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PARTITIONING DISSOLVED INORGANIC AND ORGANIC PHOSPHORUS USING ACIDIFIED MOLYBDATE AND ISOBUTANOL K. JAYACHANDRAN, A. P. SCHWAB,* AND B. A. D.

HETRICK Abstract Quantification of P-mineralization rate in soil using isotopic dilution requires that soluble inorganic 32P (32P,) be determined without interference from soluble organic 32P ("PJ. Therefore, a method is needed to partition MP, and "P0 into physically separate solutions before radiation counting. A previously suggested method of extracting P, into isobutanol with acidified molybdate was tested for efficiency of separation from P0. Inorganic P as KH2PO4 was completely recovered in the isobutanol phase with acid molybdate. Organic P (glycerophosphate) remained in the aqueous phase during the extraction. No P0 hydrolysis was observed when sodium phytate, glycerophospbate, ribonucleic acid, adenosine 2'- and 3'-monophosphate, and cytidine 2'and 3'-monophosphate were extracted, but detectable amounts of adenosine 5'-triphosphate hydrolyzed. This technique was tested on four Kansas soils, and the levels of inorganic and organic P obtained by this method were the same as those detected by conventional analyses.

agriculture in which P0 significantly contributes to P fertility. The method of Walbridge and Vitousek (1987) appears suitable, but has not been tested. The overall objective of our research was to determine the efficiency of a modification of Pons and Guthrie's (1946) isobutanol extraction for partitioning P; and P0. It was therefore necessary to determine whether all of the Pj is extracted into the isobutanol after mixing with acidified molybdate, whether all of the P0 remains in the aqueous phase during the isobutanol-acidified molybdate extraction, and whether labile P0 was hydrolyzed during the extraction process as a result of exposure to HCO3- and H2SO4. Materials and Methods The sequence of analyses used in this study to determine the efficiency of the extraction procedure is illustrated in Fig. 1. The extraction procedure for mineralization determinations is outlined by a dashed line. The remaining steps were added to this study for verification purposes. The extraction procedure was initially tested on pure P compounds with KH2PO4 as a Pj source and Phy, Gly-P, RNA, AMP, ATP, and CMP (Sigma Chemical Co., St. Louis, MO) as representative P0 sources. The top 25 cm of two agricultural soils and two native prairie soils were sampled near Manhattan, KS. The agricultural soils were Chase (fine, montmorillonitic, mesic Aquic Argiudoll) and Eudora silty clay loam (coarse-silty, mixed, mesic Fluventic Hapludol). The native prairie soils were silty and cherty clay loams of the Benfield-Florence complex (Benfield soils are fine, mixed, mesic Udic Argiustolls, Florence soils are clayey-skeletal, montmorillonitic, mesic Udic Argiustolls) and the Tully silty clay loam (fine, mixed, mesic Pachic Argiustoll). Table 1 gives selected properties of these soils as determined by standard methods for this region (Dahnke, 1980). Isobutanol saturated with water (65 mL water L-1 isobutanol) and aqueous 1 solutions saturated with isobutanol (65 mL isobutanol L- water, 50 mL isobutanol L-1 0.5 M H2SO4, or 35 mL isobutanol L-1 2.3 M H2SO4) were used for all reagents involved in the liquid extractions. Mutual saturation of the solvents was necessary to minimize volume changes during extraction. Acidified molybdate, used to complex Pj for extraction into isobutanol, was prepared by dissolving 50 g of ammonium molybdate [(NH4)6Mo7O24-4H20] in 1 L of 2.3 M H2SO4 saturated with isobutanol. Unacidified, or neutral, molybdate solution was prepared by dissolving 50 g of ammonium molybdate in 1 L of water. A stock solution with molybdate was prepared by dissolving 30 g of ammonium molybdate and 0.727 g of antimony potassium tartrate in 5 L of 1.25 M H2SO4. A similar stock solution without molybdate was prepared by dissolving 0.727 g of antimony potassium tartrate in 5 L of 0.5 M H2SO4 saturated with isobutanol. Reducing agent was prepared daily by dissolving 1.056 g L-1 ascorbic acid in 200 mL of stock solution. Samples (pure P compounds or soil extracts) were prepared in 0.5 M NaHCO3. For soils, 2 g of soil (air dried and sieved to pass a 2-m;m screen) were shaken with 40 mL 0.5 M NaHCO3 (pH 8.5) for 30 min and filtered through Whatman no. 42 paper. Pure compounds were not extracted, but aliquots of standard stock solutions were added directly to 40 mL of 0.5 M NaHCO3. A 5 mL aliquot of the HCO3 extract was added to a 125 Abbreviations: 32P,, soluble labeled inorganic P; 32P0, soluble labeled organic P; Phy, sodium phytate; Gly-P, glycerophosphate; RNA, ribonucleic acid; AMP, adenosine 2'- and 3'-monophophate; ATP, adenosine 5'-triphosphate; CMP, cytidine 2'- and 3'monophosphate.

