452-212-3111 éIEXAS AGRICULTURAL EXPERIMENT STATIONS BULLETIN N0. 146. . FEBRUARY, 1912. I E Division of Chemistry TECHNICAL BULLETIN The Forms of Phosphorus in Cotton Seed Meal BY J. B. RATHER, Assistant Chemist. POSTOFFICE College Station, Brazos County, Texas AUSTIN PRINTING coMnmY AUSTII’ TIXAS 1912 TEXAS AGRICULTURAL EXPERIMENT STATION GOVERNING BOARD. (Board of Directors A. & M. College.) p WALToN PETEET, President . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “Fort; JoHN I. GUIoN, Vice-President . . . . . . . . . . . . . . IO‘ . . . . . . . . . . . . . . . . . . ..B CHAs. DAvIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Steele’s; L. J. HART . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..San A; J. ALLEN KYLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..H R. L. BENNETT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. W. KEMPNER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “Gal En. R. KoNE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7' PRESIDENT OF COLLEGE. .. R. T. MILNIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..College S STATION STAFF. _ B. YoUNGBLoon, M. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Di 7 M. FnANoIs, D. V. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Veteri G. S. FEAPs, PH. D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .._ . . . . . . . . ..C H. NEss, M. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Horticul J. C. BURNS, B. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Animal Hus WILMoN NEWELL,'M. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Entomo A. B. CONNEB, B. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Agronp F. H. BLODGETT, PH. D . . . . . . . . . . . . . . . . . . . . .Plant Pathologist and Physio p H. L. MGKNIGHT . . . . . . . . . . . . . . . . . . . . . . . . . ..Superintendent Station ‘f W. L. BOYETT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .State Feed Insp HARPER DEAN, B. S . . . . . . . ." . . . . . . . . . . . . . . . . . . . . . . . . ..Assistant Entomol’ J. B. RATHER, M. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Assistant Ch J. B. KELLY, A. B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Assistant Ch L. C. LUDLUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Assistant A.‘ F. B. PADDOCK, B. S . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .Assistant Entomol H. H. JossoN, B. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Assistant Agrono‘ CHAs. A. FELKER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Chiet _ A. S. WARE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....Stenogra, J. M. SCHAEDEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Stenogra STATE AGRICULTURAL EXPERIMENT STATIONS GOVERNING BOARD. HIs EXCELLENCI GovERNoR O. B. CoLQUITT . . . . . . .; . . . . . . . . . . . . . . . . . ..Au LIEUTENANT GOVERNOR A. B. DAVIDSON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..C '_ COMMISSIONER 0F AGTICULTURE HoN. E. R. KoNE . . . . . . . . . . . . . . . . . . . . . .Au IRECTOR OF STATIONS. B. YOUNGBLOOD, M. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .College Stat SUPERINTENDENTS OF SUB-STATIONS. E. E. BINFORD, Beevillc Sub-Station . . . . . . . . . . . . . . . . . . ..Beeville, Bee Coui W. S. HOTCHKISS, 'I‘roup Sub-Station . . . . . . . . . . .. . . . . . ..Tr0up, Smith Cou " E. M. JorINsToN. Cooperation Rice Station . . . . . ..Beaumont, Jefferson C'o w I. S. YORK, Spur Sub-Station . . . . . . . . . . . . . . . . . . . . . . . . . .Spur, Dickens Coun. T. W. BUELL, Denton Sub-Station . . . . . . . . . . . . . . . . . . .Denton, Denton Coun; A. K. SHORT, Temple Sub-station . . . . . . . . . . . . . . . . . . . . ..Temp1e, Bell Counv A. L. PASCHALL, Lubbock Sub-Station . . . . . . . . . . . ..Lubbock, Lubbock Coun P. D. PERKINS, Angleton Sub-Station . . . . . . . . . . . . .Ang1et0n, Brazoria Coun H. C. STEWART, Pecos Sub-Station . . . . . . . . . . . . . . . . . . . ..Pecos, Reeves Coun G. T. MoNEss, Nacogdoches Sub-Station. “Nacogdoches, Nacogdoches Cou " Note.