COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCI s SIANDARD HX641401^n QP535.P1 F742 eestim RECAP m ^- -fcs QP£Jt£jL ZZfi^ intl)fCitpofilruigork College of ^tpsiiciansJ anb burgeons Hibrarp Digitized by tine Internet Arciiive in 2010 with funding from Open Knowledge Commons (for the Medical Heritage Library project) http://www.archive.org/details/studiesonestimatOOforb . Beetle , D. Wassow , •oufMBiA uNivrpsmr 0€fA«TMENT OF PHYSIOLOG WtMT fFTV^iKTM ^ OHIO AGRICULTURAL EXPERIMENT STATION J^ TECHNICAL SERIES, BULLETIN No. 8 STUDIES ON THE ESTIMATION OF INORGANIC PHOSPHORUS IN PLANT AND ANIMAL SUBSTANCES By E. B. FORBES, F. M. BEEGLE AND A. F. D. WUSSOW WOOSTER, OHIO, U. S. A., JUNE, 1915 OHIO AGRICULTURAL EXPERIMENT STATION GOVERNING BOARD THE AGRICULTURAL COMMISSION OF OHIO Columbus A. P. Sandles, President S. E. Strode C. G. Williams H. C. Price B. P. Gayman, Secretary STATION STAFF Charles E. Thorne, M. S. A., Director DEPARTMENTAL ORGANIZATION ADMINISTRATION The Director, Chief William H. Kramer, Bursar W. K. Greenbank, Librarian Horatio Markley, In charge of Exhibits W. J. Holmes, Printer Dora Ellis, Mailing Clerk Glenn Hall, Engineer AGRONOMY C. G. Williams, Chiefs P. A. Welton; B. S., Associate William Holmes, Farm Manager C A. Patton, Assistant C. A. Gearhart, B. S., Assistant E. C. MORR, Office Assistant C. H. Lebold, Asst. Foreman ANIMAL HUSBANDRY B. E. Carjiichael, M. S., Chief J. W. Hammond, M. S., Associate Geo. R. Eastwood, B. S., Assistant Don C. Mote, M. S., Assistant W. J. Buss, Assistant Anthony Russ, Herdsman E. C. SCHWAN, Shepherd (Carpenter) BOTANY A. D. Selby, B. S., Chief True Housek, B. S., Asst. (Germantown) P. K. Mathis, Office Assistant D. C Babcock, A. B., Assistant Richard Walton, B. S.. Assistant J. G. Humbert, B. S., Assistant CHEMISTRY J. W. Ames, M. S., Chief Geo. E. Boltz, B. S., Assista7it J. A. Stenius, B. S., Assistant C. J. Schollenberger, Assistant Mabel K. Corbould, Assistant T. E. Richmond, M. S., Assistant CLIMATOLOGY J. Warren Smith, Chief (Columbus)^ C. A. Patton, Observer DAIRYING C. C. Kayden, M. S., Chief A. E. Perkins, M. S., Assistant T. R. Middaugh, Office Assistant ENTOMOLOGY H. a. Gossard, M. S., Chief J. S. HousER, M. S. A., Associate W. H. Goodwin, M. S., Assistant R. D. Whitmarsh, M. S., Assistant J. L. King, B. S., Assistant FORESTRY Edmund Secrest, B. S., Chief J. J. Crumley, Ph. D., Assistant A. E. Taylor, B. S., Assistant J. W. Calland, B. S., Assistant D. E. Snyder, Office Assistant *With leave of absence. ^In cooperation with HORTICULTURE W. J. Green, Vice Director, Chief P. H. Ballou, Assistant (iJewark) Paul Thayer, M. S., Assistant C. W. Ellenwood, Office Assistant Ora Flack, Foreman of Orchards W. E. Bontrager, Foremast of Grounds C. G. Laper, Foreman of Greenhouses J. B. Keil, Orchard Assistant S. N. Green, Garden Assistant NUTRITION E. B. Forbes, Ph. D., Chief P. M. Beegle, B. S., Assistant Charles M. Fritz, M. S., Assistant L. E. Morgan, M. S., Assistant S. N. Rhue, B. S., Assistant SOILS The Director, Chief C. G. Williams, Associate in soil fertility investigations J. W. Ames, M. S., Associate in chemistry E. R. Allen, Ph. D., Associate in soil biology H. Foley Tuttle, M. S., Assistant A. Bonazzi, B. Agr., Assistant John Woodard, M. S., Assistant FARM MANAGEMENT C. W. Montgomery, Chief F. N. Meeker, Executive Assistant District Experiment Farms Northeastern Test-Farm, Strongsville. J. Paul Markley, Resident Manager Southwestern Test-Farm, Germantown Henry M. Wachter, Resident Manager Southeastern Test-Farm, Carpenter. H. D. Lewis, Resident Manager Northwestern Test-Farm, Findlay. John A. Sutton, Resident Manager County Experiment Farms Miami County Experiment Farm, Troy M. C. Thomas, Agent in Charge Paulding County Experiment Farm, Paulding C. Ellis Bundy, Agent in Charge Clermont Co. Experiment Farm. Owensville Victor Herron, Agent in Charge Hamilton Co. Experiment Farm, Mt. Healthy D. R. Van Atta, B. S., Agent in Charge Washington County Experiment Farms, Fleming and Marietta E. J. RiGGS, B. S., Agent in Charge Mahoning Co. Experiment Farm D. W. Galehouse, Agent in Charge Trumbull Co. Experiment Farm, Cortland j\r. O. BUGBY, B. S., Agent in Charge Weather Service, U. S. Department of Agriculture. s o BULLETIN OF THE Ohio Agricultural Experiment Station Number 8 Technical Semes June, 1915 STUDIES ON THE ESTIMATION OF INORGANIC PHOS- PHORUS IN PLANT AND ANIMAL SUBSTANCES BY E. B. FORBES, F. M. BEEGLE AND A. F. D. WUSSOW In connection with studies of the metabolism of plants and animals it is frequently desired to distinguish between simple inor- ganic phosphates and phosphorus in combination with organic groups. For the purpose of making such separate estimations there were published from this laboratory, by Forbes, Lehmann, Collison and Whittier, as Ohio Agr. Exp. Station Bui. 215, two different methods of inorganic phosphorus determination, one recommended for use with plant substances and the other for use with products of animal origin. Since issuing this publication, five years ago, we have come to realize that the problem was more difficult than we had known it to be, and that further evidence was required for judg- ment as to the correctness of our analytical procedures. In this spirit we have done further work on these methods, the result being the improvement and establishment of our method for animal sub- stances, and the demonstration of the imperfection of our method for vegetable products. In spite of the considerable measure of failure attending our efforts to determine inorganic phosphorus in vegetable substances, illumination of the problem was accomplished, and on this account we report the results of this study. That por- tion of this paper which deals with plant substances will be found on pages 3 to 23, and that portion having to do with animal pro- ducts on pages 23 to 40. (3) 4 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 THE ESTIMATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES The essential points of the method of Forbes and associates, as published, were (1) extraction of a 10-gram sample in 300 c. c. of 0.2 percent hydrochloric acid for three hours, (2) filtration, and precipitation of a 250 c. c. portion of the extract, with magnesia mixture and ammonia, (3) washing the precipitate with 2.5 per- cent ammonia and with 95 percent alcohol, the precipitate being then allowed to dry, (4) the separation of the inorganic phosphates from the remainder of the precipitate by breaking up the paper and precipitate in 100 c, c. of 0.2 percent nitric acid in 95 percent alco- hol, (5) filtration of the extract, pipetting out 75 c. c. of the filtrate, (6) evaporation of the alcohol, taking up with 0.2 percent aqueous nitric acid, and estimation of phosphorus in this solution by taking through the molybdate and magnesia mixture precipitates, and esti- mation as the pyrophosphate in the usual way. The result repre- sents 6.25 grams out of the original 10 grams of sample. The evidence upon which this method was based was submitted in the original publication. In the further study of this method obsei'vations were made as will be discussed. Our bases for judg- ment as to the correctness of the method were the agreement of triplicates, the completeness of recovery of added phosphates, and the completeness of extraction during the three-hour period speci- fied, as determined by subjection of the residue to further extraction. In the further study of this method especial attention was given to (1) the use of phenol in the extractive reagent, (2) the complete- ness of the 3-hour extraction, (3) improvement of the method of filtration, and other details of technique. In the original publication of the method the use of phenol in the extractive reagent was suggested in cases where the activity of enzymes seemed to aff'ect results. This was considered especially likely to be the case in estimations involving difficult and therefore protracted filtration, since it was found that the slower the filtra- tion the greater would be the amount of inorganic phosphorus found. In the working out of the details of the method as published, the duration of the extraction period received some consideration, and the 15-minute extraction of Hart and Andrews, from whose method the extractive reagent was adopted, was in our method lengthened to three hours; but our fixing upon this duration of extraction was largely arbitrary, and further evidence was deemed desirable to establish this point in a consistent manner. INORGANIC PHOSPHORUS ESTIMATION 5 Aqueous extracts of vegetable substances are often exceedingly difficult to filter. The method as published involved much difficult filtration, and with some vegetable substances was, on this account, practically unworkable. In the studies here reported these diffi- culties of filtration were removed. These points and others were considered in a series of analyti- cal determinations, as reported in the following tables, the work usually including, as a check, a test of the completeness of recovery of added inorganic phosphorus. The method, as used in this work, was as follows : ACID-ALCOHOL METHOD OF FORBES AND ASSOCIATES FOR THE DETERMINATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES Pour exactly 300 c. c. of 0.2 percent hydrochloric acid (4.6 c. c. concen- trated hydrochloric acid, sp. gr. 1.18-1.19, per liter) onto 10 grams of sample in a dry 400 c. c. Florence flask. Close with rubber stopper, and shake at inter- vals of 5 minutes for 3 hours. Filter the extract by suction into dry flasks through S. & S. No. 589 "Blue Ribbon" papers, in a Witt filtering appai-atus, or a Biichner funnel. Measure out a 250 c. c. portion of this filtered extract, and precipitate in a 400 c. c. beaker with 10 c. c. magnesia mixture and 20 c. c. ammonia, sp. gr. 0.9. Allow to stand over night, and filter through double S. & S. No. 589 "White Ribbon" papers, taking care to decant as long as possible without pouring out the precipitate. Then complete the transfer of the precipitate to the paper. Wash three times with 2.5 percent ammonia, and then three times with 95 percent alcohol. Allow the precipitate to drain, and then spread out the inner paper on the top of the funnel, and allow the alcohol to evaporate. When practically dry, place this inner paper with the precipitate into an Erlenmeyer flask. Add 100 c. c. of 95 percent alcohol containing 0.2 percent of nitric acid. Close the flask with a rubber stopper and shake vigorously until the paper is thoroughly broken up. If the precipitate is flaky, and refuses to break up on shaking, allow to stand in the acid-alcohol over night. Now filter through a dry filter into a dry flask. Pipette out 75 c. c. of the filtrate into a small beaker, and evaporate almost but not quite to dryness! Dissolve in dilute nitric acid, and filter if necessary; then determine phosphorus in the usual gravimetric way, by precipitation first with acid molybdate solution, later with magnesia mixture, and then burning to the pyrophosphate. The result obtained as above represents 6.25 grams out of .the original 10 grams of material, and so to reduce to a 1-gram basis multiply by 0.16. In the analyses reported in the following group of tables (pages 7 to 10) the above standard method was modified, in certain parts of the work, as indicated below ; 6 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 AQUEOUS HYDEOCHLORIC ACID EXTRACTION, PLUS PHOSPHATE Proceed as on p. 5 except that in place of 300 c. c. of 0.2 percent hydro- chloric acid add 250 c. c. of the same and 50 c. c. of phosphate solution contain- ing disodium phosphate equivalent to approximately 25 mg. magnesium pyro- phosphate per 50 c. c. Make up this phosphate solution with 0.2 percent hydrochloric acid. AQUEOUS HYDROCHLORIC ACID-PHENOL EXTRACTION Proceed as on p. 5 except that in place of 300 c. c. of 0.2 percent hydro- chloric acid add 300 c. c. of 0.2 percent hydrochloric acid solution containing 50 gm. phenol per liter, AQUEOUS HYDROCHLORIC ACID-PHENOL EXTRACTION, PLUS PHOSPHATE ■ Proceed as in the paragraph above except that in place 6f 300 <;. c. add 250 c. c. of 0.2 percent hydrochloric acid containing 50 gm. phenol per liter, and 50 c. c. of phosphate solution containing disodium phosphate equivalent to approximately 25 mg. magnesium pjTophosphate per 50 c. c. Make up this phosphate solution with 0.2 percent hydrochloric acid containing 50 gm. phenol per liter. PHOSPHORUS ESTIMATIONS ON REAGENTS AND PHOSPHATE SOLU- TIONS USED IN WORK REPORTED IN TABLE I, PAGE 7 Magnesium pj^rophosphate Blank 1 Aq.-HCl solutions 2 Aq.-HCl solutions 3 Aq.-HCl solutions Average Blank 1 Aq.-HCl-phenol solutions 2 Aq.-HCl-phenol solutions 3 Aq.-HCl-phenol solutions Average Phosphate solution (Aq.-HCl) 50 cc. 1 Phosphate solution (Aq.-HCl) 50 cc. 2 Phosphate solution (Aq.-ACl) 50 cc. 3 Average Phosphate solution (Aq.-HCl-phenol) 50 cc. 1. Phosphate solution (Aq.-HCl-phenol) 50 cc- 2. Phosphate solution (Aq.-HCl-phenol) 50 cc. 3. Average 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0002 0.0250 0.0248 0.0248 0.0249 0.0249 0.0249 0.0249 0.0249 INORGANIC PHOSPHORUS ESTIMATION 1 TABLE I: TEST OF THE ACID-ALCOHOL ^lETHOD OF FORBES AND ASSOCIATES FOR THE DETERMINATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES Sample Number ami troatnient Alfalfa Ai Aci.