N SOILS with high concentrations of labile organic and low inorganic P, tests of available P based solely on Pj may underestimate P availability. The high productivity of certain native prairie soils, despite their low available P,, suggests that labile P0 contributes significantly to P availability, presumably via mineralization. Organic P is generally measured indirectly as the difference between total P and P; (Watanabe and Olsen, 1965; Halm et al., 1972; Salder and Stewart, 1974; Bowman and Cole, 1978; Chauhan et al., 1978; Chater and Mattingly, 1979). Net changes in total P0 and Pj with time can then be used to estimate net P mineralization. To measure gross mineralization, an isotopic-dilution method was proposed by Sweet (1981) and later used by Walbridge and Vitousek (1987). The technique uses isotopic dilution by measuring 32Pt without interference from 32P0. This requires that 32Pt be physically separated from 32 P0 before p emission counting. Pons and Guthrie (1946) developed a liquid-extraction technique (using isobutanol with acidified molybdate) to remove the interference of P0 in the determination of Pj in plant tissue. The P; is complexed by the molybdate, the phosphomolybdate is extracted into the isobutanol, and the P0 remains in the aqueous phase. Sweet (1981) adopted this method to separate 32Pj from 32P0 in determining gross mineralization. Detailed methodology and a critical evaluation of the technique have not been published, however. We needed a method to determine the mineralization of P in soils with native vegetation or low-input K. Jayachandran and B.A.D. Hetrick, Dep. of Plant Pathology, and A.P. Schwab, Dep. of Agronomy, Throckmorton Hall, Kansas State Univ., Manhattan, KS 66506-5502. Contribution no. 90-509-J from the Kansas Agric. Exp. Stn. Received 24 Apr. 1991. * Corresponding author.
Published in Soil Sci. Soc. Am. J. 56:762-765 (1992).

762

NOTES SOIL SAMPLE


d)

763

HCOa EXTRACT
(2)

(5)

(8)

TOTAL P

P|

ISOBUTANOL EXTRACTION WITH ACID MOLYBDATE

ISOBUTANOL EXTRACTION WITHOUT ACID MOLYBDATE

TOTAL P - P| . P0 ISOBUTANOL PHASE AQUEOUS PHASE ISOBUTANOL PHASE AQUEOUS PHASE


Pi PO
N

TOTAL P Pj TOTAL P - Pj = P0 Fig. 1. The sequence of analyses used to evaluate the isobutanol-acidified molybdate extraction procedure for partitioning inorganic from organic P. The steps within the dashed lines are to be used in determining P mineralization using isotopic dilution; the remaining steps were used in this study to verify that the partitioning was complete.

mL separately funnel followed by 5 mL of acidified molybdate, 10 mL of isobutanol saturated with deionized water, and 10 mL of deionized water saturated with isobutanol. The separatory funnel was shaken for 2 min and allowed to settle. In this process, molybdenum complexed with Pf ions, and the phosphomolybdate complex was extracted into the isobutanol phase. After phase separation was complete, the more dense aqueous phase was drained from the bottom of the funnel and saved for further analysis. The isobutanol phase was washed by shaking for 1 min with 10 mL of 0.5 M H2SO4 saturated with isobutanol and the aqueous phase discarded. To determine Pj in the isobutanol, 15 mL of reducing agent without molybdate was added to the isobutanol phase, shaken for 1 min, allowed to settle, and the colorless, aqueous layer was discarded. The blue isobutanol layer was transferred to a 25-mL volumetric flask, the separatory funnel rinsed with 95% (v/v) ethanol, and the volume brought to 25 mL with 95% ethanol. Color intensity was read after 10 min at 882 nm. Standards were prepared in the same matrix as the samples. To verify that P0 was not extracted into the isobutanol phase or hydrolyzed during the extraction process, all samples were extracted with isobutanol as described above except the acidified molybdate was replaced by 2.3 M H2SO4. (The molybdate was eliminated because it interfered with the determination of total P). Inorganic P in the isobutanol phase was determined by adding 4.35 mL of 2.3 M H2SO4 to the isobutanol phase in a separatory funnel along with 8.33 mL of neutral ammonium molybdate (30 g ammonium molybdate in 1 L of water saturated with isobutanol) and 7.33 mL of deionized water saturated with isobutanol. This mixture was shaken Table 1. Selected characteristics of the four experimental soils.
Soil Agricultural Chase Eudora Prairie Benfield-Florence Tully
PH

