Determination of Organic Phosphorus in Soils R. W . PEARSOK, Iowa State College, Ames, Iowa Reagents
.A procedure for the determination of organic phosphorus i n soils is described in which the organic compounds of phosphorus are remoied from the soil by acid extraction followed b j digestion in dilute ammonium hldroxide. Organic phosphorus is found b3 difference between inorganic and total phosphorus i n the extracts. The effects of different factors on the amounts of organic phosphorus extracted are reported and comparisons are made with results obtained by the use of two other procedures. Recovery of added organic phosphorus ranged from 93.1 to 100.6 per cent, with an aFerage for four soils of 98.2 per cent. The procedure is inexpensiie and reasonablj rapid and the results obtained are reproducible within narrow limits.
AivfllhioNInf HYDROXIDE FOR EXTRACTION. Dilute 90 cc. of concentrated ammonium hydroxide to 1 liter and adjust by titration to approximately 0.5 X . SULFURIC ACIDFOR ADJCSTISGACIDITY. Dilute concentrated sulfuric acid to 35 times its volume and adjust to approximately 1.0 av. AMMOS1Uh.f ~~IOLYBDATE-SULFTRIC ACID SOLUTIOS.Dissolve 25 grams of ammonium molybdate in water heated to 60" C. and filter. Dilute 275 cc. of concentrated sulfuric acid to 800 cc. Cool both solutions and add the ammonium molybdate solution to the sulfuric acid s l o ~ l yand with stirring. Cool to room temperature and dilute with water t o 1000 cc. S T A S N O U S C H L O R I D E SOLUTIOh-. Dissolve 25 grams of stannous chloride in 100 cc. of xarm concentrated hydrochloric acid. Dilute to 1000 cc. and filter if cloudy. SDARD PHOSPHATE SOLCTIOS.Dissolve 0.2195 gram of tallized potassium dihydrogen phosphate in water and dilute t o 1000 cc. Dilute 50 cc. of this stock solution to 500 cc. This solution contains 5 p. p. m. of phosphorus and is diluted as required for preparation of final standards. Cover the 5 p. p. m. standard with a layer of toluene and store in a dropping funnel. ~-KITROPHESOL ISDICATOR. Dissolve 0.5 gram of solid pnitrophenol in 100 cc. of water and filter if cloudy. CARBONBIACICFOR DECOLORIZATION. If the carbon black s h o w a test for phosphorus, wash it on a Biichner funnel with 1 to 1 hydrochloric acid until no test can be found in the filtrate. Wash the acid out with n-ater, dry, and pulverize.
PHOSPHORUS
in organic combination occurs in relatively large amounts in many soils, amounting in some cases t o more t h a n half of the total phosphorus in the surface six inches. I n spite of the importance of this fraction, especially from the soil fertilit'y viewpoint, no method has been developed for its determination that will give entirely dependable results regardless of the composition of the soil. The method of Potter and Benton ( 6 ) and its modification by Schollenberger (6) were the most satisfactory of the earlier methods, although too long to be satisfactory for general work. This method was based on the assumption that organic compounds containing phosphorus were completely extracted by shaking in the cold with dilute ammonium hydroxide. IiarpinskiI and Zamyatina (3) in 1933 proposed a procedure using hydrogen peroxide for oxidation of the organic phosphorus compounds; the difference between the phosphorus removed by acid extraction of oxidized and unoxidized samples was assumed to represent the organic phosphorus. The chief objections to this type of procedure are the cost of hydrogen peroxide, fixation by the soil of phosphorus released from organic combination by the peroxide treatment, and difficulty in obtaining filtrates free from inorganic colloids. Also, Pet'erson ( 4 ) reported that hydrogen peroxide treatment increased the solubility of iron and aluminum, but not of calcium and magnesium, and that this increase followed the increase in phosphorus solubility. This led Peterson t o believe that peroxide treatment brought some phosphorus into solution from iron and aluminum phosphates. An attempt to fractionate the soil phosphorus was reported in 1938 by Dean (I), who believed that the organic phosphorus was soluble in 0.25 S sodium hydroxide. In t,he same year Dickman and DeTurk (8)proposed a method using hydrogen peroxide oxidation folloxed by sulfuric acid extraction for determination of organic phosphorus. The difference betiveen the phosphorus extracted from an untreated sample by 0.2 N sulfuric acid and that extracted from a sample previously oxidized FTith hydrogen peroxide was designated as organic.
