1 
 
THE DEVELOPMENT OF A METHOD FOR THE DE 
 TERMINATION OF THE AMINO-ACIDS 
 OF FEEDS 
 
 BY 
 
 THOMAS SHERMAN HAMILTON 
 B. S. University of Illinois 
 1917 
 
 THESIS 
 
 Submitted in Partial Fulfillment 
 for the requirements of the 
 Degree of 
 
 MASTER OF SCIENCE 
 IN CHEMISTRY 
 
 IN 
 
 THE GRADUATE SCHOOL 
 
 of the 
 
 UNIVERSITY OF ILLINOIS 
 
 1921 
 
UNIVERSITY OF ILLINOIS 
 
 
 THE GRADUATE SCHOOL 
 
 Decambe r eL2j ] 92 i 
 
 I HEREBY RECOMMEND THAT THE THESIS PREPARED DNDER MY 
 
 SUPERVISION BY Thomas Sherman Ha:iiilt on 
 
 ENTITLED The Deve l opment of a Metho 
 
 of the Amino-Acids o f Fe eds 
 
 BE ACCEPTED AS FULFILLING THIS PART OF THE REQUIREMENTS FOR 
 THE DEGREE OF Master of Scienc e in C hemist ry _ , 
 
 Recommendation concurred in* 
 
 Committee 
 
 on 
 
 Final Examination* 
 
 ^Required for doctor’s degree but not for master’s 
 
Digitized by the Internet Archive 
 in 2015 
 
 https://archive.org/details/developmentofmetOOhami 
 
-i- 
 
 TABLE OF COITTEOTS 
 
 THE DEVELOR.IENT OF A J.TETHOD FOR THE DETERI/.niATlOlI OF THE 
 AlvIIHO-ACIDS OF FEEDS 
 
 page 
 
 I. INTRODUCTION 1 
 
 II. HISTORICAL ^ 
 
 III. DEVELOPIviENT OF THE METHOD 6 
 
 A, Extraction of the Norqprotoin Nitrogen - -- -- -- -- - 7 
 
 (1) Extraction vdth anhydrous ether - -- -- -- -- 7 
 
 Experiment I. The completeness of extraction 
 
 with ether by the centrifuge bottle method - ^ 
 
 (h) Extraction with cold absolute alcohol ------ 9 
 
 Experiment II. Comparison of the amounts of nit- 
 rogen extracted by extractions on the Buch- 
 ner fumel and by the centrifuge bottle 
 method ------------------- 9 
 
 (3) Extraction with cold 1.0 per cent, trichlorace- 
 
 tic acid - -- -- -- -- -- -- -- -- - 10 
 
 Experiment III, Comparison of the amounts of 
 nonprotein nitrogen extracted b;/ cold 1.0 
 per cent, trichloracetic acid and by cold 
 v;ater and 0.02 per cent, hydrochloric acid - 12 
 
 Experiment IV. Determir^ation of the proper 
 strerjgth trichloracetic acid to use for 
 the extraction of nonprctein nitrogen - - - I3 
 
 (4) Separation of protein and nonprotein nitrogen - - 14 
 
 Experiment V. Determination of the amount of 
 colloidal iron necessary to precipitate 
 the proteins from the nonprotein extract - - 17 
 
 Experiment VI. Determination of suitable con- 
 ditions for the precipitation of proteins 
 from, the nonptotein extract v.dth colloid- 
 al iron ------------------ 19 
 
 (a) Neutral method ----------- 19 
 
 (b) Direct method - -- -- -- -- -- - 19 
 
-ii- 
 
 page 
 
 (c) After decomposition of trichlorace- 
 tic acid by boiling -------- 19 
 
 (dj By the use of a buffer substance - - 20 
 (e) Second precipitation - -- -- -- - ^^0 
 
 Experiment VII. Does colloidal iron precipi- 
 tate nonprotein nitrogenous compounds? - - - 22 
 
 Experiment VIII. Does dilute trichloracetic 
 
 acid hydrolyze proteins? ---------- 23 
 
 (5) ProcedTui-e finally adopted for the extraction of 
 
 . the nonprotein fraction - -- -- -- -- - 23 
 
 B. Treatment of the Residue Insoluble in Ether, Absolute 
 
 Alcohol, and Cold 1.0 per cent. Trichloracetic Acid - 2li 
 
 Experiment IX. Determination of solvents and 
 methods of extractions necessary to com- 
 pletely extrs.ct the nitrogenous compounds 
 from the residue insoluble in ether, alc- 
 ohol, and cold 1.0 per cent, trichlcrace- 
 
 tic acid ----------------- 2o 
 
 C. Extraction of Starch with Trichloracetic Acid ------ iiS 
 
 Exp er indent X. Test extraction of starch with 
 
 trichloracetic acid ------------ 3 ^ 
 
 Exper iment XI. Comparison of the nitrogen ex- 
 tracted along with the starch by hot 0.1 
 
 per cent, hydrochloric acid and by hot 2.0 
 per cent, trichloracetic acid ------ 
 
 Exper inient XII. (a) Doss the second addition 
 of two volumes to the hot 2 per cent, tri- 
 chloracetic acid extracts cause any addi- 
 tiorjal precipitation of starch? (b) Can 
 the proteins, dissolved by the hot 2 per 
 cent, trichloracetic acid, be completely 
 hydrolyzed by boiling for 3 hours with 5 
 
 per cent, hydrochloric acid? ------- 33 
 
 (1) Final procedure for the extraction of starch - - - 37 
 
 IV. EXTRACTION OF STARCE AND NOiJPROTEIN NITROGENOUS CONSTITUENTS 
 
 WITH HOT 2 PER CE1\IT. TRICHLORACETIC ACID AND THE SEPARATION 
 OF THE STARCH, PROTEIN, AND NONPROTEIN NITROGEN 3^ 
 
 Exp er iment XIII. Does colloidal iron precipi- 
 tate starch? -------- ------ 39 
 
 V. MODIFICATIONS IN TEE VAN SLYKE METHOD 
 
 40 
 

 -iii- 
 
 page 
 
 VI. THE iviSTHOD 
 
 IN DETAIL 
 
 U1 
 
 PART I. 
 
 Preparation of the Sample ------------- 
 
 Ul 
 
 PART II. 
 
 Prepara-tion of the Hydrolyzed Protein Solution - - 
 
 41 
 
 A. 
 
 Extraction with anhydrous ether ---------- 
 
 41 
 
 B. 
 
 Extraction with cold absolute alcohol ------- 
 
 4H 
 
 c. 
 
 Extraction with cold 1.0 per cent, trichloracetic 
 acid ---------------------- 
 
 ^3 
 
 D. 
 
 Extraction with dilute sodium hydroxide ------ 
 
 43 
 
 E. 
 
 Digestion with h.C per cent, trichloracetic acid - - 
 
 44 
 
 F. 
 
 Extraction with 20 per cent, hydrochloric acid - - - 
 
 44 
 
 G • 
 
 Extraction with strong sodium hydroxide ------ 
 
 45 
 
 H. 
 
 Treatment of the cold 1 per cent, trichloracetic 
 acid extracts ------------------ 
 
 46 
 
 I. 
 
 Treatment of the 2 per cent, trichloracetic acid 
 extracts -------------------- 
 
 47 
 
 J. 
 
 Determirjation of insoluble humin nitrogen - - - - - 
 
 48 
 
 PART III. Analysis of the Hydrolyzed Protein Solution - - - 
 
 48 
 
 
 Determination of ammonia (airide nitrogen) - - - - - 
 
 48 
 
 
 Soluble h-umin nitrogen - -- -- -- -- -- -- -- 
 
 48 
 
 
 Precipitation and washing of the bases ------- 
 
 49 
 
 
 Decomposition of the phospho tungstic precipitate 
 by a mixture of ether and amyl alcohol - - - - - 
 
 51 
 
 
 Determination of arginine ------------- 
 
 53 
 
 
 Determiriation of the amino nitrogen of the bases - - 
 
 53 
 
 
 Determination of the total nitrogen of the bases - - 
 
 54 
 
 
 Determination of cystine ------------- 
 
 54 
 
 
 Determination of the total nitrogen in the filtrate 
 from the bases ----------------- 
 
 55 
 
 
 Determination of the amino nitrogen in the filtrate 
 from the bases ----------------- 
 
 5b 
 
-IV- 
 
 page 
 
 Calculation of arginine, cystine, histidine, lysine, 
 and the non-amino nitrogen - -- -- -- -- -- - 
 
 (a) Arginine nitrogen ------------- 
 
 (b) Cystine nitrogen - -- -- -- -- -- -- 57 
 
 (c> Histidine nitrogen ------------ 57 
 
 (d) Lysine nitrogen -------------- 57 
 
 ( 3 ) IJon-amino nitrogen in the filtrate from 
 
 the bases --------------- 57 
 
 Purity of reagents 5^ 
 
 VII. DIITERI.'IIHATION OF THE AIvlIITO-ACIDS OF OATS, CORIJ, COTTOHSEED MEAL, 
 
 Al-ro ALFALFA 59 
 
 TABLE I. Distribution of the nitrogen of oats, corn, cot- 
 tonseed meal, and alfalfa. (Results expressed 
 in percentage of the total rh trogen of the 
 feed^ --------------------- ol 
 
 TABLE II. Distribution of the nitrogen of oats, corn, 
 cottonseed meal, and alfalfa. (Results ex- 
 pressed in percentage of the feed) ------ 6 c; 
 
 TABLE III. Comparison of results v/ith those of previous 
 
 investigators --------------- 06 
 
 TABLE IV. Nitrogen distribution of alfalfa -------- 71 
 
 VIII. SUMMARY AND CONCLUSIONS 72 
 
 IX. BIBLIOGRAPHY 73 
 
THE DEVELOPMEl^T OF A I.IETHOD FOR THE DETERIVIINATION 
 
 OF THE AMINO-ACIDS OF FEEDS 
 I. INTRODUCTION 
 
 Investigations of the past few years have shov/n very clearly that the 
 total nitrogen determination and subsequent calculation of crude protein there- 
 from, alone, as has been dona in the past and is still being done at present to 
 a large extent in feeding and metabolism experiments, is of practically no value 
 for the purpose of estimating the true nutritive value of the proteins of a feed. 
 Before the true nutritive value of the protein content of a feed can be accurate- 
 ly determined, among other things the amounts of each of the various amino-acids 
 (or at least certain ones) v/hich go to make up the proteins present must be 
 Inovm. 
 
 Notwithstanding the many almost unsurmountable difficulties connected 
 with the isolation and purification of na-turally occurring proteins, a very large 
 number of vegetable and animal proteins have been isolated, purified, and the 
 amino-acids present determined by methods available for that piurpose. The vari- 
 ety, the extremely complex character, and the ease with which proteins are 
 char^ged, make a general method for the quantitative isolation and purification 
 of all the proteins of a feed impossible. Osborne (1), in his Monograph on the 
 vegetable proteins, states, »'The solvents used to extract the proteins of seeds 
 are water, neutral saline solutions, ]0 to SO per cent, alcohol, and very dilute 
 acids and alkalies. By the successive application of these solvents the greater 
 part of the protein can be extracted from most seeds when finely ground, but the 
 residue, even after extracting as completely as possible, usuftlly contains more 
 or less nitrogen." Osborne and Mendel (2) found approximately 6 per cent, of 
 the total nitrogen of whole corn left after extractions with 10 per cent, potas- 
 
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sivm chloride solution^ 90 psr cent, alcohol, and 0.2 per cent, potassixm hy- 
 droxide solution. Miller (3), in a study of the distribution of nitrogen in the 
 alfalfa seed, makes a 0,5 par cent, potassium hydroxide extraction and leaves be- 
 tween 9 si^nd 10 per cent, of the total nitrogen in the residue. Dowell and Menaul 
 (4) report 9<i per cent, of the nitrogen compounds of pecan flour (100 mesh) was 
 soluble in 0.2 per cent, sodium hydroxide; ]0 per cent, of the total nitrogen of 
 peanuts was soluble in 5 per cent, barium hydroxide while 9^ Per cent. v?as soluble 
 in 0,2 per cent, sodium hydroxide; 62 per cent, of alfalfa (40 mesh) was extract- 
 ed by a 0.3 per cent, sodium hydroxide solution; and the mximum extraction of 
 nitrogen from kafir (60 mesh) was 36 per cent, of the total nitrogen of the san^l< . 
 It is certainly very doubtful whether extractions, which leave so much of the 
 total nitrogen unextracted in the residues, contain representative protein mater- 
 ial of the sample. 
 
 The excellent investigations of Fischer, Hausmann, Kossel, Yan Slyke, 
 Dakin, and others have made it possible to determine the various amino-acids 
 making up the protein molecule of pure proteins. But it is at present impossible 
 to calculate the amino-acid content of feeds from the amino-acid content of the 
 isolated proteins, first, because all of the proteins of no single food or feed, 
 with the possible exception of milk, have been isolated completely and in a pure 
 state; and second, because as yet there are no metliods available for the quanti- 
 tative separation and purification of all the proteins of a food or feed. 
 
 II. HISTORICAL. 
 
 Grindley, Joseph, and Slater (5) made the first attempt to determine 
 the amino-acid content of feeds directly. Tlieir method consisted simply in 
 complete hydrolysis of a finely ground sample of feed with 20 per cant, hydro- 
 chloric acid and the application of the origi:ml Van Slyke Method (6) to this 
 hydrolysate. The results v/ere expressed as percentage of the total nitrogen of 
 
- 3 - 
 
 the faad and as percentage of the feed. Soon after the above paper appeared, 
 Nollau (7) published results on »' the axnino-acid content of certain commercial 
 feedingstuff s and other sources of protein.” He also used the Van Slyke method 
 which other investigators had used so successfully in the determina,tion of the 
 chemical groups characteristic of the different amino-acids of proteins. The 
 procedure used was as follows: The samples, ViThich were ground so as to pass 
 
 through a 40 mesh sieve, were extracted with ether to remove the fat. Tns sam- 
 ples were then completely hydrolyzed by boiling with dO per cent, hydrochloric 
 acid, the insoluble residue filtered off, and the clear filtra,te analyzed accord- 
 ing to the Van Slyke method. The results were calculated on the basis of the 
 total nitrogen in the filtrate from the insoluble residue and not on the total 
 nitrogen of the sa.nrole of feed. 
 
 Later in the same year Grindley and Slater (o), using the same method 
 as that used by Grindley, Joseph, snd Slater, published additional results on the 
 same problem. The differences in the procedures used may explain to some extent 
 the lack of concordant results between those obtained by Hollau and those obtain- 
 ed by Grindley and associa,tes but the difference between many results from the 
 two laboratories is greater than the differences in procedures would warrant. 
 
 As subsequent criticism, of the application of a method designed en- 
 tirely for the p\irpose of analyzing pure proteins to the analysis of heterogene- 
 ous mixtures such as feeds, pointed out, the effects of the nonprotein nitrogen- 
 ous constituents, the carbohydrates, fats, etc. were unkno’wn. In order to de- 
 termine the effect of the nonprotein nitrogenous imterial, Grindley and Eckstein 
 (9) studied the nonprotein nitrogenous material extracted from various feeds 
 with cold water. Hart and Bentley (10) also made a study of the "vvater-soluble 
 nitrogen of soma comiTion feedings t'^offs, ” b\it used hot water instead of cold water 
 as their extracting fluid. From the fact that most of the nonprotein nitrogen 
 of the feeds sxa.mined was in the form of ammonia or oC-amino -acids, free or com- 
 
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 bined; i. e.j degradation products of proteins^ Grindley and Eckstein in part 
 
 conclude," it seams quits evident that only a small part, if any, of the 
 
 nonprotein nitrogenous constituents of foods and f eedingstuff s can in ariy way 
 interfere with the application of the Van Slyke method for the determination of 
 the chemical groups cliaract eristic of the different amino-acids of protein to 
 the estimation of the free and combined amino acids and amides of f eedingstaff s." 
 
 Gortner and his associates (11, Ik, I 3 ) have made an extensive study 
 of the formation of humin in the presence of carbohydrates during acid hydrolysis 
 These investigators have shovm that the formation of humin depends to a large 
 extent on the presence of carbohydrates and that the quantity of humin formed on 
 hydrolysis of pure proteins is greatly increased by the addition of ce.rbohydrate 
 material. From these and other investigations the chief source of error in the 
 method of aralysis used by Grindley and associates and by Nollau was thought to 
 be caused by the presence of the carbohydrates in the feeds during the hydrolysis 
 and subsequent analysis. Grindley (14) makes the sta-tement: "Tne high results 
 for humin nitrogen probably constitute the most serious of the errors involved 
 in the application of the Van Slyke method to the analysis of feeding stuffs, 
 
 " And that no claim to perfection is made for the results published by 
 
 Grindley and associates is shown by the statement of Grindley (14): "Further it 
 
 is also quite evident that the results so far obtained in this work are only 
 approximately accurate and at present are to be considered of canparative value 
 only." 
 
 Eckstein and Grindley (I5)i in an attaupt to reduce the quantity of 
 humin formed during hydrolj’-sis, made two decided improvements on the older method 
 of Grindley and associates. The first was the removal of some of the nonpro- 
 tein nitrogenous constituents by complete extractions with ether and with abso- 
 lute alcohol. The second was the treatment of the residua, remaining from the 
 ether and alcohol extractions, so that the greater part of the carbohydrates was 
 
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 separated from the main portion of the proteins before the latter are hydrolyzed. 
 The details of tne method, as given by Eckstein and Grindley ( 15 )> fol- 
 
 lows: 
 
 •'Weighed quantities of the feedingstuff are extracted with ether in 
 Soxiilet extractors and then with cold absolute alcohol on Buchner furaiels. The 
 residues thus extracted are digested for I5 hours three or four times vath 0,1 
 per cent, solution of hydrochloric acid until all the starch has been converted 
 into sugars. The residues insoluble in 0.1 per cent, hydrochloric acid are 
 boiled with cO per cent, hydrochloric acid -until the proteins which they contain 
 are completely hydrolyzed. 
 
 •Tne filtrates from the residues insoluble in 0.1 per cent, hydrochlor- 
 ic acid are neutralized with sodixm hydroxide, then faintly acidified with acetic- 
 acid, allowed to stand over night, and then filtered. The filtrates fromi the 
 precipitated proteins are concentrated vacuo to small volumes and precipitated 
 by the addition of five volumes of absolute alcohol. After standing over night 
 the precipitated proteins are removed by filtration and washed w'ith 83 per cent, 
 alcohol . 
 
 •'The filtrates from the proteins precipitated by alcohol are concen- 
 trated to small volume and enough concentrated hydrochloric acid is added to make 
 a 5 per cent, solution. The solutions are then boiled until hydrolysis is com- 
 plete. The proteins separated above b3’- neutralization and bj'- the addition of 
 alcohol are boiled with <£0 per cent, hydrochloric acid until hydrolysis is com- 
 plete, 
 
 •'As a result of the above procedure, there are obtained three differ- 
 ent fractions of the proteins of f eedingstuffs which are completely hydrolyzed; 
 namely, (1) the proteins insoluble in 0.1 per cent, hydrochloric acid; (2) the 
 proteins separated by neutralizing the mineral acid and precipitating with alco- 
 hol; (3) the soluble proteins not separa.ted by neutralizing the mineral acid or 
 precipitating with alcohol. Each of the three hydrolyzed solutions is filtered 
 and the insoluble humin substances are repea.tedly digested with 0.1 per cent, 
 hydrochloric acid and then thoroughly washed with hot water. The nitrogen in 
 these residues is considered to represent the insoluble h\xnin substances. 
 
 "The filtrates from the insoluble humin were concentrated in vacuo 
 to small volume and the ainide and soluble melanine nitrogen determined as usual. 
 The remaining parts of the analysis are continued as usual." 
 
 This method gave the followii-)g results for hur.in nitrogen, as percent- 
 e.ges of total nitrogen of the feeds: corn 3.2, wheat oats barley 
 
 3*9 psr cent. These results maj’- be compared with the humin nitrogen of the same 
 feeds by the earlier method of Grindley and associates. The humin nitrogen val- 
 ues then were corn Q.8, wheat 9 . 2 , oats 9 « 9 > barley 8.8 per cent, of the 
 
 total nitrogen. 
 
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- 6 - 
 
 The highest hiamin nitrogen reported by Van Slyke (6) for pure pro- 
 teins was 3.6 per cent, in the case of ox heraoglobin. Hartley (16) reported 
 2.5 per cent, humin nitrogen in euglobin of ox serum. Van Slyke (6) found 7*4 
 per cent, of humin nitrogen in dog's iiair. Thus the quantities of humin nitro- 
 gen obtained by Eckstein and Grindley compared very favorably with that found in 
 the analysis of some of the pure proteins. 
 
 III. DEVEL 0 PI:IEITT OF THE METHOD. 
 
 While the new method of Eckstein and Grindley decidedly lov/ered the 
 quantities of humin nitrogen, especially in the arialysis of cereals, several 
 difficulties were also introduced. Possibly the most serious of the difficul- 
 ties encountered in the use of this method was the fomation of a dark colored 
 substance during: the precipitation of the bases with phosphotungstic acid. Some- 
 times this de.rk colored substance was of a sticky nature and, \>hen present, made 
 thorough washing and a clean decomposition of the basic phospho tungstates impos- 
 sible. Neidig and Snyder (I7) encountered the same difficulty in applying the 
 method of Eckstein and Grindley to silage crops. They refer to this fraction of 
 humin as the "phosphotungstic humin" of Gortner and Holm. This fraction of humin 
 nitrogen will be referred to again later. It has been found (see Experiment 
 XII, page 33) that 5 psr cent, hydrochloric acid very probably does not ca.use 
 complete hydrolysis of the proteins soluble in the hot 0.1 per cent, hydrochloric 
 acid and are not precipitated by either neutralization or 5 volumes of alcohol, 
 by boiling for "only a short time." (From unpublished papers of Eckstein and 
 Grindley this time was 6 hours). It was also found, by the use of the Eckstein 
 a,nd Grindlej’’ method, that the total nitrogen finally recovered was alva,ys some- 
 what less than 100 par cent. As was pointed out by Neidig and Snyder ( 17 )^ the 
 per cent, humin nitrogen obtained in the analysis of forage crops by the new 
 method of Eckstein and Grindley v;as only slightly lower than that obtained in 
 
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- 7 - 
 
 the analysis of the same feeds by the original method of Grindley and associates. 
 It was not these objections to the new method of Eckstein and Grindley, alone, 
 which lead to the search for a better method. In all the previous methods for 
 the application of the Van Slyke method to the analysis of feeds the free amino- 
 acids e,nd amides as well as the combined aa'.ino -acids and aiiiides were determined. 
 That is, the free anmonia, free amino-acids, and amides, vdiich constitute the 
 main portion of the nonprotein nitrogenous material of the feed, were determined 
 along with the combined amino-acids and amides, which represent the protein 
 content of the feed. A method by which the protein and nonprotein nitrogen 
 could be separated and at the same time the distribution of each determined, if 
 desired, v;as highly desirable. 
 
 In the sea-rch for a better method a la,rge number of separate experi- 
 ments were carried out. The experiments varied from only slight changes in 
 technique or slight change in reagents used to almost complete revision of the 
 method previously used, ffeny tests gave negative results and many gave positive 
 results but for some reason could not be used. Only a fev; of the more important 
 experiments which had direct bearing on the development of the method will be 
 recorded. 
 
 A. EXTRACTION OF THE NONPROTEIN NITROGEN. 
 
 (1) Extraction with anhydrous ether. 
 
 Nollau (7), in his application of the Van Slyke method to the ar.aly- 
 sis of certain feeds, extracted the feed with ether to remove the fat. Eckstein 
 and Grindley (IE) made ether and alcohol extractions to remove some of the non- 
 protein nitrogenoiis constituents such as the nitrogenous lipoids, coloring 
 uiatters, etc. In the method finally adopted for this v/ork both anhydrous ether 
 and cold absolute alcohol extractions are made, but the method of carrying out 
 these extractions is somewhat different. Instead of the ether extraction in 
 
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-3- 
 
 Soxiilet extractors it lias been found much more convenient to UiSdce the extrac- 
 tions of the sample in a centrifuge bottle. This is the method adopted for all 
 extractions in the cold and in general is carried out as follows; The sample is 
 placed in a 5OO cc. centrifuge bottle and 100 to 200 cc. of the extracting liquid 
 added. The bottle is corked securely and placed on a mechanical shaker which 
 keeps the bottle and contents in continuous motion. Usuadly but tv/o extractions 
 are made each 24 hours; one extraction for a, 7 to S hour period is mde during 
 the -day and a second extraction for a l4 to I5 hour period is made during the 
 night. As a rule 6 or 7 extractions with a solvent are necessary to insure com- 
 plete extractions in the cold. After each extraction period the sides of the 
 bottle are washed down with a few cc. of tiie solvent or ammonia-free water in 
 case of aqueous solvents and the solution centrifuged until the solid matter 
 forms a compact mass in the bottom of the bottle. The supernatant liquid is then 
 decanted. This is the method referred to in the follov/ing pages as the centrifug* 
 bottle method of extraction. 
 
 The completeness of extraction with ether by this method is shown by 
 the following experin:ient. 
 
 EXPERIIvIENT I. 
 
 Four 30 gm, samples of alfalfa which had been gro\md to pass through 
 a bO mesh sieve v/ere ^ch extracted 7 times with 100 cc. portions of anhydrous 
 ether in a 5OO cc. centrifioge bottle in the usual mamer. The ether extracts 
 were filtered to remove ar.y solid material that may have been decanted into them, 
 the ether recovered by distillation, and the total nitrogen determined. The ni- 
 trogen extracted by ether, as percentage of the total nitrogen of the sample, 
 was 0.490 cent., 0.49«^ psr cent., 0,472 per cent., and 0.433 cent. An 
 eighth extraction v/ith 100 cc. portions of ether v/as made and the total nitrogen 
 extracted was determined. Five cc. of N/10 acid were used in the receiving 
 
■■"I 
 
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 A ai 
 
 tl 111 
 
 I* 
 
 - 
 
 ‘.‘•:--:c:y e/ 
 
 VtOKat’ 9 7 
 
 
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 . . j«-: : -?5 1-5 ' ^ OcJ 
 
 . f>...JC 
 
 
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 li;? ' ra«X? 
 
 i‘ '■^ 
 
 •;5-> 
 
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 • •. - 
 
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 .' t!..* C, f, tll~ i C >i.' 1-i" >* '•' 
 
 
 , .. . _ .J .• .'AT. jbiioft cv lO*-: O- ''i .Jii'l 
 
 4^ 
 
 ^.■ai V i t 
 
_o_ 
 
 ’ 
 
 bottles and the following amounts of N/10 alkali were required for neutraliza- 
 tion; 4.70 cc., 4.73 cc., 4.90 cc., and 4»70 cc. The reagent factor was O.H 5 
 cc. of IT/iO alkali. 
 
 Since oats is the only other feed examined which had as large a per- 
 centage of nitrogen extracted by either^ this experiment was considered sufficient 
 proof that seven 100 cc. extractions completely extracted the ether soluble ni- 
 trogen in the other feeds examined. 
 
 ( 2 ) Extraction with cold absolute alcohol. 
 
 By carrying out the alcohol extractions also in the centrifuge 
 bottle a more complete extraction is obtained and there is also the additior.al 
 advantage ti^t the residue is not iiandled between the two extractions. In the 
 following experiiLent the nitrogen extracted by alcohol according to the Eckstein 
 and Grindley method on Buchner furjcels is compared with that extracted by the 
 centrifuge bottle method. 
 
 EXPEHIlViENT II. 
 
 The four residues remaining after the ether extraction in Experi- 
 ment I were extracted 7 times in centrifuge bottles with 200 cc. portions of ab- 
 solute alcohol. The alcohol was recovered by distillation and total nitrogen 
 determined in the extract residues. As percentage of total nitrogen of the 
 sample k.Ooh per cent., 2,0'i1 per cent., 1,809 psr cent., and 2.12k per cent, of 
 nitrogen were extracted. An eighth extraction with 200 cc. portions of absolute 
 alcohol contained no nitrogen as shown by the fact that 5 cc. portions of H/lO 
 acid were plaxed in the receiving bottles and after distillation it required 
 4.75 cc., 4 . 7 *^ cc., 4 . 7 s cc., and 4.7^ cc. of N/lO alkali for ne'atralization. Thi 
 reagent factor was O.25 cc. of N/IO e.lkali. 
 
 Two more 3 O portions of the same sample of alfalfa were extracted 
 
r 
 
 
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 ■ • & 
 
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 r j. 9: ~ 
 
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 vie.--:-'. ..U !>C ■ I.. ■ 
 
 r> . -2. 
 
 
 At. i • 
 
 .1:.'' ■- 1; 
 
 a- J: : -£ 7 ^ » '*-t; 
 
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 v'■..^v '!•: {-. 
 
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 Jf .. e. ■ .TVOai; 3>‘i ix6:;oi3ic. 
 
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- 10 - 
 
 with ether as in Experiment then the residues transferred to Buchner funr.els 
 and extracted with 1400 cc. of absolute alcohol by the method of Eckstein and 
 Grindley. The total nitrogen extracted in these two cases was 0.4<^4 per cent, 
 and 0.646 per cent. Comparing these values with those obtained in the centrifuge 
 bottle method it is evident that the extraction in the centrifuge bottle extracts 
 considerably mors nitrogen than is extracted by the same volume of solvent on 
 Bucliner funnels. Since alfalfa contains more alcohol soluble nitrogen than any 
 other feed exainined, seven <200 cc. extractions were considered sufficient to ex- 
 tract all the alcohol soluble nitrogen from the other feeds if ground to the same 
 degree of fineness. 
 
