid. The ammonia trapped represented the “amide” nitrogen.’ The alkaline mixtures in the distillation flasks were filtered and the residue was washed with hot water until free from ch,l,orides; the nitrogenous residue was designated as “humin 11. The duplicate filtrates were combined, acidified, and concentrated at reduced pressure to about 100 cc. After treating with 9 cc. of concentrated hydrochloric acid and heating to boiling, 5 grams of phos hotungstic acid were added, the heating was continued for an [our, and the mixture was allowed to stand for 2 days. The use of a small fritted glass filter for collecting and washing the insoluble phosphotungstate was a marked improvement over the original method. Approximately 100 cc. of an aqueous solution containing 2.5 er cent of phosphotungstic and 3.5 per cent of hydrochloric aci& were used for washing. The precipitate was effectively transferred into a Kjeldahl flask b flushing the glass 6lter with water and finally washing with a $lute solution of alkali. The nitrogen present in the phosphotungstate precipitate was designated as “total basic” nitrogen. On account of the relatively small amount of basic nitrogen present in the ammoniated peat, no attempt was made to fractionate it further. The filtrate from the phosphotungstate precipitate was made alkaline to phenolphthalein and then acidified with acetic acid (litmus indicator). The solution was concentrated at reduced pressure, filtered into a 100-cc. flask, and made up to volume. Aliquots of 25 cc. were analyzed for “total nonbasic” nitrogen, 2 The classification of types of nitrogen as “amide,” “total basic,” ”total nonbasic,” and “primary amino nonbasic” is, of courne, arbitrary.

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and 10-cc. aliquots were analyzed by the method of Van Slyke for “primary amino nonbasic” nitrogen. As the presence of urea in the water-soluble fraction and in the 20 per cent hydrochloric acid extract of the ammoniated products resulted in spurious values3for the different types of nitrogen when determined by the above procedure, it was necessary in the investigation of these fractions to modify it markedly. Various methods with the object of effecting complete hydrolysis were unsuccessful. For example, solutions of urea in 20 per cent and 0.25 N hydrochloric acid, after boiling for 48 hours, still contained 85 and 50 per cent unchanged urea, respectively. Another method investigated involved treatment of the neutral extract with urease prior to the amide distillation. This, of course, gave reliable values for the amide nitrogen but, owing t o the added protein, the other fractions were increased appreciably. The Fosse method for the determination of urea (8) was finally adopted as one of the steps in the procedure. In this case the urea, remaining unhydrolyzed after the initial extraction, is separated with xanthydrol. This modification may also be useful in the study of other complex organic nitrogenous mixtures containing urea. The method adopted is as follows: The alkaline filtrate obtained in the separation of humin 11, subsequent to the amide determination,4 was treated with about 5 to 10 CC. of concentrated hydrochloric acid to inhibit hydrolysis of urea and distilled a t reduced pressure to dryness. The dry residue was taken up in 90 cc. of 80 per cent acetic acid, a few crystals of sodium acetate were added, and the urea was precipitated by the addition of 10 cc. of methyl alcohol containing 0.5 gram of xanthydrol. After standing for several hours, the ureaxanthydrol product was collected on a filter, washed with methyl alcohol, dried and weighed. The urea nitrogen was calculated from the weight of this precipitate and added to the amide previously determined. The filtrate and washings were then concentrated to dryness at reduced pressure, and the residue was thoroughly triturated with about 100 cc. of hot 3 per cent hydrochloric acid and filtered from uncombined xanthydrol. The solution contained the “basic” and “nonbasic” nitrogen, and was treated with phosphotungstic acid and analyzed for the other types of nitrogen in the usual manner. The analytical data of the various extracts and of the one obtained from a 48-hour digestion of ammoniated peat with 20 per cent hydrochloric acid are presented in Table 11. The water extract used for analysis was obtained by a 24-hour digestion under reflux of a 1 per cent suspension of ammoniated peat and not the original aqueous extract described in Figure 1, since the latter had lost nitrogen because of too long and drastic treatment. All values presented in Table 11,as well as those of Tables IV, VI, and VI1 are percentages of nitrogen of that in the original ammoniated product. AMMONIATEDLIGNIN.^ This was extracted with ether, water, 2 per cent hydrochloric acid, and alcohol, in a manner similar t o that described for ammoniated peat. The extraction data are presented in Figure 2 and Table 111. For a comparison between ammoniated peat and ammoniated lignin, the data on the distribution of the nitrogen in their respective water extracts and in the extracts obtained from the 48-hour digestion with 20 per cent acid were considered to be sufficient. For this work a specimen of ammoniated lignin containing 7.0 per cent nitrogen was used. A cold water extract was prepared for the determination of urea and ammonia. The data for the different types of nitro:The amide nitrogen was low and the unhydrolyzed urea was accounted for principally in the “nonbasic” fraction. 4 The amide was determined before the precipitation of urea because, as pointed out by Fosse, the presence of ammonia interferes with the urea determination. 6 The ammoniation of lignin with aqueous ammonia has recently been reported by Phillips. Goss, Brown, and Reid, J. Wash. Acad. Sei., 24, 1 (1934). They were interested in ita use aa a fertilizer and no chemical atudy waa reported.