TABLE I. SOILSUSEDIN STUDY Depth Of
Soil Kebster silt loam Carrington silt loam T a m a silt loam Fayette silt loam Orangeburg sandy loam Houston clay Red clay Rosebud s a n d s loam Holdiege very fine s a n d s loam .ilkali soil
Sampling p H Inches 0-6 7.63
Soil Group
5 . 2 4 Prairie 5 , 10 Prairie 6 , 3 5 Gray-brown Podzolic 5 , 2 2 Red and yellow 6 . 5 0 Rendzina 6 . 15 Laterite
0-12
7.29
Iowa Iowa Iowa Iowa Mississippi Uississippi Hawaii Sebraska
0-12
6.55
Nebraska Sebraska
0-6 0-6 0-1.5 0-6 0-6
. .
...
Wesenboden
Source
Chestnut
Chernozem 9 . 10 Solonetz
Procedure PRELIivrsaRY EXTRACTION OF BASES. Weigh duplicate 1gram samples (0.5 gram if the soil contains large amounts of organic matter) of 60-mesh soil into a small beaker, add 20 cc. of 0.1 N hydrochloric acid to each, and let stand for several minutes. If the soil is calcareous or contains perceptible quantities of undecomposed plant tissue, place the beaker on a steam chest for about 5 minutes. Filter through phosphorus-free paper and wash the soil n-ith small portions of 0.1 N acid until no test,for calcium can be detected in the filtrate; this generally requires six to eight washings. Make the acid filtrate to volume in a 200cc. volumetric flask and save. Transfer the acid-washed soil EXTRACTIOS OF PHOSPHORUS. jvith the filter paper to a 500-cc. Erlenmeyer flask graduated at 400 cc. Add 200 t o 300 cc. of 0.5 N ammonium hydroxide, stopper, and shake vigorously until the filter paper is thoroughly shredded. Rinse the stopper and sides of the flask and make to volume with 0.5 S ammonium hydroxide. Close the flask with a rubber stopper fitted TTith Bunsen valves as shown in Figure 1, and digest in an oven a t 89" to 91" C. for 16 to 18 hours. By the use of these valves the loss of ammonia during digestion is kept a t a minimum. After digestion is completed, cool the flask in a running mater bath to room temperature and add 5 grams of ammonium chloride, Adjust the volume in the Erlenmeyer to the 400-cc. mark, mix thoroughly by shaking, and let stand for a few minutes until the bulk of the suspended material settles out. Decant through a close, phosphorus-free filter paper, discarding the filtrate as long as any suspended material can be detected in it. Pipet aliquot, portions of the clear filtrate into two thoroughly aeathered 100-cc. beakers and add proportionate amounts of the acid
This investigation was undertaken, following a preliminary study of available methods, in a n attempt' to perfect a procedure t h a t would be independent of the t'ype of soil and a t the same time low enough in cost of operation t o be acceptable for routine rrork.
Soils Used T h e soils selected for this study, listed in Table I, were cleveloped under Tyidely different climatic conditions and on diff erent kinds of parent materials, representing nine of the great soil groups. Thus, large variations were obtained not only in organic phosphorus content but also in the general composition of the soils. All samples were ground to pass a 60-mesh screen and thoroughly mixed. 198
AXALYTICAL EDITION
APRII, 15, 1940
199
EXTRACTION OF BASESox TABLE 11. EFFECTOF PRELIMISARY ORGANIC PHOSPHORUS EXTRACTED BY AMh%oNIU&f HYDROXIDE
--
NO
Soil
extraction P.p.m. 200 300 220
Tama Webster Fayette
t o mix t h e suspension
I
thoroughly; filter, rinse the
p-nitrophenol and 1 to 1 ammonium hydroxide until the
I '3
Organic Phosphorus0 . 1 .v SlH oi% Cl HCI P.p.n?, P,p.m. 212 249 364 392 322
TABLE 111. ORGANIC PHOSPHORVS REMOVED DURING ACID EXTRACTION OF BASES Phosphorus Removed 0 1 N HzSOa
so11
0 1 ,V HC1 P . p . m.