—The main station is located on the grounds of the Agricultural an Mechanical College, in Brazos County. The postoffice address is Colll Station, Texas. Reports and bulletins are sent upon application to Director; A postal card will bring these publications. ’ [HE FORMS OF PHOSIPHORUS 1N ‘ -COTTON SEED MEAL BY J. B. RATHER, ASSISTANT Cl-lEhilSTfé ottonseed meal is used extensively as a feed and as a fertilizer. is rich in phosphorus and that element is‘ indispensable to animal d plant life. Phosphorus exists in animal and plant tissues in both organic and rganic forms. According to Forbesl “The principal need for phos- ;orus in the body is for inorganic phosphorus,” but “organic phos- orus can supply all the bodily needs for phosphorus, both or- ic and inorganic,” and “inorganic phosphorus can supply the eds of the body for inorganic phosphorus only.” If the phospho- 9; in cottonseed meal is largely inorganic, as is claimed, then it il ». ible that it is deficient in organic phosphorus, from the stand- ont of the student of animal nutrition. Nearly all the phosphorus of cottonseed meal is found to be “avail- le” when tested by the Official Method2 for available phosphoric Qid in fertilizers, This method does not distinguish between solu- -- organic and inorganic phosphorus, and the value of the former uld quite possibly be less to plants than the latter. Again, if the phosphorus were largely inorganic, the well known xic effects of certain inorganic salts of phosphoric acid would ren- ‘r a knowledge of these constituents of the meal of much value. Review 0f the Literature. The amount of work done on the nature 1 the phosphorus compounds in cottonseed meal is not extensive. Gebeck2 found .058 per cent phosphorus in the ether extract of ttonseed meal. * Stelwaag‘ found that 4.35 per cent lecithin was in the ether extract a cottonseed meal. i In 1892 Hardin“ published the results of a study of cottonseed meal. e concluded that “all the reactions which have been described seem 3 show, beyond any reasonable ‘doubt, the presence of both meta and ‘f; ophosphoric acid in the aqueous solutions of the meals examined." I The more important criteria on which Hardin based his conclu- Ons are as follows: ~ i The phosphorus passed through a semipermeable membrane. “In all the cases of direct precipitation (of the water extracts), f the filtrates were allowed to stand at 60° (1., additional precipita- fon occurred, the quantity increasing with the time of standing.” l A white precipitate soluble in nitric acid formed whenthe aqueous lutions of cottonseed meal were treated with silver nitrate solution. 5 The aqueous extracts coagulated albumen when acidified with acetic id. ' Magnesia mixture gave a precipitate largely soluble in an excess if ammonium chloride and wholly soluble in an excess of magnesium loride. it *Under the general direction of G. S. Fraps, Chemist. 4 A TEXAS AGRICULTURAL EXPERIMENT STATIONS. A sample, prepared by difiusion, precipitation with lead and decomposition of the lead salt with hydrogen sulphid strongly acid, coagulated albumen, and gave white precipitat silver nitrate soluble in an excess of the acid. After nearly =1 izing with sodium hydroxide, silver nitrate gave ainearly pure‘ precipitate, leuto-cobaltic chloride a pale reddish yellow prec' soluble in an excess of the solution. Magnesium sulphate fl no precipitate. Calcium salts produced a precipitate soluble excess of the solution. Ammonium molybdate gave a yellow 1 tate only after standing for some hours at 60° C‘. ' The product obtained by precipitation by boiling with q mixture and an excess of magnesium sulphate and ammonium; ride, gave a white precipitate with silver nitrate, did not coa albumen, and did not precipitate with ammonium molybdate] after heating and standing. ' a “The facts that the original solution of the acids of the meal p ulated albumen, and that phosphoric acid in considerable =5 remained in the solution after the removal of the ortho and pyrof phoric acid, and that this phosphoric acid was "not readily pr tated by molybdate solution, show that the aqueous solution of V; seed meal contains also metaphosphoric acid.” - The possibility that the cottonseed meal contained organic with phosphorus in the molecule does not seem to have been c0 ered by Hardin. More recently Crawford“ reported a study of cottonseed meal L, the standpoint of its toxicity and concluded that “the chief f, ous principle of cottonseed meal is a salt of pyrophosphoric ac The principal chemical experiments on which this conclusion based were as follows: A He treated the digestion products of cottonseed meal with lead J tate and decomposed the lead precipitate with hydrogen sulphide. filtrate from the lead sulphide Was markedly acid, soluble in alco but could be precipitated from alcohol solution by ether. On y removal of the alcohol a colored gum was obtained. This gum treated with absolute alcohol to remove inorganic salts, the fil ‘k evaporated and taken up with water. Lead acetate was added and ‘g lead precipitate decomposed with hydrogen sulphide. The filtr" from the lead sulphide was colorless and very acid in reaction. f? gave no protein reaction and did not reduce Fehling’s solution. After neutralization the solution gave a white precipitate 4 silver nitrate, which was insoluble in an excess of sodium phate but soluble in