for 2 min, allowed to settle, and the aqueous layer saved for Pj and total P analysis. The Pj in both aqueous and isobutanol phases was determined colorimetrically as described above. Total P in the aqueous and isobutanol phases was determined after HCIO4-HNO3 digestion. A 5-mL aliquot was added to a digestion tube along with 0.25-mL of saturated MgCl2 (167 g MgCl2-6H2O in 100 mL of deionized water) to prevent P loss during acid digestion (Brookes and Fowlson, 1981). One milliliter of 6 M HNO3 and 8 mL of 1:1 HNC>3/rIClO4 were added, the samples were digested in a block digester, and total P was determined. Organic P was calculated as the difference between total P and Pj. The Pj in the original HCO3 extract was determined colorimetrically (Olsen et al., 1954). Total P was determined by the digestion procedure described above. Organic P was calculated as the difference between total P and P,. Hydrolysis of the P0 in the alkaline HCO3 solution or the acidified molybdate solutions is a potential source of error in the extraction and separation of Pt from labile P0. This possibility was tested with the five P0 sources: Phy, GlyP, RNA,1 ATP, AMP, and CMP. Solutions containing 20 mg P L- of each compound made in 0.5 M NaHCO3, 0.4 M H2SO4, or acidified molybdate were incubated for up to 16 h at 23 C. Organic P hydrolysis was determined by measuring the Pt concentrations at the end of the incubation periods. Results and Discussion Partitioning of Pj from P0 was tested on known quantities of highly soluble KH2PO4 and a soluble P0 source, Gly-P (Table 2). Solutions contained Pi? P0, or both with a total of 24 mg PL- 1 . Complete separation of Pj and P0 was achieved for all combinations of KH2PC>4 and Gly-P when HCO3 extracts were extracted by isobutanol with acidified molybdate. When only KH2PO4 was extracted with the acidified molybdate, all of the P; was recovered in the isobutanol. Determination of total P in the aqueous phase was not possible because the presence of the molybdate interfered with the analysis. Therefore, an identical solution of Pj was extracted with isobutanol but without acidified molybdate. In this extraction, the Pj should

Organic matter

Bray P

Clay

Sand

gkg6.5 6.9

mg kg44.7 42.5 12.7 9.0

gkg- 1 350 160 170 380 110 230 200 210

23 36 25
46

7.7
6.5

764
HCO3 solutions.

SOIL SCI. SOC. AM. J., VOL. 56, MAY-JUNE 1992

Table 2. Recovery of inorganic (P,) and organic P (P0) from Phosphorus recovery Extractionf P partitioning Acid molybdate: P, in isobutanol (6) P, in aqueous (7) P0 in aqueous (7) Without acidified molybdate: Total P in aqueous (11) Pi in aqueous (12) P0 in aqueous (11 minus 12) P, in isobutanol (9) P0 in isobutanol (9) Direct analysis of HCO, extracts Total P (3) PI (4) P0 (3 minus 4)
P, + P.f
mg P L-

Table 3. Recovery of P in soil extracts by the isobutanol


extraction method.
Soil

P, + P0#

Extractiont P partitioning Acid molybdate: P, in isobutanol (6) P, in aqueous (7) P0 in aqueous (7) Without acidified molybdate: Total P in aqueous (11) P, in aqueous (12) P0 in aqueous (11 minus 12) P, in isobutanol (9) P0 in isobutanol (9) Direct analysis of HCO, extracts Total P (3) PI (4) P0 (3 minus 4)

Chase

Eudora

BenfieldFlorence

Tully

mgPL- 1

24.2 0.4
0.0
NDft

0.0 9.1 0.1 13.8 0.1 0.0 0.0 0.0 ND ND ND

37.1 0.6 34.2 0.3 12.2 0.2 7.8 0.1 0.0 0.0 0.0 0.0 ND ND ND

24.3 0.3 24.1 0.3 24.0 0.2 24.0 0.1 23.2 0.4 0.0 8.9 0.1 13.6 0.1 0.0 24.0 0.3 15.0 0.2 10.2 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 24.3 0.3 24.3 0.3 24.2 0.2 24.1 0.1 24.0 0.1 0.0 8.6 0.1 13.7 0.1 0.0 24.3 0.3 15.6 0.3 10.4 0.1

51.7 1.0 41.6 1.0 17.3 0.6 24.3 1.2 36.3 0.6 33.8 0.3 12.6 0.2 8.2 0.1 15.4 0.6 0.0 0.0 7.8 0.7 4.7 0.9 16.1 1.2 0.0 0.0 0.0 0.0 0.0 0.0

t Total P in each case was 24 mg P L~*. Numbers in parentheses refer to the steps in Fig. 1. t P, is KH2PO4-P. P0 is glycerophosphate-P. 11 Original solution contained 9 mg P, L"1 + 15 mg P0 L~'. 1 # Original solution contained 14 mg P, L-' + 10 mg P0 L- . tt Not determined because molybdate interfered with acid digestion.