 (3) Extraction with cold 1.0 per cent, trichloracetic acid. 
 
 Hart and Bentley (10) studied the ‘'character of the water-soluble ni- 
 trogen of some coiumon f eedingstuffs." In their method the air dried samples were 
 extracted with hot water. Grindley and Eckstein (9) irads a studj’’ of "the nonpro- 
 tein nitrogenous constituents of f eedingstuff s» and used cold water as the extract- 
 ing liquid. It is apparent from the results of these investigations tliat the ni- 
 trogen extracted by hot or cold water is largely in the form of hj/’drolytic pro- 
 ducts of proteins. 
 
 The finding of a reagent which would extract all the nonprotein ni- 
 trogenous constituents of a feed without altering the proteins present or extract- 
 ing considerable protein material and the quantitative separation of the proteins 
 extracted from the nonprotein nitrogenous material has bean the most difficult of 
 all the problems encountered in connection v/itli this v/ork. 
 
 Some of the various extracting liquids and the conditions under which 
 they were used are as follows: (1) cold water on Buchner funnels; (2) cold 0.2 
 per cent, hydrochloric acid in centrifuge bottles and a I5.OO cc. 0.2 per cent, 
 hydrochloric acid extraction on a Buchner funnel; (3) extraction with water and 
 
- 11 - 
 
 0.0^ per cent, hydrochloric acid in centrifuge bottles^ in which tv/o cc. 
 amiionia-free water extractions were made for I5 to eiO hours each, follov/ed by tv/o 
 H5O cc. 0,0'd per cent, hydrochloric acid extractions for 2 hour periods, followed 
 by tv/o more 2^0 cc. water extractions for I5 to 20 hour periods; (U) extraction 
 with aim-onia-free waiter and O.Oci per cent, hydrochloric acid as described in (3) 
 except that the acid extractions were continued for I5 to 20 hour periods; and 
 finally (5) seven 200 cc. extractions with 1.0 per cent, trichloracetic acid using 
 the usual centrifuge bottle method. 
 
 Water as an extra.cting liquid for nonprotein nitrogen was used both 
 by Hart and Bentley (10) and by Grindley and Eckstein (9). Water is a very good 
 solvent for the nonprotein nitrogen constituents of finely ground feeds, especi- 
 ally if the sample has been previously extracted with ether and alcohol. Even 
 when used after ether and alcohol extractions, water extracts the nonprotein ni- 
 trogen rather slov/ly a,nd incompletely. It also extracts, in some cases, consid- 
 erable protein which must be removed. Water very slightly acidified with mineral 
 acid extracts the nonprotein nitrogen more quickly and con^letely than plain 
 water but mors protein nitrogen is usually extracted also. This is a rather 
 serious objection for the quantitative precipitation of the proteins from the 
 nonprotein nitrogenous constituents has been found to be a very difficult task 
 in this work. There is also the possibility of slight hydrolysis or claange of 
 tile proteins when treated with mineral acids. It was found that, whatever cold 
 extraction liquid used, 6 ot 7 repeated extractions in the centrifuge bottle gave 
 a more complete extraction than was extracted by a corresponding volume of sol- 
 vent on Buchner funnels, 
 
 V/ater and 0.02 per cent, hydrochloric acid may be used very successful' 
 ly for the extraction of nonprotein nitrogen in the follov/ing manner; Tne sample 
 is placed in a centrifuge bottle and extracted twice with 25O cc. portions of 
 ammonia-free v/ater, makirg two extractions each 24 hours. Tlie residue is then 
 
3 .Ti : 1 
 
 . « 'O. . ' ■ ^ 
 
 i^-'. . ^ ’c- 
 
 u. «~oop»^ 
 
 n — ^ ^ aCi ftiS ^ '■ 
 
 • ^ . i i* ♦ 
 
 ‘ I-fci .u?, Ar’. 
 
 •-; J J..'®<I ..:■ • :i«F vcf 
 
 _ •. ;i 4 -.,..‘ jC'i ■.•'? ■ ^ ' 
 
 e. 1 'u 
 
 sifTS 'I'j'ia 
 
 ; i: 
 
 ..x^rc':-* 
 
 ..., ■..; tlr- 
 
 e: . - . • 
 
 *• 
 
 e-^. t-'W :' 5 i:j' 06 lj - 
 
 «/J 4i,’n$&y i --•' 5 I -’4^1:1 • i’-. r’t'ii' 
 
 - :. : ■ '■ 3 i 9 »- • • •'■-■ 
 
 ^y-'- ,X i:v:& 0 ':* ' ■• ; ■ 
 
 i 'xc c ^^9ax^ 1 ^Jpif t^oiiCvi-ii 
 *rr> riciJj^^JXS oi^IfTiSOO b'icr* *■ 
 
 . .art ■■ ;;.*aK- i'^'- 
 r" 
 
 Tj^'i :)■•. 3 c‘r7;:or' jaL*?;; '.. '‘d; zr>t “'•* 
 
 *r- ’ . ... al ^c'iq J 
 
 ...f tdfj '-^' . *,♦ Mf l 4 ‘. T-iStffD^ 
 
 t.vj 
 
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-‘I'd- 
 
 extracted twice with cc. portions of 0.02 per cent, hydrochloric acid, ex- 
 tracting for 2 hour periods. The residue is then extracted twice hiore with 
 £iuriOnia-free water as before. 
 
 It was thought that by the use of dil\ite trichloracetic acid, since 
 it is a comparatively strong acid and also, in dilute solutions, a good protein 
 precipitant, the solvent action of the acid would be retained and at the se,T.e time 
 but little protein would be extracted. In the follov/ing experiment the extrac- 
 tions of the nonprotein nitrogen with cold 1,0 per cent, trichloracetic acid and 
 with cold water and 0.02 per cent, hydrochloric acid are compared. 
 
 EXPERIIvIEiTT III. 
 
 Two IS gm. samples of oats which had been ground to pass through an 
 SO mesh sieve were extracted v/ith water a-nd 0.02 per cent, hydrochloric acid in 
 centrifuge bottles. Two 2^0 cc. extractions with ammonie.-free water were follov/- 
 ed by two 25O cc. extractio..s with 0.02 per cent, hj’-drochloric acid, wnicbi in 
 turn were follov/ed. by two more cc. air.raonia-free water extractions. Two ex- 
 tractions were mads each c:4 hours. 
 
 Two mors IS gm. samples of the same feed were extracted 6 times in 
 centrifuge bottles with 200 cc. portions of 1.0 per cent, trichloracetic acid, 
 ciakirig two extractions each 24 houi's. Each of the four extracts were filtered by 
 gravity to remove any solid mterial that may have been decanted into them, made 
 up to a definite volvtne and total nitrogen determined in duplicate portions. The 
 results, expressed as percentage of the total nitrogen of the feed were; 
 
 IT extracted by cold 
 wa.ter and 0.02 per cent. HCl 
 
 IT extracted by cold 1 
 per cent, trichloracetic acid 
 
 27.63s 
 
 26.194 
 
 27.731 aver. 27.0S5 
 28. 333 »' 28.264 
 
 •' ^7.975 
 
 14.QO7 14.629 aver. 14.76S 
 
 14.351 14.212 ” 14.2S2 
 
 14.525 
 
- 13 - 
 
 The nonprotein nitrogen in this sample of oats was^ by fo-'or previous 
 extractions with v;ater and 0,02. per cent, hydrochloric acid followed by precipi- 
 tation with colloidal ferric hydrate^ 9 *^^^ per cent, of the total nitrogen and^ 
 by eleven previous extractions with cold 1.0 per cent, trichloracetic acid fol- 
 lowed by precipitation with colloidal ferric hydrate, 10 . I 5 per cent, of the to- 
 tal nitrogen. 
 
 It is apparent from the above experiment that much more nitrogen is 
 extracted, in the case of oats at least, by the water and 0.02 per cent, hydro- 
 chloric acid than by the 1.0 per cent, trichloracetic acid. Tr.at most if riOt all 
 of the additioiml nitrogen extracted is protein and not nonprotein nitrogen is 
 shown by the fact that by precipitation of the proteins in both cases with col- 
 loidal ferric hydrate practically the same value for the nonprctein nitrogen con- 
 tent is obtained. 
 
 In order to determine the best strength trichloracetic acid to use 
 for the extraction of the nonprotein nitrogen the following e^^eriment was carriec 
 out. 
 
 EXPERIIvIEriT IV. 
 
 Five 30 sanples of oats which had been ground so as to pass 
 through a UO mesh sieve were transferred to ^00 cc. centrifuge bottles and ex- 
 tracted on the shaker with 200 cc. portions of the followirig strengths trichlora- 
 cetic acid; 0,2 per cent., 1.0 per cent., 2.5 per cent., 5 .O per cent., and 10.0 
 per cent. Eight 24 hour extractions were made. Each of the combined extracts 
 ware mads up to a definite volume, filtered, and total nitrogen determined in 
 triplicate portions. The results, expressed as percentage of the total nitrogen 
 of the feed were as follows; 
 
OZ^:bL 
 
 
 :jr- 
 
 
 -r ■’ 
 
 >t -•. .-c-.iH . ■' ai:oIi&*‘. '• 
 
 .' j^'T'il/V* r::-v 
 
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 il . 
 
 :-r.v»- .. V-" ■• 
 
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 -ra^ 
 
 "■ r 
 
 - _ ^ ., - , . •■•, ,*.-. ' : - --SO * 1 .' cs^ : va “I . iv.iv i‘ 
 
 *" . ' • oi ioC-” . • .^•'-1 *• C' , 
 
 ,;■*«, i .' ', 3 '..-■ ‘irx^ aOy^'.'*f? '' '.£^^'.jiyiJ■^lA j,*. 
 
 .-f v.ijf-J-'V; :l 1; 'l'* V *>j ir'* '.a! 
 
 - ' - - *; ' ‘ 
 
 w , i . ^ , _ ■ - .. ^. ■^.1 , J 
 
 , i> '. i i •? 
 
 l" t.J ’. :' 1 -i-'C. 
 
 r -: 
 
 i'i'ti.C' ra... 7 ■ ..i.",. I 
 
 ., . ;: ,xi :'^9 ; 
 
 
 '.Oii'J VX 9 V - 
 
 ■'■1 
 
 r\ 
 
 i'i 
 
 a-jr-.r :.- 
 
 f-vi , 
 
 - 7 .r 
 
 > 
 
 : :. - y j ■• 
 
 ^': - c -j; c • ^ 
 
 '5 *K' 
 
 it- ■ ; -..: , -'tcv 
 
 :., I 'y: 
 
 r Ca^ ^*Zi -'j 
 
 ;V-'] .'• lo i*nof:if‘c .-t >C'a < : ::o 
 
 - 3 tq . . ' •. *^ 
 
 _ 
 
 
 H &» 4 W<X-'Xv iyS** 
 
 
 
 I f. »» • 1 * 
 
 , , : ’ -.: :o 01 ; ^ 
 
 - ’, :.*,c 7 '-:, 
 
 )T c'lft- 
 
 ' i • >1 i. "nw . i-c : 
 
 
- 14 - 
 
 Per cent, trichloracetic 
 acid used for extraction 
 
 Per cent, of the total 
 nitrogen extracted 
 
 O.cO 
 
 II 
 
 I' 
 
 16.139 
 
 16.053 
 
 16.359 
 
 1.0 
 
 II 
 
 II 
 
 la .855 
 
 lh.805 
 
 ^■5 
 
 II 
 
 II 
 
 13 -WS 
 
 13-333 
 
 13.639 
 
 5.0 
 
 II 
 
 l< 
 
 15.355 
 
 15.556 
 
 15.556 
 
 10. Q 
 
 II 
 
 II 
 
 16.528 
 16.639 
 16 . 7c2 
 
 A portion of the 1 per cent, trichloracetic acid extract was precip- 
 itated with colloidal iron and the percentage of the total nitrogen of the feed 
 left in the filtrate was 11.111 per cent.^ 11.454 per cent., and 11.593 per cent. 
 Since various experiments had shov/n ttat the nonprotein nitrogen content of this 
 sample of oats was between 10 and 11 per cent, of the total nitrogen of the feed 
 and since the 1 per cent, acid extra.cted all the nonprotein nitrogen and less 
 protein nitrogen than ai'iy other strength trichloracetic acid used, the 1 per cent, 
 trichloracetic acid was chosen s,s the extraction liquid for the nonprotein nitro- 
 gen in the future experirsents. 
 
 (4) Sepa ration of protein and nonprotein nitrogen. 
 
 As was stcted above, the selection of a suitable precipitant for the 
 protein rraterial that is extre.cted with the nonprotein nitrogenous constituents 
 has been a most difficult problem. Some of the points which must be taken into 
 consideration when choosing the protein precipitant are as follows: (1) The re- 
 agent should contain no nitrogen. (£) The precipitation must be quantitative. 
 
■VV, 
 
 i 
 
 4 - 
 
 u 
 
 .1 i»xn3 
 
 jl 
 
 ' , N 
 
 * H 
 
 •' i» / 
 
 I? 
 
 >feV, 
 
 ^ , -. '•. ^ w. •- 
 
 fi... Tfl" ;C i fr> : ,r';-.'Xi> ■? i '.i'uJ.'j 
 
 ' -'3 '1C ;.K-a •• - 
 
 f* ■ ..Cl ’ i’ sac> 
 
 ft r 
 
 ■■;-3 • 
 
 
 i. , 
 
 S','--: •.' 
 
 ... -..1 I :: o - 
 
 
 
 
-1 
 
 (3) The precipitant must not precipitate, occlude, or adsorb any nonprotein 
 nitrogenous constituents. (U) The filtration must be fairly rapid and practicable. 
 (5) The reagent must not interfere in any way with the application of t'he Van 
 Slyke analysis. Some of the protein precipitants and methods of precipitation 
 tried are: (1) neutralization; (c) neutralization and boiling; (3) 10 volunes of 
 alcohol; (4) neutralization follov/ed by precipitation with 10 volumes of alcohol; 
 (5) trichloracetic acid; (6) Almen's tannic acid; (7) meta-phosphoric acid; 
 
 (S) zinc sulfate; and (9) colloidal ferric hydrate.^ 
 
 It would be useless to record the results of all these tests but it 
 may be said that no reagent tested was entirely satisfactory. Hart and Bentley 
 (10) in their study of »' the character of the water- solr.ble nitrogen of somscommon 
 f eedingstuff s, » precipitated the proteins which were soluble in hot water by 
 slightly acidifying and boiling for a few moments. This method does not precipi- 
 tate all the proteins extracted by the cold 1.0 per cent, trichloracetic acid, 
 which is used in this work for the extraction of the nonprotein nitrogen. 
 
 In some cases precipitation by neutralization followed by precipita- 
 tion with 10 volumes of alcohol serves very well. By this method the solution 
 is neutralized with sodium hydroxide solution, then made slightly acid with ace- 
 tic acid, boiled for a few minutes, and allovifed to stand over night, ijihe solutior 
 is then filtered and washed. The filtrate is concentrated under diminished 
 pressure to a small volume and precipitated by the addition of 10 volumes of ab- 
 solute alcohol. This solution is allov;ed to stand over night, filtered and 
 washed with 90 per cent, alcohol. Amorig the objections to this method and, in 
 fact, any method in which strong alcohol is used as the precipitant, are a slight 
 precipitation of starch which, when present during the hydrolysis of the protein, 
 causes an increase in the humin nitrogen and the fact that strong alcohol precip- 
 itates some nonprotein nitrogenous substances. 
 
 o 
 
 Iron Dialyzed Merck, containing 5 cent. Fe^Og. 
 
r-j- ^ 
 
 i: 
 
 ' .V ^ 
 
 J :« ■ 
 
 
 :-,^; jUi ;; ;■ 
 
 U¥ '^Arf 
 
 
 .or. .. .‘tojo*:; ^.'r 
 
 . :^-': i -. i - 
 
 ' V f: • 
 
 : :■:••. lUA 
 
 4.* -X - 
 
 \ ,'J 'iv (. 30 , 
 
 •» £.*14 I . 
 
 
 f» f •r ‘ 
 
 4» » « 
 
 
 I 
 
 
 .. 
 
 ti * *1 " * ; 
 
 ; .1 
 
 j : to . , 
 
 !.' O, 
 
 
 ; - u t . .JV .i 
 
 r; 
 
 Uf f»r* .'. _■ ' i' : . . 0 ,;-r:::iu 5 lY; 
 
 ,.' : T ^ ’ ' : ' ; r.ni 
 
 ■•P”! Oil$ •/ - .-i/-, '■ J » .» ; 
 
 ^ jocv.-tst Ou 5 :-.‘-; '--i. sr* ’Viw- 
 . . .i i., .. -I :v,c »•..•?■'• ii> \l-:f 'ritu’ii .. : ,'vJ^ 
 
 c .. .■.•Ti o-:j ic '. 
 
 " .4 
 
 % 
 
 .;■. .• -.t.- r -4 rr 1 Xt'‘ 
 
 , J VC-- '■ ' .A *■ fc: ; £. 1 ? i* &'I‘. '■ .' . 1 -s^C .'"J 
 
 . c. - S; . *;v . »♦. :^v* - . ,’ ..i >j,: >::■ it. 
 
 ■• • j: 4 .“ ('.O'*- ^ 0 .i *• ':! 
 
 I -I.- .r^-, . . 'r. i.- 9 .. 'i, .'v- 
 
 r'-'l . ^ 4 ..:..X'''.- ■ ' 
 
 ‘ .. . ■ V .... „''t . 
 
 -.'. 'I '• '■ * 
 
 . •. ..J ■.' ... r ...... x v r; ctL. . 
 
 .. ;^,i‘- . ’ -. i 'o-v. -. a.; ,;io» »l . . ■ •.wsiA 
 
 .;. , .. . .' V ‘ . . -. iv(j 
 
 . . j . ^''T. • ■■\A 1 -- - . ^.' . : --''v 
 
 *' ■ • • •■V . “D i. r- -Xv 4 
 
 ^.S. ' 
 
 1 -.J 'C 
 
 
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 ; J i ii 5 I i Otf 1 0 .'^ ' * ► 
 ’ :; ts-'J.Ll'r r^’ .c -' 
 
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 ■A 
 
 I i.i :; .t- l»- 
 
 , ,.x’. c',. .■jr.;: t-LiO.- ■ iJJr: . 
 
 L 
 
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 -« 5 T-r J*r— r i 
 
 
 ..- „ ?i 
 
-16- 
 
 Gr09rayald (IS) has shov/n that trichloracetic acid makes a quantita- 
 tive separation of the nonprotein and protein nitrogen in blood but it has been 
 found that trichloracetic acid is not nearly as good a precipitant for vegetable 
 proteins^ encountered in this work at least, as it is for animal proteins. In 
 addition, the trichloracetic acid precipitates of the cold 1 per cent, trichlora- 
 cetic acid extracts i^ave been found to be very difficult to filter. This is also 
 the chief objection to meta-phosphoric acid recommended by Folin and Denis (19) • 
 Wolff (20) ha.s shown that colloidal ferric hydrate is a suitable re- 
 agent for removing the proteins from blood in order to obtain a filtrate for the 
 quantitative determination of free amino nitrogen. Hill (21) states triat by the 
 addition of the proper ainount of colloidal iron all the proteins of milk are com- 
 pletely precipitated and caii be rapidly filtered off leaving a perfectly clear 
 colorless filtrate. Van Slyke, Vinograd-Villchur, and Losee (22) make the fol- 
 lowing statanent in regard to the use of colloidal iron; »'In experiments on Witte 
 
 peptone and partially digested proteins we have found, furthermore, that 
 
 colloidal ferric hydrate not only lets all the airiino-acids go through into the 
 filtrate, but that it also precipitates none of the int errnediary products up to 
 the albumosss, and none of these except som.e of complexity but little below that 
 
 of the original proteins " Grindley and Eckstein (9), in their study of the 
 
 nonprotein nitrogenous constituents of feeds, used colloidal ferric hydra-te to 
 precipitate the proteins from their nonprotein extract. From the results of many 
 experiments it seems that colloidal iron is not as good a precipitant for vege- 
 table proteins as it is for animal proteins. 
 
 In order to determine the proper amount of colloi'dal iron necessary 
 to precipitate the proteins from the nonprotein nitrogen extract the following 
 experiment was carried out. 
 
c J -: ! 
 
 .;aur A tier, c : 
 
 r ;..^/< I -1 i'f:* . 
 
 . ,., j g. 
 
 I*- ;: - A-oc : ' . i .*.<•« . 
 
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 '•■ p : *■; . 
 
 si 
 
 . ^ 0 "•joIu 
 “•' i/D.'T 
 
 I «i..7 at ivn'if-3.’-UOO' 
 t .* * 
 
 -V-*-. ill ' -i, . 
 
 > : 
 
 * 
 
 r.;jt 
 
 ^ ;A >;1< 
 
 
 f- 
 
 
 I 
 
 -o < 
 
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 .So ^ tit y 
 
 •'TS'i ; :r^ .oXXo* . .'’ 
 
 / 
 
 •’* t'T 9tS* 
 
 ' I 
 
 
 i:. 
 
 s. ^ 
 
 ;v. v '. *l'i - t-'.’ffili' ■•• 
 
 •r IT ..1 . 
 
 : . .! ■ ." "3 • ® • - ' 
 
 ^ 
 
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 1 
 
 ( 
 
 a. * -VI, 
 
 
 I 
 
 .i - 0- . 
 ; ^ . •• T«* ■ 
 
 
 ) 9J f/ r 
 
 beti^ 
 
 ■• ■ J-' itf-’irJi 5 
 
 ' ‘ * i{i#q opq 
 
 . r JiC'ztL: 
 
 , >'>f i/j ‘-•j/J ^ 
 . iAfxtV^s. 'ii: ' I 
 
 '.0 .#^>1 • ' ' ' ■ «*i i»0 , *4lOi*l - .^i 
 
 *1 
 
 ■■ito i 
 
 i 
 
 ft I . M iCtS 
 
 
 riA 1^'i «: t; . . 
 
 V -. *! - a ; ' t ,. *} •; a d J tiM Ttr - 
 
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- 17 - 
 
 EXPERI?/IEI'IT V. 
 
 Three 5 ^ sajuples of finely ground corn v/ere transferred to centri- 
 fuge bottles and extracted 7 times v/ith dOO cc. portions of 1 per cent, trichlora-- 
 cetic acid. The extracts were combined and made up to 6000 cc. Five hundred cc. 
 portions were used for each of the precipitations. The method of precipitation 
 in general was to bring the solution to boiling then to add the colloidal iron 
 drop by drop. During the boiling and precipitation frequent addition of hydro- 
 chloric acid were necessary to maintain the slight acidity necessary for precipi- 
 tation. After boiling for 1 minute the magnesium sulfate solution v/a,s added and 
 the solution again boiled for 1 minute. The solution ws,s then removed from the 
 flame and the precipitate allowed to settle. The solution was then filtered or, 
 if it was to be treated again, it was brought to boiling and the procedure repeat- 
 ed. After filtration the precipitate wa,s washed thoroughlv with hot water. The 
 followirig table gives the times precipitated before filtration, the amounts of 
 colloidal iron used, the temperature, and the average results of nitrogen left 
 in the filtrate. The results are expressed as percentage of the total nitrogen of 
 the saciple. 
 
 Times 
 
 pptd. 
 
 cc. of colloidal 
 iron 
 
 cc. of magnesiULii 
 sulfate soln. 
 
 Temper- 
 
 ature 
 
 IT in f ii trate 
 as percentage 
 of total N 
 
 1 
 
 25 
 
 3 
 
 cold 
 
 9.67 
 
 1 
 
 5 
 
 1 
 
 boilir^ 
 
 6.05 
 
 a 
 
 5.5 
 
 1.1 
 
 II 
 
 7.27 
 
 3 
 
 5 > 5.5 
 
 1 . 1,1 
 
 II 
 
 6.70 
 
 1 
 
 10 
 
 1 
 
 II 
 
 7.69 
 
 2 
 
 10,10 
 
 1.1 
 
 
 6.S£ 
 
 3 
 
 10,10,10 
 
 1,1,1 
 
 II 
 
 5.95 
 
 1 
 
 £0 
 
 3 
 
 n 
 
 0.36 
 
 £ 
 
 £0,10 
 
 3.3 
 
 11 
 
 5.52 
 
 3 
 
 £0,10,10 
 
 3 . 3.3 
 
 II 
 
 5.I4O 
 
 3 
 
 10,10,10 
 
 3 . 3.3 
 
 II 
 
 5 -i 4 £ 
 
 The filtrate from the last two precipitations were reprecipitated. 
 
• fca v’enil y 
 
 .- ■•■ 
 
 \K: oi 9 ; ikBff 
 
 . I| -<v. 
 
 .'V V:, tiov-.'ro •!< ■ t. 
 
 . "r-' -v. Oi' 5-es 
 
 
 -•" . -1* 
 
 Sv' r -rTyf/i *icl £:1 ■ ' >" 
 
 ^ Vh .' < • i 
 
 • •-• » i. - • - - 
 
 ^ ■ . * ' *T I: » 
 
 ic.: ..'r • ^ ^ ,: ' ■' '. .i'l'r ^ 
 
 , «ii*- lA'iSfjftjij r.L 
 
 \ 
 
 . . 4 'y •- -. -■! 
 
 '- ■ ; 
 
 inov; r::-i ^ -»n 
 
 . 'Ol .'■-.v-t 
 
 - 1 ^'; ’‘*i. '*?»•? 
 
 . ;■,, :: 
 
 * *i.“ ■■ -u ’.-..T;-.'. : 
 
 •.• ; O'"' ilvcl'f:, 
 
 ‘ “ ' /,> *i . 
 
 . r$r.':. . ’• 
 
 •:., • - ■ . , '•■-‘’t Silli r-^. 
 
 4 ' w *. . Ty » w 5 % 
 
 c ■ ■; -.r - 
 
 ' V. -r* ^ 
 
 
 
 
 ••:.*'j^J • i<"T' 
 
 j i- * -■; • li 'i ..j • 
 
 ' \ 
 
 ■• f i; .Itfie . 
 
 i 
 
 , ■ fiA/- .-.t J.: 
 
 .. .. :-::c . 
 
 *d ."^J rf.lfW’-'?! 'it 
 
 '■«^^'rc. »r. ,ic^: ' 
 
 r-.u ' jijt ■ -I -CJ- i^.'* frOV Ifi iSl ' ^ ’fch^Oilvi 
 , ' iixj. ■' ■ 'i-i 
 
 • . !^* r» ^ « • .'. i ^ r #( • -;-?■? 
 
 ;■ -..I 
 
 T -' #<i '.1 
 
 '"'S ' v' 
 
 
-lii- 
 
 using exactly tiie same amoxmts of reagents as were used for the first precipita- 
 tion. The percentages of total nitrogen reiaaining after the second precipitation 
 were 4. ’/I Pei* cent, and 4.?0 P®!* cent.^ respectively. 
 
 Tliis experiment indicated that the best method of precipitation v/ith 
 colloidal iron was three 10 cc. portions of colloidal iron, followed by three 
 3 cc. portions of magnesium sulfate solution, and allov/ing the precipitate to 
 settle between additions of the colloidal iron. 
 
 Experiment V, as well as others have indicated that, under all con- 
 ditions of precipitation tried, colloidal iron did not precipitate Vne proteins 
 from the cold 1 per cent, trichloracetic acid extracts quanti tatively . In addition 
 it had been almost impossible to obtain results which a,gree, even on four samples 
 
 of the same feed, of the same weight, run at the seme time, and in as near the 
 
 same manner as possible. The reason for this lack of agreement was thought to 
 
 be that, under the conditions for precipitation, it was practically impossible to 
 
 control very accurately the acidity of the solution. This is due, of course, to 
 the fact that trichloracetic acid in alkaline solution readily breaks up on boil- 
 ing, according to the reactions, 
 
 (1) g.Clg.COgH + NaOH = C.Clg.COgNa + H^O 
 (a) C.Clg.COglTa + H^O = G.HCI3 + NaHCOg 
 
 The chloroform vola.tilizes and the reaction goes gradually to completion towards 
 the formation of carborate. According to the method of precipitation used in 
 the previous experiments, during the boiling and precipitation with colloidal 
 iron, the solution v/as kept acid to litnms by the addition of hydrochloric acid 
 as fast as the solution became alkaline. In 8,n attempt to find, if possible, 
 more suitable conditions for the precipitation v;ith colloidal iron, experiment 
 
 VI was carried out. 
 
y 
 
 04 
 
 
 -K- 
 
 \' 
 
 ■ -vq 
 
 j> ' 
 
 
 
 .' -■ ' 
 
 «irfP 
 
 :or I 
 
 '»-*Q < 
 
 'a. Ir • ^*' {_ 
 
 tU 
 
 it:'<j . 'Ui: ** •> 
 
 
 /I*:- •, ••^.;jf. 
 
 
 T « 
 
 ' rf 
 
 n ' • ' 1 r - 1* c . ' .w - - - 
 
 : T >C - •■ a ''■ 
 
 p. ' 
 
 
 0 . -:tfj 
 
 
 ■■ <“ i 
 
 ••■ . « jjji 
 
 « . A 
 
 X . 
 
 •• 4. 
 
 . f. j . " 
 
 cl' 1 *:■•' . '^ 1 ;-- 
 
 • ..: Iri* . : ■* :... 
 