the free ammonia and nitric acid. Bari chloride gave a precipitate, ammonium molybdate gave a precipit ‘ on heating, and magnesia mixture gave at once a white precipita The acetates of lead, mercury, iron and codmum gave precipitates. Copper sulphate gave a bluish white precipitate in the neutral lution of the acid, soluble in an excess of the mother fluid. Z’ sulphate gave white precipitate, likewise soluble in an excess of solution. Egg albumen was precipitated by the acid solution, b after precipitating with magnesia mixture and dissolving the precii \ itate in acetic acid egg albumen gave only a cloud. ' i’ L. f , ' THE FORMS OF PHOSPHORUS IN COTTON SEED MEAL. 5 Copper acetate gave a bluish white precipitate, ferric chloride a white precipitate and uranyl acetate a yellow-white precipitate. Suzuki and others claim that the principal acid-soluble phosphorus aompound in rice bran,’ Wheat branf corn, oats and barley,” is a salt of an acid of the formula C2H809, which was first described by Pos- ;ernak and named phytic acid by him, Quantitative studies of the inorganic phosphorus present in cone. feeding stuifs have been made in this country by Hart and Andrews“ and by Forbes and associates". So far as we have been able to find, no determinations of the inorganic phosphorus in cottonseed meal have been published except one reported by Forbes and associates“. They got .044 per cent phosphorus by the Hart and Andrews method and .118 per cent by one of their own which they subsequently de- clared unreliable. - PRELIMINARY STUDIES. Effect 0f Washing With Ether Before Emtraction. In order to test if the fat in the meal would interfere with the extraction of the phos- phorus the following experiment was made: Three grams of meal were extracted six times with ether on a hardenedfilter, transferred to a 300 cc, flask and digested with 300 cc. 0.2 per cent hydrochloric acid for three hours with frequent shaking. The extract was decanted through a filter, 100 cc. pipetted off, evaporated, and ignited with magnesium nitrate solution and the phosphorus determined by the oificial volumetric method ( A). The phosphorus was determined in three grams of meal as above except the ether washing was omitted (B). TABLE 1—-EFFECT OF WASHING WITH ETHER ON PHOSlPHORUS EXTRACTED FROM COTTON SEED MEAL WITH 0.2 PER. CENT HYDROCHLORIC ACID. Per Cent Phosphorus Soluble in 0.2 Per Cent Hydro- chloric Acid. (A) | (B) Laboratory Washed Six i Number. Times With Without - Ether. Washing. 0196--- _ _ _ . . _ _ _ . . _ . _ _ _ . _ . _ _ _ _ __ .234 .258 9251-- .............. -_ .166 . .205 92B- _____________________ -- .218 .227 p430 __ .179 .218 9468 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . __ _ .262 .288 0523 .221 .218 Average - _ .11: j .220 The average amount of phosphorus extracted when the meal was washed with ether (A) was .213 per cent; when no ether was used (B) it was .235 per cent. The results are slightly lower with the ether washing than without. The removal of the fat does not increase the amount dissolved under the conditions of the experiment. The fat did not appreciably inter- fere with the extraction of the phosphorus. The lower results with the ether washed meal are possibly due to the lecithin removed by the treatment. ' - 6 TEXAS AGRICULTURAL EXPERIMENT STATIONS. ‘nitric acid and filtered. Seventy-five cubic centimeters of th Solubility in water, acids, and ammonia. The amount of p, rus soluble in 0.2 per cent hydrochloric acid is much lower th, lished results for water soluble phosphorus in cottonseed me following experiment was made to ascertain if the phospho ’ extracted by acid was soluble in water or ammonia: . Three grams of sample No, 9523 were extracted with 300. 0.2 per cent hydrochloric acid as already "described. The resid j the extraction was washed with 0.2 per cent hydrochloric ac: then digested for three hours with 300 cc. of water. One h cubic centimeters of the filtered extract were evaporated, i with magnesium nitrate and the phosphorus precipitated as p official volumetric method. Only a trace of the ammonium ti, molybdate appeared and the determination was not completed.‘ phosphorus remaining from the extraction with acid Was not in water. i a The residue from the extraction with water was extracted ' same manner with 0.2 per cent ammonia and the phosphorus mined in 100 cc. of the filtrate as described above. The amo phosphorus found was .638 per cent. The amount remaining the acid extraction was calculated to be .716 per cent. Two- per cent ammonia extracted nearly all of the phosphorus rem in the residue from the acid extraction. = A qualitative test on a portion of _the filtered ammonia ~ (above) showed that no precipitate formed when the solutio acidified with varying amounts of hydrochloric acid. The phos not removed from the meal by the acid extraction was not p i tated from the ammonia extract on acidifying with hydrochloric Precipitation of Meta and Pyro-Phosphoric Acids. In y.