-HCl extraction A2 Aq.-HCl extraction Averajre Bi Aq.-HCl extr. plus phosphate B2 Aq.-HCl e.xtr. plus phosphate Bs Aq.-HCl extr. plus phosphate.. Averaire Ci Aq.-HCl-phenoIextraction C'j Aq.-HCl-phenolextraction C3 Aq.-HCl-phenolextraction Average Di Aq.-HCl-phenolextr. plus phosphate. D2 Aq.-HCl-phenol extr. plus phosphate. Ds Aq.-HCl-phenol e.xtr. plus phosphate. Averag-e Magne- sium pyro- phos- phate Grams 0.0189 0.0170 0.0180 0.0188 0.0222 0.0225 0.0212 0.0182 0.0168 0.0175 0.0175 0.0210 0.0203 0.0229 0.0213 Inor- ganic phos- phorus Percent 0.0803 0.0780 Phos- phorus added (magne- sium pyro- phos- phate) Grams 0.0156 0.0156 Added phos- phorus recovered (magne slum pyro- phos- phate) Grams 0.0032 0.0038 Added phos- phorus recovered Percent 20.5 24.3 Blue Grass Al Aq.-HCl extraction A2 Aq.-HCl extraction A3 Aq.-HCl extraction Average Bi Aq.-HCl extr. plus phosphate. B2 Aq.-HCl extr. plus phosphate B3 Aq.-HCl extr. plus phosphate Average Ci /r q.-HCl-phenol extraction C2 Aq.-HCl-phenol extraction C3 Aq.-HCl-phenol extraction. A verage Di Aq.-HCl-phenol e.xtr. plus phosphate. D2 Aq.-HCl-phenol extr. plus phosphate. D3 Aq.-HCl-phenol extr. plus phosphate. Average 0.0228 0.0225 0.0224 0.0226 0.0283 0.0230 0255 0.0256 0.0365 0.0355 0.0347 0.0356 0.0493 0.0458 0.0498 0.0483 0. 1008 0. 1588 0.0156 0.0156 Brewer's Grains Al Aq.-HCl extraction I A2 Aq.-HCl extraction A3 A q.-HCl extraction Average Bi Aq.-HCl extr. plus phosphate : . B2 Aq.-HCl extr. plus phosphate B3 Aq -HClextr. plus phosphate Average Ci Aq.-HCl-phenol extraction C2 Aq.-HCl-phenol extraction C3 Aq.-HCl-phenolextraction A verage Di Aq.-HCl-phenol e.xtr. plus phosphate. D2 Aq.-HCl-phenol e.xtr. plus phosphate. D3 Aq.-HCl-phenolextr. plus phosphate. Average 0.0025 0.0021 0.0023 0.0023 0.0160 0.0148 0.0161 0.0156 0.0012 0.0010 0.0013 0.0012 0.0162 0.0158 0.0160 0.0160 Rice Polish Al Aq.-HCl extraction A2 Aq.-HCl extraction A3 Aq.-HCl extraction A verage Bi Aq.-HCl extr. plus phosphate B2 Aq.-HCl extr. plus phosphate Bs Aq -HCl extr. plus phosphate Average Ci Aq.-HCl-phenolextraction C2 Aq.-HCl-phenol extraction C3 Aq.-HCl-phenol extraction Average Di Aq.-HCl-phenolextr. plus phosphate. U2 Aq.-HCl-phenolextr. plus phosphate. D3 Aq.-HCl-phenolextr. plus phosphate. A verage 0.0038 0.0038 - 0.0035 0.0037 0.0119 0.0110 0.0111 0.0113 0.0027 0.0017 0.0024 0.0023 0.0052 0.0093 0.0138 0.0094 0.0103 0.0053 0.0165 0.0102 0.0156 0.0156 0.0030 0.0127 19.2 81.4 0.0156 0.0156 0.0133 0.0148 0.0076 0.0071 85.2 94.9 48.7 45.5 8 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE II: TEST OF THE ACID-ALCOHOL METHOD OF FORBES AND ASSOCIATES FOR THE DETERMINATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES Sample Alfalfal Blue Grass^ Rice Polish 3 Number and treatment Magne- sium pyro- phos- phate Grams Ai Aq.-HCl extraction A2 Aq.-HCl extraction A3 Aq.-HCl extraction Average Bi Aq.-HCl extr. plus phosphate B3 Aq.-HCl extr. plus phosphate Average Cl Aq.-HCl-phenol extraction C2 Aq.-HCl-phenol extraction Cs Aq.-HCl-phenol extraction Average Di Aq.-HCl-phenol extr. plus phosphate D2 Aq.-HCl-phenol extr. plus phosphate Average Ai Aq.-HCl extraction A2 Aq.-HCl extraction As Aq.-HCl extraction Average Bi Aq.-HClextr. plus phosphate B2 Aq.-HCl extr. plus phosphate Bs Aq.-HCl extr. plus phosphate Average Cl Aq.-HCl-phenol extraction C2 Aq.-HCl-phenol extraction Ca Aq.-HCl-phenol e.xtraction Average Di Aq.-HCl-phenol extr. plus phosphate D2 Aq.-HCl-phenol extr. plus phosphate Ds Aq.-HCl-phenol extr. plus phosphate Average Ai Aq.-HCl extraction A2 Aq.-HCl extraction As Aq.-HCl extraction Average Bi Aq.-HCl extr. plus phosphate B2 Aq.-HCl extr. plus phosphate Bs Aq.-HCl e.xtr. plus phosphate Average Cl Aq.-HCl-phenol extraction C2 Aq.-HCl-phenol extraction Cs Aq.-HCl-phenol extraction Average Di Aq.-HCl-phenol extr. plus phosphate D2 Aq.-HCl-phenol extr. plus phosphate Average 0.0219 0.0184 0.0160 0.0188 0.0237 0.02.35 0.0236 0.0140 0.0189 0.0149 0.0159 0.0232 0.0241 0.0236 0.0380 0.0379 0.0335 0.0365 0.0321 0.0298 0.0320 0.0313 0.0397 0.0409 0.0408 0.0404 0.0554 0.0548 0.0545 0.0549 0.0036 0.0026 0.0027 0.0030 0.0076 0.0075 0.0079 0.0077 0.0020 0.0020 0.0020 0.0020 0.0064 0.0082 0.0073 Inor- ganic phos- phorus 0.0838 0.0709 0. 1628 0. 1802 0.0134 0.0089 Phos- phorus added (magne- sium pyro- phos- phate) Grams Added phos- phorus . J. . recovered Aaaea (magne Slum pyro- phos- phate) Grams phos- phorus recovered Percent 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0048 0.0077 30. 49.3 -0.0052 0.0145 -33.3 92.9 0.0047 0.0053 30.1 34. (1) Second set of determinations; first magnesium precipitates allowed to stand an extra day before filtering, and, after filtering, an extra day in acid alcohol. With samples A-1 and A-3 only 200 c. c. of aqueous-HCl extract was used, but the figures given represent 250 c. c. as usual. (2) With samples A-1, A-2 and A-3 only 200 c. c. of the aqueous HCl extract was used, but weights given for magnesium pyrophosphate represent 250 c. c. (3) First magnesium precipitate broken up in acid alcohol with stirring rod before filtering oflf 75 c. c. aliquot. INORGANIC PHOSPHORUS ESTIMATION 9 TABLE III: TEST OF COMPLETENESS OF EXTRACTION AND INFLU- ENCE OF PHENOL IN THE DETERMINATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES First extraction Second extraction Phos- phorus Added pho.s- Excess added phorus Mag'ne- phos- phorus Mag-ne- Inor- sium extracted Sample, treatment sium g'anic slum pyro- (magrne- and sample number pyrophos- phos- phos- phate) pyro- phos- sium phate phorus phos- phate pyro- phos- phate) phate) Grams Percent Grams Grams Grams Grams Timothy: Ai 0.0064 0.0012 Aq.-HCl A2 0.0069 0.0010 extraction A3 0.0053 0.0012 Av. 0062 0.0276 0.0011 -f 0.0001 Timothy: A 4 0.0253 0.0032 Aq.-HClextr. A;, 0.0250 0.0033 plus phosphate A 6 0.0256 0.0032 Av. 0.0253 0.0153 0.0191 0.0032 -0.0010 Timothy: Bi 0.0099 0.0006 Aq.-HCl-phenol Hi' 0.0099 0.0005 extraction K3 0.0096 0.00291 Av. 0.0098 0.0437 0.0005 -0.0011 Timothy: B4 0.0249 0.0029 Aq.-HCl-phenol extr. Bs 0.0246 0.0030 plus phosphate Bo 0.0252 0.00061 Av. 0.0249 0.0153 0.0151 0.0029 —0.0012 Rice Polish: Ai 0.0186 0.0026 Aq.-HCl extraction A2 0.0192 0.0032 plus phosphate A3 0.0182 0.0034 Av. 0.0187 0.0111 0.0089 0.0031 0.0000 A4 0.0098 0.0012 Rice Polish: As 0.0096 • 0.0012 Aq.-HCl A6 0.0098 0.0012 extraction A 7 0.0098 0.0012 Av. 0.0098 0.0434 0.0012 -0.0004 Rice Polish: Bi 0.0150 0.0017 Aq.-HCl-phenol extr. B2 0.0144 0.0018 plus phosphate B3 0.0150 Av. 0.0148 0.0111 0.0110 0.0017 -0.0008 B4 0.0036 Rice Polish: B5 0.0040 0.0014 +0.0007 Aq.-HCl-phenol Be 0.0040 extraction B7 0.0036 Av. 0.0038 0.0169 iNot included in average. 10 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE IV: TEST OF COMPLETENESS OF EXTRACTION AND INFLU- ENCE OF PHENOL IN THE DETERMINATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES Treatment and sample number First extraction Second extraction Sample Magne- sium pyro- phos- phate Grams Inor- ganic phos- phorus Percent Magne- sium pyro- phos- phate Grams Kxcess phos- phorus extracted (magne- sium pyro- phos- phate) Grams Gluten feed A q.-HCl extraction Ai Aq.-HCl extraction A2 Aq.-HCl extraction A3 Av. A q.-HCl-phenol extraction Bi Aq.-HCl-phenol extraction Ba A q.-HCl-phenol extraction Bs Av. 0.0211 0.0210 0.0209 0.0210 0.0212 0.0207 0.0200 0.0206 0.0936 0.0919 0.0032 0.0032 0.0032 0.0011 0.0009 0.0011 0.0010 0.0003 -0.0024 Brewer's grains Aq.-HCl extraction Ai A q.-HCl extraction A2 Aq.-HCl extraction A3 Av. Aq.-HCl-phenol extraction Bi Aq.-HCl-phenol extraction B2 Aq-HCl-phenol extraction Bs Av. 0.0026 0.0028 0.0028 0.0027 0.00121 0.0022 0.0026 0.0024 0.0120 0.0107 0.0010 0.0010 O.flOlO 0.0010 0.0010 0.0010 0.0010 0.0010 J. 0007 - 0.0006 Timothy Aq.-HCl extraction Al Aq.-HCl extraction A2 Aq.-HCl extraction A3 Av. Aq.-HCl-phenol extraction Bi Aq.-HCl-phenol extraction B2 Aq.-HCl-phenol extraction B3 Av. 0.0062 0.0040 0.0025 0.0042 0.0105 0.0097 0.0106 0.0103 0.0187 0.0459 0.0019 0.0016 0.0018 0.0017 0.0003 0.0006 0.0004 J. 0011 -0.0010 Wheat Aq.-HCl extraction Ai Aq.-HCl extraction A2 Aq.-HCl extraction A3 Av. Aq.-HCl-phenol extraction Bi Aq.-HCl-phenol extraction B2 Aq.-HCl-phenol extraction Bs Av. 0.0092 0.0096 0.0092 0.0093 0.0040 0.0049 0.0048 0.0046 0.0415 0.0205 Wheat bran Aq.-HCl extraction Ai Aq.-HCl extraction A 2 Aq.-HCl extraction A3 Av. Aq.-HCl-phenol extraction Bi Aq.-HCl-phenol extraction B2 Aq.-HCl-phenol extraction Bs Av. 0.0143 0.0134 0.0138 0.0138 0.0145 0.0157 0.0140 0.0147 0.0615 0.0655 iNot included in average. INORGANIC PHOSPHORUS ESTIMATION 11 The recovery of added phosphorus was usually incomplete, the method proving unsatisfactory, as judged by this measure. The recovery varied from a minus quantity in one case, with blue grass (Table II, p. 8) to 100 percent in one case, with rice polish (Table III, p. 9). The variability of results with the same products, in the different sets of estimations, when considered in connection with the difficulties experienced in filtration, suggested to us that at least a part of the incompleteness of recovery of added phosphates was due to the gelatinous character of the magnesia mixture pre- cipitate, notably so in the case of alfalfa, as shown in Tables I and II, pp. 7 and 8. In the work reported in Table II the precipitates remained an extra day in acid-alcohol, and the recovery of added phosphate was more nearly complete than in the work reported in Table I. A further consideration of this factor is reported in Tables VIII to X on pp. 18 to 20. The presence of phenol gave lower results for inorganic phos- phorus with alfalfa, brewer's grains, rice polish, gluten feed and wheat ; and higher results with blue grass, timothy and wheat bran. The recovery of added phosphate was usually incomplete; it was almost always higher with phenol than without, and was practically complete with brewer's grains (Table I, p. 7), timothy hay and rice poHsh (Table III, p. 9). With timothy hay the percent of inor- ganic phosphate found with phenol was higher than without ; the recovery of added phosphate was complete with phenol, and appar- ently more than complete without phenol. It is not clear, from these results, w^hat is the nature of the effect of phenol in this estimation. Since the effect of phenol is usually to lower the inorganic phosphorus, though sometimes to increase the same, we might suppose that in the former cases cleav- age predominated, except as suppressed by phenol, while in the latter cases the inhibited processes were in the direction of syn- thesis. We have found phenol, as used in this work, to be without effect on the precipitation of magnesium ammonium phosphate from pure solutions. Its efficiency to prevent enzymatic cleavage was not experimentally demonstrated. The test of completeness of extraction of inorganic phosphates by 0.2 percent hydrochloric acid in three hours was made with, gluten feed, brewer's grains, timothy hay, and rice polish, the re- sults being given in Tables III and IV on pp. 9 and 10. In con- sidering the significance of the weight of pyrophosphate obtained from the second extraction one should bear in mind the fact that this is due largely to dissolved phosphate from the first extraction 12 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 remaining adherent to the foodstuff. After making the necessary correction of this weight by subtracting such amount of pyrophos- phate as corresponds to the inorganic phosphorus in the liquid retained by the sample, the results are very small, and are more often minus quantities than not, showing that with these four food- stuffs the 3-hour extraction is practically complete. Further tests of the completeness of the 3-hour extraction are reported in Table VI, p. 16. TEST OF THE METHOD OF R. C. COLLISON As a possible improvement upon the method thus far con- sidered the similar procedure of R. C. Collison* was studied. Colli- son's method depends, as does that of Forbes and associates, from which it was derived, on an acid-alcohol separation of inorganic phosphorus from phytin, but this separation takes the form of a direct acid-alcohol extraction of the substance to be analyzed. The two methods differ in certain other details. Coliison's method has the advantage of being much more easily workable. Unfortunately, however, as first noted by Grindley and Ross, and later substan- tiated in this laboratory, results from the use of this method are unsatisfactory in that the 3-hour extraction is either insufficient or causes a cleavage of inorganic from organic phosphorus com- pounds. A second and even a third 3-hour extraction with acid- alcohol yields considerable amounts of inorganic phosphorus. The details of the method as used are as follows : METHOD OF R. C. COLLISON FOR THE DETERMINATION OF INORGANIC PHOSPHORUS IN VEGETABLE SUBSTANCES Weigh out 10-gram portions of the samples in triplicate, and place in 400 c. c. Florence flasks, to which add exactly 300 c. c. of 94-96 percent phosphorus- free alcohol, containing 0.2 percent of hydrochloric acid (0.2 percent actual HCl), and close with rubber stopper. Shake the flasks at intervals of 5 minutes for 3 hours, and filter through dry double filters into dry flasks. Measure out 250 c. c. aliquots of the filtrates into 400 c. c. beakers; make just alkaline to litmus with ammonia, and allow to stand for 8-12 hours, or over night. Filter through double filters, and wash with slightly ammoniacal 94-96 percent alcohol. In case a small portion of the precipitate resists transfer from the beaker by the usual means the last traces may be dissolved in 5 drops of hydrochloric acid, with the assistance of a rubber-capped rod. To this acid solution add 10 c. c. of alcohol; make slightly alkaline with ammonia, and then transfer to the filter. *Jour. Ind. and Eng. Chem., TV, p. 606, 1912. INORGANIC PHOSPHORUS ESTIMATION 13 Wash several times with ammoniacal alcohol; then spread out the inner papers with the precipitate and allow to dry completely. Transfer papers and precipitates to Erlenmeyer flasks containing exactly 100 c. c. of 0.5 percent aqueous solution of nitric acid (0.5 percent actual HNO3). Close the flasks with rubber stoppers; shake until the precipitates are thoroughly broken up, and let stand over night. Filter through dry double filters into dry beakers; pipette out 75 c. c. of each filtrate and determine phosphorus in the usual way, precipitating first with acid molybdate solution, then with magnesia mixture, and weigh as the pyrophosphate. If the final solutions are highly colored, dissolve the pyrophosphates and reprecipitate. Analytical data from our test of Collison's method are set forth in the following table : 14 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 < o o o t— I H H CO O O Q O » H O H » •d i! V t~(»(M c^q »-HO0t^ O^ COLOC^J CO .