 -,A,CW \ r 
 
 J' .i» ’ ^ 1 *'■•3 
 
 '4 
 
 t 
 
 Tf 
 
 n.c'j.’ ~e^v 
 
 
 IT -n©.,_ 
 
 : , ,.i Ditx t: 'f'-t'r'li'*; 
 
 laftT 
 
 ■!* ;.;• T3;;'::X i': 
 
 4 - JSl* 
 
 ill 
 
 — y’ ' 
 
 , 
 
 
 I : * ‘-f 
 
 *c.. Til: 
 
 
 mJi 5 * '♦■ 
 
 r-» 
 
 r*. • 
 
 •1- , - i i V'- ti** : 
 
 . ;i.- . : : ' f’3 Jrisift j ,;v:i t ^oi * “ i M -' 
 
 . , .;i . .'ii'-' - --* 5 '3 i. - 
 
 "?’**'* 
 
 ' r y '’•<“ ■ ■ * ' •■*-' •'^-* ^ * t:* 
 
 ^ * -i' ! i ' 
 
 
 
-19- 
 
 EXPERIivlENT YI. 
 
 Four 25 samples of corn which had been ground to pass through a 
 UO mesh sieve were transferred to centrifuge bottles and each extracted 7 times 
 v/ith 200 cc. portions of 1 per cent, trichloracetic acid. The extracts were com- 
 bined, made up to bOOO cc., and filtered. Tnis stock solution was used for the 
 different methods of precipitation outlined below. Total nitrogen determined in 
 three I 5 O cc. portions gave an average of Id. 126 per cent, of the total nitrogen 
 of the feed in the solution. 
 
 (a) IdUTRAX IvIETHOD. Tw'O ^00 cc. portions of the stock solution were 
 precipitated ’with colloidal iron by neutralizing, boiling, and using three 10 cc. 
 portions of colloidal iron as described for experiment V. After filtration on 
 Buchner funnels the precipitates were washed thoroughly with hot water. 
 
 (b) DIRECT I-ISTHOD. Without neutralizing, two 5 OO cc. portions of 
 the stock solution were precipitated with colloidal iron by boiling and using 
 three 10 cc. portions of colloidal iron in exactly the same manner as the above 
 except that the trichloracetic acid was not neutralized. The precipitation was 
 carried out in tire presence of approximately 1 per cent, trichloracetic acid. 
 
 (c) AFTER DECOlviPOSITION OF TEE TRICHLORACETIC ACID BY BOILING. It 
 had been noticed that, during a second precipitation with colloidal iron, the 
 acidity could be maintained more nearly constant tlian during the first precipita- 
 tion. This was presumably due to the fact that most of the trichloracetic acid 
 had been broken down by the boiling during the first precipitation. Previous 
 tests had also indicated that little or no hydrolysis of protein is caused by 
 boiling with dilute trichloracetic acid. Decomposition of the acid by boiling 
 before precipitation was therefore tried. Two 5 OO cc. portions of the stock 
 solution were neutralized with sodium hydroxide solution. They were then boiled 
 gently, keeping the solution .just acid with dilute hj^drochloric acid, until there 
 
- -:si ja. 
 
 
 • ‘ r <T* ^ 
 
 ii 
 
 a ,r-,-0‘.'7 i; -i . 
 
 \J 0 . 
 
 *;:t. . 
 
 a.rt *5 6 
 
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 • . r'\'x:"\o iof: 
 
 e ' i-'l jL r 04 *: • .'tvtB .-u.c*u; ‘ 
 
 *«t ?.-.7 r.' i ■ . '' 79q~XT6 ,s.."0. .'ic^ .-"■'vO- >■*'» 
 
 , . . i • f . 't ’ • ‘- s’ ^ <* ^ ^ 
 
 \x, ^: ;o c ■'.j*-. ^;-‘'V 
 
 r iJ ‘j* , , 
 
 ..X B.: i..: 
 
 -■'■■ «..l* *r> : ■•■'■ 
 
 . Liii* ,jj . /v" , ;u X i-z.. 
 
 ,.:r" ' ) 
 
 :. :'^Ox.-:3 
 
 
 •: ' 
 
 
 ">■■ ,- '- •-' •'• 
 
 •s 30 . «J .V' -.'' '. 
 
 : * i r'4'j h-t'f: 
 
 V 1 r . J 
 
 it-. •‘r.; 
 
 
 t'. 
 
 1 
 
 -■ itf- .-rs-;; - . 
 
 f t 
 
 ■<1 
 
 .'1^. .-/vt ^ 
 
 i-.l-fc ft-.aw .. i.'x. fo« ' ‘ .' | 
 
 d2#1S . t r-juv 
 
 . : ..i j 
 
 : *. ii.' _ ‘ ' .0-t «.i< 
 
 
 
 . hlOi . 
 
 A '.- - 
 
 i.' -t ;i'*a • - ■' 
 
 •’•* ■ ■ . '■ . '■ 1 T!'''T '• ft t -t' 
 
 .’*. .Ci* 'R-r ’ : 
 
 ;s i.t- iTi.i- 
 
 "'"r’/Tv- 
 
 
 il, ■ 
 
 . ^ iV. . • 
 
 L • T 
 
 crc ^ 
 
 ■-’ i’ t .-,x-c 
 
 * «. gv. 4-- - 
 
 & vf 
 
 .5 in 
 
 »3^iVC. 
 
 
 
 I 
 
 .{ . 
 
 f , V * -U ---U- X. * •- w . .4# . . *W ^ •- * f X . •- 
 
 1 
 
 ... :v'. • '•.. ..T Jj ; . c~- 3 :. ‘'C‘ 
 
 3 . ■ £.,*. f ^ ; 1 ' 6 1 n 
 
 i :"'t i; f-r- • il .' • : 
 
 
 .::/: «.3.iii I ’ -iJ:' a<e 7 '._ 
 
 - i 7 -■ X :■ ' - ' ‘ ■:- ■ ‘ ‘ I ^ 
 
 
 
was no change in reaction. This took about IiO minutes. The solutions wore then 
 precipitated w^ith three 10 cc. portions of colloidal iron as usual. 
 
 (d) BY THE USE OF A BUFFER SUBSTANCE. It wa-s thought that by the 
 use of a buffer substance the acidity could bo accurately controlled. The mono- 
 basic sodium acid phosphate was selected as the most promising from a considera- 
 tion of its properties and the following reactions; 
 
 ( 1 ) C.Clj.COgH + NaOH = C.Clg.CO^lfe + E^O 
 
 ( 2 ) C.Clg.CO^Na + K ^0 = CHCI3 + NaHCOg (on boiling) 
 
 (3) NaHCOg + NaK^PO^ = Na^H PO^ + 
 
 ( 4 ) H2CO3 = H3O + CO3 
 
 An excess of the phosphate was considered necessary to insure a slight acidity. 
 
 One 5OO cc. portion of the stock solution v/s,s exactly neutralized to 
 litmus with N /2 sodiiom hydroxide, the exact amount being noted. Then twice its 
 equivalent of mono-basic sodium acid phosphate added. The solution was then pre- 
 cipitated with colloidal iron, using tliree 10 cc. portions as in the above cases. 
 
 (e) SECOl'JD PRECIPITATION. Since a second precipitation has alv;ays 
 removed considerable nitrogen in previous experiments, each of the above fil- 
 trates were reprecipitated v/ith colloidal iron using three 10 cc. portions of 
 colloidal iron as usual. 
 
 The filtrates from the above precipitation were each made up to a 
 definite volume and total nitrogen determined in triplicate aliquots. A condensed 
 table of the averages of the results, expressed as percentage of the total nitro- 
 gen of the feed, follows; 
 

 Ileutral metuod 
 
 Conditions of precipitation 
 
 In presence 
 of 1 pet. tri- 
 chloracetic acid 
 
 After boiling in presence 
 
 >f NaH.PO. 
 
 ^ 4 
 
 B 
 
 aver. 
 
 B 
 
 aver. 
 
 B aver. 
 
 aver. 
 
 IO.U5O 10.13^5 10.294 
 
 After first precipitation 
 
 10.410 10.585 10.49^ 
 
 11.500 10. 780 11.140 
 
 17.436 
 
 6.261 5.602 5.932 
 
 After second precipitation 
 
 5.492 5.376 5.684 
 
 5.657 6.124 5.891 
 
 7.703 
 
 Fto 21 the above results the following conclusions v;ere drawn: (1) 
 The use of the mono-basic sodium acid phospliate to maintain the acidity is not 
 
 practicable for it interferes in some way with the precipitation. ^2) ^.'?hils I 
 
 boiling the solution to bree-k down the trichloracetic acid does not seem to 
 cause hydrolysis (unless colloidal iron precipitates some hydrolytic products) I 
 the method is less desirable tlian either of the first two. (3) It is irnpossiblej 
 from the results alone, to choose one from the other of the first two methods. 
 
 As to the technique involved in the two methods, the first or neutral method is 
 slightly the more preferable because of a quicker settling of the precipitate, 
 more rapid filtration, and a clearer filtrate. (4) The second precipitation in 
 tnis experiment, as in all other cases, shows tiiat some nitrogenous substances 
 are further removed. This seems to indicate that the precipitation of the pro- 
 teins from a cold 1 per cent, trichloracetic acid extract of the common feeds is 
 either not quantitative or that it is removing some nonprotein nitrogenous sub- 
 stances , 
 
 The work of Wolff (20), Hill (21), Yan Slyke, Vinograd-Villchur, 
 and Losee (22), and of others has proven, however, very conclusively tliat col- 
 loidal iron does not precipitate the norprotein nitrogenous substances, at least 
 from animal extracts. Although the nonprotein nitrogenous compounds found in an 
 
I J 
 
 t 
 
 i 
 
 I 
 
 f 
 
 I :r ^ ; 'to 
 
 
 
 I 
 
 : -I.’ 
 
 I 
 
 K 
 
 I: 
 
 V ~ f ^ ^ I 
 
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 ■ ' . » , j 1 Tr". . 
 
 r. ,>,v . 
 
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 ■ ; r •• ; ' ^ 0 , - s <^ -- 
 
 ■ V'- . • a . . r/3 TO ^7 !j :*rMDp t'lr. : 
 
 ..C'f 
 
 
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 . ; ^5 
 
 / I'Jl'J''? V.’ 
 
 i ,• -. -•- 7 *'. • :o '- 
 
 r -. . » *. j 0 3 > 1 ' . ':i ,', eT'i' ' 
 
 , ' , .) •«' ic*«x 
 
 issm 
 
aniuial extract should not differ to any great extent, at least in regards to 
 size of the molecules, from those found in a vegetable extract, the following 
 experiiTient was carried out in order to siiow whether or not free Stfn.r.onia nitrogen 
 or free amino-acid nitrogen was precipitated by colloidal iron. 
 
 EXPERIIvIENT VII. 
 
 Two UO gm. samples of alfalfa and two oO gm. samples of corn were 
 extracted in centrifuge bottles with ether, alcohol, and 1 per cent, trichlora- 
 cetic acid in the usual manner. The free ammonia and free amino-acid nitrogen 
 in the filtered trichloracetic acid extracts, in the filtrates from the first 
 and in the filtrates from the second colloidal iron precipitation, were deter- 
 mined in a. maniier similar to that used by Grindley and Eckstein (9). Tne re- 
 sults, expressed as percentage of the total nitrogen of the feeds, v/ere as 
 
 follows; 
 
 
 In 1 pet. trichlor- 
 
 After 1st 
 
 After ^rnd 
 
 
 acetic anid extracts 
 
 pp tn. 
 
 pptn. 
 
 alfalfa 
 
 1 • g64 
 
 2.097 
 
 2. 996 
 
 Free ammonia N 
 
 
 
 
 corn 
 
 3.050 
 
 3.034 
 
 
 al fal fa 
 
 10.749 
 
 9. 315 
 
 9.044 
 
 Free amino IT 
 
 
 
 
 corn 
 
 2.936 
 
 2.7S5 
 
 2.400 
 
 The above results indicate that colloidal iron does not carry down 
 free amij^onia nitrogen or free amino nitrogen unless slight hydrolysis is taking 
 place at the same rate of precipitation which is not very probable. From the 
 fact that 40 minutes boiling of the 1 per cent, trichloracetic acid extra.cts 
 without neutralization in Experiment VI did not cause any higher nonprotein 
 values than was shown in the extracts that were not boiled it does not seem 
 probable that dilute trichloracetic acid will lij'’drolyze proteins. In order to 
 prove conclusively that this is true, Experixaent VIII was carried out. 
 
-^ 3 - 
 
 EXPSRII'.ffil?r VIII. 
 
 A sainple of oats was extracted in the cold a few times with 0,'d. per 
 cent sodium hydroxide solution. A large amount of protein was obtained by neutra] 
 ization. The precipitate was filtered off and divided as accurately as possible 
 into 3 parts. Part A v;as transferred to a flask with 100 cc. of aamionia-free 
 water; part to a flask with 100 cc. of d.O per cent, trichloracetic acid; and 
 part to a flask with 100 cc. of 5 cent, trichloracetic acid. Each solu- 
 tion was refluxed for h hours. The free amino-acid nitrogen in each solution 
 was determined by the Van Slyke nitrous acid method. The results were: 
 
 
 Pressure 
 
 Temperature 
 
 Volume 
 
 A, Boiled with ammonia-free water 
 
 752.8 
 
 di.5 
 
 0 . 125CC . , 0. l30c< 
 
 B. " •' 2 pet. trichlorace- 
 
 tic acid 
 
 II 
 
 11 
 
 0.1c:5cc., 0.125c< 
 
 C. v;ith 5 pet. trichlorace- 
 
 tic acid 
 
 II 
 
 II 
 
 0.l30cc.,0.l30c( 
 
 Tile results show no indication of hydrolysis with d or ^ per cent, 
 trichloracetic acid by boiling for 'd hours. 
 
 (5) Procedure finally adopted for extraction of noi^protein fraction 
 
 Tne best method of extracting the nonprotein nitrogenous constitu- 
 ents of a finely ground simple of feed, as shown by the above experiments, is 7 
 extractions of the sample in a centrifuge bottle, first, with 100 cc. portions 
 of anhydrous ether; second, 200 cc. portions of cold absolute alcohol; and third, 
 200 cc. portions of cold 1 per cent, trichloracetic acid. The small amount of 
 protein material extracted by the trichloracetic acid is remioved by precipitat- 
 ing with colloidal iron. For this precipitation the extract is brought to boil- 
 ing, nade distinctly aLkaline with sodiun hydroxide solution, then immediately 
 
-^ 4 - 
 
 made just acid to litmus with hydrochloric acid. Tsn cc. of colloidal ferric 
 hydrate are slo'wly added^ the solution boiled for 1 minute, 3 cc. of a solution 
 of magnesium sulfate (made by dissolving crystallized magnesium sulfate in an 
 equal volume of water) are added, and the solution boiled for 1 minute. The 
 solution is then removed from the flcime and the frecipi tS'.te allov.'ed to settle. 
 During the boiling, precipitation, and standing the solv.tion is kept acid to 
 litmus by the occassional addition of hydrochloric acid. As soon as the precip- 
 itate has settled the solution is again brought to boiling and the precipitation 
 with 10 cc. of colloidal iron and 3 cc. of magnesium sulfate solution repeated. 
 After the precipitate has settled the second time the procedure is again repeat- 
 ed. After the precipitate has settled the third time the solution is filtered 
 on iiard paper v/ith suction. The precipitate is v;ashed thorou^ly with boiling 
 aranonia-free water. The filtrate is now reprecipitated, using three 10 cc. por- 
 tions of colloidal ferric hydrate, in exactly the same manner as described for 
 the first precipi tetion. 
 
 B. TREATiOTT OF THE RESIDUE INSOLUBLE IN ETHER, ABSOLUTE ALCOHOL, 
 
 AND COLD 1 PER CEN’T. TRICHLOPJLCETIC ACID. 
 
 In the improved method of Eckstein and Grindley (I 5 ) the residue 
 left after extractions v/ith ether and alcohol was digested for I 5 hours three 
 or four times with 0.1 per cent, hydrochloric acid until all the starch liad been 
 converted into sugars. The residue insoluble in 0.1 per cent, hydrochloric acid 
 was boiled with 20 per cent, hydrochloric acid until the proteins v;ere ccanpletely 
 hydrolyzed. The proteins in the 0.1 per cent, hydrochloric acid extract were 
 precipitated by neutralization and by 5 volumes of absolute alcohol. The pro- 
 teins precipitated by neutralization and by alcohol were completely hydrolyzed 
 by boiling with 20 per cent, hydrochloric acid. The filtrate from^ the alcoholic 
 precipitate was finally hydrolyzed by boiling S hours with 5 cent, hydro- 
 
1 
 
- 25 - 
 
 chloric acid. 
 
 The first crianga to be made in the Eckstein and Grindley method was 
 the complete removal of all the nonprotein nitrogenous constituents. The proce- 
 dure finally adopted, as outlined in the last section above, consisted of extrac- 
 tions in the centrifuge bottle with ether, absol^ite alcohol, and cold 1 per cent, 
 trichloracetic acid. The small amount of protein material extracted by the tri- 
 chloracetic acid was recovered by precipitation with colloidal ferric hydrate. 
 
 The next was a minor change and consisted of \ising 10 volunies in- 
 stead of 5 vol-umes of absolute alcohol to precipita.te the proteins remaining in 
 the 0,1 per cent, hydrochloric acid extract after precipitation by neutralization. 
 Ten volumes of absolute alcohol was found to remove the proteins more completely 
 and at the same time less starch tlian was precipitated vd.th 5 voliimes of alcohol. 
 
 With these modifications it was possible to separate the nonprotein 
 nitrogen and the greater part of the carbohydrates from the main portion of the 
 proteins before the latter were hydrolyzed. In this manner the quantity of 
 htimin formed was reduced decidedly in the case of cereals, but only slightly in 
 the case of rougiiages. This latter fact has also been noticed by Eeidig and Sny- 
 der (17)* Tlie explanation of the fact that the httmin nitrogen of roughages is 
 decreased but little over that obtaixied by the direct hydrolysis of the feed, 
 used earlier by Grindley and associates, is probably because the roughages exaa;in- 
 ed had comparatively little starch and a high content of fiber v/hich is present 
 during the hydrolysis by the Eckstein and Grindley method. The fiber present 
 during the hydrolysis is the proba.ble cause of the high humin nitrogen values. 
 
 In later experiinents this has been shown to be the case or at least experiments 
 in wnich the fiber was not present during hydrolysis show much lower nitrogen 
 values. 
 

 jl 
 
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-^ 0 - 
 
 EXPERIMEIfT IX. 
 
 In order to remove the starch, fiber, and other substances which 
 might interfere with the application of the Van Slyke analysis, many changes were 
 tried. The first was a dilute alkali extraction of the residue, insoluble in 
 ether, alcohol, and 1 per cent, trichloracetic acid, to remove most of the pro- 
 teins. The strength alkali used was O.h per cent, sodium hydroxide solution. Sia 
 HOO cc. extractions in a centrifuge bottle were made, two extractions being made 
 each 24 hours as usual. At first attempts were i.oade to precipitate the proteins 
 extracted by the dilute alkali. Neutralization, trichloracetic acid, and col- 
 loidal iron were all used for this purpose but a small amount of nitrogen e.lways 
 remained unprecipitated. Farther tests showed that no substances which inter- 
 fered with the Van Slyke analysis were extracted by the dilute alkali. The alka- 
 li extract was therefore made distinctly acid with hydrochloric acid, concentrated 
 under diminished pressure to a small voluine, transferred to a digestion flask 
 with an equal volume of concentrated hydrochloric acid, and completely hydrolyzed 
 by boiling under a reflux condenser for I5 to 20 hours. 
 
 The residue insoluble in dilute alkali was then digested vrith 0.1 per 
 cent, hydrocnloric acid and the sterch removed in the usual raa.nner. Two ^0 gm. 
 samples of oats which had been ground so as to pass throu^ an 80 mesh sieve ’.\^re 
 extracted completely with ether, absolute alcohol, cold 1 par cent, trichilorace- 
 tic acid, cold 0.2 per cent, sodium hydroxide, and 0.1 per cent, hy'drcchloric acic , 
 Tne residues reiiiaining after the la.st treatment were transferred to Kjeldahl 
 flasks and the total nitrogen determined. Four and fourteen hundredths per cent, 
 and 4.3^ cent, of the total nitrogen of the feed remained. 
 
 Two more 3O gm. portions of the same sample of oats were treated in 
 the same manner as the two above with the exception that after the extraction of 
 the starch with hot 0.1 per cent, hydrochloric acid the residues were transferred 
 back to the centrifuge bottles and extracted with 200 cc. portions of strorig 
 
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 v.yj 
 
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 *:o ■: • <5 - . b'.* 
 
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 alkali. Each extraction was for a cil4 ho'or period. The first four extractions 
 v/ere w'ith O.d per cent, sodiui: hydroxide^ the fiftii v/ith O.5 per cent., the sixth 
 with 1 per cent.^ and a seventh with 5 P'^r cent. The two residues insoluble in 
 strong alkali were transferred to Kjsldahl flasks and total nitrogen determined. 
 l.Sll per cent, and 1.30c; per cent, of the total nitrogen of the feed remained. 
 
 Three more 3O gm. portions of the SO mesh oats were extracted vrith 
 ether, alcohol, cold 1 per cent, trichloracetic acid, cold O.c; per cent, sodium 
 hydroxide, and hot 0.1 per cent, hydrochloric acid in the usual manner. The resi- 
 dues were then transferred to round bottom digestion flasks and boiled 3 minutes 
 with C5O cc. portions of dO per cent, hydrochloric acid. The solutions v/ere 
 cooled, filtered, aiid washed. The treatment with dO per cent, hj/drochloric acid 
 was repes-ted again. After washing the residues thoroughly they v;ere submitted 
 to Kjeldahl analysis. Tv;o and one hundredth per cent., d.d'k per cent., and ^.37 
 per cent, of the total nitrogen remained. Tae dO per cent, hydrochloric acid 
 extracts were made up to a definite volume and total nitrogen determined in ali- 
 quot portions showed tiiat c.40 per cant., d.yd per cent., and <i.51 per cent, of 
 the total nitrogen had been extra,cted. 
 
 Since tv;o 3 •minute boilings v/ith 'dO per cent, hydrochloric acid 
 removes about 2.5 pei' cent, of the total nitrogen and a strong alkali extraction 
 about 3 per cent, of the total rhtrogen remaining in the residue insoluble in 
 ether, alcohol, cold 1 per cent, trichloracetic acid, cold 0.2 per cent, sodium 
 hydroxide, and hot 0.1 per cent, hydrochloric acid, both extractions v/ere made on 
 two 50 gra. samples of oats and tv;o 3O gm. samples of corn. The c ;0 per cent, 
 hydrochloric a.cid extraction was follov/ed by the strong alkali extraction. The 
 percentages of total nitrogen in the residues v/are 0,120 per cent, and O.IO3 
 per cent, in the case of oats and O.I32 per cent, and O.II7 per cent, in the case 
 of corn. 
 
 From the results of the above experiment the 20 per cent, hydrochlor- 
 

 1 
 
 
 1 
 
-28- 
 
 ic acid digestion follov/ed by the strong alkali extraction was adopted as part 
 of the regular procedure. Tne 20 per cent, hydrochloric acid extract was trans- 
 ferred to a digestion flask with a volume of concentrated hydrochloric acid equal 
 to the volume of the water used in the washing which had been kept separate from 
 the 20 per cent, hydrochloric acid extracts and measured. This gives a 20 per 
 cent, hydrochloric acid solution and the proteins in the acid extract are com- 
 pletely hydrolyzed by boiling I 5 to 20 hours. 
 
 At first, precipitation of the proteins extracted by the strong alka- 
 li was attempted but finally this extract v/as acidified with hydrochloric acid, 
 concentrated under diminished pressure, and hydrolyzed with 20 per cent, hydro- 
 chloric acid just the same as in-the case of the dilute alkali extract. Later 
 the extraction v;ith three 5 O cc. portions of 5 per cent, sodium hydroxide for 
 24 hour periods 'was adopted as tiie procedure for the strong alkali extraction. 
 
 It was found that this extraction removed just as much nitrogen as the more 
 elaborate method of four 0.2 per cent., one O.R per cent., one 1 per cent., and 
 one 5 cent, sodium hydroxide extractions. This smaller volume of extract 
 was much more desirable than the larger ’volume because of the smaller quantity 
 of salts introduced into the final hydrolysate ard also because the strorig alka- 
 li extract is very difficult to concentrate on account of the foaming. The 
 smaller volume of extract can be neutralized and without concentration, if nec- 
 essary, tra.nsf erred to a digestion flask with an equal volume of concentrated 
 hydrochloric acid and hydrolyzed in the usual manner. 
 
 Qualitative tests with 3O gm. samples of oats, corn, and alfalfa in- 
 dicated that by the use of the above procedure the quantity of humin was not 
 large and that during the precipitation of the bases witli phospho tungstic acid 
 only a very small aaiOunt of black substance was formed alorig with the bases. 
 
 C. EXTMCTION OF STARCH WITH TRICHLORACETIC ACID. 
 
 All the extractions in the procedure, as modified according to the 
 

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last section, were now quite satisfactory v/ith the exception of the extraction 
 of starch with hot 0.1 per cent, hydrochloric acid. Tliis extraction, as carried 
 out by the Eckstein and Grindley method, consisted of boiling the residue in- 
 soluble in ether and alcohol for I 5 hour periods with 0.1 per cent, hydrochloric 
 acid until the residue gave no starch test. Two or three I 5 hour digestions were 
 usually necessary to remove the starch completely from the cereals such as oats 
 and corn. The proteins extracted by this procedure were precipitated first, by 
 neutralization, and then, after concentration of the filtrate, with 5 volumes of 
 absolute alcohol. Two to 5 cent, of nitrogen usually remained unprecipitated. 
 The filtrate from the alcoholic precipitation was concentrated under diminished 
 pressure and completely hydrolyzed with 5 cent, hydrochloric acid by boiling 
 6 hours. 
 
 As was mentioned in a previous section, 5 volumes of alcohol does not 
 precipitate proteins as completely as 10 volumes. This change w'as made but still 
 there reirained considerable nitrogen in the filtrate. Colloidal iron was used but 
 with no better results. Vfnen tne alcdiolic filtrate ws,s concexitrated and finally 
 hydrolyzed even with 5 pe^’ cent, hydrochloric acid and boiling for only <3 hours 
 quite a large aiiount of humin resulted. 
 
 Since the proteins could not be precipitated completely free: the ex- 
 tract containir:g the hydrolyzed starch and, of course, there ms the possibility 
 of slight hydrolysis of protein, it was thaught that, if it were possible to ob- 
 tain a reagent which would dissolve the starch without hydrolyzing it, it might 
 be possible to precipitate the starch from the proteins. It is 'well knes-vn that 
 certain organic acids will dissolve starch v/ith little if any hydrolysis. For 
 example, a 1 per cent, salicylic acid solution will dissolve starch and the solu- 
 tion filters quite readily. Dr. Mitchell suggested the use of a ci per cent, 
 trichloracetic acid solution in an autoclave and the precipitation of the starch 
 

 
-30- 
 
 with. two volumes of 95 P^r cent, alcohol. Since it W5s fomd that trichloracetic 
 acid dissolved only a small amount of protein in the extraction of the nonprotein 
 nitrogen the follov/ing test expsriiiient wa.s carried out to test the solvent ac- 
 tion of trichloracetic acid on starch. 
 
 EXPERIMENT X. 
 
 Two 10 gm. samples of oats v/hich had been ground to pass tnrough an 
 SO mesh sieve were completely extracted with ether^ absolute alcohol, and water 
 and O.OS per cent, hydrochloric acid in centrifuge bottles in the usual raamer . 
 The residues were labeled A and B. Residue B v«.s then extracted 6 times v/ith 
 <£00 cc. portions of alkali in the centrifuge bottle. Each residue was then trans 
 f erred to a I 5 OO cc. round bottom digestion flask v/ith '^0 cc. of h per cent, 
 trichloracetic acid. The first idea was to heat in the autoclave but it was 
 thought later tliat the saiue thing might be accomplished by heating on the steam 
 bath. About two thirds of solution A va.s transferred to a separate flask and 
 labeled A1 while the remaining portion; was labeled A2. Solution A1 was heated 
 in the autoclave for I 5 minutes at I 5 pounds pressure while ML was placed on a 
 steam bath, and with frequent sliaking, allowed to digest until it was apparent 
 (from the disappearance of the milky color) that much of the starch had been 
 dissolved. This took about 45 minutes. Solution B was set aside until the 
 tests on solutions A1 and Ah had been made. Solutions A1 and A2 were filtered 
 on hard mper with suction while hot. Tbi.e filtration, while quite rapid in both 
 cases, was faster in solution Ah. The residues were washed a, few times with hot 
 water and then tested with potassium iodide solution for the presence of starch. 
 Residue A1 gave a slight but distinct starch test while residue Ak gave no test 
 for starch. The filtrates v/hile hot were v/ater clea,r but on cooling became milkj 
 and after ste-nding over night much starch had settled to the bottom of the flasks 
 In order to insure complete extraction of all the starch the two residues were 
 
- 31 - 
 
 transfsrred back to tneir original digestion flasks with I 5 O cc. of d. per cent, 
 trichloracetic acid. A1 was again placed in the autoclave for I 5 minutes at 
 15 pounds pressure and Ac heated on the steain bath for about 3 O minutes. At no 
 time during the second digestion of .A2 did it give a starch test, while A1 after 
 being removed from the autoclave, gave a very distinct test. The solutions were 
 filtered and washed as before. The filtrates were added to the corresponding 
 filtrate from the first filtration. Solution B was then treated on the stemn 
 bath in exactly the same manner as Aci. The 3 filtrates v;sre warmed until the 
 starch dissolved and then filtered to remove any solid material that might have 
 got into thei'a. After cooling, each filtrate was made up to 1000 cc. and total 
 nitrogen determined in duplicate aliquots. The results, expressed as percentage 
 of the feed, were; 
 
 Al - treated 
 
 in 
 
 the 
 
 autoclave, not extracted with 
 
 di lute 
 
 NaOH 
 
 16 - 5 , 
 
 14.8 pet. 
 