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gen found in the hot water and 20 per cent hydrochloric acid extracts are presented in Table IV. From the results it appears that a 20 per cent hydrochloric acid extraction of ammoniated lignin is equivalent to all the other extracts, excluding that of alcohol. This is in accord with the fact that, after a 48-hour digestion with 20 per cent acid, only 13.2 per cent of the nitrogen in the alcohol extract was water-soluble. The data show that considerably more soluble material was removed from ammoniated lignin than from ammoniated peat. For example, the solvents extracted 82 per cent (containing 87 per cent of the total nitrogen) of the former and only 35 per cent (containing 48 per cent of the total nitrogen) from the latter. ~~

TABLE 111.

~

FRACTIOXATION O F

Solvent Ether Water 2To HC1 Alcohol Total soluble Insoluble

~

~~

AMMONIATED IJIGXIN

Grams Combined N Extd. from Ammoniated Lignin Containing 1.73 g. N 0.04 0.77" 0.22 0.47 1.51 0.20

Percentage of Total N 2.5 44.6 19.6 27.3 87.0 11.7

a Includes an estimate of the loss of volatile nitrogenous compounds during the extraction.

AMMONIATED DEXTROSE.~ The ammoniation product from 700 grams of dextrose remaining in the glass capsule which contained the reactants in the bomb could be roughly divided into three parts: an upper layer of a mobile, very dark colored liquid; a small middle layer of almost black, viscous tarry matter; and a large lower layer of black granular material. VOLATILEBASES. The liquid layer was decanted (336 grams) and vacuum-distilled into a receiver cooled with ice. The distillate (217 grams) was colorless and had, among other odors, a strong indication of ammonia. During the distillation a colorless crystalline deposit of ammonium carbamate collected on the chilled walls of the receiver. The distillate was acidified with hydrochloric acid with the concomitant evolution of much carbon dioxide, distilled in vacuo from ammonium chloride, and then fractionated by boiling at atmospheric pressure under an 18-inch (45.7-cm.) Vi,ereaux column to yield: fraction I, boiling point 97.0' to 98.2" C. (4.5 grams); fraction 11, boiling point 98.2' t o 99.0" C. (7.7 grams); and fraction 111,boiling point 99.0" to 99.2" C. (50.0 grams). The residue which was mainly water was discarded after no appreciable amount of material could be salted out by sodium hydroxide. The first fraction appeared to be a mixture and was exhausted in making various identification tests. It was a colorless, mobile, very volatile liquid which had a strong, somewhat ethereal odor smelling in some ways like stale crackers; upon standing, it turned brown. It was an extremely weak base and was soluble in water and ether. It did not reduce ammoniacal silver nitrate and did not form a hydrazone with phenylhydrazine. The response to fuchsin reagent as well as the isatin test for pyrrole was very weak; on the other hand, it gave a strong pine-splinter reaction. The reduction product formed when it was treated with zinc and hydrochloric acid had an amine-like odor and gave a positive carbylamine test. From fraction I1 which contained a considerable amount Tanret [Bull. BOC. chim., [2] 44, 102 (1885)], by heating dextrose with aqueous ammonia a t looo C., obtained two "diasines" which Brandes and Stoehr (1) proved t o be methylpyrazine and 2,6-dimethylpyrazine. The latter investigators also isolated pyrazine and a small amount of pyridine. Stolte [Beilr. Chem. Physiol. (Hofmezster), 11, 19 (1907-8)] obtained 2.5ditetrahydroxybutylpyramine by allowing fructose t o stand with methyl alcoholic ammonia a t room temperature for 2 months.