-Hinm o c "S, tointo '* rt Ln--tto in T-HCMCvi eg rt o^o o ooo o t^ u a ooo S OOO O O'O'O -o OOO O X 0.) ooo o odd d ddd d ddd d 3 Ah o O 'j-j ft u m O Si a, .2 o u tfi LDCC-^CO CO mcMcotr^to-^ coo'^c-q in in woo-* ooo li u; ^ +j s C^l Cs] CO CO CO CO V !» & d oooo o oooood oooo o goooog •d o ss-a rt oooo o oooooo oooo o OOOCDOO o 1) O oooo o d d> d d <~> '^ dddd d dddddd a |S m ^ i .Sou Cfi CD -^ ir> oj cvi CO Ot^oo^^ooo OOOCV300 c^q O CM CM ^ Ol o 05 J3 -w E CV5 C^J CVI -^ -^3* ■<* ■^-T'^ to to to o c^ .-( q e-j 'J O in d ^ to £ u Ol CTV Ol 00 3 u O ft CL( S 6 ,'„ .5| „ 0) O Nr-J ■"j-to^in t^^OlO tn ^ *J E 0|^T-|0 OiOOlO •d 11 men! C p XI o oo oooo oqqq oooo •d ■a Sf fc » o' od dodd dddo dddd 5 |i. CB to too tou^to to to to to to to E ooo ooo ooq ooo , O odd odd doo ddd P M S 4-) § S c ■^coo
ddddd S ^ i-H cv) CO ^ Ln to M ^ CM CO'* in to 01 <—< CM CO -^ in to 01 T-* CM CO ■<* in to 01 1) u > > < ^ < -< « a V '0 » 3J 4) o 13 -i-t *j a n ^ rt c3 rt rt rt rt rt rt (« XIJ3X1 Xi^ J= s:s:si xijaxi rt D. ?. p. a D. 0. ft O. ft ft ft ft Cfi a. 1/1 01 01 01 0) 01 0) 01 t/i m in ooo coo ooo OOO j:^:^: - - --c-s^ sisisi J3Xlja a o. P. £ S £ D. a G ftft a C C C ft ft ft C C C 01 01 01 t. U, U [-, u) 01 4) U (U 3 3 3 s: si si 111 in m .£.2.2 3 3 3 !* i' ^ (B 01 01 I; I, 4) 0) u rt rt rt oj « 0) OOO ft ft ft ft ft P* D (U JJ +j .p 4J ft ft ft « rt rt oj u J) eeeEEE ^^^EEE S 8 S 1 E S ^^^SES o o o rt rt rt ^^^c^c2c2 kSkccckcc j> K^ [-> ra ra flS INORGANIC PHOSPHORUS ESTIMATION 15 FURTHER CONSIDERATION OF THE METHOD OF FORBES AND ASSOCIATES Since Collison's method proved unsatisfactory, further work was done, at a later date, in attempts to improve upon the method of Forbes and associates (p. 5). In this work we gave attention to the following points: (a) The completeness of extraction, (b) the effect of using much larger amounts of magnesia mixture in the precipitation, (c) the allowing of more time for the precipitation with magnesia mix- ture, (d) the facilitating of filtration by the use of the centrifuge, and (e) the use of mechanical means to break up the precipitate in acid-alcohol to insure the complete solution of the phosphate. The following tabular data set forth the results of this study, the general method being that of the recovery of known amounts of phosphate introduced into the estimation. The details were as specified on p. 5, though phenol was used only in the tests reported in Table IX, p. 19. Throughout this work the centrifuge was used to facilitate filtration of the 0.2 percent hydrochloric acid extracts. The advantage derived from this treatment was very great. Extracts which it was impossible otherwise to filter within a reason- able time were, with the aid of the centrifuge, filtered without difficulty or delay. Other constant conditions in this work were the use of an extreme amount (50 c. c.) of magnesia mixture in the preliminary precipitation (instead of 10 c. c. as usual) ; and three days' time were allowed in all cases for the completion of this precipitation. Table VI, p. 16, reports results from a test of the Acid-Alcohol Method of Forbes and associates (for details see p. 5; modified as above), with alfalfa hay, by the method of recovery of added phos- phates, and a test of the completeness of extraction. Alfalfa was selected for this test as that substance which, in our previous experience, had given us the most trouble and the poorest results. Samples 4, 5 and 6 as compared with 1, 2 and 3, show that the recovery of added phosphates was incomplete, except in the case of sample 5, in which case, as explained in the footnote below the table, on account of the accidental breaking of the first filter paper, the precipitation was finally made in the presence of the pulp from this paper. In this case the recovery was complete. This acci- dental result sustained our hypothesis that our difficulty in recover- ing added phosphates was due to the physical character of the first magnesia mixture precipitate, its gummy character rendering im^- possible the complete separation, in acid-alcohol, of the inorganic phosphates from the phytin and other substances present. This point was given further study. 16 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 Determinations 7a, 8a and 9a were made in the same way as 1, 2 and 3. Determinations 7b, 8b and 9b were second extractions of the residues from determinations 7a, 8a and 9a. The results from the second extraction equalled only the residual amount of phosphate clinging to the sample, from the iirst extraction ; that is, no more inorganic phosphate was dissolved in a three-hour extrac- tion, following the three-hour extraction regularly prescribed in the method. The extraction was complete at the end of the first three- hour treatment. TABLE VI: TEST OF INORGANIC PHOSPHORUS ESTIMATION ON VEGETABLE SUBSTANCES BY THE ACID-ALCOHOL METHOD (WITHOUT PHENOL) Sample No. Treatment Magne- sium pyro- phos- phate Grams Percent inor- ganic phos- phorus Added phos- phorus (magne- sium pyro- phos- phate) Grams Added phos- phorus recovered Sub- stance Magne- sium pyro- phos- phate Grams Percent Alfalfa hay 1 2 3 Filtrate precip. with 50c. c. magnesia mixture +25 c. c. ammonia; without added phosphate .0176 .0181 .0178 Average .0178 .0992 Alfalfa hay 4 5* 6 Same, with added phosphate .0425 .0477 .0404 .0299 .0299 .0299 .0246 .0299 .0226 82.27 100.00 75.58 Average .0435 85.95 Alfalfa hay 7a 8a 9a Without added phosphate; same as 1, 2 and 3 .0182 .0181 .0181 Average .0181 .1008 Alfalfa hay 7b 8b** 9b Second extraction of samples 7a, 8a and 9a .0062 .0060 .0058 .0060*** .0060 .0060 Average .0060 Weight of samples 10 grams; results represent one-half of this amount. *During the filtration of the first magnesia mixture precipitate the filter -paper broke. This paper was then added to the beaker containing the precipitate, was beaten up into a pulp, and the filtration continued through a new paper. ** Samples 7b, 8b end 9b are the extraction residues from 7a, 8a and 9a, with filter paper added, and also enough 0.2 percent hydrochloric acid solution to make the original volume of 300 c. c. This set was extracted for three hours to test the completeness of the previous extraction. Magnesium pyrophosphate equivalent to phosphorus in solution, from previous extrac* tion, remaining in the sample. INORGANIC PHOSPHORUS ESTIMATION 17 Considering the data in Table VII, below, with samples 1-9 the first magnesia mixture precipitates were extracted with 200 c. c. of 0.2 percent nitric acid in alcohol instead of 100 c. c. as usual, in order to test the sufficiency of the latter amount to neutralize the ammonia remaining in the precipitates, and to dissolve the phos- phates. With samples 10-12 the usual 100 c. c. of acid alcohol were used. The comparison shows that 100 c. c. of acid was probably sufficient, though the result from sample 11, for some unknown reason was low. The second and third sets of triplicates, samples 4-9, contrast results from the addition of phosphates (to be recovered) after the extraction (immediately before precipitation) and previous to the three-hour extraction. We see here no evidence of a retention of added phosphates by the solid substance of the sample. TABLE VII: TEST OF INORGANIC PHOSPHORUS ESTIMATION ON VEGETABLE SUBSTANCES BY THE ACID-ALCOHOL METHOD (WITHOUT PHENOL) Sample No. Treatment Mag- nesium pyro- phos- phate Gm. Added phos- phorus (mag- nesium pyrophos- phate) Gm. Added phosphorus recovered Substance Mag- nesium pyrophos- phate Gm. Percent Alfalfa hay 1 2 3 Ave. Filtrate precipitated with 50 c. c. mag. mixture and 20 c. c. ammonia, 200 c. c. acid alcohol used to extract magnesia mixture precipitate. .0183 .0180 .0184 .0182 .0000 Alfalfa hay 4 5 6 Ave. Same as above + 25 c. c. phosphate solution just before precipitation. .0603 .0606 .0604 .0604 .04485 .0429 94.1 Alfalfa hay 7 8 9 Ave. Same as 1. 2 and 3 except that 25 c.c. phosphate solution added, shaken for 3 hrs., filtered and filtrate precip. as 1, 2 and 3. .0466 .0466 .0472 .0299 .0284 ) .0284 f .0290 94.98 96.99 95.98 Alfalfa hay 10 11 12 Ave. Same as 7, 8 and 9, except that only 100 c. c. acid alcohol were used in extraction of magnesia mixture pre- cipitate. .0467 .0447 .0464 .0285 .0265 .0282 95.32 88.63 94.31 92.75 Alfalfa bay 13 14 15 Ave. 25 c. c. phosphate solution put in flasks-|-175 c. c. .2% nitric acid in alco- hol+2 filter papers, shaken, filtered and aliquot taken for precipitation. .0404 .0404 .0402 .03987 .0404 .0404 .0402 101.1 Weight of samples, 10 grams; results represent 0.5 of this amount. 18 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 Determinations 13-15 were made to ascertain whether or not we could get a complete recovery of phosphates from filter paper pulp. It was possible completely to recover the phosphates. This test has a bearing on work to follow, and shows that the incomplete recovery of added phosphates could not be due to the presence of filter paper pulp. The recovery of added phosphates in these estimations on alfalfa was fairly satisfactory. TABLE VIII : TEST OF INORGANIC PHOSPHORUS ESTIMATION ON VEGETABLE SUBSTANCES BY THE ACID-ALCOHOL METHOD (WITHOUT PHENOL) Sample No. Treatment Magne- sium pyro- phos- phate Grams Added phos- Added phos- phorus recovered Sub- stance Percent inor- ganic phos- phorus phorus (magne- sium pyro- phos- ■ phate) Grams Magne- sium pyro- phos-., phate Grams Percent Blue grass ■ 1 2 3 Average Filtrate precip. with 50 c. c- magnesia mixture -^25 c- c. ammonia, plus paper pulp .0373 .0371 .0386 .2078 .2067 .2151 .2099 Blue grass 4 5 6 Average Same +25 c. c. phosphate solution .0624 .0623 .0633 .0250 .0250 .0250 .0251 .0252 .0247 100.04 100.08 98.80 99.97 Rice polish 7 8 9 Average Same as 1, 2 and 3 .0070 .0070 .0070 i0390 Rice polish. 10 11 12 Average Same+25c. c phosphate solution .0295 .0295 .0250 .0250 .0225 .0225 .0225 90.00 Weight of samples 10 grams; results represent 0.5 of this amount INORGANIC PHOSPHORUS ESTIMATION 19 table ix: test of inorganic phosphorus estimation on vp:getable substances by the acid-alcohol method (with phenol) Sam- ple No. Treatment Mag- nesium pyro- phos- phate Gm. Percent inor- ganic phos- phorus Added phos- phorus (mag- nesium pyro- phos- phate) Gm. Added phos- phorus recovered Substance Mag- nes i u m pyro- phos- phate Gm. Per- cent Rice polish 1 2 3 Ave. Extracted with 0.2% hydrochlo- ric acid + 50 u m. phenol per liter: filtrate precipitated with mag- nesia mixture + 25 c.c. ammonia. .0080 .0072 .0446 .0401 .0424 Rice polish + phosphate 4 5 6 Ave. Same .0543 .0461 .0443 .0768 .0768 .0768 .0467 .0385 .0367 60.81 50.13 47.79 52.91 Middlings 7 8 9 Ave. Same .0132 .0133 .0129 .0131 .0724 Middling-s -f phos- phate 10 11 12 Ave. Same .0830 .0864 .0847 .0768 .0768 .0768 .0699 .0733 .0716 91.01 95.44 93.23 93.23 Soy beans 13 14 15 Ave. Same .0102 .0105 .0102 .0103 .0574 Soy beans+ phosphate 16 17 18 Ave. Same .0790 .0801 .0823 .0805 .0768 .0768 .0768 .0687 .0698 .0720 89.45 90.89 93.75 91.36 Weight of samples 10 grams; results represent 0.5 of this amount. 