 A2 - treated 
 
 on 
 
 the 
 
 steam bath, » »' '• 
 
 II 
 
 II 
 
 3 . 8 , 
 
 4.0 " 
 
 B - treated 
 
 II 
 
 11 
 
 " " , extracted ” 
 
 II 
 
 It 
 
 2.0, 
 
 1.7 «' 
 
 T’.vo cOO cc. aliquots of the filtered extracts were treated with 'd 
 
 volumes of 95 cent, alcohol to precipitate the starch. After standing 3 days 
 the starch precipitates were filtered off on ijard paper with suction end v/ashed 
 with alcohol {^d volumes 05 cent, alcohol; 1 volume of v/ater"^. Each of the 
 sterch precipitates ware transferred to Kjel^d^il flasks and total nitrogen de- 
 termined. The duplica.tes were averaged and showed 0.10 per cent., 0.12 per cent, 
 and 0.10 per cent, of the total nitrogen of the feed in the starch precipitates 
 Al, A2, and B, respective!;/. 
 
 The res'olts from the above experiment indicate that very little ni- 
 trogen is extracted by hot 2 per cent, trichloracetic acid, especially if the 
 greater part of the protein material has been removed previously by a dilute 
 alkali extraction; that the extraction of starch can be carried out very quickly 
 
-32- 
 
 and easily by heating on the steam bath for a comparatively short period of time; 
 and that two volumes of alcohol precipitates most of the sts-rch without precipitat > 
 ing but little nitrogen. 
 
 In order to compare the total nitrogen extracted by hot 0.1 per cent. 
 h 3 '’drochloric acid and hot c; per cent, trichloracetic acid the following experi- 
 ment was carried out. 
 
 EXPERLMEITT XI. 
 
 Four 16 gm. samples of 30 mesh oats were transferred to centrifuge 
 bottles and extracted in the follo'wing manner: Two samples, A and B, were ex- 
 
 tracted with two 250 cc. portions of ammonia -free water, followed by two cc. 
 portions of 0,02 per cent, hydrochloric acid, which in turn were followed by 2 
 more 25 O cc. portions of amraonia--free water. Two extractions were nade each 24 
 hours. Two samples, C and D, were extracted 6 times v/ith 200 cc. portions of 
 1 per cent, trichloracetic acid. Two extractions were mads each 24 hours. (See 
 Experiment III for this part of the experiment. Experiment III sdiowed that the 
 water and 0.02 per cent, hydrochloric acid extracted 27.975 cent, of the to- 
 tal nitrogen of the feed while the 1 per cent, trichloracetic extracted only 
 14.575 cent.). The residues were then extracted 6 times vvith cold dilute 
 sodium hydroxide to remove most of the proteins. Residues A and C were then di- 
 gested with 2 per cent, trichloracetic acid on the steam bath until the starch 
 was removed in the manner described in the preceeding sxj)eriraant. Residues B 
 and D were extracted in exactly the same rnamier as A and C with the exception 
 that 0.1 per cent, hydrochloric acid was used instead of 2 per cent, trichlorace- 
 tic acid. The extracts were made up to a definite volume and total nitrogen de- 
 termined in duplicate aliquots. The avererage results, expressed as percentage 
 of the total nitrogen of the feed, were; 
 

 
 j 7 - f V i 
 
 : P-r: .li, ‘ -'f-f 
 
 "I’lq X i.:c'5 C « te i . ■ ■ . . . 
 
 V,. 
 
 11 . :^tf 
 
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 - .•'N;; - ■ •' 
 
 
 
 
 ■.J X ; J- 
 
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 t I . *- •' .v 4 .> *c« : b£ 
 
 . . . • ■ . :7 . c-fi 
 
 . A X. • - • • - . ... j— *> — ' - 1 fc/. ‘ aO A . 
 
 
 Lo. iXt -on; ;' • 1 :K 
 
 • -» S. » . • 
 
 
 • X . V 
 
 '. OJ> '- ^ ti ./ 
 
 .-<2 i-'.-.'Iuu*. 
 
 c» 4 !w “• •wVCftrfl 
 
 .. . 7 . '' * '.X A *« ; • '•■•:-■-•#? -■': -li . aAtJa e-nsw'Cl 
 
 ■cz n- \ '.0 ^a^c&ir!; . < •- - ... tioA o.noUcc'xf _ . '■ 
 
 fT' V t rr sji.nt’ifi, r. ft'iitoi tno . k'Ccuani. ^ fri^ ^2^ tt: 
 
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 ■ ■ :;/»r 
 
- 33 - 
 
 Total nitrogan extracted v/ith the starch 
 I ' 
 
 I by hot c; pet. 
 trichloracetic 
 acid 
 
 from the residues left after extractions 
 
 with cold and 0.0^ pet. HCl 
 and cold dilute NaOH 
 
 (A) pet. 
 
 by hot 0.1 pet . 
 HCl 
 
 (B) c..<£70 pet. 
 
 with cold 1 pet. trichloracetic acid 
 and cold dilute KaOK 
 
 I (C) 1.91b pet. I (D) 3.5Q5 pet 
 
 Experiment III showed that over I3 per cent, more nitrogen was re- 
 moved from A and B than fran C and D, yet the hot h per cent, trichloracetic acid 
 removed less rutrogen from C tnan from A, which had over I3 per cent, more ni- 
 trogen in it, and the hot d per cent, trichloracetic removed only 1.91b psr cent, 
 nitrogen from C while hot 0.1 per cent, hydrochloric acid removed 3.595 cent, 
 from D, which had the same amount of nitrogen. 
 
 In order to determine whether or no t a second addition of two volnmes 
 of alcohol to the hot c per cent, trichloracetic acid extracts causes an;t’' addi- 
 tional precipitation of starch and also in order to determine whether or net the 
 small amouiit of protein extracted by the hot 2 per cent, trichloracetic acid was 
 completely hydrolyzed by boiling the concentrated alcoholic filtrate from the 
 starch precipitate with 5 Psr sent, hydrochloric acid for 6 hours, the following 
 experiment was carried out. 
 
 EXPERIivIElTT XII. 
 
 Five 25 gm. portions of corn w'ere weighed out and labeled 1 , 2 , 3 ^ 
 and 5 * Each portion was transferred quantitativeljr to a 5OO cc. centrifuge bot- 
 tle and extracted with cold 1 p?r cent, trichloracetic acid, v/ith dilute sodium 
 hydroxide and with 2 per cent, trichloracetic in the usual manner, iphe trichlora 
 cetic acid and dilute alkali extracts v/ere discarded v/hile the extracts contain- 
 

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 ; f i 
 
 V 
 
 
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 b 
 
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 w .1 
 
 
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 I 
 
 li 
 
 ii. 
 
 I 
 
 I 
 
 i 
 
 ■i -rf t. 
 
 
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 -3U- 
 
 ing the starch were treated as follows: Each of the 2 per cent, trichloracetic 
 
 acid extracts, without neutralization, were concentrated -under diminished pres- 
 sure to about 175 cc. The concentrated solutions were then transferred to liter 
 beakers, upon which marks had been placed to indicate cc., iiade up to the 25O 
 cc. mark, heated on the steam bath until much of the starch had dissolved, and 
 then 500 cc. of 95 psr cent, alcohol added. The solutions were allowed to stand 
 3 days, the starch precipitates filtered on Buchner funnels fitted with hard 
 paper, and the precipitates washed w'ith a 1:2 water-alcohol solution. The alco- 
 holic filtrates were concentrated under diminished pressure to about ^0 cc. One 
 hundred cc. portions of ammonia-free waiter were added and the solutions concen- 
 trated again to about ^0 cc. This procedure v/as repeated three or four times in 
 order to remove all the alcohol. The very concentrated solutions appeared turbid 
 but the turbidity disappeared on dilution v/ith water. Sokations 2, 3, and 5 were 
 transferred to dOO cc. beakers, upon v;hich marks had been placed to indicate 
 130 cc., the solutions made up to ISO cc., and 3OO cc. portions of 95 psr cent, 
 alcohol were added. Upon the addition of about 50 cc. of alcohol the solutions 
 became clear, just as was the case on dilution of the concentrated solutions with 
 water. At no time and in neither of the samples was there the least turbidity or 
 precipitation of starch. Therefore no attempt was imde to precipitate solutions 
 1 and 4 a secorjd time. The alcoholic solutions of 2, 3, and 5 were repeatedly 
 concentrated to small volumes under diminished pressure to remove all the alco- 
 hol. Each of the concentrated solutions were then transferred to 5*^0 cc. round 
 bottom digestion flasks. Solutions 1 and 2 were made up to I25 cc., the acidity 
 being 5 per cent, hjrdrochloric acid. Solutions 3, 4 , and 5 were :is.de up to 200 
 cc., the acidity being 20 per cent, hydrochloric acid. 
 
 All the solutions were then placed on the hot plats under reflux 
 condensers and boiled for 6 hours. The current v;as then turned off, samples 1 
 and 3 removed and cooled to room temperature (2S degrees C.}. By means of an 
 
i. r,'.-: ^ i • • Li:. 
 
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 accurately calibrated burette two 4 cc . samples were ranoved from each of solu- 
 tions 1 and 3. By means of the same burette o cc. of v/ater were added to each 
 
 4 cc. portion. Amino nitrogen was determined in a 10 cc. portion of each of the 
 1<£ cc. solutire. In running the determination the niachine was allowed to stand 
 
 5 mimtes and then shook for 1 minute. The results were*. 
 
 l-I 
 
 II 
 
 3-1 
 
 II 
 
 7 41 • 5 • 
 
 ti 
 
 cip 
 
 0.40 cc. 
 0.36 
 
 0.82 
 
 0.60 
 
 Solutions and flasks 1 and 3 were weighed and then replaced on the 
 hot plate and boiled for 2 hours longer. The solutions were cooled to 28^ and 
 any loss in weight made up with water. Amino nitrogen run in the same manner as 
 the above showed the following results: 
 
 l-I 
 
 II 
 
 3-1 
 
 II 
 
 741 . 5 nim« 
 
 II 
 
 11 
 
 II 
 
 29' 
 
 II 
 
 II 
 
 II 
 
 0.40 cc. 
 
 0.40 
 
 0.62 
 
 0.62 
 
 Tests for completeness of hj/drolysis after 10 hours boiling showed 
 the following results 
 
 l-I 
 
 II 
 
 3-1 
 
 II 
 
 737 • ^ 
 
 II 
 
 II 
 
 II 
 
 22 '- 
 
 23' 
 
 II 
 
 II 
 
 0.37 cc 
 0.37 
 
 0.56 
 
 0.70 
 
 Tests for completeness of loydrolysis after I5 hours boiling showed 
 the following results; 
 
 l-I 
 
 737.4 mm. 
 
 20'^ 
 
 0.42 
 
 II 
 
 II 
 
 210 
 
 0.44 
 
 (— 1 
 1 
 
 II 
 
 20° 
 
 0.59 
 
 II 
 
 II 
 
 II 
 
 0.59 
 
 ninety cc. of concentrated hydrochloric acid were then added to 1, 
 making a 20 per cent, hydrochloric acid solution, and the solution boiled for 10 
 hours. Tests for complete hydrolysis showed: 
 
 i 
 
lI 
 
 y 
 
 s 
 
 j 
 
l-I 
 
 II 
 
 -36- 
 
 0.70 cc. 
 0.06 
 
 7UU.U iim. 
 
 II II 
 
 The volmna of triis solution was IbO cc. while the volume of 3 was 
 dOO cc. If the above results are multiplied by d 00 /l 60 the cc. of nitrogen is 
 about the same as the amount of nitrogen evolved in 3 after I5 hours boiling. 
 
 Two facts are brought out by this experiment; First, the proteins 
 which are extracted by hot d per cent, trichloracetic acid from the residues pre- 
 viously extracted v;ith dilute alkali, are completely hydrolyzed by boiling 6 nours 
 with dO per cent, hydrochloric acid. See'ond, I5 hours boiling with 3 cent, 
 hydrochloric acid show no more hydrolysis than b haurs boiling. Considering 0.60 
 cc. IJ as indicating co.mplete hydrolysis in a solution of 200 cc. then O.I4O cc., 
 the N evolved after 6 , 8 , 10 and 13 hours boiling with 5 per cent, hydrochloric 
 acio., in a solution of 1^5 cc. v^ould indicate that a 5 pci* cent, hydrochloric 
 acid solution hydrolyzes the proteins present to about 40 per cent. The fact, 
 that, if tne 5 P®- cent, hydrochloric acid solution is made up to 20 per cent, 
 hydrochloric amid and hydrolyzed for 10 hours more, the amino nitrogen then be- 
 comes the ssa:s as that v/hich indicated complete hydrolysis in the other 20 per 
 cent, hydrochloric acid solutions, also indicates that 5 cent, hydrochloric 
 
 acid does not cause complete hydrolysis. 
 
 The constancy of the amino nitrogen values obtained on testing the 
 5 per cent, hydrochloric acid solution for completeness of liylrolysis after 6 , o, 
 10 , and 15 hours boiling is the probable reason vfny Eckstein and Brindley made 
 the mistake of considering the proteins in their similar fraction completely hy- 
 drolyzed by boiling for 0 hours with 5 per cent, hydrochloric acid. 
 
 From the appearance of the solution boiled for 6 nours and the one 
 boiled for I5 or ^3 hours with 20 per cent, hydrochloric acid it is possible that 
 less humin, and if so probably less hum.in nitrogen, is fom^ed at the end of 6 
 hours. Since hydrolysis appears to be complete at the end of o hours there is, 
 however, no use of boiling for a longer time in tlie case of corn at least. Since 
 there is probably little more than 1 per cent, of the total nitrogen of the sample 
 

- 37 - 
 
 in this fraction for any feed examined^ no appreciable error would be introduced 
 by not boiling longer than b hours, 
 
 Idot only do tlie results of Experiments X, XI, and XII favor the ex- 
 traction of the staren with trichloracetic acid over 0.1 per cent, hydrochloric 
 acid, but the ruanipulation in case of the former reagent is by far the more pre- 
 ferable. Qualitative Van Slyke analyses of oats and corn, using the trichloraceti 
 acid method of removing the starch, gave basic phosplio tungstates, which were wliite 
 or cream colored granular precipitates, entirely free from a,ny black colored sub- 
 stances. This alone would v/arrant the adoption of the trichloracetic acid method. 
 
 (1) Fir.al procedure for the extraction of starch. 
 
 The procedure finally adopted for the extraction of starch was as 
 follows: The residue insoluble in ether, absolute alcohol, cold 1 per cent, 
 
 trichloracetic acid, and cold 0.t£ per cent, sodium hydroxide is transferred to a 
 1500 cc. ro'and bottom digestion flask with 5 OO cc. of ^.0 per cent, trichloracetic 
 acid and placed on a. steam bath. Y/lth frequent shaking the solution is allowed to 
 digest until it is apparent (from the disappeara^nce of the milky color) that 
 much of the starch has been dissolved. This usually requires about 1 hour. The 
 solution is filtered while hot through a Sucliner funnel fitted with hard paper. 
 
 Tlie residue is washed a few times with boiling v/ater and the residue again treat- 
 ed with cc. of c! per cent, trichloracetic acid for about a half hour period. 
 The hot solution is filtered and washed as before and if the starch is not all 
 removed this treatment is repeated. The extract is concentrated tuider diminished 
 pressure to about kOO cc. and then the starch precipitated by the addition of two 
 voluiaes of 95 cent, alcohol. The solution is allowed to stand until the pre- 
 cipitate settles (about d days) and then filtered. The precipitate is washed 
 w'ith alcohol (h volumes of 95 cent, alcohol; 1 volume of water). The alco- 
 holic filtrate is concentrated repeatedly ur.der diminished pressure to remove 
 all the alcohol and is then completely hydrolyzed by boiling 8 hours with 20 per 
 
cent, hydrochloric acid. 
 
 -3d- 
 
 IV. EX'IEACTION OF STARCH AI^D NOIJPROTEIH NITROGENOUS CONSTITUENTS WITH HOT 
 2 PER CENT. TRICHLORACETIC ACID AIT) THE SEPARATION OF THE STARCH, PRO- 
 TEIN AND NONPROTEIN NITROGEN. 
 
 It has been shown during the course of this work that cold 1 per cent, 
 trichloracetic acid extracts all the nonprotein nitrogenous compouinds; that the 
 starch can be readily extracted with hot 2 p er cent, trichloracetic acid; that 
 2.0 per cent, trichloracetic acid will not hydrolyze protein on boiling for the 
 length of time required to extract the starch; and that hot 2 par cent, trichlor- 
 acetic acid extracts but little protein. It v/as thought tha.t it might be possible 
 to extract the sample of feed directly with hot 2 per cent, trichloracetic acid, 
 separate the starch, protein nitrogen, and norprotein nitrogen and in this manner 
 save much time and reagents by doing away v/ith the ether, absolute alcohol, cold 
 1 per cent, trichloracetic acid, and cold 0.2 per cent, sodium hydroxide extrac- 
 tions. In cases, where the distribution of the nitrogen in the nonprotein frac- 
 tion v;as riot desired, it was thought that the proteins might be precipitated 
 leaving the starch and nonprotein nitrogen. The total nitrogen determiriation on 
 this fraction would give the percentage of norprotein nitrogen while the protein 
 precipitate v/ould be saved for ai'ialysis. V/ith these id^-s in mind several exper- 
 iments and tests were mads with the following general results; 
 
 First, if the residue left, after extraction with hot 2 per cent, 
 trichloracetic acid, is dried and then extracted with ether and alcohol in the 
 usual manner, almost the same aarount of nitrogen is extracted as is extracted from 
 the fresh sample by ether and alcohol. Of course, this amount of nitrogen is very 
 small but the extraction of fats with ether h-'s been shovm to be very helpful in 
 the application of the Van Slyke ar.alysis. 
 
 Second, while the precipitation of the starch 'With alcohol was almost 
 a.s complete as in other cases, as shown by v/eights of the starch precipita.te, the 
 
 i 
 
- 39 - 
 
 precipitate contains considerably more nitrogen than is the case when the nonpro- 
 tein and much of the protein nitrogen is removed by previous extrs-ctions. 
 
 Tnird, colloidal iron precipitation of the proteins extracted by a 
 direct 2 per cent, trichloracetic acid extraction leaves an average of about lb 
 per cent, of the total riitrogen in case of cats and about 12 per cent, in case 
 of corn. Fran a large number of experiments the nonprotein nitrogen of oats and 
 corn has been shov/n to be about I 3 psi* cent, and 10 per cent.^ respectively. 
 
 Fourth, a colloidal iron precipitate of the proteins extracted by 
 hot trichloracetic acid contains very little if any starch. This was shovm by 
 the following experiment. 
 
 EXPERBIEhT? XIII. 
 
 The starch was removed from about 20 gm. of finely ground corn with 
 2 per cent, trichloracetic acid by the direct extra-ction of the sample. 
 
 A. The proteins were precipitated fron the 2 per cent, trichloracetic 
 acid extract with colloidal iron in the usual marmier. The precipitate gave no 
 starch test with potassium iodide solution. It was thought that the color of the 
 colloidal iron precipitate might mask the color test but on mixing a very small 
 amount of starch paste with a portion of the precipitate the test was very clea-r. 
 
 B. About 2 gn;. of the colloidal iron precipitate, obtained in (A) 
 were boiled for about 3 O minutes with dilute hydrochloric acid, the solution neu- 
 tralized, filtered, and the filtrate tested for reducing sugars with freshly pre- 
 pared Fehling's solution. IJo reducing sugars v/ere present. 
 
 C. Two gm. of starch ware dissolved in 225 cc. of 2 per cent, tri- 
 chloracetic acid by boiling. This is the approximate proportion of starch to acid 
 in the usual experimient. This solution was precipitated with colloidal iron in 
 
 the usual manner, the precipitate filtered and washed. On testing v/ith potassium 
 iodide solution, the precipitate gave absolutely no test v\hile a, very small por- 
 
i ^ 
 
 r‘ 
 
 j 
 
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 • .if ■ .< » i ..J _ . 
 
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 ■ . ' •■ ( 
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 :.e i- . - : v :r r; i : ,: , i' •:. ; . ’• - ^ •: • . ^ 
 
 ^■'. . . iv, C_ . 'tire: /j; .. . »><- •: . .w ,, . .. 'iv' f *’’. r 
 
 •:. i*, v i'.:> T , :: rTT I .i .'IIi: :: , * " ;c* 
 
 . ' . -.'If . . :.• *: 
 
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 ' ' iw . ^ 
 
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 w£ .*■ c ^ r. . '"r*' ' ' •., _ ** j ; • ,,v'Iv? ‘■.".i-JiJ J.'.iJl 
 
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 tion of the filtrate highly diluted gave a very clear test for starch. Both the 
 filtrate and the colloidal iron precipitate were boiled for 3O minutes with dilute 
 hydrochloric acid^ neutralized and filtered. Two cc. of the filtrate from the 
 original filtrate gave by boiling 'd. minutes with freshly prepared Fehling's 
 solutions, quits a large precipitate of cuprous oxide. The filtrate from the 
 hydrolysis of the colloidal iron precipitate was made up to ^0 cc. and a 5 cc. 
 portion gave absolutely no cuprous oxide and a 15 cc. portion gave just a trace 
 when tested in the usual rxanner with freshly prepared Fehiing's solutions. 
 
 ?/here tiiue is more imxportant than accuracy^ the direct extraction of 
 the feed with hot 2 per cent, trichloracetic acid, followed by the precipitation 
 of the proteins vnth colloidal iron, nay perhaps be used to an advantage. The 
 filtrate from the colloidal iron may be. concentra,ted and the starch precipitated 
 with two volumes of alcohol, leaving the rionprotein nitrogen, the distribution of 
 which nay be detennined if desired. 
 
 V. MODIFICATIONS OF THE VAN SLYKE METHOD 
 
 In the application of the Van Slyke method to the analysis of feeds, 
 the original method (6) modified as to the method of decomposing the basic phos- 
 photungstates (23) was used. Some slight modifications, most of which were made 
 necessary by the larger amount of protein material present, have been made. A 
 few' other minor changes in the technique have been found advisable. Plimmer's 
 (2h) method and a schenae for aeration long used in this laboratory is used for 
 the arginine determination, Denis' modification of Benedict's method for organic 
 sulfur is used for cystine. If considered necessary’-, an e2pla.rjation for any 
 further change will be given in the detailed procedure oi\tlined below. 
 
-Ill- 
 
 VI. THE METHOD IN DETAIL. 
 
 PART I. PREPARATION OF THE SA/IPLE. 
 
 Twenty five to 100 pconds of an average sample of the feed is obtained 
 usually on the rijarket. The sample, except in the case of roughages, is mixed 
 thoroughly and quartered down until about a 2 to 4 gallon sample rerriains. In 
 case of roughages the entire sample is cut in small pieces by passing it through 
 a motor driven cutter. This satrrple is then mixed and qua.rtered down until a. H 
 to 4 gallon sarnple remains. The 2 to 4 gallon sample in all cases is then coarse- 
 ly ground, thoroughly mixed, and quartered dovv-n again until 2 to 6 quarts remain. 
 This final sample is then ground so tnat the entire sample passes through a 40 
 to 60 mesh sieve. It is then mixed thoroughly and stored in air-tight Sui’e Seal 
 jars. The total nitrogen determined very carefully in each sample of the feed 
 is the basis of all calculations since all nitrogen determira.tions are expressed 
 in percentage of the total nitrogen of the feed. 
 
 Four portions of the finely ground sample of feed are us'oally run at 
 the same time. The samples are of exactly the same weight and the weight is 
 always such that each portion contains approximately 6 gn. of protein. Duplicate 
 or triplicate total nitrogen determinations are always made whenever possible. 
 
 Each portion is treated as fellows: 
 
 PART II. PRSPAPuS.TION OF THE HYDROLYZED PROTEIN SOLUTION. 
 
 A. EXTPA.CTION WITH AI3IYDR0US ETHER. 
 
 The sample of feed is quantitatively transferred to a 5 OO cc. centri- 
 fuge bottle and <^00 cc. of anhydrous ether added. The bottle is corked securely 
 and the cork wired in place. The bottle is then placed on a mechanical siiaker 
 v/hich keeps the bottle and contents in centinusou motion. Two extractions are 
 made each dM- hours; one extraction for a 7 to 6 hour period is made during the 
 
r 
 
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 rnA *! ^ 
 
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 ■ .: ..c -.. .■ vi ^ -.T I . v 
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-Uii- 
 
 day and a second extraction for a 14 to I 5 hour period is made during the night. 
 After each extraction the sides of the bottle are washed down with a fev/ cc. of 
 ether and the solution centrifuged until the solid material forms a compact mass. 
 The perfectly clear supernatant liquid is decanted into a properly labeled flask. 
 This treatment with ether is rex^eated 6 times, using 100 cc. portions of anhydrous 
 ether for each extraction. The combined ether extracts are filtered by gravity 
 turoiigh Whatimn filter paper No. 1, using a 4 inch funnel. The flask and filter 
 paper are washed thoroughly v/ith ether. Any solid matter collected on the filter 
 paper is transferred back to the insoluble residue which is treated as directed 
 below in (B). The ether filtrate is acidified vath a few drops of sulfuric acid, 
 the ether recovered by distillation from a Kjeldahl flask, and the total nitrogen 
 determined in the residue ranaining in the flask. 
 
 B. EXTRACTION 1.YITH COLD ABSOLUTE ALCOHOL. 
 
 Tv;o }vundrad cc. of absolute alcohol are added to the residue insoluble 
 in ether. The bottle is corked securely and placed on the shaker. The time of 
 the extraction during the day should be 7 to S hours and the time of extraction 
 during the night should be 14 to I 5 hours. After each extraction the sides of 
 the bottle are washed down with a few cc. of alcohol and the solution centrifuged 
 until the solid matter forms a compact mass. The perfectly clear supernatant 
 liquid is decanted into a labeled flask. This treatment with alcohol is repeated 
 5 times using each time £00 cc. of absolute alcohol. The residue is washed twice, 
 using each tiir.e 100 cc. of absolute alcohol, placing on the shaker for 1 hour, 
 centrifuging, and decanting. The alcohol extract is filtered by gravity through 
 a vrnataan filter paper No. 1, using a 4 inch funnel and the flask and funnel 
 washed with absolute alcohol. The alcoholic filtrate is acidified with a few 
 drops of sulfuric acid, the alcohol recovered by distillation fran a Kjeldahl 
 flask, and the total nitrogen determined in the residue remaining in the flask. 
 
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 Any solid uiatter collected on the filter paper is added to the insolable residue 
 which is treated as described below in (C). 
 
 C. EXTRACTION WITH 1.0 PER. CENT. TRICHLORACETIC ACID. 
 
 The residue insoluble in alcohol is extracted in the centrifuge bottl« 
 with HOO cc . of 1.0 trichloracetic acid. The time of the extraction during the 
 day should be | to H hours and the time of extraction during the night should be 
 14 to 15 hours. After each extraction the sides of the bottle are washed dovvn 
 with a few cc . of aujiaonia-free water and the solution centrifuged until the solid 
 matter forms a compact mass. The supernatant liquid is decanted^ allowing, if 
 possible, no undissolved material into the decantates. The residue is then ex- 
 tracted 6 times with 200 cc. portions of 1.0 per cent, trichloracetic acid, mak- 
 ing two extractions each 24 hours. Tne residue is then vmshed two or three timep; 
 using 100 cc. portions of ammonia-free water, placing on the shaker for 1 hour, 
 centrifuging, and decanting. The residue is treated s.s directed below in (D) 
 and the extr-act as directed below in (H). 
 
 D. ETI'RACTiaT WITH DILL^E SODIUivI HYDROXIDE. 
 
 The residue insoluble in 1.0 per cent, trichloracetic acid is now' ex- 
 tracted in the centrifuge bottle w'ith 200 cc. portions of dilute alkali. The 
 time of extraction during the day is 7 to hours, using 0.2 per cent, sodium 
 hydroxide, and the tLme of extraction during the night is 14 to 1 5 hours, using 
 0.1 per cent, sodium hydroxide. After each extraction the sides of the bottle, 
 are washed dovmi with a few cc. of aiomonia-free vrater, the solution centrifuged 
 and decanted as described for the previous extracts. If the alkali extracts tend 
 to foam a few drops of alcohol are added before the solution is centrifuged. Six 
 extractions with dilute alkali are made and then the residue is washed with 100 
 cc. portions of ammonia-free water in the manner described above until practically 
 
fraa of alkali. As soon as tiia extracts are decant 9 d_, they are made distinctly 
 acid with hydrochloric acid. The residua insoluble in dilute sodium hydroxide is 
 treated as directed below in (E) . The dilute alkali extract is concentrated under 
 diiTiinished pressure to about I5O cc., then transferred to a digestion flask with 
 an equal volume of concentrated hydrochloric acid, and completely hydrolyzed by 
 boiling on the hot plate for I5 to £0 hours under a reflux condenser. The com- 
 pletely hydrolyzed proteins in this solution are treated as directed below in (J). 
 
 E. DIGESTION WITH h.O PER CENT. TRICHLORACETIC ACID. 
 
 NOTE: - This extraction is unnecessary in the case of feeds having 
 little or no starch present. 
 