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of water, a brownish colored oil was separated by salting out with sodium hydroxide, which was distilled from solid sodium hydroxide into two fractions: fraction 1, 0.5 gram, boiling point 100.5" C.; fraction 2, 1.5 gram, boiling point 144" C. The former was similar to fraction I of the original distillate and was analyzed as follows: Calcd. for CsHsNzO: C 53.53' H 7 19. N 25 00 Found: C , 56.63, 56.87'; H , 7.'96, ?.66; I N , b4.23, 24.10

Although the analytical results are in poor agreement with the calculated values for hydroxymethyldihydropyrazine, the formation of such a compound is not illogical. The purity of the sample may well be questioned, since the difficulties of handling such a small amount of volatile material are obvious. Fraction 2 was converted into a picrate by warming with 2 grams of picric acid in 15 cc. of alcohol; the yield was 2.6 grams of canary yellow crystals, melting point 117' to 125' C. The material was a mixture of picrates, and a homogeneous product was obtained only after recrystallization from various solvents (petroleum ether-benzene mixture, alcohol, and water). The melting point of 172" to 173' C. and the analytical results identified the compound as the picrate of 2,bdimethylpyrazine (1). When the picrate was dried in an evacuated Abderhalden apparatus a t 100" C. for 2 hours, the dimethylpyrazine was completely removed and pure picric acid remained; the analysis is as follows: Calcd. for CIZHUNIOI: C, 42.71; H, 3.29; N, 20.78 Found: C, 42.84, 42.93; H, 3.25, 3.27; N, 21.13, 20.64

The low boiling point of the free base (144' C.; the literature gives 155") is probably due to the presence of monomethylpyrazine (boiling point 136" C.) and perhaps other similar material. TABLE IV.

DISTRIBUTION OF NITROGEN IN EXTRACTS OF AMMONIATEDLIGNIN (In per cent) Types of N

-Extracts Water 3.2 45.4 25.4b 0.7 1.6 11.9 5.0 6.9

-

20% HCI a

59.9 33.9 3.4 3.2 19.2 8.3 10.9

"

The sum of the percentage of nitrogen in humin I and in the fraction insoluble in 20% HC1 is 40.1% of the total. b Includes 18.1%urea nitrogen and 2.9% ammonium nitrogen.

From fraction I11 about 8 cc. of oil were obtained by salting out with sodium hydroxide. This was distilled a t atmospheric pressure into two fractions: fraction A, boiling point 97' to 100' C. (1 cc.) and fraction B, boiling point 125' to 128' C. (6 cc.). Fraction A appeared to be essentially the same as fraction I, and B appeared to be a mixture of pyrazine (boiling point 118' C.), methylpyrazine (boiling point 136"), and dimethylpyrazine (boiling point 155"). Analysis indicated a composition essentially in agreement with methylpyrazine : Calcd. for CSHSNI: C 62 40; Found: C, 62.23; H,'7.99

R,8.39

I n an effort to separate the mixture into its components, only dimethylpyrazine was isolated as the picrate. WATEREXTRACT. The dark granular material and the viscous tarry portion were removed from the capsule and ground together into a coarse powder. The 400 grams so obtained were extracted three times under the reflux, each for 6 hours with 400 cc. of water. During the first extraction, ammonium bicarbonate (about 3 grams) collected in the condenser. The insoluble material was combined with the solid matter of the upper liquid layer which had been de-