Tama Webster Fayette
3 39 4
7
Soil Tama Webster Fayette Carrington
.
.
Factors Affecting Amount of Organic Phosphorus Extracted The folloJving five factors were studied separately during the development of this procedure: (1) preliminary extraction of bases, (2) length of digestion period, (3) volume of digesting solution, (4) concentration of digesting solution, and ( 5 ) temperature of digestion. The point of maximum recorery of organic phosphorus was debermined for each of these factors as given below. 1. The values for organic phosphorus found in two soils without extraction of bases and after extraction with different reagents are given in Table 11. It is apparent a t once that larger amounts of organic phosphorus were found after extraction of replaceable bases than before they Tvere removed, particularly when hydrochloric or sulfuric acid was used as the extracting reagent. This agrees with the results reported by Schollenberger ( 6 ) ,~ d i ofound the use of hydrochloric acid
P. p.
m.
4 20 2
superior to extraction with salt solutions. Hydrochloric acid was adopted because fewer subsequent washings were required than with sulfuric acid. Some organic phosphorus is removed by the acid extraction, as shown in Table 111, although in most cases the amount is small. However, since in some soils this fraction is significant, aliquots of t'he acid extract are added to the ammonium hydroxide extract used for analysis. TABLE IV. ORGANIC PHOSPHORUS EXTR.4CTED PERIODS OF DIGESTION
cc. of standard X concentration of standard (p. p. m.) X cc. of x dilution of X DETERMISATION O F TOTAL PHOSPHORUS I N EXTRACT.Evaporate the second aliquot to dryness with 1 cc. of 10 per cent phosphorus-free magnesium nitrate, and ignite in a muffle furnace at 600' C. until a perfectly white ash is obtained. Dissolve the residue in 4 cc. of 1.0 N sulfuric acid and dilute to about 40 cc. with water. Adjust the acidity and add ammonium molybdate as directed for inorganic phosphorus. Prepare phosphorus standards of the required concentrations, usually between 0.10 and 0.25 p. p. m., and adjust the acidity, using the same amount of sulfuric acid as in the soil extract; add ammonium molybdate, develop the colors in standard and unknox-n solutions, and determine phosphorus as before. Organic phosphorus is found by subtracting inorganic phosphorus from total phosphorus in the ammonium hydroxide and acid extracts.
7
0.1 N HgSOr P.p.m. 235 396 323
9 Hours P,p.n.
...
288
...
198
\VITH
Organic Phosphorus 12 Hours 15 Hours P,p,m. P.p.m. 237 240 367 386 345! 298 22 1 237
DIFFERENT 18 Hours P,p,m. 242 390 341 239
2. The effect of time of digestion on recovery of organic phosphorus from four soils is shown in Table IV. I n each case the amount of phosphorus found increased with time of digestion to the 15-hour mark, beyond which very little increase was obtained. il 16- or 18-hour digestion period is convenient, since samples weighed out and ext,racted with acid in the afternoon can digest overnight; the determination can then be completed the next morning. By arranging the work in this way six to eight determinations in duplicate can be completed each day. TABLE V.