 The residue insoluble in dilute sodium hydroxide is transferred to a. 
 1500 cc. round bottom digestion flask with 5^0 cc. of k.O 'pei* cent, trichlora.ee tic 
 acid and placed on a stea^n bath. With frequent shaking the solution is allowed 
 to digest until it is apparent (from the disappearance of the milky color) that 
 much of the starch has been dissolved. This usually takes about 1 hour. The 
 , solution is filtered wiiile hot through a Buchner funnel fitted with liard paper. 
 
 The residue is washed a few times with boiling v/ater and the residue again treated 
 with cc. of 2.0 per cant, trichloracetic acid for aboiit a half hour period. 
 
 Tna hot solution is filtered and washed as before and if the starch is not all 
 removed this treatment is repeated. The residue insoluble in hot 2.0 per cent, 
 trichloracetic acid is treated as directed below in (E) and the extract as direct- 
 ed below in (I). 
 
 F. EXTRACTION WITH c :0 PER CENT. HYDROCHLORIC ACID. 
 
 'ihe residue insoluble in 2.0 per cent, trichloracetic acid is trans- 
 ferred to a round bottom digestion flask with 25O cc. of 20 per cent, hydrochloric 
 acid. The solution is heated to boiling on a wire gauze and boiled for 3 minutes. 
 
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 cooled to room temperature, and filtered through hard paper on a Buchner funiiel 
 with suction. This procedure is repeated once. The residue is washed e£ or 3 
 times with dO per cent, hydrochloric acid and than with ami..onia-free water until 
 practically free from acid. The wasnings with water are kept separate from the 20 
 per cent, hydrochloric acid extract. The 20 per cent, hydrochloric acid extract 
 and washings are transferred to a digestion flask with a volu'ne of concentrated 
 hydrochloric acid equal to the volume of the washings. iJiie proteins in this 
 solution are completely li^'-drolyzed in the usual manner. The completely hydrolyzed 
 prcteins are treated as directed below in ( J) . The residue is treated as directed 
 below in (G) . 
 
 G. EXTRACTION WITH STRONG SODIUIvl HYDROXIDE. 
 
 The residue insoluble in 20 per cent, hydrochloric acid is transferred 
 back to the original ^00 cc. centrifuge bottle with ammonia-free water. The so- 
 lution is centrifuged until the solid rraterial forms a compact mass and the super- 
 natant liquid decanted into a properly labeled flask. The residue is then ex- 
 tracted for three 2 k hour periods V7ith 5O cc. portions of 5 per cent, sodium hy- 
 droxide. After each extraction period the sides of the bottle are washed dom 
 with a few cc. of aimTiOnia-free water, the solution centrifuged until the solid 
 matter forms a compact mass, and the supernatant liquid decanted, the extracts 
 being added to the wrater which had been used to make the transfer to the centri- 
 fuge. Tne residue is washed 2 or 3 times in the centrifuge bottle with 100 cc. 
 portions of armionia-free water in the usual manner. After each extraction the 
 extracts are a-cidified v/ith hydrochloric acid. Ttie extract is concentrated, if 
 necessary, then transferred to a digestion flask v/ith an equal volume of concen- 
 trated hydrochloric acid, and completely hydrolyzed by boiling for IR to 20 hours 
 under a reflux condenser on the hot plate. The completely hydrolyzed proteins 
 in this solution are treated as directed belov/ in (J). The residue insoluble in 
 
r k 
 
in strong sodixun hydroxide is transferred to a Kjeldahl flask and total nitrogen 
 determii.ed. 
 
 H. THEATlIEl^ 07<’ THE 1 PEH CEHT. TRICHLOHACETIC ACID EXTRACTS. 
 
 The 1 per cent, triculoracetic acid extract is brought to boiling^ 
 niade distinctly alkaline with sodium hydroxide solution, then acid to litrnus with 
 hydrochloric acid. Add very slowly 10 cc. of colloidal iron^ and boil for 1 
 minute. Three cc. of a solution of crystallized magnesium sulfa-te (made by dis- 
 solving crystallized fiagnesi-arn sulfate in an equal volume of v\’ater) is added to 
 coagulate the excess of colloidal ferric hydroxide, and the solution again boiled 
 for 1 minute. The solution is allowed to stand until the precipitate settles, 
 then, without filtration, bring to the boiling point and reprecipitate just as 
 before. Allow the precipitate to settle and reprecipitate a third time. During 
 the boiling, precipitation, and standing the solution gradually becomes alkaline 
 due to the decomposition of the trichloracetic acid. The slight acidity is main- 
 tained by the occassional addition of hydrochloric acid. After the precipitate 
 has settled the third time the solution is filtered through ha,rd paper on a 
 Buchner funnel with suction. The solution should be poured on the filter so as 
 to filter practically all of the liquid before much of the precipitate is poured 
 on to the filter. The precipitate is vra.shed thoroughl 2 ;' with hot v/ater. The fil- 
 trate is now reprecipitated again in exactly the same manner as described above 
 and filtered on a Buchner funnel fitted with a fresh hard paper. The filtrate is 
 now cooled, made up to cc. ( 3 OOO cc . if necessary) and total nitrogen de- 
 
 termined in three cc. portions. Transfer the colloidal iron precipitates to 
 a digestion flask v/ith 20 per cent, hydrochloric acid and completely hydrolyze 
 by boiling on a hot plate under reflux condensers for I 5 to 20 hours. The com- 
 o 
 
 Iron Dialyzed Merck, contains 5 cent. Fe^Og. 
 
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- 47 - 
 
 pletelj'’ liydrolyzed proteins in this solution are treated as directed below in (J). 
 
 I. TRE.miEi'JT OF THE ci PER CEIIT. iRICHLORACETIC ACID EXTPJICTS. 
 
 The <L per cent, trichloracetic acid extract is concentrated under 
 diminished pressure^ without neutralizing, to about 175 then transferrec 
 
 to a beaker upon which a mark has been placed to indicate 25 O cc., made up to the 
 mark, heated on the steam bath until most of the starch has dissolved, and H vol- 
 umes of 95 cent, alcohol added. The solution is allov/ed to stand until the 
 precipitate settles completely (2 or 3 days), is then filtered on a Buchner funnel 
 fitted with hard paper and washed with alcohol of the same strength (2 volumes 
 of 95 cant, alcohol: 1 volunne of water). Fne starch precipitate is transferrec 
 to a Kjeldahl flask and total nitrogen determined. The filtrate is concentrated 
 repeatedly under diminished pressure until all the alcohol is removed. It is then 
 transferred to a round bottom digestion flask with an equal volume of concentrated 
 hydrochloric acid and completely hydrolyzed by boiling for 6 to 10 hours. The 
 hydrolyzed proteins in this solution are treated as described below in (J). 
 
 J. DETERIcIIlIATIOH OF INSOLUBLE HUI.IIN I^^ITR0GE^T. 
 
 The completely hydrolyzed proteins from (D), (F), (G), (H), and (I) 
 above, are filtered tlirougii the same Buchner funnel and washed a few times with 
 hot water. The residue is then transferred back to a digestion flask with 0.1 
 per cent, hydrochloric acid and refluxed two or three hours. The solution is 
 filtered, washed with hot water, and the residue again boiled for 1 hour with 0.1 
 per cent hydrochloric acid. After this treatment the residue is washed thoroughly 
 with hot water and the insoluble hurain nitrogen determined by submitting this 
 residue to Kjeldahl ana,lysis. The filtrate and washings from the above are united 
 and axialyzed according to the Van Slyke method as described in Part III below. 
 

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PART III. ANALYSIS OF THP HYDROLYZED PROTEIITS. 
 
 DETERIvlINATlON OF AvLiOMA (A/IID NITROGEtl) . Concentrate the filtrate 
 and washings from the determination of the insoluble hurnin nitrogen in Claissen 
 flasks luider diminished pressure until all the hydrochloric acid possible has 
 been driven off. Wash down the sides of the flask with ajuiionia-free water and 
 concentrate again almost to drjaiess. Transfer to a kOO cc. measuring flask with 
 amiv.orua-free vvater and make up to the mark. Now transfer to a clean one-liter 
 Claissen flask witn 100 cc. of 95 per cent, alcohol. For the determination of 
 the ammonia arrange the Claissen flask, and an ordira-ry one-liter, distilling 
 flask as shown by Van Slyke, (o)> Fig. 1. Now add 10 per cent, suspension of 
 calcium hydrate until alkaline and then add kO cc. in excess. Tne apparatus is 
 then joined together as shown in the figure and evacuated to a pressure of ^0 mm. 
 or less. The amount of n/iO acid added to the larger receiving flask should be 
 bO cc. and the amount added in the trap should be 3 O cc. The Glaissen flask is 
 then placed in a bath at U5“5*^'^> the solution boiled for a half-hour. In 
 
 case distillation starts too rapidly a little air is let in from the stop-cock 
 in the neck of the Claissen. 
 
 hhen the distillation is firhshed, the flask is lifted from the water 
 bath, and the vacuum is released by first allowing a very small stream of air to 
 enter through the side am and then through the capillary and finally opening 
 the stop-cock in the side arm. The n/iO acid from the receiving flask and the 
 small guard flask is washed into an 800 cc. titration bottle and titrated bade 
 with n/i 0 sodium hydroxide using alizarine sulforjate as indicator. 
 
 SOLUBLE HIIvlIN NITROGEN. Drring the distillation Lmiiiediately above all 
 the black coloring matter as soluble huirin is absorbed by the undissolved lime. 
 Filter through hard paper on 3 ii'icn Buchner furmel using suction. V/ash with 
 ammonia-free water and transfer the insoluble residue to a beaker vvith about 850 
 cc . of armonia-free water. With frequent stirring heat gently for about I 5 min- 
 
-49- 
 
 utes. Filter and wash thoroughly with ammonia-free water. Repeat this treat- 
 ment once. The total nitrogen determined in the residue is coxisidered to be 
 the soluble humin nitrogen. 
 
 PRECIPITATIOI'I OF THE BASES (CYSTIHE, LYSI!TS, ARGININE, AND HISTIDINE) . 
 The filtrate from the soluble humin is neutralized with hjrdrochloric acid, re- 
 turned to the vacuum distilling flask, and concentrated to about 65 cc. Wash 
 the concenti’ated solution into an accurately calibrated 200 cc. measuring flask 
 and dilute to the mark. Mix thoroughly. Now, by means of a 100 cc. pipette 
 calibrated to deliver exactly 100 cc., transfer 100 cc. of the well mixed solu- 
 tion to a clean flask and wash out txie pipette with water, back into the 200 cc. 
 measuring flask. Add IS cc. of concentrated hydrochloric acid to each 100 cc. 
 portion and shake. Place I5 gra. of purified phospho tungstic acid in each of 
 two 3OO cc. Erlexjmeyer flasks upon which marks have been placed to indicate 
 200 cc. Dissolve the phospho tungstic acid in a sirall volume of water. Now to 
 each flask containirig the phosphotuiigstic acid, add, very slowly with constant 
 shaking, a 100 cc. portion of the acidified hydrolyzed protein solution. Each 
 solution is diluted with water up to 200 cc. and heated in a water bath until 
 the precipitate of the bases is nearly or quite dissolved. The temperatui’e of 
 the solutions should be increased very gradually. The bases reprecipi tate on 
 cooling e.s crystalline or granular phospho tungstates. Hie solutions are allowed 
 to stand 4h hours for the precipitates to form for in less time the precipita- 
 tion of histidine may be incomplete. The precipitates should not be allowed to 
 stand much longer than 4^ hours. 
 
 WASHING OF THE BASES. It. is necessary that the phosphotungst ic acid 
 precipitate shall.be washed entirely free of the mother liquors and the amino- 
 acids of the unprec ip i table fraction. It is also necessary/ ths.t as small an 
 amount of the washing solution as possible shall be used, in order that the 
 precipitate, which is slightly soluble in the washing solution shall not dissolv( 
 
f 
 
- 50 - 
 
 tc an appreciable extent during the washing. 
 
 The phospho tungstic precipitate of the bases is filtered and washed 
 in the following rnani^er; A hardened filter paper is cut to the proper size to 
 fit accurately as usual against the bottom of a, ^ inch Buchner funnel. T^e pre- 
 cipitate is poured onto the filter thus prepared and the mother liquors are dravm 
 off as completely as possible by a steady and moderately strong suction^ the 
 precipitate being pressed down by a flattened rod. Do not allow the precipitate 
 to be sucked dry. The v/ashing is carried out v/ithout at any time releasing the 
 suction. Successive portions of 10 cc. of a washing solution^ containing 3.5 
 per cent, hydrochloric acid and 2.5 P^r cent, phospho tungstic acid, are poured 
 onto the well packed precipitate, which is stirred up with each portion by means 
 of a flat- tipped rod, so tiiat all parts of the precipitate are reached and all 
 lumps well broken up. If a steady suction is maiiitained all the time, this can 
 be done without danger of loosening the filter from the floor of the funnel. The 
 first three or four portions of the washing solution are used to dislodge the 
 last granules of the precipitate from the flask in which the latter are formed. 
 The succeeding portions are added from a pipette in a fine stream. In case the 
 first four washings leave a few granules of the precipitate still in the flask, 
 they are allowed to remain there, as they are already sufficiently v/ashed and the 
 subsequent portions of the washing solution used to wash the precipitate, in the 
 me.nner just described. 
 
 The washing solution should be cooled to 0^ before it is poured onto 
 the precipitate. It lias been found that the solubijity of the phospho tungstates 
 of the hexose bases is only about one-fourth as great at as it is at room 
 temperature, consequently, when the washing solution is used at or near 0^ the 
 danger of dissolving appreciable amount® of the precipitate is reduced to a min- 
 imum. The number of washings necessary in each case to free the phospho tungstic 
 precipitate completely from the mother liquors is determined, according to the 
 
-^ 1 - 
 
 raethod of Tan Slyke, by testing the successive washings for calciuni. It has been 
 found that 6 washings with 10 cc. portions are usually sufficient to remove the 
 calcium. 
 
 DECOI'^POSITION OF THE PH) SPHOTUHGSTIC PRECIPITATE BY A MIXTHPJE OF 
 ETHER AND AWYL ALCOHOL. Hie precipitate of basic phospho tungstates is removed 
 from the filter by a spatula and washing, and transferred to a half-liter separa- 
 tory funnel, using 200 to 3 OO cc. of water to effect the transfer. Vrnen the pre- 
 cipitate has been removed as completely as possible by mechanical means, the fil- 
 ter paper is spread out on the bottom of a dish and v/ashed Vvith water made alkaline 
 v/ith a few drops of sodium hydroxide. This dissolves the portions of precipitate 
 imbedded in the fibers of the filter paper. In case any granules of the precipi- 
 tate have remsiined in the flask in which the precipitate was originally formed, 
 these are either washed or dissolved out. These alkaline washings should be 
 neutralized v/ith dilute hjrdrochloric acid before being added to the main portion. 
 Seven and one-half cc. of concentrated hydrochloric acid are added, and the m»ix- 
 ture is snaken with 1:1 amyl alcohol-ether, using so much of the later that it 
 all floats in a layer above the water after the precipitate has gone into solu- 
 tion. Usually about 100 cc. of the ether -amyl alcohol suffices, and one or two 
 mirmites shaking results in complete solution of the precipitate. If too little 
 of the ether-amyl alcohol has been used, some of it will, after taking up phos- 
 photungstic acid, sirk as an oil below the aqueous phase. In case this happens, 
 more of the extracting mixture is added, until all floats in one layer at the 
 top. 
 
 In some cases the aqueous and ether -amyl alcohol layers do not sep- 
 are.te readily v/ith a clean boundary betv/een them. This effect is due to the pres- 
 ence of a slight amount of humin which may have escaped previous adsorption by 
 calcium nydrate. In this case the unadsorbed huinin is carried do\m by the basic 
 phosphotungstates, and fouls the solution when their precipitate is decomposed 
 
as above described. In order to clear the solution up^ it is all, without sep- 
 aration of the aqueous and ether -cuiiyl alcohol layers, passed through a Buclaner 
 funnel v;ith suction. The residue is washed, first with amaonia-free water and 
 than with the aiayl alcohol-ether mixture, using no mors washing solutions than 
 absolutely necessary. The residue together with the filter paper is transferred 
 to a Kjeldahl flask and total nitrogen determined. This fraction of nitrogen is 
 referred to as the "unadsorbed humin nitrogen." The filtrate is returned to the 
 separatory funnel, tne two layers allowed to separate, and the aqueous layer 
 drawn off into a second !half-liter separatory funnel. The aqueous layer is then 
 extracted with three more successive portions of ether-amyl alcohol, using each 
 time a volume of the mixture equal to about one-fourth the volume of the water 
 solution (about ^0 cc.). Fiiially the combined ether-amyl alcohol extracts are 
 shaken out twice with water to remove traces of bases that might have been carried 
 into the extract; this portion of water is then shaken once or twice with fresh 
 arijj'l alcohol -ether, axid combined with the main solution of the bases. Tae latter 
 should be free of phosphotungstic acid as demonstrated by the absence of a precip- 
 itate when a few drops are added to a saturated solution of barium hj^drate in a 
 snail test-tube. The amyl alcohol-ether solution is transferred to a Xjeldahl 
 flask, a few pieces of pumice a^nd a few drops of sulfuric acid added; the ether 
 is driven off by heating on the steam bath; the axryl alcohol is distilled off, 
 recovering the alcohol; and then the total rutrogen determined in the residue in 
 the usual manner. The nitrogen in this fra-ction is referred to as the "nitrogen 
 soluble in amyl alcohol-ether mixture." The solution of the bases is now concen- 
 trated to dryness under diminished pressure in order to drive off the free hydro- 
 chloric acid. Add a small amount of ammonia-free v;ater to the residue and thor- 
 oughly and comipletely loosen the particles adhering to the sides of the flask with 
 a properly bent glass rod. Wash off the glass rod and ’wash dovm the sides of the 
 flask v/ith a small amount of water. Concentrate to about 10 cc. and then transfer 
 
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 ths concentrated solution of the bases to a 100 cc. measuring flask^ by means 
 of a bent glass rod and small portions of araraonia-free v^ater. Ivlake the solution 
 up to the mark and mix thoroughly. If a residue settles out on standing, filter 
 through a small dry quantitative filter paper into a clean dry flask. Disca.rd 
 the first cc. of the filtrate and use the reriiainder for the various determinatiom 
 of the bases below. V/ash the residue on the filter paper thoroughly ;vith ammonia- 
 free ^'sater and discard the washings. The thorou.^ly v/ashed residue, together witl 
 the filter paper, is submitted to Kjeldahl analysis and the nitrogen referred to 
 as the "nitrogen in residue filtered from solution of the bases." 
 
 DETERIvIIl'ATIOiJ OF ARGIFIIJE. Of the 100 cc. of solution containing 
 the bases, two 25 cc. portions are placed in 5 OO cc. short necked Kjeldahl flasks 
 v/hich are connected with upright Liebig condensers, Cc^rryir^ at the top Folin 
 bulbs connected to the condensers by rubber stoppers. Fifteen cc. of K/10 acid 
 are placed in each of the Folin bulbs. Twenty five cc. portions of 50 P-h cent, 
 sodium hj^'dr oxide solution and severa-l bits of porosis plate are added to the 
 Kjeldahl flasks. The solutions are then boiled gently on wire gauzes for ex- 
 actly five and one-half hours, then let the water out of the condensers and boil 
 for one-half hour longer. A small stream of p^urified air is passed through the 
 apparatus during the entire six hours. This is simply done by passing the air 
 from compressed a-ir pipes through a purifying train and admitting it to the 
 apparatus by means of a capillarj'- placed in the stopper of the Kjeldahl flasks 
 and reaching to the bottom of the flasks. The acid in the Folin bulbs is trans- 
 ferred to titration bottles with neutral water and titrated back with k/iO alka- 
 li, using alizarine suiforiite as indicator. 
 
 DETEEivillATION OF THE iluIITO KITHOGEiT OF lEE BASES. Two 5 cc. portions 
 of the solution containing the bases are used for the amino-acid nitrogen deter- 
 rnii'^tion which is run in the usual manner in the Van Sl^/ke machine. The deter- 
 mination is continued for a laalf hour period. A blsmik deterrairiation of the 
 
reagents aiust be run for the same length of time. 
 
 DETERJ'/illJATION OF THE TOTAL iMITROGEN OF TEE BASES. Two 10 cc. por- 
 tions of the 100 cc. solution containing t^e bases are used for the total nitro- 
 gen determination. The digestion wi tii sulfuric acid must be continued for six 
 hours after the solutions becoiiie clear. 
 
 DETEEIvlINATION OF CYSTINE. The amount of cystine present in the bases 
 is obtained from the content of the solution in organic sxilfur. Take 1^ cc. of 
 each of the solutions containing the bases for the determination of cystine. 
 
 Transfer the measured portion of the solution containing the bases to 
 fused silica dishes of 7 to 10 cm. diameter and add 10 cc. of Denis' modifica- 
 tion of Benedict's solution. Add about 0.1 gm. of pure sucrose. The mixture 
 is concentrated to :?ryness on the water bath. It is then heated over a free 
 flame, gradually increasing the temperature until the mixture is thoroughl;^ 
 charred. Then place the dish in a muffle furnace heated to a dull red heat for 
 10 to 15 minutes. Allow to cool. Add 10 cc. of 10 per cent, hydrochloric acid 
 and dissolve the contents by warming and stirring for 3 to 5 minutes. Although 
 the solution may appear to be perfectly clear,” filter thrcu.gh a good grade of 
 quantitative filter paper and wash tlioroughly with hot v®,ter. Dilute the solution 
 to about 150 cc. and heat bo boiling. In order to make sure that an excess of 
 barium chloride is present, while boiling, add dnop by drop, 10 cc. of a filtered 
 10 per cent, solution of barium chloride. Allow to stand 'd days and if the pre- 
 cipitate is perfectly white, filter on weighed Gooch crvicibles. If the precipi- 
 tate produced by barium chloride is yellow indicating impurities the precipitate 
 must be purified by the use of sodium carbonate in the usual manner. If this 
 procedure is followed carefully, purification of the barium sulfs.te precipitate 
 is rarely necessary. 
 
 The barium sulfate is washed and v/eighed as usual. The weight of 
 barium sulfate obtained must be corrected for the amount of sulfur found in the 
 
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 reagents by blanl: analyses. Tiiat is^ for each series of cystine deterrairiations 
 two blarik anedyses on the reagents are run at the same time. In the reagents 
 used by Van Slyka the correction v/as I.5 mgs. of barixjm sulfate. Reagents re- 
 quiring a much larger correction should not be usedj as the cystine present of- 
 ten yields only a few milligrams of barium sulfate. It ha.s been found that^ by 
 repui'ifying the sodium chloride used in Derds* reagent and using fused silica 
 dishes instead of porcelain, the blanks are exceptionally low and constant, 
 usually varying between 0.] and O.9 mgs. 
 
 DETSEivilllATlOlT OF THE TOTAL NITROGEII U THE FILTRATE FROM THE BASES. 
 
 To the combined filtrate and w'ashings from the phospho tungstate precipitate of 
 the bases, 5O cent, sodium hydrate solution is added until the solution 
 turns slightly turbid by precipitation of lime and it is almost neutral to lit- 
 mus paper. In some cases a slight precipitate is formed before the solution is 
 neutral and it readily redissclves by the addition of a fe-.; more drops of alkali. 
 In adding the alkali, it is essential that the neutral point should not be 
 passed by more than a few drops, as otherwise a precipitate, vmich usually dis- 
 solves readily upon the addition of a little auetic acid, may form which will 
 not dissolve. The solution is placed in a Claissen flask a-nd concentrated under 
 diminished pressure until salts begin to crystallize out. The solution is then 
 washed into a 200 cc. measuring flask and diluted to the mark. Allovi the solu- 
 tion to stand over night and if a precipitate settles out filter the solution 
 through a dry filter paper into a dry flask. Total nitrogen is deterniined in 
 the filtrate in three 25 cc. portions. In each determination use one and one- 
 half times the us\ial amount of reagents and continue the digestion for 6 hours 
 after the solution has become clear. Under these conditions the phospho tungstic 
 acid does not interfere at all with the accuracy of the determination. The 
 residue which was filtered from the solution of the filtrate from the bases is 
 
 washed thorouglily with v/ater and su'Dinitted to Kjeldahl ar^..lysis. The nitrogen in 
 
• ^ . i i'*c. , . V. '.' i^ir.: ■ 
 
 la, ^-| ^1 Mil ’i'l 
 
 irr.'S‘frf> t9ca:«^lfum||P 
 
 ' iH » ■ 
 
r 
 
 -56- 
 
 this fraction^ which lias always been found to be exceedingly smll, is referred 
 to as the ’'nitrogen in the residue filtered from the solution of the filtrate 
 from the bases.’' 
 
 DETEExMIMTIOlI OF THE iUyllUO NITHOGEil IN THE FILTRATE FROM THE BASES. 
 
 Tan cc. portions of the filtrate from the bases are used for the ai:iino-acid 
 determinations, which are run in the usual manner for six minutes using the 
 large burette. 
 
 CALCULATION OF ARGININE, CYSTINE, HISTIDINi:, LYSIIE NITROGSI'J, AND THE 
 NON-AMINO -AC ID NITROGEN IN THE FILTRi-TE FROM 'THE BASES. All determinations are 
 expressed in percentage of the total nitrogen of the feed and in percentage of 
 the feed. The determinations on the solution of the bases and on the solution 
 of the filtrate from the bases are corrected for the solubility of the bases 
 accordirjg to the tables of Van Slyke. It is to be remembered tliat the hydrols’-sed 
 proteins from an entire sample were made up to 200 cc. and two 100 cc, portions 
 taken for precipitation which was carried out, as far a-s quantities of reagents 
 and volumes are concerned, according to the method of Van Slyke. The standard 
 acid, while referred to as n/ 10, is in realitj^ sliglitl*/ less than that. One cc. 
 of the acid is equivalent to 0.0014 grams of nitrogen. 
 
 (a) ARGININE NITROGEN. The grams of uncorrectad arginine nitrogen 
 in tlie portion of the sample taken for precipitation of the bases is equal to 
 the cc. of acid neutralized multiplied by 0.0014> multiplied by 2 (sines only 
 half of the nitrogen of arginine is determined as ammonia), multiplied by IOO/25 
 (since 25 cc. portion of the 100 cc. solution of the bases \<.'ere taken for argin- 
 ine determination). Fne grams of corrected arginine nitrogen in an entire sample 
 is equal to the grams 0 f uncorrected arginine rdtregen obtained above plus O.OO32 
 .(the solubility correction for arginine) multiplied by 2OO/1OO (since half of the 
 t:00 cc. solution of the hydrolyzed proteins of the whole sample was taken for 
 precipitation v/ith phospho tungstic acid). 
 
r V iT*/> - ■' ,TV r.' .,»• .f-'V.fv .;• 
 
 t. - 1 
 
 r>WJ,(^.: ^ • Kj 
 
 W " ■- ^'i 
 
 L_fe. ,t>z?i'f-,f •'? U;h'l»y^;;i^'^j^4., •• vM(». ». ,:iv 9|§’ 
 
 v-4 
 
 i*V. \l * 
 ' I . ' 
 
 * -\ 
 
 • ai 04 
 
 ».i 
 
 ■,»r 
 
 ■ > «vV ^ m: - v^’'^;. . <y ^^j,™ 
 
 i : ■: iV .4 HfT \'fr 
 
 »-. ■■>• *' •■•■'»* ; ■' /jutL>rr ■■ ■'•'• '’’^•'l^M '" ‘ ' HP''''''* ' iJ 
 
 '■ ■ . / ■. . ■ :• 
 
 
 
 ■** . 
 
 
 
 W !}’.j .. '■ >.;> ■t;l' V-Ttfrl^vUC*. j}j,1 
 
 ^'.r; •;’\ ' ’•’'« “ v‘ "''■*; ■*’’■ • . ' . * ■■ . '^vJFSf?? 
 
 X'-i '!» '- »- 0 >- .. 4 h 
 
 j\T .n ct> '■ ' ' .J '•'/' '‘•'■'r '■ . '^k*',, 
 
 [U- o.< <V< 4 1 .c^. ..• • U;.iS 
 
 ,v>- .‘‘•••■i <v4' •?'ji^)8f0|jB' -'v Jk5^ •■ IP 
 
 
 iv '>^ p 'i>,v ,;'■; « ..' ■' . l'i> • :9 1 * < <4 .*1 •. v*\44' 
 
 -f*' .t ■ , . V ' ■£. ■ r . » ' •^* ’ ' 
 
 ' ■I'* t .-.. ■*; 
 
 ,x < »."..v ..-• -.n •» £ , 
 
 'I'fWKiT^ 
 
 ' 'iv' , ^*1 . 
 
 i)U(ij?pi‘ 
 
 nA- ?'3fi‘/,i..«tt, -S&'i’i -t: ;<! 
 
 L--.^' ’ ’ *£‘‘ ‘ ' '•'•(<■■'■*''' ''^'. '■ 2 ^ 
 
 • .’ ■ ‘ ■ *.. . ' ■' ■"'' ' ' k % .-.tfi t ; ■ ; ;.j|)l ‘ ^v]. 
 