20 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE X: TEST OF INORGANIC PHOSPHORUS ESTIMATION ON VEGETABLE SUBSTANCES BY THE ACID-ALCOHOL METHOD (WITHOUT PHENOL) Sub- stance Sample No. Treatment Magne- sium pyro- phos- phate Gram Percent inor- ganic phos- phorus Added phos- phorus (magne- sium pyro- phos- phate) Gram Added phos- phorus recovered Magne- sium pyro- phos- Percent phate Gram Soy beans 1 2 3 Average Usual method, plus iilter paper pulp;\vithout phosphate .0102 .0103 .0101 .0102 .0568 Soy beans 1 2 3 Average Same, plus phosphate .0336 .0345 .0347 .0261 .0261 .0261 .0234 .0243 .0245 90.03 93.10 93.87 92.33 Mid- dlings 1 2 3 Average Same, without phosphate .0135 .0131 .0139 .0135 .0752 Mid- dlings 1 2 3 Average Same, with phosphate .0432 .0430 .0418 .0261 .0261 .0261 ,0297 .0295 .0283 113.79 113.02 108.42 111.74 Oat straw 1 2 3 A verage Same, without phosphate .0062 .0058 .0061 .0060 .0334 Oat straw 1 2 3 Average Same, with phosphate .0312 .0319 .0318 .0261 .0261 .0261 .0252 .0259 .0258 96.55 99.23 98.85 98.21 Weight of samples 10 grams; results represent 0.5 of this amount. Table VIII, p. 18, sets forth results of estimations on blue grass and rice polish, with and without added phosphate, with filter paper pulp added, in the first precipitation, to maintain a readily permeable condition in the precipitate. With blue grass the results may be considered perfect. With rice polish the recovery of added phosphates was 90 percent efficient. The loss amounted to 0.0025 gm. magnesium pyrophosphate pel* determination. INORGANIC PHOSPHORUS ESTIMATION 21 Table IX, p. 19, is a record of determinations on rice polish, wheat middlings and soy beans, with and without added phosphate, with phenol added to the extractive reagent to prevent possible enzyme action involving phosphorus compounds, and with filter paper pulp added to facilitate solution of the phosphates in the pre- cipitate. The recovery of added phosphates was unsatisfactory; the effect of the phenol on the physical condition of the first mag- nesia mixture precipitate being of such a nature as to hinder the dissolving out of the included phosphates. Table X, p. 20, sets forth results from determinations on soy beans, wheat middlings and oat straw, with and without added phos- phate, with filter paper pulp, and without phenol added to the ex- tracting sample. With oat straw the results were satisfactory. With soy beans the recovery of added phosphates was incomplete, while with wheat middlings we seem to have recovered 3 milligrams more phosphate than was added. In this case we can not ascribe the imperfection of the result, as usual, to the physical condition of the magnesia mixture precipitate. Here, it seems, that there must have been a cleavage of inorganic from organic phosphorus, either enzymatic or as a result of the extractive treatment. Anderson* has shown that 0.2 percent hydrochloric acid is not sufficiently con- centrated to prevent enzymatic hydrolysis of the phytin of wheat bran, and, since middlings contains bran, this observation must apply to middlings as well. How general may be enzymatic hydrolysis of organic phosphorus compounds in our 0.2 percent hydrochloric acid extractive reagent we are unable to say. In the case of wheat middlings only has the extent of the hydrolysis been shown, in our work, to be sufficient more than to offset the various factors tending to give low results for inorganic p^iosphorus. Our second three-hour extractions of timothy, rice polish, gluten feed and brewer's grains showed no considerable additional inorganic phosphorus resulting either from solution or hydrolytic decompo- sition of organic compounds. CONCLUSIONS FROM WORK ON INORGANIC PHOSPHORUS ESTIMATION IN VEGETABLE SUBSTANCES (1) A three-hour extraction with 0.2 percent hydrochloric acid in water appears to accomplish practically complete solution of the inorganic phosphates of finely ground vegetable substances, but in the case of wheat middlings was shown to allow enzymatic hydroly- sis of organic phosphorus, with the liberation of inorganic phos- phate. *Anderson, R. J.: Journ. Biol. Chem. XX (1915) 483-491. 22 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 (2) The introduction of filter paper pulp into such an extract materially assists in the maintenance of an easily penetrable con- dition in a magnesia mixture precipitate from the same. (3) It was found possible completely to recover phosphates from filter paper pulp alone as used in this work. (4) The use of the centrifuge very greatly facilitates the fil- tration of dilute aqueous-acid extracts of vegetable substances. (5) There has appeared no reason to doubt the completeness of the precipitation of the inorganic phosphates from the 0.2 percent hydrochloric acid solution, through the use of magnesia mixture and ammonia. (6) The separation of the inorganic phosphates from the phytin and other constituents of the magnesia mixture precipitate, through the agency of 0.2 percent nitric acid in 95 percent alcohol, is attended by difficulties which have not yet been overcome. That these difficulties are largely physical, as determined by the bulky and often gummy nature of the magnesia mixture precipitate, seems to be a fact. That they are in part of a chemical nature, and due to the cleavage of phytin or other organic phosphorus compounds of the magnesia mixture precipitate through the agency of enzymes appears also to be true. (7) The use of phenol (50 gm. per liter) in the extractive reagent was shown not to affect the precipitation and estimation of phosphates in pure solutions. In the estimation of inorganic phosphorus in extracts of vegetable substances the presence of phenol appeared to favor the recovery of added phosphates. Phenol, when used in this way, sometimes increased but more commonly decreased the inorganic phosphorus. In extracts of certain vege- table products the presence of phenol increased the difficulty, rather commonly experienced, in breaking up the magnesia mixture pre- cipitate in acid alcohol. (8) Modification of the acid-alcohol method of Forbes and associates by the introduction of filter paper pulp into the extract from which the phosphates are to be precipitated, the use of exces- sive amounts of magnesia mixture in this first precipitation, and allowing unusual duration of time for this precipitation gave appar- ently perfect results, as judged by recovery of added phosphates, in certain cases, but unsatisfactory results in others. (9) Incompleteness of recovery of added phosphates w^s shown not to be due to retention of phosphates by the solid substance of the sample. INORGANIC PHOSPHORUS ESTIMATION 23 (10) We are unable to recommend this method, or any other, as rehable for the estimation of inorganic phosphorus in vegetable substances generally. (11) The acid-alcohol extraction of the method of R. C. Collison is either incomplete, in three hours, or else causes a cleavage of organic compounds of phosphorus, with the liberation of inorganic phosphate. THE ESTIMATION OF WATER-SOLUBLE INORGANIC PHOS- PHORUS IN ANIMAL SUBSTANCES The method of determination of inorganic phosphorus in animal substances, as published from this laboratory, is an adaptation of the usual magnesia mixture method for phosphorus estimation to the conditions of work with water-extracts of animal products. The original points, therefore, in our procedures, as specified for various animal tissues, are mechanical and chemical details of extraction, filtration and precipitation. Considerable care has been bestowed upon the quantitative proof of the correctness of these details, es- pecially as providing for complete extraction, rapid filtration, and prevention of the cleavage of the organic phosphorus compounds from which the inorganic are to be separated. These studies are in the nature of comparisons of the neutral molybdate method of Emmett and Grindley, the barium chlorid method of Siegfried and Singewald and the magnesia mixture method of Forbes and asso- ciates, usually checked by the recovery of known amounts of added phosphate ; and they appear to result in the establishment of the last-named method as reliable and workable with a wide range of animal t:[ssues. In the use of this method One. should recognize the fact that it seeks to estimate the water-soluble inorganic phosphates only. This figure would be at least practically the same as total inorganic phosphates for most animal tissues and products,' but in the case of tissues such as bone, w:hich contain large amounts of inorganic phosphates which are insoluble in w^ater, this method is inapplicable. ,TJhe work on^animal tissues was done in three series of determi- nations, one each in the years 1912, 1913 and 1914. The remainder of this paper sets forth the details of this work. The discussion of th6 results of the work of 1912-1913 will be found on pages 33 and 34, and of 19 U on pages 38 to 40. ^^ riv-i- or---: f;T . THE WORK OF 1912 --■ ?~ The work 'Of 1912 .consisted of a comparison of th6 methods of Emmett. an^ Grindley, Siegfried and Singewald, and Forbes and associates with muscle, blood and brain. The muscle used was a 24 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 vacuum-dried product prepared in the laboratory of Dr. H. S. Grind- ley, of the University of Illinois. The following schedule shows methods of precipitation, amounts of extracts used, number of re- peats, and amount of standard phosphate solution added to certain of these determinations : A Neutral Molybdate Precipitation (Emmett and Grindley) A-1 500 c. c. extract A-2 500 c. c. extract A-3 250 c. c. extract + 25 c. c. phosphate solution A-4 250 c. c. extract + 25 c. c. phosphate solution 1. Muscle 2. Blood 3. Brain B-1 500 c. c. extract B-2 500 c. c. extract B-3 250 c. c. extract + 25 c. c. phosphate solution (Siegfried and Singewald) B-4 250 c. c. extract + 25 c. c. phosphate solution B Barium Chlorid Precipitation Magnesia Mixture Precipitation (Forbes, et al) C-1 500 c. c. extract C-2 500 c. c. extract C-3 250 c. c. extract + 25 c. c. phosphate solution C-4 250 c. c. extract + 25 c. c. phosphate solution The work with muscle was performed on a cold water extract prepared as specified below: OUTLINE FOR THE PREPARATION OF A COLD WATER EXTRACT OF DESICCATED FLESH FOR THE DETERMINATION OF INORGANIC PHOSPHORUS Weigh out about 45 grams of the vacuum-dried meat, and divide it among sixteen 150 c. c. beakers. To each beaker with its contents add about 3-5 c. c. of distilled water. Break up any lumps and stir well with a glass rod until the mass forms a thick paste. Add 50 c. c. of distilled water to each beaker and stir thoroughly for 15 minutes. Allow the insoluble portion to settle for a few minutes (3-5) and decant the supernatant liquid through wet 11 cm. filters. Collect the filtrates in 250 c. c. Florence flasks. Take care that the funnels touch the sides of the necks of the flasks. Drain the residues thor- oughly, keeping as much of them in the beakers as possible. Treat these residues with 25 c. c. of distilled water, stirring for 5-7 minutes, and then allow- ing 3-5 minutes for the solid particles to settle before filtering. Decant, etc., as described above. Repeat this last treatment until the filtrate measures about 220 c. c. Then transfer the entire residue to the filter and wash twice with about 8-10 c. c. of distilled water. Allow all the liquid to pass through the filter before adding the next extract. Whenever the major portion of the residue has become mechanically transferred to the filter, return it to the beaker, using great care not to break the filter paper. Take the sixteen filtrates of about 250 e, c. each and transfer all of them to a measuring flask. Wash out each Florence flask twice, using about 5-8 c. c. of distilled water each time. Make the extract up to 5000 c. c. and mix it thoroughly without too much mechanical agitation. INORGANIC PHOSPHORUS ESTIMATION 25 With blood, the comparisons were made on an extract prepared with the aid of ammonium sulphate, the details of procedure being as specified below: METHOD FOR THE PREPARATION OF HOT WATER-AMMONIUM SULPHATE EXTRACT OF BLOOD Weigh about 50 grams of fresh blood, or its equivalent of oxalated blood, into each of six 400 c. c. beakers. To each beaker add a few c. c. of distilled water and work up the blood and water with a glass rod. Make up to about 200 c. c. with boiling distilled water. Place over a flame and gradually bring to boiling, with constant stirring. When boiling begins add to each beaker 20 c. c. of 20 percent ammonium sulphate solution. Boil with constant stirring for about 10 minutes. Decant onto sand on linen. When the liquid is through lift the coagulum oif from the sand and transfer to a mortar. Grind the coag- ulum to a smooth paste and transfer from mortar to beaker with boiling dis- tilled water. Make up to about 80 c. c. with the same. Stir for 8 minutes and pour contents again onto the sand filter. After the extract is through return the coagulum to the mortar and grind a second time, transferring to the beaker as before with boiling distilled water. Repeat this process of 8-minute extrac- tions of the coagulum in hot water and filtration as above directed, without further grinding, until the filtrates measure about 750 c. c. each. Wash out each beaker twice with 8-10 c. c. hot distilled water, completing the transfer of the coagulum and extract to the sand. Wash the coagulum on the sand twice with boiling water from a wash bottle. At all times allow the filter to drain well between additions of extract or wash water. Combine the six filtrates of about 800 c. c. each, washing out the containers of each twice with distilled water. Make the extract up to 5000 c. c. and mix. Still a different method of manipulation, with the aid of am- monium sulphate, was necessary for the extraction of brain. The details are as specified below: DIRECTIONS FOR THE PREPARATION OF HOT WATER-AMMONIUM SULPHATE EXTRACT OF BRAIN Weigh out about 10 grams of brain into each of ten 250 c. c. beakers. To each beaker add a few c. c. of distilled water and work up the brain and water with a glass rod. Make up to about 100 c. c. with