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posited upon concentration, and the mixture in turn was subjected to two further extractions with 400 cc. of water for 15 hours each. All of the aqueous extracts were combined and concentrated in vmuo. The distillate was salted out with potassium hydroxide to yield an additional 2 grams of mixed pyrazines, and the residue, a dark brown viscous sirup, was placed in a vacuum desiccator over phosphorus

culty of separation of the insoluble portion, much as though the removal of the hydrochloric acid was resulting in a peptizing action. The combined extract and washings were passed through a folded filter, concentrated by vacuum distillation, evaporated further on the water bath, and finally desiccated over sodium hydroxide in vmuo. SODIUMHYDROXIDE EXTRACT.The same procedure was used as that described for the similar extraction with the RELCTION PRODUCT FRDM TOO . D m O S E M TOO &WONIA(l) ammoniated peat. One gram of material yielded 4.8 mg. of volatile nitrogen. A one-fifth aliquot of the extract gave mraotion 1.17 mg. of nitrogen. The 10 per cent sodium-hydroxideinsoluble residue contained 130.7 mg. of nitrogen. The nitroIMOlUble Soluble e5-M N 1 l ~ O . O Nl gen accounted for in the volatile, soluble, and insoluble fracI tions was 94 per cent of that originally present in the 20 per Alcohol ICrtnotion cent hydrochloric-acid-insoluble fraction. DISTRIBUTION OF NITROGEN IN VARIOUS EXTRACTS. The I 1 Inrolubls Soluble general method described in the foregoing for classification of Z E m 6 $ A a a n . e %n I the types of nitrogen in peat was used for ammoniated dexmtraotion trose except that the “total basic” nitrogen, which was apI 1 preciably more in this case, was further fractionated into IMolUblO “primary amino basic” and “nonprimary amino basic” nitroaa-6 N; 1S.M CI I gen. The change in procedure affected only the phosphomotion lop ” tungstate precipitate and was that described by Morrow and Gortner. It involved removal of the phosphotungstic acid by precipitation from alkaline solution with barium chloride. The “primary amino basic” nitrogen and total nitrogen were

I , .

-

(L) B.oauae of tha hekrogeneoua natura of the emanlltnd dartroae i t .aa l m p a s i b l e to at a m o t nitrogen detal‘WLMtiOD.

TABLE V. FRACTIONATION OF AMMONIATED DEXTROSE

( 8 ) It l a of i n t e n a t that tb n t i o of ohlorina t o nitrogen i o a b r t

stoiohiatrlc. ( 3 ) s o note 1 , Figure 1.

FIGURE 3. FRACTIONATION OF AI^^^^^^^ DEXTROSE

pentoxide for several weeks. A urea determination on this residue gave 16.5 grams of urea or 7.7 grams of urea nitrogen corresponding to 32.7 per cent of the nonvolatile watersoluble nitrogen. Since it was believed that the above determination for urea did not represent a true value of the amount of urea originally present because of hydrolysis, the following experiment was carried out: A sealed glass tube containing 10 grams of dextrose and 10 grams of ammonia was heated in a steel bomb (containing ammonia t o equalize the pressure) at 180’ C. for 24 hours. The tube was cooled in liquid air, opened, allowed to warm slowly in a Dewar flask; after the excess ammonia was gone, it was transferred to a vacuum desiccator where it was held over sulfuric acid for 2 days. The resultant solid material was pulverized and the whole leached with small portions of water until the filtered extracts gave a volume of 100 cc. Aliquots withdrawn immediately gave urea by the xanthydrol method equivalent to 0.96 per cent of the original dextrose. Three days later, after removal of some precipitated material by filtration, aliquots gave urea by the urease method equivalent to 0.80 per cent and by the xanthydrol method equivalent to 0.80 per cent of the ori nal dextrose. This compares with 2.36 per cent urea in t%e original large-scale experiment.

ALCOHOL EXTRACT. Since a preliminary extraction with ether of the water-insoluble fraction (270 grams) failed to remove any appreciable amount of material, it was set with 750 cc. of 95 per cent alcohol under the reflux for 15 hours. This extraction was repeated six times, using 600-cc. portions of solvent. The combined extracts were filtered, concentrated, and desiccated. HYDROCHLORIC ACID EXTRACT. The insoluble material from the alcohol extraction was dried a t 75” to 80” C. and then refluxed with 800 cc. of 20 per cent hydrochloric acid for 48 hours. The insoluble material was separated by centrifugalizing, repeatedly washed with distilled water, and again centrifugalized, Each successive washing led to greater d f i -

Solvent Water Alcohol

:$$

E%H Total soluble Insoluble

Grams Combined N Extd : f r i m Ammo&ated Dextrose Con- Percentage of taining 80.1 g. N4 Total N 39.2b 49.0 5.4 6.7 4.7 5.9 2.7 64.3 35.7