‘ ‘determine how meta and pyro-phosphoric acids would act und, conditions of the determination of inorganic phosphorus b} method of Forbes and associates the following experiment was --f About 0.5 gram of meta-phosphoric acid and 1 gram of y, Reagent) sodium pyrophosphate were dissolved in 200 cc. wa t. a. graduated flask. Ten cubic centimeters of each solution were w? in beakers with 150 cc. water and precipitated with 20 cc. am, and 15 cc. magnesia mixture, adding the magnesia mixture dr‘ drop and allowing to stand over night. The resulting precipi which were scarcely visible in all cases, were washed on a filter; 2.5 per cent ammonia, and with alcohol, the filter and preci‘ digested with 100 cc. of 95 per cent alcohol containing 0.2 per; P‘. . trate were evaporated nearly to dryness, taken up with water“ the phosphorus determined by the official volumetric method; a quots of the original solutions were digested with hot dilute r acid for about 16 hours and the phosphorus determined by the. cial volumetric method. The results are shown below." l Milligrams Phosphorus Percentage i Recovered. Phosphorus Recover Added 1 2 " Meta-Phosphoric Acid 8.25 0.40 0.36 4.6 ' Pyro-Phosphoric Acid 7.10 0.03 0.00 0.2 THE Foams on PHosPHoRUs IN CoTToN SEED MEAL. 7 . The amount of phosphorus in solution was about the same as would be in 150 cc. of the 0.2 per cent hydrochloric acid extract of cotton- seed meal. Four and six-tenths per cent of the meta-phosphoric acid was recovered and 0.2 of the pyro-phosphoric acid. These results may be due to ortho-phosphoric acid present in the salts as an impurity. - The amount of phosphorus recovered shows that the method of Forbes and associates will not include any appreciable amount of meta and pyro-phosphoricacid in the determination of inorganic phosphorus, if these acids are present in the plant tissue examined. QUANTITATIVE STUDY OF THE PHOSPORUS SOLUBLE IN WATER. Six samples of cottonseed meal, average quality, probably of upland origin, and collected from different parts of the State were used in this investigation. For the determination of inorganic phosphorus in feeds. the method of Forbes and associates” was used. However, there are some objections to this method. It depends upon the as- sumption that the organic phosphorus precipitated by magnesia mix- ture is insoluble in 95 per cent alcohol containing 0.2 per cent nitric acid. Although it was found that magnesium phytate was insolu- ble under the above conditions, it has not yet been shown that phytin is the only acid soluble phosphorus compound in plants. Other or- ganic phosphorus compounds may be present which are precipitated by magnesia mixture but behave differently towards the acid alcohol. Meta and pyro-phosphoric acid, if present, would be estimated as or- ganic phosphorus, by this method. This should be borne in mind in considering the analyses by the Forbes method. . The amount and nature of the water soluble phosphorus were de- termined in the following manner: Determination. 0f Total Phosphorus. (A) One gram of cottonseed meal was ignited with 5 cc. magnesium nitrate solution, and the phosphorus determined by the official volumetric method, Phosphorus Soluble in Water. (B) Three grams of cottonseed meal were digested in 300 cc. acid for three hours with frequent shak- ing, the extract decanted through a filter and the phosphorus deter- mined in 100 cc. by evaporating, igniting with magnesium nitrate and completing as in the official volumetric method. Inorganic Phosphorus Soluble in Water. (C) The solution was prepared as described above. One hundred and fifty cubic centimeters were precipitated in a beaker with 20 cc. strong ammonia and 15 cc. magnesia mixture. The precipitate was allowed to stand over night, filtered and washed several times with 25 per cent ammonia and with 95 per cent alcohol until free from ammonia. _ After allowing to dry the precipitate and filter was transferred to an erlenmeyer flask containing 100 cc. of 95% alcohol containing 0.2 per cent nitric acid. The flask was closed with a rubber stopper, shaken vigorously and allowed to stand over night. The mixture was then filtered, 75 cc. of the filtrate evaporated almost to dryness, dis- solved in ‘dilute nitric acid and the phosphorus determined by the official volumetric method. The results are shown in Table 2. . after Wh'en the word inorganic phosphorus is used the above limi 8 TEXAS AGRICULTURAL EXPERIMENT STATIONS. TABLE z-rroTAL, WATER-SOLUBLE, AND INORGANIC PHOSPHORUS s01. vi IN WATER. i; (A) (B) Total Phos- Phosphorus Pho H? Laboratory phorus. Soluble in Number. Water. (a) Wa .1 919s _ .886 .698 i 9251 ____ __ .786 .458 P298 ‘ __ .947 .567 9430 .978 .589 9468 ____ __ 1.070 .698 9523 _ .934 .568 Average ___________________________ -_ .933 .563 Average of 30 Texas Meals (b)..