boiling water. Place over a flame and gradually bring to boiling, with constant stirring. After boiling has begun add to each beaker 20 c. c. of 20 percent ammonium sulphate solution. Boil for about 10 minutes. Allow to settle for a moment and decant liquid onto sand on linen. In case the extracts do not filter readily, carefully push the coagulum to one side or return to the beakers. Add to the beakers containing the coagulum 50 c. c. of 0.1 percent ammonium sulphate solution; stir for one minute and decant the liquid onto the filter. Repeat this process of one-minute extractions of the coagulum in 0.1 percent ammonium sulphate solution, and filtration as above directed, until the filtrates measure about 450 c. c. Wash out each beaker twice with 8-10 c. c. of hot 0.1 percent ammonium sulphate solution, completing the transfer of the coagulum and extract to the sand. 26 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 Wash the coagulum twice with the above wash solution from a wash bottle. At all times allow the filter to drain well between additions of extract or wash solution. Combine the 10 filtrates, washing out the container of each of the filtrates twice with 5-8 c. c. of distilled water. Make the extract up to 5000 c. c, and mix. The three methods compared, for the precipitation of inorganic phosphorus in the extracts prepared as above, and for the final esti- mation of the phosphorus, were as specified below : EMMETT AND GRINDLEY NEUTRAL AMMONIUM MOLYBDATE METHOD FOR THE DETERMINATION OF INORGANIC PHOS- PHORUS IN WATER EXTRACTS OF FLESH Measure out the number and volumes of extracts indicated in the schedule on p. 24. Evaporate, with frequent stirring, on the water or steam bath to approximately 20 to 25 c. c. While hot, filter into 300 c. c. beakers, using doubled 11 cm. No. 589 (Blue Ribbon brand) S. and S. papers. Wash beakers, precipi- tates, and filters thoroughly with hot water. The volume of the resulting fil- trate' and washings should be about 125 c. c. Add 10 grams of ammonium nitrate and heat upon the water bath to 60° C. Then add 10 c. c. of nitric acid (sp. gr. 1.20); stir, and add 125 c. c. of clear ammonium molybdate solution. (Neutral ammonium molybdate is prepared by adding ammonia to the ordinary molybdate solution, using litmus paper as an indicator. This work should be done very carefully, and both red and blue litmus paper used). Reheat, bring- ing temperature to 60° C. Keep at this temperature for 15 minutes. Stir vigorously every few minutes during this time. Remove from the bath and allow the solutions to stand 2 hours in a warm place. Decant the clear super- natant liquid through doubled 11 cm. No. 589 (Blue Ribbon brand) S. and S. filters. Transfer the remaining liquid and precipitate to the filters, using a 10 percent ammonium nitrate solution. Wash precipitates and beaker four or five times with small volumes of the ammonium nitrate solution. Dissolve the yellow precipitate upon the filter, and that in the precipitating beaker, with dilute ammonium hydroxid (2.5 percent) and hot water, collecting the filtrate in a 250 c. c. beaker. Wash thoroughly; neutralize the solution with nitric acid (1.20 sp. gr.), and make up to approximately 150 c. c. Add 5 grams of am- monium nitrate; heat upon the water bath to 60° C. and then carefully add, while stirring, 5 c. c. of concentrated nitric acid and 50 c. c. of clear acid molybdate solution. Digest at 60° C. for 15 minutes, stirring occasionally. From here on continue the determination of phosphorus as usual, weighing the phosphorus as magnesium pyrophosphate. SIEGFRIED AND SINGEWALD METHOD, AS USED BY EMMETT AND GRINDLEY, FOR THE DETERMINATION OF INORGANIC PHOS- PHORUS IN WATER EXTRACTS OF ANIMAL SUBSTANCES Measure out the number and volumes of extracts specified in the schedule on p. 24. To each portion add 50 c. c. of a 10 percent barium chlorid solution and 10 c. c. of 10 percent ammonium hydroxid. Stir the solutions every 15 minutes for a period of one hour, allow to stand undisturbed for at least 12 hours, and then filter (decanting at first, as much as possible) through INORGANIC PHOSPHORUS ESTIMATION 27 double quantitative filters. Wash the beakers, precipitates and filters repeat- edly, with small quantities of wash water containing 10 c. c. of the barium chlorid solution and 10 c. c. of the dilute ammonium hydroxid per liter. Place the upper filters containing the precipitates in the beakers in which the precipi- tation occurred, and digest at room temperature with 35 c. c. of dilute nitric acid (sp. gr. 1.20) with frequent stirring. Filter the acid solution through the second filter, which was not removed from the funnel, and wash the beakers and filters thoroughly with hot water. Neutralize the filtrates with ammonia, slightly acidify with nitric acid; add 10 grams of ammonium nitrate, dilute to about 125 c. c. and heat upon the water-bath to 60^ C. Add 100 c. c. of acid ammonium molybdate and continue the phosphorus determination from here on as usual. MAGNESIA MIXTURE METHOD OF FORBES AND ASSOCIATES FOR THE DETERMINATION OF INORGANIC PHOSPHORUS IN EXTRACTS OF ANIMAL TISSUES Measure out the number and volumes of extracts specified in the schedule on p. 24. Add 10 c. c. magnesia mixture, stirring freely; allow to stand 15 minutes, and add 25 c. c. of ammonia, sp. gr. 0.90; cover and allow to stand over night. On the next morning filter, and wash the precipitate with 2.5 percent ammonia water. Dissolve the precipitate on the filter paper with dilute nitric acid into the same beaker in which the first precipitation was made, and wash the papers thoroughly with hot water. Render the resulting solutions nearly neutral; add 5 grams of ammonium nitrate; heat to 65^ C; add 50 c. c. of official acid molybdate solution, and keep at 60" for two hours. Then continue in the usual way for the gravimetric estimation of phosphorus as the pjT:o- phosphate. , The numerical results of this comparative study of methods are reported in the following table, these data being discussed, together with those from the work of 1913, on pages 33-34. 28 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE XI: COMPARISON OF METHODS OF ESTIMATION OF INORGANIC PHOSPHORUS IN ANIMAL PRODUCTS Phos- phorus added (magne- sium pyro- phos- phate) Grams Magne- sium pyro- phos- phate obtained Grams Inor- ganic phos- phorus Percent Added phosphorus recovered Sample Method, and sample number Volume of extract used c. c. Magne- sium pyro- phos- phate Grams Percent Muscle Ai Neutral Molybdate A2 Method (Emmett As and Grindley) A4 Average 500 500 250 250 0.0419 0.0419 0.0824 0.0814 0.0814 0.0811 0.5018 0.4956 0.4987 0.0405 0.0402 0.0403 96.2 Muscle Bi Barium Chlorid B2 Method (Siegfried Bs and Singewald) B4 A verage 500 500 250 250 0.0419 0.0419 0.0836 0.5090 0.0826 0.5029 0.0850 0.0848 0.5059 0.0435 0.0433 0.0434 103.6 Muscle Ci Magnesia Mixture C2 Method Forbes and C3 Associates) C4 Average 500 500 250 250 0.0419 0.0419 0.0792 0.0794 0.0798 0.0806 0.4822 0.4834 0.4828 0.0402 0.0410 0.0406 96.9 Blood Ai Neutral .Molybdate A2 Method (Emmett As and Grindley) A4 A verage 500 500 250 250 0.0419 0.0419 0.0380 0.0394 0.0568 0.0584 0.0281 0.0292 0.0286 0.0375 0.0391 0.0383 91.4 Blood Bi Barium Chlorid B2 Method (Siegfried B;; and Singewald) B4 Average 500 500 250 250 0.0419 0.0419 0.0088 0.0094 0.0166 0.0164 0.0065 0.0070 0.0067 0.0121 0.0119 0.0120 28.6 ■ Ci Mag-nesia Mi.xture Cs •Ri^/t ! Method (Forbes and Cs ^^°°^ 1 Associates) C4 Average 500 500 250 250 0.0419 0.0419 0.0210 0.0212 0.0514 0.0518 0.0156 0.0157 0.0156 0.0409 0.0413 0.0411 98.1 Ai Neutral Molybdate A 2 Method (Emmett As Brain and Grindley) Ai A verag'e 500 500 250 250 0.0280 0.0327 0.0419 0.0540 0.0419 0.0533 0.0776 0.0907 0.0841 0.0237 0.0230 0.0233 55.6 Brain Bi Barium Chlorid B2 Method (Siegfried Bs and Singewald) B4 A verage 500 500 250 250 0.0419 0.0419 0.0034 0.0038 0.0158 0.0170 0.0094 0.0105 0.0099 0.0140 0.0152 0.0146 34.6 Brain Ci Magnesia Mixture C2 Method (Forbes and Cs Associates) C4 Average 500 500 250 250 0.0419 0.0419 0.0216 0.0210 0.0498 0.0507 0.0599 0.0582 0.0590 0.0392 0.0401 0.0396 94.5 Notes: Weights of samples used: muscle (vacuum dried sample)— 45.7822 grams: blood (fresh pig's blood)— 376.2846 grams; brain (fresh calf brain)— 100.5238 grams; extracts made up to 5000 c c INORGANIC PHOSPHORUS ESTIMATION THE WORK OF 1913 29 The work of 1913 consisted of a further comparison of the methods of Emmett and Grindley, and of Forbes and associates, with muscle, blood and brain, the consideration of the method of Siegfried and Singewald being discontinued. The following schedule indicates the estimations made. 1. Muscle 2. Blood 3. Brain Extract of samples as weighed B Extract of samples as weighed plus 25 c. c. phosphate sol- ution Ai Neutral molybdate precipitation A << « « A3 Ai Magnesia mixture precipitation A « « K A « « it Bi Neutral molybdate precipitation Tin " " " B. " " " Bi Magnesia mixture precipitation B„ " " " Phosphorus was also determined by both methods of precipita- tion in the phosphate solution used ; and blank determinations were made, in triplicate, on the reagents. DIRECTIONS FOR THE PREPARATION OF COLD WATER EXTRACT OF MUSCLE A Weigh out 10-12 grams of fresh muscle and divide as nearly equally as possible between two small beakers. Moisten the samples with a few c. c. of distilled water, and break up lumps with a glass rod. Add 50 c. c. of water to each beaker and stir contents for 15 minutes. Allow insoluble residue to settle for 3-5 minutes; then decant the liquid from each beaker through filters into beakers; allow to drain, and add 25 c. c. of water. Stir for 7-8 minutes, and after allowing to settle, decant onto the same filter. Continue this treat- ment, using each time 25 c. c. of water, until the filtrates measure about 230 c. c. each. Allow the filters to drain completely between extractions. Whenever the major portion of the residue has become mechanically transferred to the filter return it to the beaker, using care not to break the filter paper. After the last extraction throw the entire contents of each beaker onto the filter, and, when drained, wash twice with small quantities of distilled water. Combine the two extracts, and use for the precipitation of the phosphates under A. B Weigh out same quantity of flesh as specified above, and divide as nearly equally as possible between two small beakers; work up with a few c. c. of distilled water; add 25 c. c. of aqueous solution of disodium phosphate, equivalent to about 40 mg. magnesium pyi'ophosphate, dividing as nearly equally as possible between the two beakers, and proceed as directed under A. The extract thus obtained is ready for precipitation as under B. 30 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 METHOD FOR THE PREPARATION OF HOT WATER-AMxMONIUM SULPHATE EXTRACT OF BLOOD A Weigh out 30-35 grams of fresh blood, or the equivalent of oxalated blood, into a 400 c. c. beaker. Add a few c. c. of distilled water, and work up the blood and water with a glass rod. Make up to about 150 c. c. with boiling distilled water. Place over a flame, and gradually bring to boiling, with con- stant stirring. When boiling begins add 20 c. c. of 20 percent ammonium sul- phate solution. Boil, with constant stirring, for about ten minutes. Decant onto a filter of sand on linen, receiving the filtrate in an 800 c. c. beaker. When the liquid is through, lift the coagulum from the sand, and transfer it to a mortar. Grind to a smooth paste and transfer from mortar to beaker with boiling distilled water. Make up to about 50 c. c. with the same; stir for 8 minutes and pour contents again onto the sand filter. After the extract is through, return the coagulum to the mortar, and grind a second time, trans- ferring to the beaker as before with boiling distilled water. Repeat this process of 8-minute extractions of the coagulum in hot water, and filtration as above directed, without further grinding, until the filtrate measures about 450 c. c. Wash out each beaker twice with 8-10 c. c. of hot water, completing the trans- fer of the coagulum and extract to the sand. Wash the coagulum on the sand twice with boiling water from a wash bottle. At all times allow the filter to drain well between additions of extract or wash w^ater. This extract of about 500 c. c. is ready for precipitation as under A. B Weigh out same quantity of blood as specified above. Work up with a few c. c. of distilled water; add 25 c. c. of an aqueous solution of disodium phosphate equivalent to about 40 mg. magnesium pyrophosphate, and proceed as directed under A. The extract thus obtained is ready for precipitation under B. METHOD FOR THE PREPARATION OF HOT WATER-AMMONIUM SULPHATE EXTRACT OF BRAIN A Weigh out about ten grams of brain into a 250 c. c. beaker. Add a few c. c. of distilled water, and work up the brain and water with a glass rod. Make up to about 100 c. c. with boiling water; place over a flame, and gradually bring to boiling, with constant stirring. After boiling has begun add 20 c. c. of 20 percent ammonium sulphate solution; boil gently for about ten minutes; allow to settle for a moment, and decant liquid slowly onto a filter of sand on linen,* receiving the extract in an 800 c. c. beaker. Add to the beaker containing the coagulum 50 c. c. of 0.1 percent ammonium