51.0 1.0 41.1 1.0 18.0 0.6 23.8 1.2 36.3 0.6 34.1 0.3 12.6 0.2 8.1 0.1 14.7 0.6 7.0 0.7 5.4 0.9 15.8 1.2

t Numbers in parentheses refer to the steps in Fig. 1. P, = inorganic P; P0 = organic P. t Not determined because molybdate interfered with acid digestion.

remain in the aqueous phase because the molybdate is not present to complex it. Indeed, all of the P remained in the aqueous phase and was recovered as P; (Table 2). When Gly-P was extracted with acidified molybdate, no Pj was detected in the isobutanol (Table 2). As discussed in more detail below, this indicates that the Gly-P did not hydrolyze during the extraction. In the absence of acidified molybdate, all of the Gly-P remained in the aqueous phase as P0. These results suggest that the P0 was not extracted into the organic phase and the Gly-P was not hydrolyzed. Mixtures of KH2PO4 and Gly-P were extracted to test the efficacy of the procedure to partition Pj from P0. Initial1concentrations were 9 mg Pj L"1 plus 15 mg P0 L- or 14 mg Pj L-1 plus 10 mg P0 L-1. All the PJ was recovered in the isobutanol after extraction with acidified molybdate, and P0 was neither hydrolyzed nor extracted into the isobutanol. In all cases, the amounts of Pj and P0 recovered agreed with the concentrations determined by direct analysis of the HCO3 extract. When this extraction technique was further tested on Phy, RNA, AMP, ATP, and CMP, no P0 was detected in the isobutanol phase. This confirms that the proposed partitioning technique yields complete separation of P; and P0 without causing hydrolysis of P0. The separation procedure was tested on HCO3 extracts of four soils from different sites near Manhattan, KS (Table 3). The Pj, P0, and total P recovered by liquid extraction were compared with those obtained after direct Pj analysis of the HCO3 extracts followed by total -P analysis without partitioning (Olsen et al., 1954; Watanabe and Olsen; 1965). In all four soils, the concentrations of Pj and P0 as determined by the proposed P-separation method were similar to those found by the Olsen et al. (1954) method.

Cleavage of organic, phosphoester bonds by HCO3 or H2SO4 to convert P0 to P; would confound the results. This possibility was tested by determining the formation of P; after (i) incubating P0 in HCO3 or H2SO4 solutions and (ii) extracting solutions of organophosphates with isobutanol in the presence of acidified molybdate. Hydrolysis of the P0 to Pj was not observed under any of these conditions for AMP, CMP, Phy, or Gly-P, even after 16 h of incubation. However, ATP hydrolyzed to the extent of 0.3% in 15 min, 1% in 1 h, 6% in 4 h, and 11% after 8 h. From these observations, it may be concluded that hydrolysis will not be a problem except for the most labile P0 forms, and this will be small if the solutions are extracted in <4 h. If hydrolysis of labile P0 were to be a problem, an approach similar to the method of Dick and Tabatabai (1977) could be used to prevent interference from hydrolyzed P0. Quantification of P-mineralization rate in soil using isotopic dilution requires that soluble 32P; be determined without interference from soluble 32P0. Therefore, a method is needed to partition 32P; and 32P0 into physically separate solutions before counting 3 emissions from the 32P. Organic solvents like isobutanol, benzene, and octan-1-ol to separate Pf from labile P0 compounds have been used for plant materials, biological materials, plain C steels, and water samples (Pons and Guthrie, 1946; Martin and Doty, 1949; Schaffer et al., 1953; Lueck and Boltz, 1956; Helyar and Brown, 1976), but had not been confirmed for separation of soil P. While Sweet (1981) and Walbridge and Vitousek (1987) applied the modified Pons and Guthrie's (1946) method to soils, its efficacy apparently was not tested. The P-separation procedure described in our study provides a methodology for partitioning Pj and P0 in soils. The observed separations were complete and will be suitable for P-min-

NOTES

765

eralization experiments. Although molybdate-catalyzed hydrolysis of labile P0 compounds has been observed (Marsh, 1959; Parvin and Smith, 1969), hydrolysis of labile P0 was not observed for labile P0 compounds except for ATP after long exposure periods. ACKNOWLEDGMENTS The authors thank Dr. R. David Jones for preliminary research, and Drs. Jan E. J^each and Charles W. Rice for their critical evaluation of this study. This study was supported in part by National Science Foundation LTER program, Grant no. BSR-8514 327.