-__- 1.148 t. Under conditions of experiment. b. Bull. 112 Texas Station. ~ v The total phosphorus (A) varied from .786 per cent to 1.070_ cent and averaged .933 per cent. The average of 30 analyses of '1‘ meals made at this Experiment Station is 1.148. These samples j therefore low in phosphorus. The phosphorus slouble in water (B) varied from .458 per cent; .698 per cent and averages about 6O per cent of the total phospho é Hardin“ has shown that the amount of phosphorus removed Water depends on the conditions of the experiment and that by? peated washing with water most of the phosphorus goes into so“: tion. The inorganic phosphorus (C) varies from .023 per cent .T_I .042 per cent and averages .039 per cent, This does not include me Q phosphoric acid and pyro-phosphoric acid if they are present. He tions are implied. These analyses show that there is very li i ortho-phosphoric acid in the Water extracts of the meals examined: PHOSPHORUS SOLUBLE IN ACID AND AMMONIA. Based on the preliminary work described on a preceding page ‘_ following method of work was used: f} Acid Soluble Phosphorus. Three grams of cottonseed meal were ~25‘ * gested with 0.2 per cent hydrochloric acid as described in the of the Water extract. The total (A) and inorganic (ortho) ~5Y phorus (B) were determined as described for the water extract. i“ acid-alcohol insoluble matter obtained in the determination of the -.. organic phosphorus was washed with acid alcohol, ignited with =. _ nesium nitrate, taken up with 1O cc. of nitric acid and 2 cc. of hyd 1f chloric acid and the phosphorus determined by the official volumet - method (C). . Ammonia Soluble Phosphorus. The cottonseed meal residue fr‘ (- the extraction with 0.2 per cent hydrochloric acid was washed 1 i water, put in a flask and digested for three hours with 0.2 per‘|}:_ ammonia with frequent shaking. The extract was filtered and t" total phosphorus "determined as already described for the water an acid extracts (D). One hundred and fifty cubic centimeters of 1.. extract were treated with 2O cc. strong ammonia and 15 cc. magnesi mixture. The precipitate was allowed to settle over night, filter and washed with 4 per cent ammoniaj The filter and precipitate we l_ ~ p b F. t I THE FoEMs OF PHOSPHORUS IN COTTON SEED MEAL. 9 ignited with magnesium nitrate and the determination completed as i‘ lready described for total phosphorus (E). g The results of the work are shown in Table 3. TABLE 8—PHOSPHORUS EXTRAOTED FROM COTTON SEED MEAL BY ACI 1' AND BY AMMONIA. l 2. Phos l phorus In-. 1. Acid-Soluble Phosphorus. soluble in 4 2. Ammonia Soluble Acid (by ‘ Phosphorus. dit.). 1 (A) (B) ' (C) i (D) l (E) Total Organic i Magnesia. Laboratory Phos- Total. Inorganic Magnesia ‘ Total. | Precipi~ F Number. phorus. (a). . Ppt. i tate. g1’ 0190 ______ __ sss .258 .039 .144 .628 l .068 l __________ -- 0251 _______ __ .780 .205 .025 .144 .581 l .000 i .......... -- i 029s ....... -_ .047 .227 .043 .144 .720 1 .729 l _________ .._ % o4s0.---_---- .078 .218 .039 .144 .700 l .740 .437 9468 ....... -_ 1.070 .288 .056 .205 .782 1 .860 .725 062s ...... -- .034 .210 .051 .131 .714 y .653 4 .603 avenge“ .934 .234 .042 .152 y .608 l .708 .......... -- t (a) According to method of Forbes et a1. 0' The phosphorus soluble in 0.2 per cent hydrochloric acid (A) Lvaried from .205 per cent to .288 per cent, and averaged about 25 iipbl‘ cent of the total phosphorus- p The total water soluble phosphorus (Table 2) is more than twice as ;great as the total acid soluble phosphorus, averaging .563 per cent and {.235 per cent respectively. The Inorganic Phosphorus. The inorganic phosphorus soluble in 0.2 per cent hydrochloric acid (B) varied from .025 per cent to .056 per cent and averaged about 4 per cent of the total phosphorus. Since meta-phosphoric acid and pyro-phosphoric acid are not precipi- tated by magnesia mixture under the conditions of the experiment they would not be included in these figures and the results would be too low if they were present. We will show on another page that we have no proof of their presence in cottonseed meal. The sum of the inorganic (B) and the organic phosphorous precipitated by magnesia mixture (C) subtracted from the total acid soluble (A) phosphorus represents nucleins, meta-phosphoric acid and pyro-phos- phoric acid (if present), and unknown compounds. This averages .040 per cent. Assuming that this consisted entirely of meta and pyro-phosphoric acid the total inorganic phosphorus would then be .082 per cent, less than ten per cent of the total phosphorus. These results are not in accord with the theory that the phosphorous of cottonseed meal is principally inorganic. A comparison of Table 3 and Table 2 brings out the fact that there. is a.s much inorganic phosphorus soluble in water as there is in 0.2 per cent hydrochloric acid. The use of hydrochloric acid in the de- termination of inorganic phosphorus in cottonseed meal is therefore unnecessary. i Magnesia Precipitate Insoluble in Acid Alcohol. The phosphorus in the magnesia precipitate (C) insoluble in acid-alcohol varies from .131 per cent to .205 per cent and comprises the larger portion of the acid soluble phosphorus. a A 9523 the determinations are therefore probably too low. Nev . the precipitations Were freed from lead, evaporatedt ignited " 10 TEXAS AGRICULTURAL EXPERIMENT STATIONS. Ammonia Soluble Phosphorus. The phosphorus removed H ammonia averaged .708 per cent While the phosphorus remainingi the hydrochloric acid extraction Was .698 per cent (calculated i? ference). Ammonia extracted all of the phosphorus remaining _ the acid extraction. The differences in the table are probably z p ical errors. The precipitates of ammonia-soluble phosphorus by nesia mixture (E) Were very ‘difficult to filter. They ran t ‘ the filters repeatedly and finally clogged up the filters s’ tration Was impossible. With sample 9523 a clear filtrate w_ tained. The other filtrates Were opalescent and apparently wer free from the magnesia precipitate. With the exception of s ‘ less, they indicate that most of the ammonia soluble phospho 3 precipitated by magnesia mixture. This precipitate is probabl; v organic. i ‘ PRECIPITATION BY COPPER AND LEAD. The amount of phosphorus in cottonseed meal precipitate lead subacetate and by copper acetate from Water solution Was tained in the following manner: Three grams of meal Were dig for three hours with 300 cc. of Water and 200 cc. of the extract 5f cipitated With lead subacetate solution 100 cc. of the filtrates 1 magnesium nitrate solution and the phosphorus determined by 1 official volumetric method. The phosphorus not precipitated by leaid averaged .028 per c (average of 6 analyses). Q Sample 9523 Was treated as above, but copper acetate was used L stead of lead subacetate. The amount of phosphorus remaining in solution was .022 per ce These reagents precipitated almost all of the phosphorus from wa‘ solution. SEPARATION AND PURIFICATION OF PHOSPHORUS I COMPOUNDS. The meal Was extracted in a manner similar to that described in =3 analytical Work. The method of purification of the acids was simil to that used by Hart and Tottingham”, except that We used al hol at one stage of the Work to precipitate an alcohol insoluble co pound. The method Was as follows: TWo kilograms of cottonseed meal Were digested with 8000 cc. of 0. per cent hydrochloric acid for 3 hours with frequent shaking. T extract was strained through cheese cloth and the residue Washed Well With Water. The residue Was digested with 8000 cc. 0.2 pe cent ammonia for 3 hours With frequent shaking and allowed to set. tle, and Washed With water by ‘decantation. - Copper acetate was addedi to the acid extract of the cottonseed meal in sufficient quantity precipitate most of the phosphorus compounds, the precipitate was; Washed well with Water, decomposed with hydrogen sulphide, filtered,}, and evaporated to a syrupy consistency. The brownish syrup was? dissolved in a small amount of Water and filtered from an insolublei THE Foams OF PHosPHoRUs IN COTTON SEED MEAL. 11 ipbrownish residue. Ninety-five per cent alcohol was then added to the gwater solution in sufficient amount to make the solution about 80 per zicent alcohol. A white fiocculent precipitate formed and quickly set- ijtled. It Was filtered and washed with 95 per cent alcohol, dissolved {in weak, hot ammonia, the ammonia removed by boiling and alcohol again added. The precipitate was again washed, dissolved in weak ‘gammonia, made neutral with acetic acid and precipitated with copper iacetate. The copper acetate precipitate was washed well with water, iidecomposed with hydrogen sulphide and filtered. The filtrate was igconcentrated and made alkaline with sodium hydroxide and barum tchloride added in excess. The barium precipitate was washed free ifrom alkali with water, suspended in water and the barium precipi- isvtated with sulphuric acid. The filtrate from the barium sulphate fwas precipitated with copper acetate, washed and decomposed with §1hydrogen sulphide. The filtrate from the hydrogen sulphide precipi- itate was exaporated to a syrupy consistency. (Product A.) A The filtrate from the first precipitation with alcohol in the prepa- ération of Product A was evaporated on a water-bath to remove the igalcohol, taken up with water and made alkaline with sodium hydrox- ide. Barium‘ chloride was added and the resulting precipitate was iwashed with water. The barium salts were decomposed with sulphuric acid and filtered. The filtrate was again made alkaline and precip- iitated with barium chloride, This process was repeated two or three times and the acid finally precipitated with copper acetate in acid olution, the copper salt decomposed with hydrogen sulphide, filtered, and evaporated to a syrupy consistency. (Product B.) ‘ ' The ammonia extract was made acid with hydrochloric acid, the precipitate allowed to settle and the liquid decanted through a filter. v his extract was precipitated with copper acetate, alcohol and barium p hloride exactly as described for the acid extract. (Produce C.) PROPERTIES OF THE PRODUCTS. - Product A (alcohol insoluble) was a colorless gummy substance with a strong acid reaction. It darkened on heating at 100° C. i, It was precipitated by copper acetate, lead subacetate, barium hloride and silver nitrate. The silver salt had a pinkish tinge. The cid was precipitated from water solution by alcohol and the result- ng precipitate was dilficultly soluble in water and weak hydrochloric acid, and-soluble in hot dilute ammonia and sodium hy'droxide_ The product was separated from a sample which had been purified “y precipitation with barium chloride and copper acetate. Since this pulletin went to press, further work has made it appear probable that = roduct A is the lime or magnesia salt of Product B. Y An alcohol insoluble substance similar to Product A was found 5| the ammonia extract, but smaller in amount than that found in Je acid extract. " Properties of Products B and C’. Product B (acid soluble) and roduct C (ammonia soluble) had similar properties and will be de- . ribed together. They were thick, almost’ colorless, gummy sub- i the precipitate in acetic acid the solution did not coagulate al‘ 12 TEXAS AGRICULTURAL: EXPERIMENT STATIONS. stances, which ‘darkened in the air and 0n heating at 100° were strongly acid. Million and Biuret tests gave no reacti_ protein. Product B fused with sodium gave no reaction for ni‘ Upon precipitating Product B with magnesia mixture and dis‘ until allowed to stand over night and then only slightly. Portions of the acid and ammonia soluble products were =- ized with sodium hydroxide and placed in diffusion shells. "a twenty-four hours the water on the outside of the shells gave precipitates with ammonium molybdate when heated above 6y Portions of the "difiused substance gave white precipitates; silver nitrate and coagulated albumen. In Table 4 are collected for comparison the above described»; tions together with additional ones, and those of the products ob by Crawford and by Hardin. . 1 13 THE Foams OF PHOSPHORUS IN COTTON SEED MEAL. .0 ,1... .-. 68858.0 6332x522 QWEB . 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A possible explanation of the fact that the magnesia preci did not coagulate albumen when acidified with acetic acid 5 acetic acid may not be strong enough acid to liberate the oth from its salt. x The cobaltamine used in the test given in, the table was pr by us in the following Way: A few grams of cobalt chlorid‘ dissolved in about 500 cc. water, a little hydrochloric acid adde solution made alkaline with ammonia and allowed to stand in (a low dish for several days, ammonia being added occasionally. precipitate which formed was washed with weak ammonia _ using. In the test with ammonia molybdate the precipitate began to f0 about fifteen minutes and continued to precipitate for ab0utf_ hours. At the end of that time the precipitation was not comp shown by heating the filtrate to a higher temperature (85-90°C) an additional yellow precipitate formed. r Since Products B and C are both precipitated by magnesia i ture, and since most of the phosphorus in theacid and ammoni tracts was precipitated by magnesia mixture, it appears probable; these products are the principal phosphorus compounds in cotto meal. A ANALYSIS OF THE SILVER SALTS OF THE PRODUC The acids were diluted with water and neutralized with amm boiled until free from ammonia and precipitated with silver ni solution. The silver salts were washed with water, dried in the it powdered and dried for several days in a vacuum desiccator f, sulphuric acid. i, Product A was pinkish white, and Products B and C were w, Analysis of these salts gave the following results: Carbon. Hydrogen. Silver. Phospho Product A (alcohol insoluble) 4.80 , 1.23 57.98 10.16 Product B (acid extraction) 5.42 0.86 61.40 9.82 Product_ C (ammonia extrac- Vi, tion) . . . . . . . . . . . . . . . . . . .. 