sulphate solution. Stir for one minute, keeping over flame and at the boiling point; decant the liquid onto the filter. Repeat this process of one-minute extractions of the coagulum in 0.1 percent ammonium sulphate solution, and filtration as above directed, until the filtrate measures about 450 c. c. Wash out the beaker twice with 8-10 c. c. of hot 0.1 percent ammonium sulphate solution, completing the transfer of the coagulum and extract to the sand. Wash the coagulum twice with the above wash solution from a wash bottle. At all times allow the filter to drain well between additions of extract or wash solution. This extract of about 500 c. c. is ready for precipitation under A. *It is desirable to prevent the extract or coagulum from coming in contact with the linen before passing through the sand. To this end pour extract slowly onto center of sand or into a cup-shaped depression. INORGANIC PHOSPHORUS ESTIMATION 31 B Weigh out same quantity of brain as specified above; work up with a few c. c. of distilled water; add 25 c. c. of an aqueous solution of disodium phosphate equivalent to about 40 mg. of magnesium pyrophosphate, and pro- ceed as directed under A. The extract thus obtained is ready for precipitation under B. NEUTRAL AMMONIUM MOLYBDATE METHOD OF EMMETT AND GRINDLEY FOR THE DETERMINATION OF INORGANIC PHOS- PHORUS IN WATER EXTRACTS OF FLESH Treat 3 of the extracts prepared according to the directions on the pre- ceding pages under A, and 3 of those prepared according to B as follows: Evaporate, with frequent stirring, on the water or steam bath, to approximately 20-25 c. c. While hot, filter into 300 c. c. beakers, using doubled 11 cm. No. 589 (Blue Ribbon brand) S. and S. papers. Wash beakers, precipitates, and filters thoroughly with hot water. The volume of the resulting filtrate and washings should be about 125 c. c. Add 10 grams of ammonium nitrate and heat upon the water bath to 60° C. Then add 10 c. c. of nitric acid (sp. gr. 1.20); stir, and add 125 c. c. of clear neutral molybdic solution. (Neutral ammonium molybdate is prepared by adding ammonia to the ordinary molybdic solution, using litmus paper as an indicator. This work should be done very carefully, and both red and blue litmus paper used). Reheat, bringing temperature to 60° C. Keep at this temperature for 15 minutes. Stir vigorously every few minutes during this time. Remove from the bath and allow the solutions to stand 2 hours in a warm place. Decant the clear supernatant liquid through doubled 11 cm. No. 589 (Blue Ribbon brand) S. and S. filters. Transfer the remaining liquid and precipitate to the filters, using a 10 percent ammonium nitrate solution. Wash precipitates and beakers four or five times with small volumes of the ammonium nitrate solution. Dissolve the yellow precipitate upon the filter and that in the precipitating beaker with dilute ammonia (2.5 percent) and hot water, collecting the filtrate in a 250 c. c. beaker. Wash thoroughly. Neutralize the solution with nitric acid (1.20 sp. gr.), and make up to approximately 150 c. c. Add 5 grams of ammonium nitrate; heat upon the water bath to 60° C. and then carefully add, while stirring, 5 c. c. of concentrated nitric acid and 50 c. c. of clear acid molybdic solution. Digest at 60° C. for 15 minutes, stir- ring occasionally. From this point continue the determination of phosphorus as usual, weighing the phosphorus as magnesium pyrophosphate. MAGNESIA MIXTURE METHOD OF FORBES AND ASSOCIATES FOR THE DETERMINATION OF INORGANIC PHOSPHORUS IN EXTRACTS OF ANIMAL TISSUES Treat three of the extracts prepared according to the directions on the pre- ceding pages under A, and three of those prepared according to B as follows: Add 10 c. c. magnesia mixture, stirring freely. Allow to stand 15 minutes; add 25 c. c. ammonia, sp. gr. 0.90; cover, and allow to stand over night. On the next morning filter, and wash the precipitate with 2.5 percent ammonia water. Dissolve the precipitate on the filter paper and that remaining in the beaker in which the precipitation was made with dilute nitric acid (1:1) and hot water, receiving the solution in 400 c. c. beakers. Neutralize the nitric acid with ammonia; make slightly acid with nitric acid; add 5 grams ammonium nitrate, and precipitate in the usual way with molybdate solution. Continue in the usual way for the gravimetric estimation of phosphorus as the p>Tophosphate. 32 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 The numerical results of the work of 1913 are reported in the following group of tables, these data being discussed, together with those from the work of 1912, on pages 33 and 34. TABLE XII: COMPARISON OF METHODS OF ESTIMATION OF INORGANIC PHOSPHORUS IN ANIMAL PRODUCTS Added Phos- phos- Magne- Inor- phorus phorus Added Blank Sample Treatment, and Weight of sium pyro- phos- phate ganic phos- added (magne- sium recovered (magne- sium phos- phorus estima- tions on rea- sample number sample phorus pyro- phos- phate) pyro- phos- phate) ered gents Grams Grams Percent Grams Grams Percent Grams Neutral Moljbdate Ai 13.5485 0.0276 0.0568 Neutral Molybdate A2 11.5760 0.0240 0.0578 Neutral Molybdate A3 13.3025 0.0259 0,0543 Average 0.0563 Same plus phosphate Bi 11.9395 0.0648 0.0407 Same plus phosphate Bl' 11.5370 0.0636 0.0403 Same plus phosphate B3 12.6880 0.0654 0.0398 Muscle Average 0.0417 0.0403 96.6 Magnesia Mixture A 4 14.5075 0.0303 0.0582 Magnesia Mixture A 5 10.0850 0.0219 0.0605 Magnesia Mixture A h 11.4130 0.0229 0.0559 Average 0.0582 Same plus phosphate B4 11.7065 0.0617 0.0373 Same plus phosphate B.i 11.3530 0.0606 0.0369 Same plus phosphate Br, 12.1045 0.0621 0.0368 Average 0.0417 0.0370 88.7 Neutral Molybdate Ai 29. 1950 0.03241 0.0309 0.0020 Neutral Molybdate A2 35.0955 0.03591- 0.0285 0.0016 Neutral Molybdate A3 28.4170 0.02571 0.0252 0.0018 Average 0.0282 0.0018 Same plus phosphate Bi 36.5820 0.07181 0.0348 Same plus phosphate Bi' 27.3368 0.06621 0.0385 Same pius phosphate B3 32.8275 0.06991 0.0367 Blood A verage 0.0417 0.0367 88.0 Magnesia Mixture Ai 30.9601 0.01571 0.0141 0.0030 Magnesia Mixture A5 32.'<669 0.01461 0.0125 0.0037 Magnesia Mixture Ae 29.2820 0.01511 0.0144 0.0038 Average 0.0137 0.0035 Same plus phosphate B4 35.6872 0.05451 0.0370 Same plus phosphate B:, 35.2757 0.05651 0.0392 Same plus phosphate Bi; 36.1812 0.05321 0.0354 Average ff.0417 0.0372 89.2 Magnesia Mixture A4 10.1650 0.02461 0.0575 0.0010 Magnesia Mixture A5 10.3040 0.02511 0.0679 0.0010 Magnesia Mixture Ae 10.9550 0.02671 0.0679 0.0006 Average 0.0678 0.0009 Same plus phosphate B4 10.5095 0.05121 0.0257 Same plus phosphate Bs 9. 2880 0.04831 0.0257 Same plus phosphate Be 10.5465 0.05141 0.0258 Brain Average 0.0417 0.0257 96.6 Magnesia Mixture 2A4 7.7011 0.01761 0.0662 0.0008 Magnesia Mixture 2A,i 9.2368 0.02091 0.0652 0.0006 Average 0.0007 Same plus phosphate 2B4 10.7215 0.05071 0.0263 Same plus phosphate 2E,^ 9.2277 O.O474I 0.0264 Same plus phosphate 2B(; 10.5182 0.05021 0.0263 Average 0.0267 0.0263 98.9 iBlanks deducted. INORGANIC PHOSPHORUS ESTIMATION 33 TABLE XIII: TEST OF EFFECTS OF HEAT AND AMMONIUM SULPHATE IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN BLOOD Blood of Pig Used; Extracted with 3.33 Percent Ammonium Sulphate Solution; Each Extract About 500 c. c. in Volume No.. Weight of sample Treatment Magnesium pyrophosphate Inorganic phosphorus Grams Grams Percent 1 2 Ave. 33.0990 34.4230 Extract evaporated; boiled; filtered 0.0217 0.0222 0.0183 0.0180 0.0181 3 4 Ave. 33.3960 31.3340 Extract evaporated; filtered 0.0216 0.0218 0.0180 0.0194 0.0187 5 6 Ave. 33.7026 33.2960 Extract precipitated directly 0.0108 0.0108 0.0089 0.0090 0.0089 CONCLUSIONS FROM WORK OF 1912 AND 1913 ON INORGANIC PHOSPHORUS ESTIMATION IN ANIMAL SUBSTANCES The neutral molybdate method of Emmett and Grindley, the barium chlorid method of Siegfried and Singewald, (provided a suffi- cient excess of barium chlorid is used) and the magnesia mixture method of Forbes and associates all gave satisfactory results, which were practically identical, on vacuum-dried muscle. The barium chlorid method was found inappHcable in the pres- ence of ammonium sulphate, and hence was not useful on extracts of blood and brain prepared with the aid of this reagent. The neutral molybdate method gave results on blood which were apparently too high, a decomposition of organic phosphorus seeming to result from the heat used during the concentration of the extract. Some difficulty was experienced in the recovery of inorganic phos- phorus added to blood. The recovery was slightly greater with the magnesia mixture than with the neutral molybdate method. As compared with the magnesia mixture method the neutral molybdate method gave, on extract of brain, prepared with the aid of ammonium sulphate, higher results for inorganic phosphorus, with lower recovery of added phosphates, (Table XI, p. 28). The difficulties of filtration are greater with the neutral molybdate than with the magnesia m.ixture method. Readily filterable extracts of brain may be prepared by the use of 3.33 percent ammonium sulphate solution in place of 0.1 percent ammonium sulphate in each place where the latter is specified in the 34 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 published magnesia mixture method (see p. 25) ; and the hinder- ing effect of the added amount of ammonium sulphate on the precipi- tation of phosphorus by magnesia mixture may be overcome by the substitution of 50 c. c. of magnesia mixture for the 10 c. c. as speci- fied, and allowing the precipitate to stand 3 days before filtering. With these modifications the magnesia mixture method is readily workable on brain ; concordant results are obtained, and added phos- phate is all recovered. The work on brain reported in Table XII, p. 32, was done by this modified method. In the trial reported as Samples A4, 5 and 6, and B4, 5 and 6, Table XII, p. 32, the phosphate was all recovered but 0.0009 gm. magnesium pyrophosphate. In the trial reported as Samples 2A4, 5 and 6, and 2B4, 5 and 6 the loss was 0.0004 gm. magnesium pyro- phosphate. This set was precipitated in a refrigerated room. The neutral molybdate method can not be used satisfactorily with extracts prepared as suggested above, with the aid of ammon- ium sulphate, and it is not practicable to prepare cold water extracts of brain as in the neutral molybdate method, which has been used principally with flesh. The test of the influence of heat on inorganic phosphorus esti- mation in blood, as set forth in Table XIII, p. 33, shows that the high results on blood obtained by the neutral molybdate method must be due to the cleavage of organic phosphorus by the heat used in the evaporation of the extract. While the duration of heating used in this method is much greater than in the preparation of hot water-ammonium sulphate extracts of tissues as in the magnesia mixture method, this test raises the question of the existence and magnitude of such cleavage. This point has received further con- sideration. The recovery of added phosphates from the extract of muscle by the magnesia mixture method is usually practically complete (see Table XI, p. 28). In the last analyses, however, (Table XII, p. 32), the recovery of added phosphate was appreciably incomplete, though the determination without the added phosphate was higher than by the neutral molybdate method, where the recovery of added phosphate was practically complete. The low recovery of added phosphate from both blood and muscle, as reported in this table, suggests that the conditions were not perfect for the precipitation of this amount of magnesium ammonium phosphate. These imper- fections were ehminated in the later work. With brain the recovery of added phosphate was complete, since special measures (added amounts of magnesia mixture, and increased time allowed for pre- cipitation) were taken to insure complete precipitation. INORGANIC PHOSPHORUS ESTIMATION 85 THE WORK OF 1914 In tlie previous work on inorganic phosphorus estimation on animal tissues three methods have been compared, namely, the Neutral Molybdate Method of Emmett and Grindley, the modified Barium Chlorid Method of Siegfried and Singewald, and the Mag- nesia Mixture Method of Forbes and associates. Satisfactory com- parisons of these methods have been made on muscle, the results, with this tissue, being practically identical ; and certain important limitations to the applicability of the two methods first mentioned, to tissues other than muscle, have been established. It was now desired to test, by the method of recovery of added phosphates, the accuracy of the Magnesia Mixture Method, in its latest form, with animal tissues of diverse character, and also to study individually, a number of details of this method, namely (1) the influence of heat, as specified, (2) the method of filtration, (3) the completeness of extraction, (4) the influence of ammonium sulphate, as specified, (5) the effects of varying amounts of ammonium sulphate and (6) the effects of diflferent methods of use of ammonium sulphate. In the test of the accuracy of the Magnesia Mixture Method, determinations were made, in triplicate, on blood, brain, flesh and liver, with and without the addition of known amounts of inorganic phosphate. The detailed directions followed in this test are on pages 35-37, and the results are set forth