EFFECTOF VOLUME OF EXTRACTING SOLUTION ON ORGANIC PHOSPHORUS EXTRACTED Xeigh t of
Soil
Tama Webarer Carlington 3Iarshall
Sample Grnms 1 0 0 5 1 0 1 0
- 4 5 .V SIIaOH per Sample of Soil-100 cc. 200 cc. 400 cc. 600 cc. P , p , m . P.p.m. P.g.m. P.p.m 230 242 226 194 367 392 390 262 240 217 237 246 239 180 214
3. The data obtained from a study of the effect of volume of ammonium hydroxide used on the amount of organic phosphorus extract'ed are presented in Table V. Values for organic phosphorus increased with dilution in every case to 400 cc., although in general there was less difference between 200 and 400 cc. than between 100 and 200 cc. S o significant difference was found between 400 and 600 cc. Since the dilution a t 400 cc. is rather high, any error in sampling or in the actual determination is greatly magnified. For this reason the accuracy of the results obtained can be seriously impaired by careless technique, especially in the color comparison between standard and unknown solutions. The use of a photoelectric colorimeter for determination of phosphorus in the solution would reduce the chances of error a t this point in the procedure. 4. The amounts of organic phosphorus found were considerably higher when 400 cc. of 0.4 ammonium hydroxide
VOL. 12, NO. 4
INDUSTRIAL AND ENGINEERING CHEMISTRY
200
OF AMMONIUM TABLE VI. EFFECTOF CONCENTRATION HYDROXIDE ON ORGANIC PHOSPHORUS EXTRACTED
0.2 N NHiOH P.p.m. 225 167 123
Soil Tama Webster Carrington
Organic Phosphorus 0.4 N 0.5 N "4OH NHkOH P.p.m. P.p,m. 248 247 390 394 237 237
1.0 N "4OH P.p.m. 247 388 240
were used for digestion than when the same volume of a 0.2 N solution was used, as shown in Table VI. No significant difference was found in the phosphorus removed by 0.4 N , 0.5 A', and 1.0 iV ammonium hydroxide. Half-normal solutions were adopted because less acid is required to neutralize the aliquots for inorganic phosphorus than if 1.0 Ar solutions are used. OF DIGESTION ON TABLE VII. EFFECTOF TEMPERATURE ORGANIC PHOSPHORUS EXTRACTED FROM THREESOILS 70' C. P.p.m.
Soil Tama Webster Fayette
,
207 295 287
Phosphorus Extracted 87' C. 900 c. P.p.m. P.p.m. 240 249 392 363 342 339
c.
920 P.p.m. 248 388 334
5 . The effect of temperature of digestion on organic phosphorus recovered from three soils is shown in Table 1'11. The optimum range is about 89" to 92" C. Recovery decreases with decreasing temperatures below 87" C., and increases slightly with increasing temperature above 87 '. Although no data are presented for temperatures above 92", erratic results are obtained a t temperatures of 94" to 95", probably due to decreased concentration of the ammonium hydroxide, and possibly also to some hydrolysis of organic phosphorus compounds. TABLE VIII. ORGANIC PHOSPHORCS CONTENT O F TWELVE SOILS5
As determined b y hydrogen peroxide and ammonium hydroxide digestion procedures) Organic Phosphorus HzOz NH4OH digestion Soil procedure procedure P. p . m. P. p . m. Rosebudb 96 127 100 Holdrege 92 156 118 Alkali soilb 32 26 Houston 99 54 Lateritec Orangeburgc 5 18 256 245 Shelby 270 Carrington 257 254 Marshall 256 Tama 223 243 Fayette 356 342 378 390 A ' ebsterb 0 Extractions of T a m a , Fayette, and Webster were also made according t o procedure outlined b y Schollenberger, and determinations of phosphorus In extract were made using procedure described in this paper. The following values for organic phosphorus were found: T a m a , 97 p. p. m.; Fayette, 220 p. p m : and Webster, 166 p. p. m. b Alkaline (see Table I). c Contains large amounts of free iron oxides.