5.09 1.14 60.03 10.15 The empirical formulae corresponding to the above‘ analyses i’ Product A: C_,H12Ag_.,P,_O15; Product B: C6HmAg,P,O,,; i Product C: C_,H10Ag5P3O13. ~ It appears that, since the compounds described on the precel pages have properties very similar to meta and pyro-phospho acids, conclusions that the latter are present in cottonseed meal ' no value when based on these reactions. Therefore we have no pr that cottonseed meal contains either pyro or meta phosphoric acida These results are not in accord with the conclusions of other inv ’ tigators (5 and 6), based on qualitative reactions which are descri in detail on preceding pages, that cottonseed meal contains pyro .1} meta-phosphoric acids in considerable amounts. However valuab the qualitative tests for phosphoric acids may be in a mixture q inorganic compounds, conclusions based on them in "an unkno i _ organic mixture should be drawn with caution. .; THE FoRMs or PHOSPHORUS IN CoTToN SEED MEAL. 15 From the Work described above it appears that the acid and 140011121 soluble products have very similar properties difiering only their solubility in hydrochloric acid and the percentage of silver i the silver salts. , We are notprepared to state at this time that the above products 5e pure chemical compounds, but it seems certain that they are ee from all but traces of inorganic phosphates since the copper ’ts of these acids are soluble in water to some extent, in hydro- loric and sulphuric acids and the preparation. of the products (eludes precipitation in dilute solutions of both of these acids. The all amount present Would probably be removed by the treatment ven, since over nine-tenths of the preparations were lost in purifi- tion. They also appear to be free from lime and magnesia since a t of the product obtained from the first precipitation with copper, er Washing, decomposition with hydrogen sulphide and concen-l tion of the filtrate from the copper sulphide, showed only traces these metals and they would no doubt be removed by the second Q id precipitation. The study of the Products A, B, and C, is being continued. . I LITERATURE CITED. .1. Forbes, Ohio Agricultural Experiment Station, Bulletin 201, O e 149. ;2. U. S. Department of Agriculture, Bureau of Chemistry, Bulle- | 107 (revised). :3. Gebeck, ‘Landwirtschlaftlichen Versuchs-Stationen 42, pages 278. _ "4. Stellwaag, ibid, 37, page 148. 5. Hardin, South Carolina Agricultural Experiment Station, Bul- " in 8 (new series). .6. Crawford, Journal of Pharmacology and Experimental Thera- utics 1, page 51.’ 7. Suzui, Tokio Imperial University, Bulletin of College of Agri- . ture 7, page 495. 8. Hart and Andrews, American Chemical Journal 30, page 470. 9. Hart and Tottingham, Wisconsin "Agricultural Experiment tion, Research Buletin 9. :10. Posternak, Rev. Gen. Bot. 1900, 12, 5 and 65. 711. Hart and Andrews, New York State Agricultural Experiment tion, Bulletin 238. v12. Forbes and Associates, Ohio Agricultural Experiment Station, nlletin 215. SUMMARY AND CONCLUSIONS. » . The fat in cottonseed meal did not interfere with the extraction the phosphorus with 0.2 per cent hydrochloric acid. . The phosphorus soluble in Water was more thanltwice as much ;that soluble in 0.2 per cent hydrochloric acid. i The same amounts of inorganic phosphorus (Forbes Method) a e found in the acid and in the aqueous extracts. i. The phosphorus soluble in 0.2 per cent hydrochloric acid was t 25 per cent of the total phosphorus. 1_6 _ TEXAS AGRICULTURAL EXPERIMENT STATIONS. 5. The inorganic phosphorus (Forbes’ Method), in the s1’ cottonseed meal examined was less than 5 per cent of_ the' W phorus. i 6. The phosphorus in the hydrochloric acid extract of w‘ meal capable of being precipitated by magnesia mixture ‘ soluble in acid-alcohol was about 16 per cent of the total ph 7. After extraction with 0.2 per cent hydrochloric acid ~' phorus remaining was insoluble in water but nearly completely’ in 0.2 per cent ammonia. _ 8. Magnesia mixture precipitated most of the phosphorus -»_' by ammonia. , 9. Practically all of the phosphorus was precipitated I aqueous extract of cottonseed meal by lead subacetate and by} acetate. ' . A _ 10. The principal compounds containing phosphorus w" arated, which give the same reactions as those relied upon for (‘i the presence of meta and pyro-phosphoric acid. f 11. We have no evidence that the samples of cottons examined contain either pyro-phosphoric acid or meta-phospho