in Tables XIV and XV on pages 41 and 42. The results of the further analytical proving of the details of this method were all made on blood. The data are to be found- in Tables XVI-XXI on pages 43 to 48. WATER-SOLUBLE INORGANIC PHOSPHORUS IN ANIMAL TISSUES From samples of finely divided tissue prepare extracts as specified under A, B, C or D, and determine inorganic phosphorus as specified under E. A. PREPARATION OF COLD WATER EXTRACT OF FLESH Weigh out 10-12 grams of fresh muscle, and divide as nearly equally as possible between two small beakers. Moisten the samples with a few c. c. of distilled water, and break up lumps with a glass rod. Add 50 c. c. of water to each beaker and stir contents for 15 minutes. Allow insoluble residue to settle for 3-5 minutes; then decant the liquid from each beaker through filters into beakers; and add 25 c. c. of distilled water to the residue in the beakers. Stir for 7-8 minutes, and after allowing to settle, decant onto the same filter. Continue this treatment, using each time 25 c. c. of water, until the filtrates measure about 230 c. c. each. Allow the filters to drain completely between extractions. Whenever the major portion of the residue has become mechanic- ally transferred to the filter return it to the beaker, using care not to break 36 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 the filter paper. After the last extraction throw the entire contents of each beaker onto the filter, and, when drained, wash twice with small quantities of distilled water. Combine the two extracts, and determine inorganic phos- phorus as under E. B. PREPARATION OF HOT WATER-AMMONIUM SULPHATE EXTRACT OF BLOOD Weigh out 30-35 grams of fresh blood, (entire portions as caught from the animal) into a porcelain mortar. Grind and transfer to a 400 c. c. beaker with hot distilled water. Make up to about 150 c. c. with boiling distilled water. Place over a flame, and gradually bring to boiling, with constant stirring. When boiling begins add 20 c. c. of 20 percent ammonium sulphate solution. Boil, with constant stirring, for about ten minutes. Decant onto an 18 cm. filter paper, receiving the filtrate in an 800 c. c. beaker. When the liquid is through, lift the coagulum from the paper, being very careful not to break the paper filter, and transfer it, along with that remaining in the beaker, to the mortar. Grind to a smooth paste and transfer from mortar to beaker with boiling 3.33 percent ammonium sulphate solution. Make up to about 50 c. c. with the same, stir for 8 minutes, and pour contents again onto the filter paper. After the extract is through, return the coagulum to the mortar and grind a second time, transferring to the beaker as before with boiling 3.33 percent ammonium sul- phate solution. Repeat this process of 8-minute extractions of the coagulum in hot 3.33. percent ammonium sulphate solution, and filtration as above directed, without further grinding, until the filtrate measures about 450 c. c. Wash out each beaker twice with 8-10 c. c. of hot 3.33 percent ammonium sulphate solu- tion, completing the transfer of the coagulum and extract to the filter paper. Wash the coagulum on the paper twice with boiling 3.33 percent ammonium sulphate solution from a wash bottle. At all times allow the filter to drain well between additions of extract or wash solution. This extract of about 500 c. c. is ready for precipitation as described under E. C. PREPARATION OF HOT WATER-AMMONIUM SULPHATE EXTRACT OF LIVER Weigh by difi'erence from closed weighing bottles 15-20 gram portions of finely ground liver into 400 c. c. beakers. Add a few c. c. of cold distilled water, and beat up with a stirring rod to separate the particles of tissue. Add enough boiling distilled water to make the volume about 150 c. c; place over a flame and bring to boiling. Add 10 c. c. of 20 percent ammonium sulphate solution, and continue to boil for 10 minutes. Remove from the flame, allow to settle, for a moment and decant the boil- ing-hot liquid onto 18 cm. paper filters. Add 50 c. c. of boiling water and stir for 8 minutes, without further heating over a flame, and decant onto the filter again. Repeat this addition of 50 c. c. of hot distilled w^ater, stirring, and decanting eight times, returning the coagulum to the beaker as soon as any considerable amount collects upon the filter. With the eighth portion of water throw the entire contents of the beaker onto the filter and wash twice with hot water from a wash bottle. At all times allow the filter to drain well between additions of extract or wash water. This extract of about 600 c. c. is now ready for precipitation as described under E. INORGANIC PHOSPHORUS ESTIMATION 37 D. PREPARATION OF HOT WATER-AMMONIUM SULPHATE EXTRACT OF BRAIN Weigh out about ten grams of brain into a 250 c. c. beaker. Add a few cubic centimeters of distilled water, and work up the brain and water with a glass rod. [Make up to about 100 c. c. with boiling water; place over a flame, and gradually bring to boiling, with constant stirring. While boiling vigor- ously (not before) add 20 c. c. of 20 percent ammonium sulphate solution; boil gently for about 10 minutes; allow to settle for a moment, and decant liquid slowly onto a filter of acid-washed glass maker's sand on linen, receiving the extract in an 800 c. c. beaker. Add to the beaker containing the coagulum 50 c. c. of a hot 3.33 pei-cent ammonium sulphate solution. Stir for one minute, keeping over flame and at the boiling point; decant the liquid onto the filter. Repeat this process of one-minute extractions of the coagulum in hot 3.33 per- cent ammonium sulphate solution, and filtration as above directed, until the filtrate measures about 450 c. c. Wash out the beaker twice with 8-10 c. c. of hot 3.33 percent ammonium sulphate solution, completing the transfer of the coagulum and extract to the sand. Wash the coagulum twice with the above wash solution from a wash bottle. At all times allow the filter to drain well between additions of extract or wash solution. This extract of about 500 c. c. is ready for precipitation as directed under E. PRECAUTIONS In making extracts of brain it is desirable that the analyst give careful attention to the handling of the sample. The coagulum is very soft. It should be stirred only enough to keep it in motion. If roughly handled in returning from the sand filter to the beaker it becomes too much broken up and holds onto a great deal of liquid. To prevent the extract or the coagulum from coming into contact with the linen before passing through the sand pour the extract slowly into a slight depression in the center of the sand, or, better yet, onto a thin film of absorbent cotton l^^ inches in diameter, laid over a de- pression in the sand. ,The coagulum remains on the cotton, and its return to- the beaker is thereby facilitated. If the cotton is not broken up by needless stirring it can be taken out of the beaker with a glass rod and returned to the sand each time a partial extract is to be filtered. Care is necessary to prevent loss through bumping, on account of sand in the beakers during the last extrac- tions. Each partial extract should be boiling hot at the time filtration begins. E. MAGNESIA MIXTURE METHOD FOR THE DETERMINATION OF WATER-SOLUBLE INORGANIC PHOSPHORUS IN EXTRACTS OF ANIMAL TISSUES To the extracts prepared according to the preceding directions add 50 c. c. magnesia mixture, stirring freely. Allow to stand 15 minutes; add 25 c. c. ammonium hydroxid, sp. gr. 0.90; cover, and allow to stand three days. Filter, and wash the precipitate with 2.5 percent ammonia water. Dissolve the pre- cipitate on the filter paper and that remaining in the beaker in which the pre- cipitation was made with dilute nitric acid (1:1) and hot water, receiving the solution in 400 c. c. beakers. Neutralize the nitric acid with ammonium hydroxid; make slightly acid with nitric acid. Add 5 grams ammonium nitrate, and precipitate in the usual way with molybdate solution. Continue in the usual way for the gravimetric estimation of phosphorus as the pyrophosphate. 38 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 DISCUSSION OF RESULTS OF WORK OF 1914 The data in Tables XIV and XV, pages 41 and 42, show that as tested by the recovery of added phosphates, the Magnesia Mix- ture Method, in the form stated on pages 35-37, gives results appar- ently characterized by a high degree of accuracy. The recovery of added phosphates was 96 percent efficient with liver, 97 percent with flesh, 99 percent with brain and 100 percent with blood. In consideration of the close agreement of triplicates, the high percentage of recovery of added phosphates, and the amounts of coagulum from which the phosphates were recovered, these results are considered a satisfactory demonstration of the reliability of the method. In the further scrutiny and analysis of the method, however, it was deemed advisable to test individually certain of its details. Blood was selected for this work, since the ready decomposition of its phosphocarnic acid was considered likely to reveal possible im- proprieties of procedure. The results of these studies on blood are set forth in Tables XVI to XXI on pages 43-48. Table XVI, page 43, gives results from a study of the effects of heat and ammonium sulphate in this estimation. A cold-water ex- tract of steer blood was used. This extract was obtained through the use of a centrifuge. In sets A and C the phosphates were precipitated direct, with magnesia mixture, with and without ammonium sulphate added (in the cold) before precipitation. The results were practically identi- cal, and show that, in the cold, ammonium sulphate does not affect inorganic phosphate determination in blood. Sets B and D were boiled containing different amounts of am- monium sulphate. The boiling and precipitation of inorganic phos- phates in 1.25 percent solution of ammonium sulphate (20 c. c. of 20 percent ammonium sulphate, as specified) gave weights of mag- nesium pyrophosphate half of a milligram greater than those obtained from boiling and precipitation in a 3.33 percent solution of ammonium sulphate. These results were, in both cases, appreciably lower than those obtained from A and C, with and without ammonium sulphate, but without boiling. These results show, therefore, that ammonium sulphate, in the cold, is without influence on the inorganic phos- phorus estimation, but that boihng and ammonium sulphate to- gether, as used, not only do not split off inorganic from organic phosphorus compounds, but, as shown by the lower results obtained, cause a coagulation and precipitation of organic phosphorus in the INORGANIC PHOSPHORUS ESTIMATION 39 water extract which when not so precipitated remains in solution until precipitated by the magnesia mixture, after which it may be hydrolyzed by nitric acid in the later steps of the phosphorus esti- mation. Considering the possibility that the lower results above noted as obtained in the presence of ammonium sulphate might be due to the mechanical inclusion of phosphates in the coagulum, another set of determinations was made, as reported in Table XVII, p. 44. The grinding of the coagulum with sand, to allow of more complete ex- traction and washing, gave exactly the same result as did the wash- ing of the coagulum by decantation, in the usual way. Therefore, the extraction, as usually carried out, is complete, and coagulation by boiling and ammonium sulphate does not lock up inorganic phos- phate by mechanical inclusion. Further, as in the previous set of analyses, lower results were obtained with boiling and ammonium sulphate than with direct precipitation in the cold, though the re- covery of added phosphates was perfect in both cases. This rein- forces our previous observation as to the precipitation of organic phosphorus from cold-water extracts of blood, along with the inor- ganic phosphates. Thus, boiling and ammonium sulphate are needed to coagulate a certain water-soluble organic phosphorus frac- tion of blood in the estimation of inorganic phosphorus by the Magnesia Mixture Method. The results in Table XVIII, p. 45, show that acid alcohol (0.2 percent nitric acid) will dissolve the organic phosphorus which is precipitated, along with the phosphates, by magnesia mixture alone, in cold-water extracts of blood ; a separation of the organic from the inorganic phosphorus in this precipitate, by the use of this reagent, therefore, is not possible. In Table XIX, p. 46, we have results from tests made to deter- mine ( 1 ) whether hot water or ammonium sulphate should be used in the completion of the extraction of the coagulum from the boiling with ammonium sulphate, and (2) whether, in the extraction of blood, the partial extracts should be filtered through sand on linen or through filter paper. Lower results (and, therefore, in the light of the previous evi- dence, more nearly correct results) were obtained when a 3.33 per- cent solution of ammonium sulphate, rather than hot water, was used in the completion of the extraction of the coagulum. The re- covery of added phosphates was also higher under these circum- stances. Filtration of the blood extracts through paper was found preferable to filtration through sand on linen. 