 '»S.''"i,'._'i«..f 5 
 
 j, ,■,!■• j ■ . . 'll -. ’ V ; ' I ’ !?■ ^ 2 '' ' ■? '^' * ‘ 
 
 -i . i' '• .• ^* 1 . 10 ;:^, . \^h . K * ^ '• a* ■ I’d ■■) :' 
 
 Si..; :c# . ^vr-;-'.'^, 'f. >J'' .. •• > ; .V- ■ • y H ' vi •'• -5 . / y 
 
 -•■v«' — t 
 
 ,i •'•■ •./ 'rr‘‘ » uN«V ’'’5 < AT-* ' '< 1 ^<>1 
 
 ji.^.ilj .|i ^ , - jQy- - ■--■-•fi ii r i ilf't ^-i--, [ . II '■ - 1 n H U... I. l■^^ll ]ii- 
 
“ 57 - 
 
 (b) CYS'i'I:\iE i'JlTKuGE-v. las gx'cuiis oi uncorrected cystine m trogen in 
 t.ie portion of tne solutiox^ talien for precipitation is eq-ual to tne grsms of 
 bariuii sulfate multiplied by 0 . 0 b 0 ou 9 , multiuixei by 1UO/15 (since cc . of tne 
 lub cc. solution of tne bases were taken for cj’'stins det eriiunation) . rhe grams 
 Oi corrected cystine nitrogen in an exitire saxxxple is eo;ucl to tne grsans of un- 
 corrected cystine nitrogen obtained above plus U.UU<io (tne solubility cori action 
 for cystine) multiplied by cUO/lOO. 
 
 (c) HISTiDIi'.E i'ilTJiOGK'i . fbie non-amino nitrogen of me bases comes 
 from tiie argiuinej wnicn contains txiree-fourtns of its nitrogen in a foriu wnicn 
 does not react witn nitrous acid, and from tne nistiaine, wnicn contains two- 
 thirds of its nitrogen in non-amino form, rherefore to calculate the histidine 
 nitrogen, subtract thre --fo-irtns of the uncorrected arginine nitrogen in the por- 
 tioxi taken for phospho tungstic acid precipitation from the total non-amino rxitro- 
 gen (difference betvxeen total nitrogen and amino nitrogen), axxd multiply the dif- 
 ference by 3/2, or 
 
 Histidixie N = 3/2 (non-amino N - 3/4 arginine N) 
 
 = 1.5 non-amino IJ - I.I25 arginine N 
 
 This gives the gra-ms of uncorrected histidine nitrogen in the portion taken for 
 phospho tungstic acid precipitation. The grams of corrected histidine nitrogen 
 in an entire sample is erual to the grams of uncorrected histidine nitrogen ob- 
 tained above plus 0.003d, multiplied by 2OO/1OO. 
 
 (d) JLYSli'^E hli’HOGEi!. Tne grams of unccrrected lysine nitrogen in the 
 portion taken for phospho tungstic acid precipitation is e-iual to the difference 
 between the grcmxs of total nitrogen a.nd the sum of the arginine, histidixie, and 
 cystine nitrogen, ike grams of corrected lysine nitrogen in an entire sample 
 
 is equal to the grams of unccrrected lysine nitrogen obtained above plus O.OOOp 
 (the solubility correction for lysine), nniltiplied by 2OO/1OO. 
 
 (e) hm-AidIh'O NITlKOGElT If) THE FILTEATE FROM THE BASES. The grams of 
 uxicorrected non-amino nitrogen in the filtrate from the bases in the portion 
 

 y .; ■• ..r ',1^* .y',” ,fH.T^ 
 
 '■, T ■■ • 
 
 
 
 V. 
 
 ■r^^- 'i 
 
 
 -''y'r S'- '■ 'i ^'< 
 
 • ’<■ ■,•;.;■ -iJ" ?j(tti 
 
 ;S V \‘ 
 
 {V, . .; ... .; ■•' yM,^ 
 
 -A.. • 
 
 .-. i 
 
 . * ■' * J . V 
 
 • I , » w . /i' •«. j I . . \ 
 
 >»'j;**’. ' ifri. •' .V ♦' A ' I ^ 
 
 y., , . 
 
 'f VTK »? 
 
 
 
 S’ 
 
 > , f , \ ■ ’ * 
 
 ■,%^aiP>.'-/6^: ‘ f • ‘j a'; . , ^ 
 
 It 
 
 
 • (J ■' ' ■ '• -'y.?* ■'*^'; s^^.. 
 
 .. «W'M • • 1 — ,; . H ’.'. "'y, ■ 
 
 'v*^/S^^v'"w',.. .’« -• s •’ f '■ i7 jS'.^ '/■ K** i 
 
 . a 
 
 
 .,.#■•< t>. f ,.' -. V' 
 
 i ' " ^.' - -.v/ V ■ < /*->i,%rt ^ 
 ^' • ‘ ■ ' ,. ■’.. \-H 
 
 > r 
 
 . •' in-^v * ) vi '?.-: . 7 > ,, 1 
 
 
 1 ^, yt - 
 
 i of 
 
 ^'.'yV. v>’7y; 
 
 ' ■ '' f ''.’ i 'i,i ■ . 
 
 
 s6n ititf i ■ ; T/ftiJ 3^ >■ ^ 
 
 V»- /'•"• .’ , '- ■ i •■•-'. , t' y .... ■’• 
 
 7’'v ■'•' ,4- «'».'.x / .4 y?,< ,%.: f . - s 4> ..4sji,ik^ 
 
 ’ J'-" - '‘V' V ”, '•-«■■...■ \ 'i y"' ^ •' , ;■■ ■ ..kiJS’Viil?,. ' 
 
 i, ■• ■••' . • .‘ ' ’.• . •.'■•Je.. . • •,.’ !’V'< ’ «A’---‘v V' ’. .; 
 
 L>VK^ .Jt'-'-^’'. .•vv-.'fr’ ■■ .3V- ■ «^ .*1, . '«.'J. ■■'■'i' ' ' ‘ / MWBftT. '' 
 
 .:.,yr,#>- ; ' . iftiw*:. .a LT?b(j *rii #>(•*» ^-. . .-, - : 4*, 'BBMRT . 
 
 f 
 
 
 . y-'.ii. •'• • ■■•■■V'-'''*V^.\S;. .j. : ■•‘^^'•'■'it{<'4'S ;,iV." ••’*:' ' '•'ST' >•••.! 
 
 f Jli/ ti. lA 
 
taken for precipitation v/ith phospho tungstic acid is equal to the difference 
 between the grams of uncorrected total nitrogen and the uncorrected amino nitro- 
 gen in the filtrate fran the bases. The grains of corrected non-amino nitrogen 
 in the filtrate from the bases in an entire sample is equal to the grams of un- 
 corrected non-amino nitrogen obtained above minus 0.0049 (the solubility correc- 
 tion), multiplied by SOO/lOO. 
 
 PURITY OF REAGEilTS. Since some of the calculations are based on dif- 
 ferences between determinations, it is imperative that the latter shall be as 
 accurate as possible. Every reagent used for any determination involved is 
 tested by blank analysis. Blank determinations are always run at the same time 
 all amino-acid, sulfur, and total nitrogen determinations are made and suite.ble 
 corrections made for the reagents used. 
 
 Som.e reagents should be repurified. The sodium chloride and the 
 sugc,r used in the determination of sialfur are carefully repurified. The phos- 
 photungstic acid used for the precipitation and washing of the bases is repuri- 
 fied according to the method of Winterstein (24) and the tec'hnique found most 
 suitable for this purif ica,tion is as follows; I5 grams of phospho tungstic acid, 
 50 cc. of 0.2 per cent, hydrochloric acid, and 9O cc. of ether are placed in a 
 separatory funnel and shaken until the solid phospho tungstic acid ]aas completely 
 dissolved. On standing about 3 minutes three layers separate. The clear lower 
 layer, consisting of phospho tungstic acid in ether is carefully drawn off into 
 a, casserole or evaporating dish. Add a drop or tv/o of concentrated hydrocriloric 
 acid to the contents of the funnel, in order to break up any emulsion that had 
 not been completely broken up by the previous treatment, and shake. Allow to 
 stand and again v/ithdraw the ether-phospho tungstic acid layer, if one forms, 
 into the casserole or evaporating dish. Repeat this procedure until no more of 
 this layer is obtained. The reinaining two lajrers in the separatory funnel may 
 be used for the purification of a second I5 gram portion of the phospho tungstic 
 
-59- 
 
 acid. 
 
 The ethereal solution is allowed to evaporate on the steaia bath at a 
 low temperature until a thick syrup remains. Then set avi/ay from the heat and 
 direct sunlight and allow the remaining ether to evaporate spontaneously when 
 crystals of purified phcsphotungstic acid are obtained, iphe latter process may 
 be hastened by placing the evaporating dish in front of an electric fan and 
 blowing a current of air over the dish. The yield is about 6 ^ per cent. All 
 the liquors remaining from the purification should be ^ved and the phosplio tungs- 
 tic acid which they contain recovered later. 
 
 VII. DSTSRIvlIITATION OF THE AIvlINO-ACIDS OF OATS, COEIT, COTTONSEED 
 
 IvlEAL AITD ALFfJ.FA. 
 
 By the detailed method outlined above in section VI, 6 samples of 
 oats, 6 of corn, 8 of cottonseed meal, and U of alfalfa were analyzed. The re- 
 sults are given in Table I, in which the results are expressed in percentage of 
 the total nitrogen of the feed, and in Table II, in which the results are ex- 
 pressed in percentage of the feed. The oats contained l.oHO per cent, nitrogen, 
 the corn l.UOyU per cent., the cottonseed meal 6.79S per cent., and the alfalfa 
 2.b2S per cent. The. weights of feeds taken for each of the various analyses 
 were 60 grair4S in case of oats and corn, 3O grams in case of alfalfa, and I5 
 grams in cas^ of cottonseed meal. Tne oats and corn v;ere ground so as to pass 
 through an 80 mesh sieve, the alfalfa through a 60 mesh sieve, and the cottonseed 
 meal through a UO mesh sieve. 
 
 A brief examination of Tables I and II will show that all solutions, 
 residues, precipitates, and other fractions, obtained in the preparation of the 
 hydrolyzed protein solution and in the subsequent Van Slyka analysis of that sol- 
 ution, were analyzed for their nitrogen content. In other words, no fraction, 
 v/hich might in any way contain any portion of the origira,! sample of feed taken 
 
X\> 
 
 * ‘•''•‘i ■» 
 
 , '.t-/ 
 
 iU-t 
 
 
 
 
 
 I 
 
 ' 
 
 -t 
 
 ■I 
 
 J 
 
 r 
 
 L 
 
 
 ..'•a 
 
 iiJlikni 
 
 
 ', ■ > 
 
 > 
 
 ' 'T'i.'* *V^ ' 
 
- 60 - 
 
 <«5 
 
 TABLE I. 
 
 B 
 
 DISTRIBU'ilOIT OF THE ITITROOEiN OF OATS, C 0 E 1 \', COTTOIJSEED 
 imj . AlsT) ALFALFA. 
 
 (Expressed as Percentage of Total Nitrogen'! 
 
 D 
 
 E 
 
 H 
 
 Nonprotein Nitrogen 
 
 
 
 
 Results of the 
 
 -1 
 
 Solu- 
 
 Solu- 
 
 In fil- 
 
 Total 
 
 Insol- 
 
 Solu- 
 
 Acid 
 
 Argin- 
 
 Cys- 
 
 Histi- 
 
 — 
 
 ble 
 
 ble in 
 
 trate 
 
 nonpro- 
 
 uble 
 
 ble hu- 
 
 amide 
 
 ine 
 
 tine 
 
 dine 
 
 
 in 
 
 alco- 
 
 from 
 
 tein 
 
 hmin 
 
 min nit- 
 
 nitro- 
 
 nitro- 
 
 nitro- 
 
 ni tro- 
 
 
 ether 
 
 hoi 
 
 colloid- 
 
 nitro- 
 
 nitro- 
 
 rogen 
 
 gen^ 
 
 gen^ 
 
 gen^ 
 
 gen^ 
 
 
 
 
 al iron 
 
 ge n 
 
 gen 
 
 
 
 
 
 
 
 — — 
 
 0.655 
 
 1.172 
 
 11.522 
 
 13.3^9 
 
 2 .IS 73 
 
 2.783 
 
 ll.Olb 
 
 11.427 
 
 0.961 
 
 6.504 
 
 r 2 
 
 0.597 
 
 1.163 
 
 11,126 
 
 12.386 
 
 2.968 
 
 3.^31 
 
 11.033 
 
 11.652 
 
 1.008 
 
 5.651 
 
 s 
 
 0.553 
 
 1.041 
 
 11.766 
 
 13.360 
 
 3.481 
 
 1.498 
 
 11 . 780 
 
 11.888 
 
 0.976 
 
 5.543 
 
 >1 
 
 0.555 
 
 1.375 
 
 11.140 
 
 13.070 
 
 2.393 
 
 1.71^8 
 
 11 . 566 
 
 11.474 
 
 0.951 
 
 5.942 
 
 18 
 
 0.513 
 
 1.307 
 
 10.360 
 
 12.180 
 
 2.611 
 
 2.914 
 
 11.350 
 
 11.892 
 
 0.894 
 
 5.840 
 
 1 
 
 0 . 54 U 
 
 1.294 
 
 10.360 
 
 12.69s 
 
 3.255 
 
 2.926 
 
 11.73s 
 
 11.554 
 
 0.876 
 
 5.283 
 
 7 
 
 Aver. 0.569 
 
 1.225 
 
 11.129 
 
 12.926 
 
 3.013 
 
 2.516 
 
 11.422 
 
 11.647 
 
 0.944 
 
 5.796 
 
 3 
 
 
 0,050 
 
 0.997 
 
 9.311 
 
 10.35s 
 
 1.571 
 
 1.375 
 
 11,729 
 
 8.620 
 
 1.099 
 
 r 5 . 2 i *5 
 
 i8 
 
 0.792 
 
 2.341 
 
 8.093 
 
 11.226 
 
 1.796 
 
 2.602 
 
 12.265 
 
 8.868 
 
 1.165 
 
 5» 460 
 
 )8 
 
 0,65a 
 
 2.369 
 
 3.395 
 
 11.422 
 
 1.499 
 
 2.284 
 
 12.225 
 
 8.867 
 
 1.186 
 
 4.906 
 
 )0 
 
 O.cil^O 
 
 0.239 
 
 6.336 
 
 7.315 
 
 0.702 
 
 2.685 
 
 11.241 
 
 8.782 
 
 0.925 
 
 4.748 
 
 )8 
 
 o.Ocia 
 
 O.3O5 
 
 7.316 
 
 8.149 
 
 0.758 
 
 2.596 
 
 12.218 
 
 8.762 
 
 0.935 
 
 3.928 
 
 )3 
 
 0.139 
 
 1.953 
 
 3.356 
 
 10.503 
 
 1.084 
 
 2.27s 
 
 9.3331 
 
 8. 451 
 
 1.071 
 
 4.702 
 
 )2 
 
 Aver. 0 . 3 ci 6 
 
 1.363 
 
 8.135 
 
 9.329 
 
 1.235 
 
 3.303 
 
 11.936 
 
 8.725 
 
 1.072 
 
 4.832 
 
 .0 
 
 "T 
 
 O.ORl 
 
 0.570 
 
 ^.943 
 
 5.534 
 
 2.609 
 
 3-462 
 
 9.455 
 
 18. 672 
 
 0.961 
 
 5.436 
 
 ;8 
 
 0,039 
 
 O.6I8 
 
 4.870 
 
 5-577 
 
 2.609 
 
 5.117 
 
 9.689 
 
 19.050 
 
 0.902 
 
 0.330 
 
 )2 
 
 0.202 
 
 0.652 
 
 5.053 
 
 5.907 
 
 2,492 
 
 5.459 
 
 9.929 
 
 18.467 
 
 1.068 
 
 7.543 
 
 '6 
 
 0.109 
 
 0.614 
 
 5.531 
 
 6.254 
 
 2.623 
 
 4.477 
 
 8.892 
 
 18.398 
 
 1.123 
 
 7.240 
 
 10 
 
 0.031 
 
 O.5O0 
 
 5.245 
 
 5.S32 
 
 2 . 981 
 
 2,415 
 
 9.249 
 
 17.520 
 
 1.051 
 
 8.534 
 
 JO 
 
 0.129 
 
 0.489 
 
 5.722 
 
 6.340 
 
 2,930 
 
 2.650 
 
 Q.3I8 
 
 19.443 
 
 0.Q48 
 
 6.366 
 
 i6 
 
 0.031 
 
 0.420 
 
 6.097 
 
 6.598 
 
 2.763 
 
 2-334 
 
 9.002 
 
 17.987 
 
 0.707 
 
 9.351 
 
 J6 
 
 0.0U6 
 
 0.506 
 
 7 .012 
 
 7.564 
 
 2.772 
 
 2.746 
 
 9.764 
 
 20.102 
 
 O.78I 
 
 6.464 
 
 ?3 
 
 Aver. 0,095 
 
 0.51*7 
 
 5-559 
 
 b .201 
 
 2.722 
 
 3.582 
 
 9.412 
 
 18. 705 
 
 0.943 
 
 7.171 
 
 f 3 
 
 
 
 
 
 
 
 
 
 
 
 — * 
 
 0.577 
 
 1.940 
 
 16.466 
 
 18.983 
 
 3.632 
 
 4.433 
 
 6.943 
 
 7.949 
 
 0.924 
 
 4.355 
 
 +5 
 
 0-577 
 
 1.600 
 
 16.301 
 
 18.478 
 
 3.6902 
 
 3.512 
 
 7.104 
 
 8.064 
 
 1.062 
 
 3-655 
 
 3 l 
 
 0 . 5^4 
 
 1 . 9 S 3 
 
 17.289 
 
 19.801 
 
 3.597 
 
 5.132 
 
 5.204 
 
 7.523 
 
 0.986 
 
 3-782 
 
 •‘L. 
 
 0.522 
 
 1.864 
 
 16.712 
 
 19.09s 
 
 3.791 
 
 4.796 
 
 7.204 
 
 8.446 
 
 0.991 
 
 3-931^ 
 
 
 Aver. 0.550 
 
 1.848 
 
 16.692 
 
 19.090 
 
 3.690 
 
 4.481 
 
 7.364 
 
 7 . 99 b 
 
 0 .Q 91 
 
 ^. 9^1 
 
 53 
 
 ^ Co 
 
 rrected 
 
 for solubility of the bases 
 
 
 
 
 
 
 ^ The writer is indebted chi 
 
 sfly to 
 
 Nao Uyei 
 
 , assistant chemist, for the 
 
 
 
 following arialyses of corn. 
 
 
 
 
 
 
 
 The write 
 
 r is inde 
 
 bted to 
 
 W. B. Nevens for 
 
 the following analyses of 
 
 Dll- 
 
 
 cottonseed raea].. 
 
 
 
 
 
 
 
 
 ".f 
 
L^3LE I. riSTRI3UiI0ir OF THE jlTROaEiN OF OATS, COEN, COTTONSEED 
 ;.3:/X AlO) ALFALFA 
 
 (Eijjressed as Parcentage of Total ITitrogen' 
 
 A 
 
 3 
 
 C 
 
 D 
 
 E 
 
 F 
 
 G 
 
 H 
 
 I 
 
 J 
 
 7 . 
 
 L 
 
 
 N 
 
 0 
 
 p 
 
 0 
 
 H i 
 
 honprote 
 
 in Nitrogen 
 
 
 
 
 Resiilts of the 
 
 Van SI 
 
 yke Analysis 
 
 
 
 
 Nitrogen 
 
 lost in 
 
 ” Solu- 
 
 ble 
 in 
 
 ether 
 
 Solu- 
 ble in 
 alco- 
 hol 
 
 In fil- 
 trate 
 from 
 colloid- 
 al iron 
 
 Total 
 nonpro- 
 tein 
 nitro- 
 ge n 
 
 Insol- 
 ubl a 
 hum in 
 nitro- 
 gen 
 
 Solu- 
 ble hu- 
 min nit- 
 rogen 
 
 Acid 
 
 amide 
 
 nitro- 
 
 gen^ 
 
 Argin- 
 
 ine 
 
 nitro- 
 
 gen* 
 
 Cys- 
 
 tine 
 
 nitro- 
 
 gen* 
 
 Histi- 
 dine 
 ni tro- 
 gen* 
 
 , Ly- 
 sine 
 k nit- 
 
 K X 
 
 rogen 
 
 Amino 
 acid N 
 in fil- 
 trate 
 from 
 bases^ 
 
 Non-a- 
 mino 
 acid N 
 in fil- 
 trate 
 from 
 bases 
 
 Total non- 
 protein + 
 results of 
 Van Slyke 
 analysis 
 
 N in res- 
 idue after 
 treatment 
 if.lth strong 
 NaOH 
 
 In alc- 
 ohol ppt. 
 of hot 2 
 pet . 
 
 CCI3.CO2H 
 
 extracts 
 
 Unadsorbed 
 hwflin (fil- 
 tered from 
 sol. during 
 dacomp . of 
 bases) 
 
 In axyl 
 alcohol 
 ether | 
 extract 
 
 1 
 
 1 
 
 J 
 
 
 
 
 
 
 
 
 
 
 
 OATS (Contains 1. 
 
 oSO pet . N' 
 
 
 
 
 
 0.655 
 
 1.172 
 
 11.522 
 
 I3.349 
 
 2.273 
 
 2.723 
 
 ll.Olo 
 
 11.427 
 
 0.961 
 
 6.504 
 
 £.122 
 
 41.992 
 
 4.102 
 
 97-195 
 
 0.120 
 
 0.109 
 
 0.552 
 
 0.525 
 
 0.^07 
 
 1.163 
 
 11.126 
 
 12.Sdb 
 
 2. 962 
 
 3.231 
 
 11.023 
 
 11.652 
 
 1.002 
 
 5.651 
 
 2.221 
 
 41.921 
 
 2.964 
 
 96.245 
 
 0.0'^5 
 
 0.102 
 
 0.235 
 
 0.771 
 
 0.553 
 
 l.OUl 
 
 11.766 
 
 13.360 
 
 3.1421 
 
 1.492 
 
 11.720 
 
 11.222 
 
 0.976 
 
 5.543 
 
 3.445 
 
 42.174 
 
 3.209 
 
 97-954 
 
 0.136 
 
 0.142 
 
 0 . 506 
 
 0.759 
 
 0.555 
 
 1.375 
 
 ll.lUO 
 
 13.070 
 
 2.293 
 
 1.742 
 
 11 . 566 
 
 11.474 
 
 0.951 
 
 5.942 
 
 ; 2.326 
 
 41.922 
 
 4.922 
 
 96.946 
 
 0.165 
 
 0.123 
 
 0.251 
 
 0.029 
 
 0.513 
 
 1.307 
 
 10.360 
 
 12.120 
 
 2.611 
 
 2.914 
 
 11.350 
 
 11.292 
 
 0.2P4 
 
 5.240 
 
 3.426 
 
 42.O06 
 
 3.522 
 
 96.751 
 
 0.142 
 
 0.153 
 
 0.340 
 
 0.755 
 
 0.5LL 
 
 1.29U 
 
 10.S60 
 
 12.69s 
 
 3.255 
 
 2.926 
 
 11.73s 
 
 11.554 
 
 0.276 
 
 5.223 
 
 2.727 
 
 42.622 
 
 3.704 
 
 97.503 
 
 o.i6s 
 
 0.123 
 
 0.233 
 
 0.732 
 
 Aver. 0.569 
 
 1.225 
 
 11.129 
 
 12.926 
 
 3.013 
 
 2.516 
 
 11.422 
 
 11.647 
 
 0.944 
 
 5.796 
 
 2.241 
 
 42.137 
 
 3. 260 
 
 97.100 
 
 0.132 
 
 0.127 
 
 0.664 
 
 0.746 
 
 7 ... s T 
 
 u 
 
 V 
 
 
 
 In resi- 
 
 In resi- 
 
 Total 
 
 To 
 
 Total 
 
 
 
 due fil- 
 
 due fil- 
 
 ni tro- 
 
 nitro- 
 
 
 1 
 
 tered 
 
 tered 
 
 gen 
 
 gen ac- 
 
 
 
 from so- 
 
 from so- 
 
 lost 
 
 counted 
 
 
 1 
 
 lution 
 
 lution of 
 
 
 for 
 
 
 1 
 
 of bases 
 
 filtrate 
 
 
 
 
 J.. 
 
 
 from btises 
 
 
 
 
 
 0.363 
 
 0.024 
 
 1.699 
 
 92.294 
 
 — 
 
 
 0-297 
 
 0.033 
 
 2.oqq 
 
 36 . 344 
 
 
 1 
 
 O.OS3 
 
 0.015 
 
 1.707 
 
 99.661 
 
 
 
 0.141 
 
 0.025 
 
 2.234 
 
 99.120 
 
 
 
 0.191 
 
 0.020 
 
 1.607 
 
 9S.362 
 
 
 
 0.179 
 
 0-033 
 
 2.074 
 
 99-577 
 
 
 
 0.209 
 
 0.025 
 
 1.903 
 
 99.004 
 
 
 ALFALFA (Contains 2.6dS pet 
 
 0.577 
 
 1.940 
 
 l6*4bo 
 
 12.923 
 
 3.622 
 
 4.423 
 
 6.943 
 
 7.949 
 
 0.924 
 
 4.356 
 
 4.334 
 
 3S.349 
 
 3.863 
 
 93-866 
 
 2.663 
 
 Extrac- 
 
 0.999 
 
 0.609 
 
 
 Not 
 
 0.144 
 
 4.415 
 
 98.221 
 
 0.577 
 
 1.600 
 
 16.301 
 
 12.472 
 
 3.6902 
 
 3.512 
 
 7.104 
 
 8.064 
 
 1.062 
 
 3-655 
 
 4.959 
 
 37.681 
 
 3.059 
 
 91.264 
 
 2.335 
 
 tion 
 
 1.110 
 
 0.216 
 
 
 de- 
 
 0.639 
 
 4.900 
 
 96.164 
 
 0.524 
 
 1.922 
 
 17.229 
 
 19. 801 
 
 3.597 
 
 5.132 
 
 2.204 
 
 7.523 
 
 0.926 
 
 3-782 
 
 4.002 
 
 37.312 
 
 1.196 
 
 91.541 
 
 2.930 
 
 not 
 
 1.274 
 
 O.3OI 
 
 
 term- 
 
 0.604 
 
 5.109 
 
 96.650 
 
 0.522 
 
 1.264 
 
 16.712 
 
 19.098 
 
 3-791 
 
 4.796 
 
 7.i!04 
 
 3.446 
 
 0.991 
 
 3.931^ 
 
 4.4342 
 
 32.726 
 
 1.927 
 
 93-404 
 
 2.146 
 
 made 
 
 1.261 
 
 0.717 
 
 
 ined 
 
 0.311 
 
 4.501 
 
 97.905 
 
 Aver. 0.550 
 
 1.242 
 
 16.692 
 
 19.090 
 
 
 4.481 
 
 7.364 
 
 7.996 
 
 o.qqi 
 
 3-931 
 
 4.434 
 
 32.032 
 
 <i.5ll 
 
 92.5<;0 
 
 2.519 
 
 
 1.161 
 
 0.611 
 
 4 
 
 
 
 0.441 
 
 4.732 
 
 97-25<: 
 
 m. 
 
 0.050 
 
 0.997 
 
 9.311 
 
 10.352 
 
 1.571 
 
 1.375 
 
 11.729 
 
 2.620 
 
 1.099 
 
 5.245 
 
 2.424 
 
 46.090 
 
 5. 600 
 
 94.111 
 
 O.5051 
 
 0.513 
 
 0:930 
 
 0.521 
 
 
 0.129 
 
 O.O65 
 
 2.723 
 
 96.234 
 
 — 
 
 0.792 
 
 2.341 
 
 8.093 
 
 11.226 
 
 1.796 
 
 2.602 
 
 12.265 
 
 2.268 
 
 1.165 
 
 5.46b 
 
 :2.41s 
 
 46.790 
 
 0.224^ 
 
 93.474 
 
 0.176 
 
 0.163 
 
 0.930 
 
 0.527 
 
 
 0.206 
 
 1.4681 
 
 3-U7O 
 
 96.944 
 
 
 0.652 
 
 2.369 
 
 8.395 
 
 11,422 
 
 1.499 
 
 2.224 
 
 12.225 
 
 2.867 
 
 1.186 
 
 4.906 
 
 I2.473 
 
 45.059 
 
 2.251 
 
 90.172 
 
 0.147 
 
 0.335 
 
 1.999 
 
 0.515 
 
 
 o.a30 
 
 0.002 
 
 3.234 
 
 101.406 
 
 
 0.240 
 
 0.239 
 
 6.836 
 
 7.315 
 
 0.702 
 
 2.625 
 
 ii.24i 
 
 2.722 
 
 0.925 
 
 4.74s 
 
 1.847 
 
 47.750 
 
 2.763 
 
 94.758 
 
 0.107 
 
 0.265 
 
 3-626 
 
 0.372 
 
 
 0.149 
 
 0.133 
 
 4.7I8 
 
 99.470 
 
 
 0.022 
 
 O.3O5 
 
 7-816 
 
 2.149 
 
 0.758 
 
 2.596 
 
 12.218 
 
 2,762 
 
 0.925 
 
 3.922 
 
 2.221 
 
 47.962 
 
 6.270 
 
 94. 449 
 
 0.113 
 
 0.073 
 
 2.791 
 
 0.531 
 
 
 0.164 
 
 0.045 
 
 3-717 
 
 92.166 
 
 
 0.129 
 
 1.958 
 
 2.356 
 
 10.503 
 
 1.024 
 
 2.272 
 
 9.8331 
 
 8. 451 
 
 1.071 
 
 4.702 
 
 1.214 
 
 46 . 57 4 
 
 q-599 
 
 Q2.909 
 
 0.137 
 
 0.305 
 
 5.250 
 
 0.412 
 
 
 0.209 
 
 0.075 
 
 6-994 
 
 99.903 
 
 
 Aver. 0.326 
 
 1.368 
 
 8.135 
 
 9.229 
 
 1.235 
 
 2.303 
 
 11.936 
 
 8. 725 
 
 1.072 
 
 4.832 
 
 2.200 
 
 46.704 
 
 7.216 
 
 96.052 
 
 0.136 
 
 0.276 
 
 2.698 
 
 0.421 
 
 
 0.191 
 
 0.065 
 
 3.247 
 
 99-899 
 
 
 
 
 
 
 
 
 
 
 
 
 COTTONSEED MEAL^ 
 
 (Contains b 
 
 • 796 pet. N'l 
 
 
 
 
 
 
 
 0.021 
 
 0.570 
 
 4.943 
 
 5-534 
 
 2.609 
 
 '3.462 
 
 ^ 9.455 
 
 12.672 
 
 no.qpi 
 
 5-486 
 
 4.240 
 
 39-981 
 
 3-871 
 
 93-671 
 
 0.h20 
 
 
 2.274 
 
 0.920 
 
 
 0.20b 
 
 0.036 
 
 3-7I6 
 
 97.38? 
 