Organic phosphorus was determined in twelve soils by both the hydrogen peroxide method proposed by Dickman and DeTurk (2) and the ammonium hydroxide digestion method. Three determinations were also made using the Schollenberger procedure for extraction (6). These determinations were all made in duplicate and the results are presented in Table VIII. I n the case of the Rebster, Carrington, and Shelby soils, reproducible results were not obtained with the peroxide procedure and the values given in Table VI11 are averages of three different determinations. Difficulty was also experienced, particularly with the Shelby and Carrington soils, in retaining the inorganic colloidal material on the filter after decomposition of the organic matter, even though a close paper was used and the filtrate was poured back several times. I n several cases the color developed in the aliquots for inorganic phosphorus had a greenish tint and accurate comparisons
with a standard were not possible. I n general, however, results obtained with the peroxide procedure and with ammonium hydroxide digestion agree very well for acid soils relatively low in free iron oxides. Lower values for organic phosphorus were obtained with the peroxide method than with ammonia digestion in alkaline soils and soils containing relatively high amounts of free iron oxides. Lower values in the former case are due to the inefficiency of hydrogen peroxide in alkaline solution, and in the latter case to fixation of released phosphorus by iron oxides in the soil. S o attempt was made to correct for the amount of phosphorus fixed after its release from organic combination by the hydrogen peroxide. This difficulty is not encountered when the organic compounds containing phosphorus are extracted before release of the phosphorus, since it has been found ( 7 ) that phosphorus in organic combination is not fixed by soils. Shaking for 2 hours in 4 per cent ammonium hydroxide according to the procedure of Schollenberger (6) gave values much lower than those obtained with the other two procedures. Longer periods of shaking did not improve recovery, indicating that the organic phosphorus soluble in cold ammonium hydroxide is completely removed in 2 hours, as was reported by Schollenberger ( 6 ) . OF ADDEDORGANIC PHOSPHORCS TABLE IX. RECOVERY
--Hydrogen Peroxide. Procedu~e
--Ammonia
r
P P Soil added found -P. p. m Carrington ... 291 416 141 Webster No. 2 ... 492 322 760 Shelby . . . 266 386 172 5 Orangeburg ... 65 84
r
recoyered .
Digestion-ProceGure
7
...
126
...
Recovery
%
....
88.6
....
268
83.2
120
70.0
60
71.4
...
...
.... ....
reP P covadded found ered -P. p . m.262 ... 161 422 160 . . . 501 808 322 307 . . . 243 . . . 408 164 165 . . . 14 92 84 78
...
...
...
Recovery
%
...
99.1
...
95.3 100:6
...
93.1
Recoveries of added organic phosphorus, using both the hydrogen peroxide and ammonium hydroxide digestion procedures, are given in Table IX. Aliquots of a standard solution of nucleic acid from yeast were added to each soil in amounts equivalent to the organic phosphorus shown in the second and sixth columns. Nucleic acid was used because the recent work of Wrenshall and McKibben (8) indicates that a large share of the native soil organic phosphorus is in the form of nucleic acid or nucleotides. The results obtained with the hydrogen peroxide procedure were somewhat erratic and the values reported are averages of several determinations. The average percentage recovery for the four soils obtained with the ammonium hydroxide digestion procedure was 98.2, which is considered satisfactory. The fact that added organic phosphorus can be quantitatively recovered shows definitely that the use of ammonium chloride for flocculation of inorganic colloidal material does not cause precipitation of measurable amounts of phosphorus-containing organic compounds.
Literature Cited (1) Dean, L. A., J . A g r . Sci., 28, 234-44 (1938). (2) Dickman, S. R., and DeTurk, E. E., Soil Sei., 45, 29-41 (1938).
(3) Karpinskii, N. P., and Zamyatina, V. B., Proc. Gedroiz Inst. Fertilizers (Moscow), 2, 13347 (1933). (4) Peterson, P. P., Wis. Agr. Expt. Sta., Research Bull. 19 (1911). (5) Potter, R . S., and Benton, T. H., Soil Sci., 2, 291-8 (1916). (6) Schollenberger, C. J., Ibid., 6, 365-95 (1918). (7) Stewart, R., "Organo-Phosphates as Potential Fertilizers", Proc. 2nd Dearborn Conference (Chemurgic Council), pp. 3748, 1936. (8) Wrenshall, C. L., and McKibben, R. R., Can. J . Research, 15B, 475-9 (1938). JOGRNAL Paper KO.5-680 of the Iowa Agricultural Experiment Station, Ames, Iowa.
Project No. 617.