40 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 Table XX, p. 47, reports a further test of the desirability of using ammonium sulphate in the completion of the extraction of the coagulum from the preliminary boiling with ammonium sulphate. As in the previous work the results obtained favored the use of the 3.33 percent solution, since this procedure led to lower results for inorganic phosphorus and more nearly perfect recovery of added phosphates. Table XXI, p. 48, sets forth results from a comparison of the use of different amounts of ammonium sulphate in the coagulation and extraction of blood. No advantage could be demonstrated as due to the use of solutions of ammonium sulphate more concentrated than the 3.33 percent solution used in the preceding tests ; that is, the use of 3.33 percent solutions gave lower, and apparently more nearly correct results than were obtained with a 1.25 percent solu- tion, while further increase of the concentration of the ammonium sulphate solution did not lead to further decrease in inorganic phosphate. CONCLUSIONS FROM WORK OF 1914* ON INORGANIC PHOS- PHORUS ESTIMATION IN ANIMAL SUBSTANCES (1) The Magnesia Mixture Method gives satisfactorily agree- ing results on blood, brain, liver, and flesh, with a recovery of 96-100 percent of added phosphates. (2) Neither ammonium sulphate, nor boiling and ammonium sulphate together, as used in the Magnesia Mixture Method, were found to cause a splitting off of inorganic from organic phosphorus in blood. (3) The use of heat and ammonium sulphate, as in the Mag- nesia Mixture Method, gives lower results than are obtained without heat and ammonium sulphate, though the recovery of added phos- phates is perfect ; and evidence was obtained that these lower results were due not to inclusion of phosphates in the coagulum obtained by the use of heat and ammonium sulphate, but to the precipitation of water-soluble organic phosphorus compounds which, without the use of heat and ammonium sulphate, yield up their phosphorus as inorganic phosphate, under the influence of the nitric acid used in the subsequent steps of the inorganic phosphorus estimation. (4) It was found advisable to wash the coagulum with 3.33 per- cent ammonium sulphate rather than with hot water. A more con- centrated solution was shown not to be necessary. (5) In the case of blood, the filtration of the extract through paper was found preferable to the filtration, through sand on linen, which is necessary in the case of brain. *For conclusions from the work of 1912 and 1913 see pag-es 33 and 34. INORGANIC PHOSPHORUS ESTIMATION 41 (6) The methods of determination of inorganic phosphorus in blood, brain, flesh and liver as outlined on pages 35 to 37, including the details of extraction as well as of actual estimation of phos- phates, were provisionally adopted in 1914 as official methods of the Association of Official Agricultural Chemists. For conclusions from the work on vegetable substances see page 21. TABLE XIV: TEST OF MAGNESIA MIXTURE METHOD FOR INORGANIC PHOSPHORUS IN ANIMAL TISSUES BY RECOVERY OF ADDED PHOSPHATES A=Without Phosphates B— With Added Phosphates Added phosphorus Phosphorus added (magnesium recovered Sample and Weight Magnesium Inorganic determination of pj'rophos- phosphorus pyrophos- Magnesium No. sample phate phate) pyrophos- phate Percent Grams Grams* Percent Grams Grams Blood Ai 31.30 0.0069 0.00614 A 2 30.00 0.0069 0.00641 As 25.00 0.0051 0.00568 Average 0.00607 Bi 26.10 0.0558 0.0501 B2 28.20 0.0563 0.0501 Bs 30.50 0.0572 0.0505 Average 0.0497 0.0502 101.00 Ai 33.70 0.0064 0.00529 A2 33.60 0.0060 0.00505 As 31.20 0.0060 0.00544 Averag'e 0.00526 Bi 30.40 0.0550 0.0493 B2 32.20 0.0557 0.0496 B3 35.80 0.0568 0.0500 Averag-e 0.0496 0.0496 100.00 *AU blanks deducted. 42 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE XV: TEST OF MAGNESIA MIXTURE METHOD FOR INORGANIC PHOSPHORUS IN ANIMAL TISSUES BY RECOVERY OF ADDED PHOSPHATES A=Without Phosphates B=With Added Phosphates Weight of sample Grams Magnesium pyrophos- phate Grams* Inorganic phosphorus Percent Phosphorus added (magnesium pyrophos- phate) Grams Added phosphorus recovered Sample and determination No. Magnesium pyrophos- phate Graas Percent Brain Ai Ao A3 Average Bi B2 B3 Average 8.5600 7.7011 9.2368 10.7215 9.2277 10.5182 Lost 0.0176 0.0209 0.0507 0.0474 0.0502 0.0636 0.0630 0.0633 - 0.0266 0.0263 0.0264 0.0263 0.0263 98.87 Flesh Ai A2 A3 Average Bi B2 B3 Average 13.4653 10.3444 11.1769 10.9638 11.7942 11.3154 0.0272 0.0207 0.0223 0.0694 0.0725 0.0708 0.0562 0.0557 0.0555 0.0558 0.0496 0.0474 0.0488 0.0481 0.0481 96.97 Liver Ai A2 A3 ' Average Bi B2 B3 Average 16.2155 13.8000 14.4309 14.9094 14.9658 16.2232 0.0627 0.0552 0.0519 0.1045 0.1054 0. 1094 0.1077 0.1144 0. 1002 0. 1064 0.0496 0.0475 0.0482 0474 0.0477 96.16 *A11 blanks deducted. INORGANIC PHOSPHORUS ESTIMATION 43 TABLE XVI: TEST OF EFFECTS OF BOILING AND VARYING AMOUNTS OF AiMMONIUM SULPHATE IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN STEER BLOOD BY THE MAGNESIA MIXTURE METHOD— COLD WATER EXTRACTS Sample No. Treatment Volume extract c. c. Mag- nesium pyro- phosphate Gms. Phos- phorus Mgs. Ai A2 A3 Average Extract precipitated direct with magnesia mixture 300 300 300 0.0091 0.0087 0.0087 2.535 2.424 2.424 2.461 Bi B2 B3 Average Extract brought to boiling; ammonium sulphate added to make 1.25 percent solution; then boiled for ten minutes 300 300 300 0.0079 0.0078 0.0081 2.201 2.173 2.257 2.210 Ci C2 C3 Average Same as A, with ammonium sulphate to make 1.25 percent solution added before precipitation 300 300 300 0.0085 0.0085 0.0086 2.368 2.368 2.396 2.374 Di D2 A verage Same as B, with ammonium sulphate added to make 3.33 percent solution 300 300 300 0.0075 0.0074 0.0073 2.090 2.062 2.034 2.062 All of the above extracts and filtrates were precipitated by adding 50 c. c. of magnesia mixture to the cool solution, and then, after standing a short period, 25 c. c. of ammonia (sp. gr. .96). 44 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE XVII: TEST OF COMPLETENESS OF EXTRACTION AND EFFECTS OF BOILING AND AMMONIUM SULPHATE IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN CALF BLOOD BY THE MAGNESIA MIXTURE METHOD- COLD WATER EXTRACTS Volume of ex- tract c. c. Magne- sium pyro- phos- phate Grams Phos- phorus added (magne- sium pyro- phos- phate) Grams Added phosphorus recovered Sample No. Treatment Magne- sium pyro- phos- phate Grams Percent Al A 2 A3 Average Extract precipitated direct with magnesia mixture 300 300 300 0.0107 0.0105 0.0103 0.0105 A4 As A6 Average Same as A l, 2, 3 + 25 c. c. of phosphate solution 300 300 300 0.0619 0.0618 0.0620 0.0619 0.0517 0.0514 98.42 Bi B2 Bs' Average Extract brought to boiling; 20 c. c. of 20^ am- monium sulphate added, and boiled for 10 minutes, filtered and washed by decantation 300 300 300 0.0083 0.0086 0.0091 0.0087 B4 Bs Be Average Same as Bi, 2, 3 but coagulum ground with fine sand for more complete extraction and washing 300 300 300 0.0090 0.0084 0.0088 0.0087 Ci C2 C3 Average Same as Bi, 2, 3 + 25 c. c. phosphate solution 300 300 300 0.0603 0.0601 0.0604 0.0603 0.0517 0.0516 99.86 C4 Co C6 Average Same as B4, s, 6+25c.c. phosphate solution 300 300 300 0.0601 0.0609 0.0602 0.0604 0.0517 0.0517 100.00 All of the above extracts and filtrates were precipitated by adding 50 c. c. of magnesia mixture to the cool solution, and then, after standing a short period, 25 c. c. of ammonia (.sp. gr. .96). INORGANIC PHOSPHORUS ESTIMATION 45 TABLE XVIII: TEST OF EFFECTS OF BOILING AND AMMONIUM SUL- PHATE IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN STEER BLOOD BY THE MAGNESIA MIXTURE METHOD— COLD WATER EXTRACTS Sample No. Treatment Volume of extract c. c. Mag- nesium pyro- phosphate Grams Phos- phorus Mg. Ai A2 A3 Average Extract precipitated direct with magnesia mixture 200 200 200 0.0075 0.0070 0.0070 0.0072 2.089 1.950 1.950 1,996 A4 A5 A6 Average Extract precipitated as Ai. 2. sand precipitate dissolved in acid alcohol (.2% nitric acid) and phosphorus determined in aliquots of this solution 200 200 200 0.0076 0.0073 0.0065 0.0071 2.117 2.034 1.811 1.987 Bi B2 B3 Average Extract boiled for 20 minutes with 20 c. c. of 20% ammonium sulphate, filtered and precipitated direct 200 200 200 0.0062 0.0058 0.0096* 0.0060 1.727 1.616 1.671 *Precipitate fused during ignition; not included in average. All of the above extracts and filtrates were precipitated by adding 50 c. c. of magnesia mixture to the cool solution, and then, after standing a short period, 25 c. c. of ammonia, (sp. gr. .96). 46 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE XIX: TEST OF METHODS OF USE OF AMMONIUM SULPHATE AND OF METHODS OF FILTRATION IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN STEER BLOOD BY THE MAGNESIA MIXTURE METHOD— HOT WATER- AMMONIUM SULPHATE EXTRACTS Sample No. Treatment Weight of sample Grams Mag- nesium pyro- phos- phate Grams Phos- phorus added (mag- nesium pyrophos- phate) Grams Added phorus re Mag- nesium pyrophos- phate Grams phos- covered Percent 1 2 3 Average Sample extracted in usual way with 20 c. c. of 20^ ammonium sulphate; filtered through sand on linen 34.2 26.6 30.7 30.5 1.0 0.0079 0.0061 0.0064 0.0068 0.000223 4 5 6 Average Same as 1, 2, 3 -j- 25 c. c. phosphate solution 30.7 33.1 38.2 0.0583 0.0582 0.0590 0.0537 0.0537 0.0537 0.0515 0.0508 0.0505 0.0509 94.78 7 8 •9 Average Sample extracted as usual; then subsequent extractions made with 3.33% hot ammonium sulphate; filtered through sand on linen 28.4 34.2 39.1 33.9 1.0 0.0040 0.0060 0.0069 0.0056 0.000165 10 11 12 Average Same as 7, 8, 9-|-25c. c. of phosphate solution 37.6 40.9 32.4 0.0590 0.0590 0.0576 0.0537 0.0537 0.0537 0.0528 0.0522 0.0522 0.0524 97.57 13 14 Average Sample extracted usual way, + 25 c. c. phos- phate solution: filtered through paper in- stead of sand on linen (4, 5 and 6) 32.6 33.9 0.0583 0.0596 0.0537 0.0537 0.0510 0.0520 0.0515 95.90 15 16 Average Sample extracted usual waj'-r25 c. c. phos- phate solution; subsequent extraction with 3.33^ ammonium sulphate; filtration through paper instead of sand on linen (10, 11. 12) 31.5 37.4 0.0600 0.0603 0.0537 0.0537 0.0548 0.0541 0.0544 101.303 The above filtrates were precipitated by adding 50 c. c. of magnesia mixture to the cool solution, and then, after standing a short period, 25 c. c. of ammonia (sp. gr. .96). INORGANIC PHOSPHORUS ESTIMATION 47 TABLE XX: TEST OF METHODS OF USE OF AMMONIUM SULPHATE IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN STEER BLOOD BY THE MAGNESIA MIXTURE METHOD— HOT WATER-AMMONIUM SULPHATE EXTRACTS Treatment Weight of sample Grams Magne- sium pyro- phos- phate Grams Phos- phorus added (magne- sium pyro- phos- phate Grams Added phosphorus recovered Sample No. Magne- sium pyro- phos- phate Grams Percent Ai A2 A3 Average Sample extracted with 20 c. c. of 20* am- monium sulphate as usual: all subse- quent extractions made with hot water 32.0 32.8 34.6 33.1 1.0 0.0078 0.0080 0.0083 0.0080 0.00024 A4 As Ae Average Same as Ai, 2. 3 + 25 c.c. phosphate solution 33.5 34.1 26.3 31.3 0.0573 0.0569 0.0556 0.0566 0.0496 0.0491 98.99 Bi B2 B3 Average Same as Ai, 2. 3 only all subsequent ex- tractions made with S.SS'/o ammonium sulphate instead of water 33.7 33.6 31.2 32.8 1.0 0.0064 0.0060 0.0060 0.0061 0.00019 B4 B5 Be Average Same as Bi, 2. 3+25c. c. phosphate solution 30.4 32.2 35.8 32.8 0.0550 0.0556 0.0568 0.0558 0.0496 0.0497 100.20 Blank i 3 0.0005 0.0005 0.0004 *A11 blanks deducted. All of the above filtrates were precipitated by adding 50 c. c. magnesia mixture to the cool solution, and then, after standing a short period, 25 c. c. of ammonia (sp. gr. .96). 48 OHIO EXPERIMENT STATION: TECHNICAL BUL. 8 TABLE XXI: TEST OF VARYING AMOUNTS OF AMMONIUM SULPHATE IN THE ESTIMATION OF INORGANIC PHOSPHORUS IN STEER BLOOD BY THE MAGNESIA MIXTURE METHOD— HOT WATER-AMMONIUM SULPHATE EXTRACTS Sample No. Treatment Weight of sample Grams Mag- nesium pyro- phos- phate* Grams Phos- phorus added (mag- nesium pyro- phos- phate) Grams Added phos- phorus recovered Mag- nesium pyro- Percent phos- phate Grams Ai A2 A3 Average Sample extracted with 3.32'i ammonium sulphate throughout 31.3 30.0 25.0 28.76 1.0 0.0069 0.0069 0.0051 0.0063 0.00022 A4 As A6 Average Same as Ai, 2, 3 + 25 c. c. phosphate solution 26.1 28.2 30.5 28.3 0.0558 0.0563 0.0572 0.0564 0.0497 0.0502 101.00 Bi B2 B3 Average Sample extracted with 4i ammonium sulphate throughout 27.8 27.0 30.8 28.5 1.0 0.0071 0.0069 0.0074 0.0071 0.00025 B4 Bo Be Average Same as Bi, 2, 3 + 25 c. c. phosphate solution 31.4 31.2 41.3 34.6 0.0584 0.0579 0.0576 0.0580 0.0497 1 0.0493 99.20 Ci C2 C3 Average Sample extracted with 5% ammonium sulphate throughout 28.1 28.2 30.5 28.9 1.0 0.0065 0.0070 0.0067 0.0067 0.00023 , C4 Cs C6 Average Same as Ci, 2. 3 + 25 c. c. phosphate . solution 30.5 26.9 45.5 34.3 0.0582 0.0575 0.0589 0.0582 0.0497 0.0503 101.21 C7 500 c. c. of 5* ammonium sulphate -+- Cs 25 c. c. phosphate solution Average 0.0498 0.0496 0.0497 0.0497 j 100.00 All of the above filtrates precipitated by adding 50 solution, and then, after standing for a short period, 25 c. *A11 blanks deducted. c. magnesia mixture to the cool of ammonia (sp. gr. .96). COLUMBIA UNIVERSITY LIBRARIES This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the rules of the Library or by special arrange- i ment with the Librarian in charge. 1 DATE BORROWED DATE DUE DATE BORROWED DATE DUE c2e(n4t)Mioo QP535.P1 Forbes F742