 
 0,029 
 
 O.6I8 
 
 4.870 
 
 5-577 
 
 2.609 
 
 5.117 
 
 9.629 
 
 19.050 
 
 0,902 
 
 6.330 
 
 3.060 
 
 40.539 
 
 3.432 
 
 96 . 3O5 
 
 0.260 
 
 
 1.562 
 
 0.906 
 
 
 0.215 
 
 0.039 
 
 2.922 
 
 99.^87 
 
 
 0.202 
 
 0.652 
 
 5.053 
 
 5.907 
 
 2.492 
 
 5-459 
 
 9.929 
 
 18.467 
 
 1,068 
 
 7.543 
 
 3-570 
 
 38.852 
 
 1.901 
 
 95.122 
 
 0.30a 
 
 Extrac- 
 
 1 .652 
 
 0.722 
 
 
 O.22O 
 
 0.106 
 
 3-O74 
 
 99.262 
 
 
 0.109 
 
 0.614 
 
 5-531 
 
 0.254 
 
 2.623 
 
 4.477 
 
 2.8q2 
 
 IS. 398 
 
 1.123 
 
 7.240 
 
 4.470 
 
 39-828 
 
 0.1611 
 
 93-466 
 
 0.233 
 
 tion 
 
 3.044 
 
 1.135 
 
 
 0.245 
 
 0.130 
 
 4.787 
 
 98.253 
 
 
 0.021 
 
 
 5.245 
 
 5.832 
 
 2.921 
 
 2.415 
 
 9.249 
 
 17.520 
 
 1.051 
 
 8.524 
 
 4.462 
 
 41.958 
 
 2.621 
 
 96.733 
 
 0 . 4302 
 
 not 
 
 1.192 
 
 0.691 
 
 
 0.422 
 
 0.150 
 
 2.291 
 
 99.624 
 
 
 0.129 
 
 0.489 
 
 5.722 
 
 6.340 
 
 2.930 
 
 2.650 
 
 9.3I8 
 
 19.443 
 
 o.q48 
 
 6.366 
 
 4.998 
 
 41.950 
 
 2.290 
 
 97.833 
 
 0.625 
 
 ifiade 
 
 1.019 
 
 O.7SO 
 
 
 0.320 
 
 0.096 
 
 2.960 
 
 100.193 
 
 
 0.021 
 
 0.420 
 
 6.097 
 
 6.592 
 
 2.763 
 
 2.334 
 
 9.CO2 
 
 17.987 
 
 0.707 
 
 9.351 
 
 3.600 
 
 39.204 
 
 3.027 
 
 94.633 
 
 0.719 
 
 
 0.772 
 
 i.oa6 
 
 
 0.096 
 
 O.O05 
 
 2.672 
 
 97.311 
 
 
 0.046 
 
 O.5O6 
 
 7.012 
 
 7 - 564 
 
 2.772 
 
 2.746 
 
 9.764 
 
 20.102 
 
 0.721 
 
 6.464 
 
 _ 5-874 
 
 43-481 
 
 3-454 
 
 101.402 
 
 0.529 
 
 
 1.364 
 
 1.068 
 
 
 0.133 
 
 0.068 
 
 3.222 
 
 105.624 
 
 
 Aver. C.O95 
 
 0.547 
 
 5-559 
 
 6.201 
 
 2.7H2 
 
 3-582 
 
 9.412 
 
 12.705 
 
 0.943 
 
 7.171 
 
 ^ 4.209 
 
 40.724 
 
 2.874 
 
 96.543 
 
 0.430 
 
 
 1.611 
 
 0.922 
 
 
 0.<;40 
 
 0.026 
 
 3.229 
 
 99.832 
 
 
 xui ouxuuxxj. ui x,ne o^ses 
 
 The writer is indebted chiefly to Nao Uyei, assistant chemist, for the 
 ^ following arialyses of corn. 
 
 The writer is indebted to W, B. Nevens for the following analvsas of 
 cottonseed maaj. 
 
 Not included in the average 
 
 2 Determination lost; average result substituted to make up total 
 

 TABLE II. DISTRIBUTION OF THE NITROGEN OF OATS, COEN, COTTONSEED 
 
 MEAL AND ALFALFA 
 
 (Expressed as Percentage of Feedingstuff ) 
 
 A 
 
 B 
 
 C 
 
 D 
 
 E 
 
 F 
 
 G 
 
 H 
 
 I 
 
 J 
 
 Nonprotein 
 
 Nitrogen 
 
 
 
 
 Results of the 
 
 Solu- 
 
 'ble 
 
 in 
 
 ether 
 
 Solu- 
 ble in 
 alco- 
 hol 
 
 In fil- 
 trate 
 from 
 
 colloid- 
 al iron 
 
 Total 
 
 nonpro- 
 
 tein 
 
 nitro- 
 
 gen 
 
 Insol- 
 uble 
 hum in 
 nitro- 
 gen 
 
 Solu- 
 ble hu- 
 Viin nit- 
 rogen 
 
 A-cid 
 
 aiTjide 
 
 nitro- 
 
 gen^ 
 
 Argin- 
 
 ine 
 
 ni tro- 
 gen 
 
 Cys- 
 
 tine 
 
 nitro- 
 
 gen^ 
 
 Histi- 
 
 dine 
 
 nitre- 
 
 /-r 
 
 gen 
 
 
 0.0110 
 
 0.0197 
 
 0.1936 
 
 0.2242 
 
 0.0482 
 
 0.0467 
 
 O.I85O 
 
 0.1919 
 
 0.0161 
 
 0.1092 
 
 0.0100 
 
 0.0195 
 
 O.IS69 
 
 0.2164 
 
 0.0498 
 
 0.0542 
 
 0.1862 
 
 0.1957 
 
 0.0169 
 
 0.0949 
 
 0.0093 
 
 0.0175 
 
 0.1977 
 
 0.2244 
 
 0.0584 
 
 0.0251 
 
 0.1979 
 
 0.1997 
 
 0.0164 
 
 0.0931 
 
 0.0093 
 
 0.0231 
 
 O.I672 
 
 0.2195 
 
 0.0466 
 
 0.0293 
 
 0.1943 
 
 0.1917 
 
 0.0159 
 
 0.0998 
 
 O.OOSo 
 
 0.0220 
 
 0.1740 
 
 0.2046 
 
 0.0436 
 
 0.0469 
 
 0.1906 
 
 0.1997 
 
 0.0150 
 
 0.0981 
 
 0.0091 
 
 0.0217 
 
 0.1624 
 
 o.aX33 
 
 0.0546 
 
 0.0491 
 
 0.197s 
 
 0.1941 
 
 0.0147 
 
 0.0887 
 
 Aver. 0.0096 
 
 0.0206 
 
 O.IS7O 
 
 0.2170 
 
 0.0506 
 
 0.0422 
 
 0.1916 
 
 0.1956 
 
 0.0158 
 
 0.0973 
 
 
 0.0007 
 
 0.0140 
 
 0.1310 
 
 0.1456 
 
 0.0221 
 
 0.0194 
 
 0.1651 
 
 0.1213 
 
 O.OIR5 
 
 0.0738 
 
 0.0111 
 
 O.O33O 
 
 0.1139 
 
 O.I58O 
 
 0.0253 
 
 0.0366 
 
 0.1726 
 
 0.1246 
 
 0.0164 
 
 0,0 1 08 
 
 0.0093 
 
 0.0333 
 
 0.1162 
 
 0.1608 
 
 0.0211 
 
 0.0321 
 
 0.1721 
 
 0.1246 
 
 0.0167 
 
 0.0690 
 
 O.OO3U 
 
 0.0034 
 
 0.0962 
 
 O.IO3O 
 
 0.0099 
 
 O.O378 
 
 0.1582 
 
 0.1236 
 
 0.0130 
 
 0,06b8 
 
 0.0004 
 
 0.0043 
 
 0.1100 
 
 0.1147 
 
 0.0107 
 
 0.0365 
 
 0.1720 
 
 0.1233 
 
 0.0139 
 
 0.0553 
 
 O.OOciy 
 
 0.0276 
 
 0.1176 
 
 0.1472 
 
 0.0153 
 
 0.0321 
 
 0.1334^ 
 
 0.1189 
 
 0.0151 
 
 0.0662 
 
 Aver. 0.0046 
 
 0.0193 
 
 0.1145 
 
 0.1363 
 
 0.0174 
 
 0.0324 
 
 0.1680 
 
 0.1228 
 
 0.0151 
 
 0.0680 
 
 
 
 0.0014 
 
 0.0367 
 
 0.3360 
 
 0.3761 
 
 0.1773 
 
 o.a 35 £ 
 
 0.6426 
 
 1.2689 
 
 0.0653 
 
 0.3728 
 
 0.0061 
 
 0.0420 
 
 0.3310 
 
 0.3790 
 
 0.1773 
 
 0.3476 
 
 0.6585 
 
 1.2946 
 
 0.0613 
 
 O.43O2 
 
 0.0137 
 
 0.0443 
 
 0.3434 
 
 0.4014 
 
 0.1694 
 
 0.3710 
 
 0.6746 
 
 1.2550 
 
 0.0726 
 
 0.5126 
 
 0.0075 
 
 0.0417 
 
 0.3459 
 
 0.4250 
 
 0.1783 
 
 O.3O43 
 
 0.6043 
 
 1.2503 
 
 0.0763 
 
 0.4920 
 
 0.0055 
 
 0.0345 
 
 0.3565 
 
 0.3963 
 
 0.2026 
 
 0.1641 
 
 0.6286 
 
 1.1906 
 
 0.0714 
 
 0.5830 
 
 0 . 0036 
 
 0.0332 
 
 0.3669 
 
 0.4309 
 
 0.1991 
 
 0.1801 
 
 0.6333 
 
 1.3213 
 
 0.0644 
 
 0.4326 
 
 0.0055 
 
 0.0265 
 
 0.4144 
 
 0.4464 
 
 0.1878 
 
 O.I5S6 
 
 0.6118 
 
 1.2224 
 
 0.0480 
 
 0.6356 
 
 0.0032 
 
 0.0344 
 
 0 . 47 95 
 
 0.5140 
 
 0.1884 
 
 0.1866 
 
 0 .bb 35 
 
 1.3661 
 
 O.O63I 
 
 0.4393 
 
 Aver. 0.0065 
 
 0.0372 
 
 0.3778 
 
 0.4214 
 
 O.I85O 
 
 0.2434 
 
 0.6396 
 
 1.2712 
 
 0.0641 
 
 0 . 4^73 
 
 
 0.0152 
 
 0.0510 
 
 0.4327 
 
 0.4969 
 
 0.096s 
 
 0.1178 
 
 0.1S25 
 
 0.2089 
 
 0.0243 
 
 0.1145 
 
 0.0152 
 
 0.0420 
 
 0 . 42 o 4 
 
 0.4356 
 
 0.09702 
 
 0.0923 
 
 0.1S67 
 
 0.2009 
 
 0.0279 
 
 0.0961 
 
 0.0136 
 
 0.0522 
 
 0.4544 
 
 0.5204 
 
 0.0945 
 
 0.1349 
 
 0.2156 
 
 0.1977 
 
 0.0259 
 
 0.0994 
 
 0.0137 
 
 0.0490 
 
 0 . 439 ^i 
 
 0.5019 
 
 0.0996 
 
 0.1260 
 
 0.1893 
 
 0,2220 
 
 0.0260 
 
 0.103>' 
 
 Aver. 0.0145 
 
 0.0466 
 
 0.4397 
 
 0.5017 
 
 0.0970 
 
 0.1178 
 
 0.1935 
 
 0.2101 
 
 0.0260 
 
 0.1033 
 
 ^ Corrected for 
 
 solubility of the 
 
 bases 
 
 
 
 
 
 
 y The wr 
 
 iter is 
 
 indebted chiefly to Nao Uyei, assistant chemist; 
 
 for the 
 
 following analyses of corn 
 
 
 
 
 
 
 
 The vvTiter is 
 
 indebted to W. B. 
 
 Nevens 
 
 for the 
 
 following an^.lyses of 
 
 cotton- 
 
 seed meal. 
 
 
 
 
 
 
 
 ■ _p 
 

 IA3LE II. 
 
 DISTRIBUTIO:; OF THE NITROGEN OF OATS, COEN, COTTONSEED 
 .'.SAL AND ALFALFA 
 
 (Expressed as Percentage of Feedings tnff) 
 
 G 
 
 H 
 
 H 
 
 Solu- 
 
 tle 
 
 in 
 
 ether 
 
 Korjrotein 
 
 SolU' 
 tie in 
 alco- 
 ol 
 
 Nitrogen 
 
 In fil 
 trate 
 from 
 
 colloid- 
 al iron 
 
 Total 
 
 nonpro- 
 
 tein 
 
 nitro- 
 
 gen 
 
 Insol' 
 uble 
 hum in 
 
 nitro- jrogen 
 gen 
 
 Solu- 
 ble hu- 
 r.in nit- 
 
 Aoid 
 
 amide 
 
 nitro- nitro- nitro 
 
 Results of the Van SI yVa Arfl.lysis 
 
 gen^ 
 
 Total non- 
 protein + 
 results of 
 Van Slyke 
 analysis 
 
 N in res- 
 idue after 
 treatment 
 vith strong 
 NaOH 
 
 O.CllO 
 
 O.OIQ7 
 
 0.1936 
 
 0.2242 
 
 0.0432 
 
 0.0467 
 
 O.I85O 
 
 0.1919 
 
 0.0161 
 
 0.1092 
 
 0.0366 
 
 0.7054 
 
 O.O69O 
 
 1.632s 
 
 0.0020 
 
 0.0018 
 
 0.0094 
 
 0.0088 1 
 
 0.0100 
 
 0.0195 
 
 0.18o9 
 
 0.2164 
 
 0.0498 
 
 0.0542 
 
 O.I862 
 
 0.1957 
 
 0.0169 
 
 0.0949 
 
 0.0474 
 
 0.7052 
 
 0,0498 
 
 1.6169 
 
 0.0009 
 
 0.0018 
 
 0.0140 
 
 0.0130 ' 
 
 0.0093 
 
 0.0175 
 
 0.1977 
 
 0.2244 
 
 0.0584 
 
 0.0251 
 
 0.1979 
 
 0.1997 
 
 0.0164 
 
 0.0931 
 
 0.0578 
 
 O.7O85 
 
 0.0640 
 
 1.6456 
 
 0.0023 
 
 0.0025 
 
 0.0095 
 
 0.0128 1 
 
 0.0093 
 
 0.0231 
 
 0.1372 
 
 0.2195 
 
 0.0486 
 
 0.0293 
 
 0.1943 
 
 0.1927 
 
 0.0159 
 
 0,0998 
 
 0.0400 
 
 0.7044 
 
 O.OS'^S 
 
 1.6287 
 
 0.0028 
 
 0.0021 
 
 0.0143 
 
 o.oi‘=6 ’ 
 
 0.0080 
 
 0.0220 
 
 0.1740 
 
 0.2046 
 
 0.0438 
 
 0.0439 
 
 0.1906 
 
 0.1997 
 
 0.0150 
 
 0.0981 
 
 0.0575 
 
 0.7067 
 
 0.0602 
 
 1.6255 
 
 O.OO25 
 
 0.0026 
 
 0.0057 
 
 0.0127 ' 
 
 0.0091 
 
 0.0217 
 
 0.1624 
 
 0.2133 
 
 0.0546 
 
 0.0491 
 
 0.1972 
 
 0.1941 
 
 0,0147 
 
 0.0887 
 
 0.0468 
 
 0.7071 
 
 0.06^2 
 
 1.6380 
 
 0.0028 
 
 0.0021 
 
 0.0140 
 
 0.0124 1 
 
 Aver. 0.0096 
 
 0.0206 
 
 O.I87O 
 
 0.2170 
 
 0.0506 
 
 0.0422 
 
 0.1913 
 
 0.1956 
 
 0.0158 
 
 0.0973 
 
 0.0477 
 
 0.7079 
 
 0.0648 
 
 1.6312 
 
 0.0022 
 
 0.0021 
 
 0,0112 
 
 0.0125 
 
 OATS (Contains l.bbO pet. N) 
 
 In alc- 
 ohol ppt. 
 of hot '<L 
 pet. 
 
 CClp.COgH 
 
 extracts 
 
 R 
 
 Nitrogen l os t in' 
 
 Unadsorbed 
 humin (fil- 
 tered from 
 sol. during 
 deconp. of 
 Bases'! 
 
 In a.T.yl 
 alcohol 
 ether 
 extract 
 
 0.0007 
 
 0.0111 
 
 0.0093 
 
 0.0034 
 
 0.0004 
 
 0.00^7 
 
 Aver. 0.0046 
 
 0.0140 
 
 O.O33O 
 
 0.0333 
 
 0.0034 
 
 0.0043 
 
 0.0k7o 
 
 0.0193 
 
 0.1310 
 
 0.1139 
 
 O.llBH 
 
 0.0962 
 
 0.1100 
 
 0.1176 
 
 0.1145 
 
 0.145s 
 
 O.I58O 
 
 O.loOS 
 
 O.IO3O 
 
 0.1147 
 
 0.147s 
 
 0.1383 
 
 0.0^21 
 
 0.0253 
 
 o.oai 
 
 0.0099 
 
 0.0107 
 
 0.0153 
 
 0.0174 0.0324 
 
 0.0194 
 
 0.0366 
 
 0.0321 
 
 0.0378 
 
 0.0365 
 
 0.0321 
 
 0.1651 
 
 0.1726 
 
 0.1721 
 
 0.1582 
 
 0.1720 
 
 0.1334^0 
 
 0.1680 0.1228 
 
 1213 
 
 124H 
 
 1243 
 
 1236 
 
 1233 
 
 1189 
 
 0.011^5 
 
 0.0164 
 
 0.0167 
 
 0.013O 
 
 0.0139 
 
 0.0151 
 
 0.0151 
 
 0.07^ 
 0 . 07 6s 
 0.0690 
 0.0668 
 0.0553 
 0.0662 
 
 0.0680 0.0310 
 
 [0.0341 
 
 0.0340 
 
 0.0348 
 
 0.0260 
 
 0.0313 
 
 0.0255 
 
 CO Ri# (Contains 1.4074 Pct. N) 
 
 0.6487 
 
 0.6585 
 
 0.6342 
 
 0.67<i0 
 
 6750 
 
 6555 
 
 0.6573 
 
 0.0788 
 
 0.0124 
 
 0.1161 
 
 0.1233 
 
 0.0967 
 
 0.0929 
 
 0.1016 
 
 1.3845 
 
 1.3156 
 
 1.3817 
 
 1.3336 
 
 1.3293 
 
 1.3076 
 
 1.3518 
 
 0.0014 
 
 0.0061 
 
 0.0137 
 
 0.0075 
 
 0.0055 
 
 0.0086 
 
 0.0055 
 
 0.0032 
 
 Aver. 0.0065 
 
 0.0337 
 
 0.0420 
 
 0.0443 
 
 0.0417 
 
 0.0345 
 
 0.0332 
 
 0.0265 
 
 0.0344 
 
 0.037. 
 
 0.3360 
 
 0.3310 
 
 0.3434 
 
 0.3459 
 
 0.3565 
 
 0.3689 
 
 0.4144 
 
 0.4765 
 
 0.3778 
 
 0.3761 
 
 0.3790 
 
 0.4014 
 
 0.4250 
 
 0.3963 
 
 0.4309 
 
 0.4484 
 
 0.5140 
 
 0.4214 
 
 0.1773 
 
 0.1773 
 
 0.1694 
 
 0.1733 
 
 0.2026 
 
 0.1991 
 
 0.1878 
 
 0.1884 
 
 O.I85O 
 
 0.2352 
 
 0.3473 
 
 0.3710 
 
 0.3043 
 
 0.1641 
 
 0.1801 
 
 0.1586 
 
 0.1866 
 
 0.2434 
 
 0.6426 
 0.6535 
 0.6743 _ 
 0.6043 ll 
 0 . 6286 
 0.6333 
 
 0.6113 
 
 0.6635 
 
 0.6396 
 
 .2639 
 .2946 
 ,2550 
 ,2503 
 .1906 
 ■381 3 
 ,2224 
 .3661 
 
 ,2712 
 
 0053 
 061 3 
 0726 
 0763 
 0714 
 0644 
 0430 
 0631 
 
 0.0641 
 
 0.3728 
 
 0.4302 
 
 0.5126 
 
 0.4920 
 
 0.5630 
 
 0.4326 
 
 0.6356 
 
 0.4393 
 
 0.4373 
 
 0.2832 
 
 O.2O5O 
 
 0.2426 
 
 0.3038 
 
 0.3032 
 
 0.3399 
 
 0.2447 
 
 0.2905 
 
 0.2860 
 
 COTTONSEED MEAL^ (Contains 
 
 
 00711 
 0.0025 
 0.0021 
 0.0015 
 0.0016 
 0.0019 
 
 0.0019 
 
 0.0072 
 
 0.0023 
 
 0.0047 
 
 0.0037 
 
 0.0010 
 
 0.0043 
 
 0.0039 
 
 2.7170 
 2.7550 
 2.6404 
 2.7067 
 2. 3514 
 2.8509 
 2 . 6643 
 2.9550 
 
 .7676 
 
 0.2223 
 
 0.2332 
 
 0.1292 
 
 0.0109: 
 
 0.1822 
 
 0.1556 
 
 0.2098 
 
 0.2347 
 
 0.1953 
 
 6.3659 
 6. 4543 
 6.4690 
 6.5319 
 6.5740 
 6.6080 
 6.4313 
 6.3913 
 
 6.5611 
 
 0.0152 
 
 0.0152 
 
 0.0133 
 
 0.0137 
 
 Aver. O.CI45 
 
 0.0510 
 
 0.0420 
 
 0.0522 
 
 0.0490 
 
 0.0436 
 
 0.4327 
 
 0.4264 
 
 0.4544 
 
 0.4392 
 
 0.4337 
 
 0.4939 
 0. 4356 
 0.5204 
 0.5019 
 
 0.5017 
 
 0.0968 b.1178 
 O.O97O2 0.0923 
 
 0.0945 
 0.0996 
 
 0.0970 0.1178 
 
 0.1349 
 
 0.1260 
 
 0.1825 
 
 0.2069 
 
 O.IS67 p.20oq 
 0.2156 
 
 0.1893 
 
 0.1935 
 
 0.1977 
 
 0.2220 
 
 0.2101 
 
 0.0243 
 
 0.0279 
 
 0.0259 
 
 0.0260 
 
 0.0260 
 
 0.1145 
 
 o.oqoi 
 
 0.0994’ 
 
 0.1033? 
 
 0.1033 
 
 * Corrected for solubility of the bases 
 y The writer is indebted chiefly to Nao Uyei, assistant chemist, for the 
 ^ following analyses of corn 
 
 The writer is indebted to 1^. B. Nevens for the following ane.lyses of cotton' 
 
 ALFALFA. (Contains 2.628 Pct. N) 
 
 0.1139 1.007s 
 O.I3O3 D.9902 
 0.1053 D.9806 
 0.116521.0193 
 
 0.1165 0.9995 
 
 0.1015 
 
 0.0604 
 
 0.0314 
 
 O.O5O6 
 
 0.0660 
 
 2. 4668 
 2.3934 
 2.4057 
 2.4547 
 
 2.4314 
 
 6.796 pct. N^ 
 
 0.0131 
 
 0.0131 
 
 0.0281 
 
 0.0519 
 
 0.0393 
 
 0.0823 
 
 O.O38O 
 
 0.0073 
 
 0.0074 
 
 0.0072 
 
 0.0052 
 
 0.0075 
 
 0.0059 
 
 0.006s 
 
 0.0150 
 
 0.0177 
 
 0.0205 
 
 0.0158 
 
 0.02922 
 
 0.0465 
 
 0.0439 
 
 0.0400 
 
 0.0292 
 
 Extrac- 
 tion not 
 made 
 
 0.1545 
 
 0.1062 
 
 0.1127 
 
 0.2069 
 
 0.0810 
 
 0.0693 
 
 0.0525 
 
 0.0927 
 
 0.1095 
 
 0.0666 
 
 0.0016 
 
 0.0536 
 
 0.0771 
 
 0.0470 
 
 0.0530 
 
 0.0697 
 
 0.0726 
 
 0.0627 
 
 0.0700 
 
 0.0614 
 
 0.0770 
 
 0.0564 
 
 0.0622 
 
 Extrac- 
 tion not 
 made 
 
 0.0263 
 
 0.0292 
 
 0.0335 
 
 0.0331 
 
 O.O3O5 
 
 0.0160 
 
 0.0214 
 
 0.0079 
 
 0.0188 
 
 0.0161 
 
 ^ Not included in the average 
 
 2 Determination lost - Average result substituted to ma’xe up total 
 
 seed meal . 
 
 s 
 
 T 
 
 u 
 
 V 
 
 
 method of aralvsis 
 
 Total 
 
 
 
 In resi- 
 
 In resi- 
 
 Total 
 
 Total 
 
 
 
 due fil- 
 
 due fil- 
 
 nitro- 
 
 nitro- 
 
 
 
 tered 
 
 tered 
 
 gen 
 
 ^:en ac- 
 
 
 
 from so- 
 
 from so- 
 
 lost 
 
 counted 
 
 
 
 lution 
 
 lution of 
 
 
 for 
 
 
 
 of bases 
 
 filtrate 
 from bases 
 
 
 
 
 
 
 
 O.OOol 
 
 0.0004 
 
 O.O285 
 
 1.6614 
 
 
 
 0.0050 
 
 0.0006 
 
 0.0352 
 
 1.6521 
 
 
 
 0.0014 
 
 0.0003 
 
 0.0236 
 
 1.6743 
 
 
 
 0.0024 
 
 0.0004 
 
 0.0375 
 
 1 . 6662 
 
 
 
 0.0032 
 
 0.0003 
 
 0.0270 
 
 1.6525 
 
 
 
 0.0030 
 
 0.0006 
 
 0.0343 
 
 1.6729 
 
 
 
 0,0035 
 
 0.0004 
 
 O.O3I9 
 
 1 . 663*^ 
 
 
 
 
 
 
 
 0.0027 
 
 0.0009 
 
 O.O3S3 
 
 1.3628 
 
 
 
 0.0029 
 
 0.02071 
 
 0.0438 
 
 1.3644 
 
 
 
 0,0032 
 
 0.0001 
 
 0.0455 
 
 1.4271 
 
 
 
 0.0021 
 
 0.0019 
 
 0.0663 
 
 1.3999 
 
 
 
 0.0023 
 
 0.0006 
 
 0.0523 
 
 I.38I6 
 
 
 
 0.0029 
 
 0.0011 
 
 0.0984 
 
 1.40b0 
 
 
 . 
 
 0.0027 
 
 0.0009 
 
 0.0541 
 
 I.406O 
 
 
 
 
 
 
 
 0.0140 
 
 0.0024 
 
 0.2525 
 
 6.6134 
 
 
 
 0.0146 
 
 0.0026 
 
 0.2027 
 
 6.7432 
 
 
 
 0.0150 
 
 0.0072 
 
 0.2089 
 
 6.6779 
 
 
 
 0.0167 
 
 0.0083 
 
 0.3253 
 
 6.6773 
 
 
 
 0.0291 
 
 0.0102 
 
 0.1965 
 
 6.7704 
 
 
 
 0.0258 
 
 0.0065 
 
 0.2011 
 
 6.8O91 
 
 
 
 0.0065 
 
 0.0044 
 
 0.1820 
 
 6.6132 
 
 
 
 0.0090 
 
 0.0046 
 
 0.2190 
 
 7.1102 
 
 
 
 0.0163 
 
 0.0058 
 
 0.2235 
 
 6.7346 
 
 
 
 
 
 0.0038 
 
 0.1160 
 
 2.5828 
 
 
 
 Not 
 
 0.0168 
 
 0.1288 
 
 2.5272 
 
 
 
 deter- 
 
 0.CI59 
 
 0.1343 
 
 2.5400 
 
 
 
 mined 
 
 0.0099 
 
 O.II83 
 
 2.5729 
 
 
 
 
 o;oii6 
 
 0.1244 
 
 2.555s 
 
 
 I 
 
-C6- - 
 
 for analysis^ v/as discarded without its total ni trogen content liaving first been 
 determined. 
 
 For convenience^ the results on the distribution of nitrogen in the 
 feed, are divided into nonprotein nitrogen, nitrogen distribution as shown by 
 the Van Slyke analysis, and the nitrogen lost in the method of analysis. 
 