2.2c 51.5 28.6

0 This is an indirect value and was obtained as the sum of the water soluble and -insoluble components, including an estimate of the water-volstile nitronen (see note b ) . .~.. ~~~. b 1nccides an-estimate of the loss during the extraction of volatile nitrogenous compounds, such as pyrazines (3 grams nitrogen) and free ammonia (13 grams nitrogen) resulting from the partial hydrolysis of urea and ammonium salts which it was impractical to analyze for directly. e Includes volatile nitrogen. ~

TABLE VI. DISTRIBUTION OF NITROGEN IN EXTRACTS OF AMMONIATEDDEXTROSE Tvoes of .I’

Consecutive Extracts Alto202 hol H 1 0.0 0.3 1.1

Water

9.6O.

2.7 0.7 12.4 4.3 0.8 1.2

0.6 3.3 0.1 1.8 0.2 0.1 0.1

2.7

1.0

...

0.8 0.3

...

0.1

Total 1.4 29.lb 7.0 0.8 18.PC 4.8 0.9 1.4

0 A determination made on a previously unhydrolyzed fraction gave a value of 0.2% for “amide” which is, accordingly, a maximum for the amount of ammonium nitrogen that might he present. b Includes an estimate of 13 grams of water-volatile nitrogen (see Table which lor convenience is classified as “amide” nitrogen. ‘)e Includes an estimate of 3 grams of water-volatile nitrogen (pyrasines, see Table V).

determined on aliquots of the combined filtrate and washings. The precipitated barium phosphotungstate carried down a small amount of nitrogen which was classified as humin 111. A summary of the results is given in Table VI. AMMONIATED STARCH.The ammoniated starch (5.4 per cent nitrogen) was first extracted with cold water, and the ammonium nitrogen was found to be 5.1 per cent and the urea nitrogen 4.5 per cent of the total nitrogen. Then two extracts of this material were made for a study of the distribution of nitrogen by the methods used above for other ammoniated products. I n the first, the material was digested under the

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INDUSTRIAL AND ENGINEERING CHEMISTRY

reflux for 48 hours with 20 per cent hydrochloric acid, and in the second it was digested under reflux with 300 cc. of mater for 24 hours. The latter was then hydrolyzed with 20 per cent hydrochloric acid for 48 hours. The subsequent procedure in each case was the same as described above for ammoniated peat. The results are tabulated in Table VII.

Discussion of Results Under the same conditions of ammoniation, peat,’ lignin, and starch yielded dark brown, amorphous solids which were similar in appearance and contained 12.2,9.1, and 5.4 per cent nitrogen, respectively. Ammoniated dextrose contained a considerable amount of fluid matter which was in the main water. It was impossible to obtain a homogeneous sample of this dark brown mixture for analysis; the results of this work, however, show that the percentage of nitrogen in the material must be very high. The fractionation of the above mentioned ammoniated products by solvents was, in general, quite similar. The ammoniated peat and lignin were successively extracted with ether, water, alcohol, and dilute and strong hydrochloric acid; the ammoniated peat was also finally extracted with 10 per cent sodium hydroxide. On ammoniated dextrose, since it was impractical, the preliminary extraction with ether was omitted as well as the extraction with dilute hydrochloric acid. A water and strong hydrochloric acid extract of ammoniated starch yielded sufficient information for the purposes of these experiments. In every case the extraction with water removed the greater portion of the soluble nitrogen. For example, about 65 per cent of the soluble nitrogen was extracted from ammoniated peat, about 50 per cent from ammoniated lignin, and 75 per cent (estimated) from ammoniated dextrose. An extraction of the ammoniated peat with 20 per cent hydrochloric acid was roughly equivalent to the composite extractions with the various solvents, except alkali. The final extraction with alkali removed iin appreciable amount of material; this fraction, however, could be given only a cursory examination. The various fractions, on concentration, yielded dark brown sirups or amorphous solids. A superficial examination of these residues for chemical individuals was made. Thus far the only common organic product found present in the different ammoniated materials was urea; ammoniated dextrose (and perhaps starch) was also found to contain pyrazines (as shown in the section on experimental procedure). After a certain amount of manipulation of the residue from the aqueous extract of ammoniated peat, an appreciable amount of urea was isolated. Urea in the aqueous extracts of the other ammoniated substances was identified by the very specific urease test and then determined quantitatively. The percentage of urea based on the original raw material used in the ammoniation was found to be 3.7, 2.4, 0.5, and 2.4 in ammoniated peat, lignin, starch, and dextrose, respectively; or represented on a different basis, the urea nitrogen in the first three was 15.5, 18.1, and 4.5 per cent, respectively, of the total nitrogen. Without doubt the formation of urea in the above materials is due to the interaction of ammonia and carbon dioxide, and the amount formed is dependent on the quantity of carbon dioxide liberated during the ammoniation. Since peat is rich in carboxyl groups, it seems reasonable to suppose that a considerable portion of the liberated carbon dioxide arises from a simple decarboxylation, a reaction which is favored here not only by the high temperature but also by the alkaline medium. In the case of the carbohydrates it is evident that the formation of carbon di7 I t should be emphasized that only one specimen of peat, which wan ammoniated under one set of conditions (those originally used by Scholl and Davis) was studied in this work. I t is obvious that different types of peat ammoniated under a variety of conditions would not give the same results.