 Criticism has been made of the application of the Van Slyke method to 
 the aiialysis of feeds on the basis of the possible interference of some of the 
 nonprotein nitrogenous constituents. In the metnod used in this work ariy possi- 
 ble interference of nonprotain nitrogenous compounds is completely obviated by 
 the complete rauoval of those compounds by extractions with ether, shsolute 
 alcohol, and cold 1 per cant, trichloracetic acid. Although the exact role, 
 played in protein metabolism by all of the nonprotein rntrcgenous constituents 
 of a feed, is at present unknown, it is of interest to note that the four feeds 
 analyzed vary narkedly in their ncnprotein nitrogen content. Alfalfa has the 
 highest percentage of norprotein nitrogen with an average of 19*09 cent., 
 •while cottonseed meal has the lowest with an average of o.20 per cent. Oats 
 and corn ijave intermediate values of 1^.93 cent, and 9*^3 psi* cant., respec- 
 tiveljr. Judging from theso values, which agree vary well with those of Grindley 
 and Eckstein (Q), roughages have a higiier nonprotein nitrogen content and con- 
 centrates a lower content than the cereals. 
 
 jjnder the heading ‘’nitrogen lost in method of analysis'’ are found the 
 results of the total nitrogen determira.tions on the various fractions, the 
 amino-acid content of which escapes analysis by the method of preparing the hy- 
 drolyzed protein solution and by the Van Slyke analysis of this hydrolyzed pro- 
 tein solution. The total nitrogen lost is shown in Column U. The nitrogen 
 lost in the preparation of the hydrolyzed protein solution is siiown in Column 0, 
 representing the nitrogen left In the residue after treatment ’with the last ex- 
 tracting fluid - 5 per cent, sodium hydroxide, and in Column P, giving the nitro- 
 
1 
 
- 63 - 
 
 gen in the alcohol precipitate (starch) of the hot c per cent, trichloracetic 
 acid extract. The total nitrogen lost in the preparation of the hydrolyzed pro- 
 tein solution v/as 0.2^9 cent, in ca.se of oats, 0.U12 per cent, in case of 
 corn, O.U 3 O per cent, in case of cottonseed meal, and 2.519 P®* c®at. in case of 
 alfalfa. The extraction of starch was not made in the cases of cottonseed meal 
 and alfalfa because of the sriall amounts present. 
 
 The nitrogen lost in the ar.alysis of the hydrolyzed protein solution 
 by the Van Slyke method, consists of (0) the unadsorbed hunin, filtered from the 
 amyl alcohol-ether aqueous solution during the decomposition of the bases; (E) 
 the nitrogen extracted by the amyl alcohol-ether mixture; (S) the nitrogen in 
 the residue filtered from the solution of the bases; and (T) the nitrogen in the 
 residue filtered from the solution of the filtrate from the bases. As percent- 
 age of the total nitrogen of the feed the total nitrogen lost, as shown in 
 Column U, Table I, is l.QO per cent, in case of oats, 3.ci9 cant, of cotton- 
 seed meal, 3.65 per cent, of corn, and Ij.73 P®!* cent, of alfalfa. 
 
 Tne terms used in the literature for desigriating the humin nitrogen 
 fractions are confusing. According to Van Slyke' s original method (6): "Dur- 
 ing the distillation (of ammonia) e.ll of the black coloring matter, or melanin, 
 v;hich is formed during the hydrolysis of the proteins, is adsorbed by the undis- 
 solved lime." The latter is filtered off, washed, and submitted to Kjeldahl 
 analysis. The results are reported as melanin nitrogen. This is the only frac- 
 tion of humin reported by him. In the first s-ltempt to apply the Van Slyke 
 method to the analysis of feeds, Grindley, Joseph, and Slater ( 5 ), used the 
 original method directly. The results of their melanin nitrogen included, 
 therefore, any nitrogenous substances in the insoluble residue of the f eed a.s 
 wall as the melanin or humin formed duririg the hydrolysis. ITollau (7) filtered 
 off the insoluble residue remainirig after the direct hydrolysis of feeds and 
 analyzed the filtrate according to tne Van Slyke method. His results v.’ere based 
 

 (»!. 
 
 f ; ,. 
 
 . X 
 
 
 ■ T*- 
 
 F»: 
 
 i ':<* 
 
 ■ *aV5Ji}-^ ''-, -V' j-i-« 
 
 M 
 
 m 
 
 
 '.'mi 
 
 ''■fftj 
 
 >• ' 
 
 ^ Ki 
 
 
 
 
 r.l 
 
 
 ''tfA i--’; 'T.e^ 
 
 .in 
 
 
 H: 
 
 t 
 
 \ 
 
 f .Ti-'. 
 
 ' .I'M 
 
 iiv . ... 
 
 (:i 
 
 ' ; 
 
 i 
 
 r 
 
 :S 
 
 
 f* .A? 
 
 
 'ii/iM 
 
 ■f 
 
 fi 
 
 J . <a y .j 
 
 -,V;»’ 
 
 ./• a'' 
 
 
 < 
 
 
 
 
 
 4 .' -!•*■ -'■■••» •* .'. fji.’ ■• '.'.V^.v» t 
 
 i'^ /'lii. 
 
 t' ■•■li^ 
 
 ' V ‘Vx' 
 
 '. i<; *r 
 
 'i T ’ 
 
 
 I ■ .fi;,io. ■. J tr- 'i> i4 'ri I'-vs 
 
 . , ; *,.■:■ {»^r . 
 
 .■„■/•■ *;*.'■ 
 
 ■ /J 
 
 V ■ /, 
 
 V..' 
 
 /■ 
 
 . . . 1 
 
 
 V i, j ii.:v’j,s ^<j *A 
 
 ■ .. !('- -C* i??- 
 
- 6 ) 4 - 
 
 on the percentage of the total viitrogen in this filtrate and not on the total 
 nitrogen of the feed. Attention v/as called to this fact and to tho introduction 
 of certain errors by this procedure by G-rindley and Slater (6) v/ho refer to their 
 melanin fraction as humin^ which is probably tne better r^ame for this substarice. 
 Gortner ( 1 ^) divided the humin fraction into ••acid-insoluble" and "acid-soluble" 
 (a-bsorbed by lime) humin^ the sum of the two being the total hurnin nitrogen. 
 Gortner and Holm (I3) report the two fractions under separate headings as insol- 
 uble humin nitrogen and soluble hurnixi nitrogen. Other workers have reported 
 this humin nitrogen fraction under such various headings as ••h'un;in N absorbed 
 by lime" (Osborne^ Van Slyke, Leavenworth, and Vinograd, "humin nitrogen 
 
 absorbed by magnesia" (Miller, 3), and as "melanin nitrogen" divided into "a b c 
 fraction" and "lime fraction" (Neidig and Sryder, I7). In this work the ®::- 
 pressions insoluble and soluble humin have been retained. The humin nitrogen of 
 Table III is the sum of the insoluble and the soluble humin rhtrogen. It is 
 possible tixat the nitrogen, or parts of it at least, in the fractions listed in 
 Columns Q, B, S, and T of Tables I and II properly belongs to the total humin 
 nitrogen figures but this has not been done. 
 
 Other fractions of hurnin nitrogen have been reported. In Van Slyke's 
 improved method (^3) of decomposing the basic phosphotungstates bj'- the amyl 
 alcohol-ether method, the follov/ing statement is made: 
 
 "In some cases the aqueous and ether-amyl alcohol layers do not sep- 
 arate readily v/ith a clean bomidary between them. This effect is due to the 
 presence of e, slight amount of humin which may loavs escaped previous adsorption 
 by calcium hydrate. In this case the ur.adsorbod humin is carried dov/n v/ith the 
 basic phosphotungstates, a,nd fouls the solution when their precipitate is de- 
 composed as above described (amyl alcohol-ether method). In order to clear the 
 solution up, it is all, without separation of the aqueous and ether-amyl alcohol 
 layers, passed through a Buchner funnel with suction." 
 
 It is evident that Van Sl3/ke recognized a portion of humin substances 
 in the basic phosphotungstates. Osborne, Van Slyke, Leavenworth, and Vinograd 
 (25) in one case purified their basic phosphotungstates by reprecipitation with 
 
phoschc tungstic acid. Tney decomposed t..eir basic precipitate by the amyl alco- 
 hol-other method and state that "all the coloring matter which accompanied the 
 bases was extracted by the amyl alcohol and etner." The nitrogen taken up by 
 the organic solvents was determii:ied and reported by them as "humin N in amyl alco- 
 hol extract." Gortner ana Holm (I3) separated, besides the "acid-insoluble 
 humin" and the "acid-soluble humin," a "phosphotungstic humin." This ;vcis de- 
 termined by submitting the barium phos oho tungstate precipitate'^ to Kjelaahl 
 analysis. This fraction of ijumin nitrogen is undoubtedly the "humin IT in amyl 
 alcohol extract" of Osborne, Van Slyke, Leavenworth, and Vinograd and the "un- 
 adsorbed hurnin" of Van Slyke. ihis fraction is again divided by Miller (3) into 
 
 "humin IT insoluble in amyl alcohol" and "humin iT in smyl alcohol extract." Nei- 
 
 dig and Sr^yder ( 1 ]) also found tliat a dark-colored substance usual_y formed along 
 with the bases when the latter were precipitated with phosphotungstic acid and 
 
 refer to it as the "phosphotungstic humin" of Gortner and Holm. Until the final 
 
 method wvs adopted, this dark-colored substance, v/hich v^as sometimes of a sticmy 
 nature, was often er.countered. With this substance present it was almost im- 
 possible to wash the ba.sic pliospho tungstates thoroughly. the method firally 
 adopted the basic phospho tungstates of all four of the feeds analyzed were white, 
 gray, or slightly cresm-colored gra-nula.r precipitates entirely free from any 
 dark-colored material. However, during the decomposition of the basic phospho- 
 tungstates by the amyl alcohol-etner method, a scum, sometimes of a dark color, 
 is formed makings filtration necessary. Tii is residue is washed c5;-refully with 
 armnonia-free water, smyl alcohol, and ether, and submitted to Kjeldahl analysis. 
 The resLilts are reported in Column Q under the heading "UTiadsorbed humin," the 
 nsme used by Van Slyke. The clear aroyl alcohol-ether extract of 
 
 ^ These authors used Van Sijhce's original barium: hydroxide method of liber- 
 atine the basses. 
 
,1' 
 
 'J 
 
 I 
 
 I - ^ . - 
 
 '*'■■ r -.Y^' : - < ■[,'. 
 
 r 
 
 y 
 
 I 
 
 j" ’■■ 'i, . ■> 
 
 T .:• f 
 
 1 ^, , i »■ 
 
 
 
 
 <KS‘A 
 
/f 
 
 -OD- 
 
 tr-e phosphot’angstic acid is also subaiitted to Kjeldahl aralysis and tne nitro- 
 gen content reported n* Colmm R as the nitrogen in axnyl alcohol -ether extract. 
 
 On concentrating the solution of the bases a small gray residue has been found 
 to settle out. Van Slyke, in the original method of freeing the bases by means 
 of barium hydrate, stated that this residue v/as barium pliospho tungstate, which 
 was to be filtered off and evidently discarded, Tliis residue has alwaj/s been 
 encountered hi this v/ork even when the amyl alcohol-ether method of decomposing 
 the basic phosphotungstates v;as employed. According to the detailed procedure 
 this residue is filtered off, washed carefully and its total nitrogen determined 
 Tile results are shown in Column S. After making the filtrate from the bases up 
 to volume (tlOO cc.) ind allov/ing to stand over night occasionally a residue, 
 similar to the one in the solution of the ba.ses, separe^tes out. This is usually 
 very small in amount, but when present the nitrogen has been determined. The 
 results are shov/n in Column T, vo attempt has been made to determine the nature 
 of the nitrogenous constituents in these fractions. The total nitrogen has been 
 determined in than entirej-y for the purpose of showing the accura-cy or inaccuracj 
 of the method as applied to the aioalysis of feeds. 
 
 As percentage of the total nitrogen of the feed, the humin ri trogen oi 
 oats is cent., of corn 3.5^ psr cent., of cottonseed ineal 0.3O per cent, 
 
 and of alfalfa ].}6 per cent. 
 
 The completeness with which the nitrogen is extracted from the finely 
 ground feeds is shown in Column P, Table I, which gives the percentages of the 
 total nitrogen of the feeds remaining in the residues a-fter the last extra.ction 
 with 5 per cent, sodium hydr&xide solution. The nitrogen in the residue from 
 oats, as shovm by the a-verage, is 0.13^ per cent, of the origir.al amount present, 
 from corn 0,136 per cent., from cottonseed meal O.U3O per cent., and from al- 
 falfa £.519 per cent. 
 
The nitrogen distribution as shovv-n by the Van Slyke ar^ilysis includes 
 the insoluble huiain nitrogen, the soluble huniin nitrogen, acid-air.ide nitrogen, 
 arginine nitrogen, cystine nitrogen, histidine nitrogen, lysine nitrogen, amino- 
 acid nitrogen in the filtrate from tne bases, and the non-amino -acid nitrogen 
 in the filtrate from the bases. Tiie question has been raised in several papers 
 on the analysis of the proteins of feeds by the Van Slyke method that the various 
 fractions ordiriarily designated "arginine nitrogen," "histidine nitrogen," etc., 
 may not be accux’ately described by these terms, on account of the heterogeneous 
 nature of the nitrogenous constituents as well as other substances present. In 
 the total absence of exqxerimental evidence on this point, it seams fair to assume 
 the.t the above terms are as properly applied to the fractions of nitrogen ob- 
 tained in the analysis of feeds by the above described method as to the corres- 
 ponding fractions obtained in the analysis of pure proteins. 
 
 It is of interest to note the variation in the basic lutrogen of the 
 different feeds, since v/e knov/ more of the requirements of animals for the basic 
 amino-acids tlxan for a.ny other group, line argirdne nitrogen varies from about 
 d per cent, of the total nitrogen in alfalfa to 18.] per cent, in cottonseed 
 meal. The cystine nitrogen is about the same in ail cases but these results 
 ma^/, perhaps, be a little lov/. The histidine nitrogen is lowest in alfalfa with 
 8. value of 3.9 Por cent, of the total nitrogen and cottonseed meal is highest 
 with a value of ].£ per cent. Lysine rdtrogen is lowest in corn with a "value of 
 ci.c: par cent., sli^tly higher in oats with d.8 per cent., and highest in alfalfa 
 with U .43 pel* cent. The total basic nitrogen as percentage of total nitrogen 
 is 31 . 03 per cent, in cottonseed meal, cl .83 per cent, in oats, 1]*35 cent, 
 in alfalfa, and lb. 83 par cent, in corn. 
 
 In Table III the average results obtained in Table I are compared 
 with the results on the same feeds obtained by Grindlay and associates reported 
 by Grindley (14) and by ITollau (7). 
 
„ ^ J U 
 
 a 
 
 1 
 
 V 
 
 i;, . *.i .f ,<>v ,y ;. 
 
 ■I'jf 7,h Si- 
 V 
 
 - ’,wj' V V 
 
 • ' • *v» 
 
 •‘“W ¥ ■ 
 
 '■ . \ « 
 
 . j * ' ’ 
 
 1^* V ,; 
 
 £'■’ •'■ 
 
 *\> 
 
 'V/ti 
 
 4- 
 
 
 
 
 4 ■ ' v‘ • 
 
 * "' - ’•■; -s ". .>:• r,; .'2;> ,-i^ ;^j 
 
 ■■' , , • - ,■ 
 
 
 < ' 
 
 
 »fk.. r.’ 
 
 
 •‘' *-. V • ' ,' ' 
 
 . '*«. 
 
 
 r-^ 
 
 !l i •; .’ fti' \ ■ 
 
 ' ■•■' i 
 
 
 
 ■"J ■■ 
 
 ♦ .'■■ ♦' 
 
 -f ■*.»■.> 
 
 
 ■ i 
 
 
 'W'r-'- 
 
 
 y} 
 
 
 y 
 
 'U ».'■ •■ 
 
 
 ¥■ 
 
 ■ ■?•/-:. vv. ,. 
 
 -I ■ .■ ' , *' 
 
 'v ‘ ' J 
 
 ■ 'Wl'*'"'': jra 
 
 ■> •:< 
 
 
 
 
 
 • kJ 
 
 ,. S*-'4 
 
 ' ■' ' ’. I '■ J*T» T'X' 
 
 
 
 M I 
 
 ' , . y* ' ■* ■' \ ' , I * »‘ ^ . ' ... • '.i 
 
 • V*-»v 
 
 
 vi : h ’i,’^^ ' 
 
 ■.i .';''',n. V ■'.'i 
 
 m. 
 
TABLE III. - G0:,1PAEIS0N OF RESULTS WITH THOSE OF PREVIOUS IHVESTIGATORS 
 (Results expressed as percentage of total nitrogen) 
 
 ■^IbT 
 
 -OOSS’B pu-0 
 
 iCsip 'T.lp 
 
 I 
 
 eiq-Gj, 
 
 uiojj; 
 
 TIB I TOE 
 
 5 lbT se^BT 
 
 -OOSSB pUB 
 
 I 
 
 raoj^ 
 
 TIBI TOE 
 
 ^Ibl BBYSX 
 -OOSSB pUB 
 iCsipuTai) 
 
 I 
 
 tnojji 
 
 n.BiTOE 
 
 5 lcl seqBt 
 
 -OOSSB piTB 
 
 iCoXpuiap 
 
 I 
 
 STOBJ, 
 
 U!0 
 
 n. 
 
 Ph 
 
 < 
 
 Pi 
 
 W 
 
 CO 
 
 o 
 
 o 
 
 o 
 
 s 
 
 o 
 
 o 
 
 CO 
 
 EH 
 
 -oo- 
 
 =}• (S'< O O -=J O VI O" 
 i — > O o -=T .-H O I — 
 
 O U'l i'-O I— -U- J O 
 
 "!y 
 
 r-i 
 
 Y) 
 
 O 
 
 '£> O ex' r-H U ^ J- 
 
 fH^ O (T- O' -=t o LPi U > X) '-O I'- VJ 
 
 'VD 
 
 O O O lU O r-l ' o ^ 
 
 rH 
 
 O 
 
 '-o I-— I'- -=!■ f — ^ .r\j O'' 
 
 O U 1— o -=1' 
 
 '■O ,vj 
 
 u^. 
 
 -=T 
 
 vD 
 
 ■O 
 
 O' 
 
 Li ^ 'O -U LOv 1^ — 'O C\J I 
 ^ i'~ lI'>vO -4" X) ^ I 
 
 • • ••••••!• 
 
 O i — 0 ^ O -=t \J LT\ I 
 f-H <-4 I 
 
 O 
 
 O' 
 
 v6 
 
 cr. 
 
 rH O 
 
 rH 
 
 i-H 
 
 3 O 
 
 O'. 35 cr 
 
 NX 
 
 3- NX 
 
 i'- O' 
 
 rH 3 
 
 ^ rH 
 
 ax ax 3 
 
 -rO 
 
 C5X vO 
 
 CO o 
 
 r*H 
 
 I-— 3' 
 
 Q 3 O 
 3 
 
 o ax NX 
 
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 As a whole, the determinations of the different investigators do not 
 agree well, althoudi in some instances the agreement is quite satisfactory. 
 
 The results reported in Table I agree with those of Grindley and associates 
 slightly batter than they do with those of IJollau. The lack of concordant re- 
 sults is due, chiefly, to differences in methods used. Grindley and associates 
 hydrolyzed the finely gro-ond feeds, determined the 'acid-airi de nitrogen in the 
 hydrolysate, filtered off the hurain nitrogen, and proceeded to analyze the fil- 
 trate according to the Van Slyke method. Their results were based on the total 
 nitrogen in the feed. Noliau removed the fat by extracting the finely ground 
 feed witn ether. The sairples v/ere then completely hydrolyzed and the insoluble 
 humin filtered off. The total nitrogen determined in the filtrate was the basis 
 for calculation of results of the -nitrogen distribution a-s obtained "by the Van 
 Slyke procedure. By the iinproved method described above the nonprotein nitrogen- 
 ous constituents, most of the carbohydrates and the fiber are rs-noved before 
 hydrolysis. The objectionable features of the previous methods are thereby ob- 
 viated completely or at least greatly reduced. Considering the differences in 
 procedure, Nollau's results for humin nitrogen should be lov/er than the results 
 for the humin nitrogen of Grindley and a.ssociates. In genvsral this is true. 
 Nollau's procedure should^also lead to correspondingly higher results for the 
 remaining nitrogen values, considered on the basis of the total nitrogen of the 
 feed. V/itli a few sxceptiais this is found to be true, but the results are usu- 
 ally higher than this difference of procedure would warrant. The lower results 
 for armconia nitrogen, the amino-acid nitrogen and non-amino-acid nitrogen in the 
 filtrate from the bases of the method used in this work are expected because of 
 the removal of the nonprotein nitrogen. There a,re some differences in the re- 
 sults tiiat cannot be e^splained on the basis of differences of procedure. For 
 example, Nollau reports no lysine in oats v\foile 2.84 psr cent, was found, he re- 
 

 / 
 
 1 
 
- 70 - 
 
 ports 6.53 per cent, of lysine in corn while only c..'d per cent, w&s fo'und, and 
 again he reports no non-aniino-acid nitrogen in the filtrate from the bases in 
 com while '] ,'d.cL per cent, was found. The results IToliau obtained for cystine 
 are in all cases much higher than those reported in Table I but it is very prob- 
 able that the cystine nitrogen reported in Table I is lass than half the origir.al 
 amount present. 
 
 Miller (3) in his study of the distribution of nitrogen in the alfalfa 
 seed precipitates the protein from a O.5 P 52 * cent, potassinm hydroxide extract 
 with acetic acid and arialyses the precipitate which contains only bO per cent, 
 of the total nitrogen of the seed according to the Van Slyke ara.lysis. The re- 
 sults obtained therefore, cannot be considered as representing the distribution 
 of nitrogen in the entire seed. Dowell and Menaul (I4) report on the "Nitrogen 
 Distribution of the Proteins Extracted by Dilute Alkali from Pecans, Peanuts, 
 Kafir, and Alfalfa." Their method, \'\ 4 iicji was similar to that used by Miller, v/as 
 in case of alfalfa as follows: "Alfalfa which was ground to pass a l| 0 -mesh sieve 
 was extracted with O.3 per cent, sodium hydroxide, and 62 per cent, of the nitro- 
 gen compounds were extracted. Sixty one per cent, of the rhtrogen extracted was 
 precipitated v/hen the solution was inade sli^tly acid with acetic acid. The puri- 
 ty of the precipitated proteins was found to be 85 per cent., using the factor 
 b.25«" 
 
 The results of Table I on alfalfa are compared with those of Dowell 
 and Menaul, in Table IV. Tiio agreement is obviously very poor. 
 

-71- 
 
 TABLE IV. - NITBOGEI'I DISTRIBUTION OF ALFALFA 
 
 
 Dov/ell and 
 Menaul'^ 
 
 From 
 
 Table 
 
 IvlioN 
 
 S.SO 
 
 S.17 
 
 Him: in N 
 
 7. SO 
 
 7-3S 
 
 Arginine N 
 
 11.01 
 
 6.00 
 
 Histidine N 
 
 6 .h& 
 
 3 -^'3 
 
 Cystine N 
 
 0.65 
 
 o.qQ 
 
 Lysine N 
 
 'do 
 
 4.43 
 
 Mono-anino N 
 
 53-^3 
 
 33.03 
 
 Non--amino N 
 
 s . 4s 
 
 > C*! 
 
 ^ Average of ci analyses; results expressed as per- 
 centage of total nitrogen of the protein prep- 
 aration. 
 
 ^0 Average of 4 analyses; results expressed as per- 
 centage of total nitrogen of the alfalfa. 
 
 ‘ Dowell and llenaul rfiake the ststement: "We Imve taken advantage of 
 
 the fact that all proteins are soluble in basic solutions to separs.te them from 
 the other subste.nces in foods and feeds which make it impracticable to apply 
 the Van Slyke method to determine the nitrogen distribution." While the precip- 
 itated proteins in case of pecans and peanuts represented a slightly larger per- 
 centage of the total nitrogen of the food stuff, the proteins precipitated from 
 the alkali extract of alfalfa represented less than 40 psr cent, of the total 
 nitrogen of the alfalfa. Evidently a considerable fraction of the proteins of 
 these vegetable naterials are net soluble in basic solvents. Yet these authors 
 conclude that "the extractions of the proteins v.uth dilute alkaline solutions 
 may enable us to obtain the araino-acid composition of foods and feeds by means 
 of the Van Slyke method." With such large percentages of the nitrogen of a 
 food or feed remaining in the unauialyzed residue, the significance of the re- 
 sults obtained on the extracted proteins may be seriously questioned and it is 
 doubtful whetner the proteins of feeds may be quantitatively separated from the 
 other constituents by any such simple procedure as this. 
 
- 7 ^- 
 
 VIII. SU!.:, 1 AEY AND CONCLUSIONS 
 
 1 . A ruethod has been developed for treating a sample of feed so that 
 the proteins which it contained are obtained in solutions sufficiently free from 
 interfering substances so that the Van Slyke method for the determination of cer- 
 tain amirh-acids in purs proteins may be applied. 
 
 c. The completeness vath v;hich this treatment removes the nitrogen 
 from the finely ground feed v/as, as shovm by the avera.ges, per cent, of 
 
 the oats, 99 *^o 4 poi* cent, of the corn, 99*570 per cent, of the cottonseed meal, 
 and 97 * 4 o 1 per cent, of the alfa.lfa. 
 
 3. The objectionable parts of previous procedures for the applica- 
 tion of the Van Slyke method to the determirjation of amino-acids of feeds have 
 been obviated coinplstely or at laast greatly reduced by the following features 
 of the method used in this work: 
 
 (aj The nonprotsin nitrogenous constituents are removed by extractions 
 v/ith absolute ether, cold absolute alcohol and cold 1.0 per cent, tricliloracetic 
 
 ^ I 
 
 acid. 
 
 (b) The starch is removed by a hot 2.0 per cent, trichloracetic acid 
 
 extra ction. 
 
 (c) The fiber is not present during the hydrolysis of the proteins. 
 
 4. The amino-acid contents of oats, corn, cottonseed mee.l, and al- 
 falfa, as determined by the Van Slyke Method, a-re reported. 
 
 5. By further application of available methods for the estimation of 
 other amino-acids to hydrolyzed protein solutions, prepared in a manner similar 
 to that described for this work, it may be possible to obtain further important 
 knowledge concerning the nutritive value of the proteins of foods and feeds. 
 

 . .... . 
 
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- 73 - 
 
 IX. BIBLIOGRAPHY 
 
 (1) Osborne, T. B., The Vegetable Proteins, 1909, 13* 
 
 (H) Osborne, T. B. and Mendel, L. B., J. Biol. Chear ., 1914, xviii, 1. 
 
 (3) Miller, H. G., J. Aiu. Chem. Soc .. 1921, xliii, OOo. 
 
 (4) Dowell, C. T. and Menaul, P., J. Biol. Chem.. 1921, xlvi, 437* 
 
 (5) Grindley, H. S., Joseph, W. E., and Slater, M. E., J. Ann Cheiri. Soc .. 1915, 
 
 xxxvii, 177 ^* 
 
 (6) Van Slyke, D. D., J. Biol. Chem ., 1911-12, x, I 5 . 
 
 (7) Hollau, E. H., J. Biol. Chem .. 1915> xxi, 6 II. 
 
 (S) Grindley, H. S. and Slater, M. E., J. Am. Chem. Soc ., 1915> xxxvii, c'(ocl. 
 
 (9) Grindley, H. S. and Eckstein, H. C., J. Am. Chem. Soc ., 1916, xxxviii, 1425 . 
 
 (10) Hart, E. B. and Bentley, W. H., J. Biol. Chern ., 1915^ xxii, 477* 
 
 (11) Gortner, R. A. and Blish, M. J., J. An. Chem. Soc .. 1915^ xxxvii, I 63 O. 
 
 (12) Gortner, R. A., J. Biol. Chem.. 1916, xxvi, 177* 
 
 (1 3 ) Gortner, R. A. and Holm, G. E., J. Am. Chem. Soc ., I 917 , xxxix, 2477* 
 
 ( 14 ) Grindley, H. S., Proc. Am. Soc. Animal Production. I 916 , l33* 
 
 ( 15 ) Eckstein, H. C. and Grindley, H. S., J. Biol. Chem ., 1919> xxxvii, 373* 
 
 ( 16 ) Hartley, P., Biochem. J ., 1914, viii, 541. 
 
 ( 17 ) Neidig, R. E. and Snyder, R, S.. J. Am. Chem. Soc., 1921, xliii, 951* 
 
 (lii) Greenwald, I., J. Biol. Chem.. 1915, 3 cxi, 6 I; Ibid., 191&^ xxxiv, 97* 
 
 ( 19 ) Folin, 0. and Denis, W., J. Biol. Chem .. I 916 , xxvi, 491* 
 
 (20) Wolff, C. G. L., J. Physiol .. 1Q15> xlix, S9. 
 
 ( 21 ) Hill, R, L., J. Biol. Cha^i .. 1915, xx, 175* 
 
 ( 22 ) Van Slyke, D. D., Vinograd-Villchur , M., and Losee, J. R., J. Biol. Gnem .. 
 
 1915 . xxiii, 377 . 
 
 ( 23 ) Van Slyke, D. D., J. Biol. Chem .. 1915, xxii, 2S1 . 
 
 (24) Winterstein, E., Ze i t s ciir . f . pby s i 0 1 . Che;n . . 1901-02, xxxiv, I 53 . 
 
 ( 25 ) Osborne, T. B., Van Slyke, D. D., Leavenworth, C. S., and Vinograd, M., 
 
 J. Biol. Chem .. 1915^ xxii, 259. 
 

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