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oxide can be accounted for only by a profound degradation, perhaps involving a Canizzaro reaction. Some of the carbon dioxide liberated in the ammoniation of peat probably has a similar origin. Because of the complex nature of the ammoniation of these products, the calculation of the fraction of carbon dioxide converted t o urea is speculative. In view of the high temperature and tremendous excess of ammonia, the urea side of the equilibrium would seem t o be favored. The various fractions were analyzed for different types of nitrogen by the Van Slyke procedure. In spite of the fact, as pointed out by Morrow and Gortner, that such analytical figures for certain materials are arbitrary, they have nevertheless been found useful. The presence of urea in the aqueous extracts was found to interfere when the analytical procedure was carried out in the usual manner. This trouble was avoided by a preliminary treatment of the extracts with xanthydrol which effectively removed all the urea. The results obtained on the distribution of the nitrogen in the extracts of ammoniated peat, lignin, dextrose, and starch are recorded in Tables 11, IV, VI, and VII, respectively. Since the major portion of the soluble material was found in the water extract, the investigation of this particular fraction proved most valuable. The fractions in the aqueous extract classified as humin I, humin 11, “total basic,” and “total nonbasic” nitrogen were relatively small. Most of the water-soluble TABLEVII. DISTRIBUTION OF NITROGEN IN EXTRACTS OF AMMONIATED STARCH -Extracts water

Types of N

0

-

20% “ 2 1

Includes the fraction of ammoniated starch insoluble in the solvent

nitrogen (about 75 per cent) in ammoniated peat was found in the “amide” fraction, of which 62.3 per cent was accounted for as urea and 9.7 per cent as ammonium salts; the remainder, 28 per cent, is of an unknown nature which appears to be a complex mixture and represents 7 per cent of the total nitrogen. One is forced to conclude from an inspection of these tables that the major portion of these ammoniated materials is a complex mixture of organic nitrogenous products, in which no single compound, except urea, is present in appreciable quantities. This heterogeneous unknown mixture was mainly segregated in the “amide” (7 per cent) and the “nonamino nonbasic” (4.1 per cent) fractions. All of these materials on ammoniation yielded a complex polymerization product which is insoluble in the usual solvents and in strong acid and strong alkali. For example, the insoluble portion of ammoniated peat and dextrose was about 40 and 25 per cent, respectively, of the raw material used in the ammoniation. These insoluble residues contained a considerable amount of nitrogen. Since starch and dextrose as well as lignin are readily ammoniated, and since a mean of the results obtained on fractionation (and in the distribution of the nitrogen) very roughly approximate those obtained from ammoniated peat, it a p pears probable that both the carbohydrate and lignin units of peat are extensively ammoniated.

Literature Cited (1) Brandes and Stoehr, J. prakt. Chem., 54,481 (1896). (2) Fosse, Ann. inst. Pasteur, 30,525 (1916). (3) Fox and Geldard, IND.ENG.CHEM.,15, 743 (1923). (4) Morrow and Gortner, Soil Sci., 3, 297 (1917). (5) Soholl and Davis, IND.ENQ.CHEM.,25, 1074 (1933). R E C ~ I VDecember ~D 18, 1934.