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PARTITIONING DISSOLVED INORGANIC

AND ORGANIC PHOSPHORUS USING

ACIDIFIED MOLYBDATE AND ISOBUTANOL

K. J A Y A C H A N D R A N , A . P . S C H W A B , *A N D B. A . D. H E T R I C K

Abstract

Quanti f i cat ion of P - m i n e r a l i z a t i o n rate in soil using isotopic dilutionrequires that soluble inorganic

32P (

32P, ) be determined without inter-

ference from so luble organic32

P ("PJ. Therefore, a method is neededto part i t ion

MP, a nd "P 0 into physically separate solutions before ra-

diat ion count ing. A previously suggested method of extracting P , intoi sobutanol with ac idif ied molybdate w as tested fo r ef f ic iency o f sep-aration from P 0. Inorganic P a s K H 2PO 4 w a s com pletely recovered inth e i sobutanol phase with ac id molybdate. Organic P (glycerophos-p h a t e ) remained in the aqueous phase during th e extraction. N o P0

hydrolysis was observed w hen sodium phytate , g lycerophospbate , ri -bonucleic a c id , adenosine 2'- and 3' -mo no pho spha t e , a n d cytidine 2' -an d 3' -monophosphate were extracted, but detectable amounts o fadenosine 5' -tr iphosphate hydrolyzed. This technique was tested onfour Kan sas so i l s, an d the levels o f inorganic an d organic P obtainedby this method were th e same as those detected by convent ional anal -yses.

I N SOILS with high concentrations of labile organic

and low inorganic P, tests of available P basedsolely on P j may underestimate P availability. The

high productivity of certain native prairie soils, de-

spite their low available P,, suggests that labile P0

contributes significantly to P availability, presumablyvia mineralization. Organic P is generally measuredindirectly as the difference between total P and P;

(Watanabe andOlsen, 1965; Halm et al., 1972; Salderand Stewart, 1974; Bowman and Cole, 1978; Chau-

han et al., 1978; Chater and Mattingly, 1979). Netchanges in total P0 and Pj with time can then be used

agriculture in which P0 significantly contributes to Pfertility. The method of Walbridge and Vitousek (1987)appears suitable, but has not been tested. The overallobjective of our research was to determine the effi-ciency 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 af t er mixing with aci-dified molybdate, whether all of the P0 remains in the

aqueous phase during the isobutanol-acidified molyb-

date extraction, and whether labile P0 was hydrolyzed

during the extraction process as a result of exposure

to HCO3- and H2SO4.

Materials and MethodsT h e s e q u e n c e of a n a l y s e s u s e d in th is s t u d y to d etermin e

the efficiency of the extraction proc ed ure is i l l u s tra te d inFig. 1. The extraction proc ed ure for min era l iza t ion d eter-min a t ion s is out l in ed b y a d a s h e d line. T h e r e m a i n i n g stepsw e r e a d d e d t o this study for verification purposes. T h e e x -traction procedure w a s in i t ia l ly tested on pure P compoundsw i t h KH2PO4 a s a Pj source a n d Phy, Gly-P, R N A , AMP,ATP, and CMP (Sigma C h e m i c a l Co., St. Louis, MO) asrepresentat ive P0 sources.

The t op 25 cm of t wo a g ric u l tura l soils and t wo nat i ve

prairie soils were s a m p le d n e a r M a n h a t t a n , KS. The agri-cul tural soils were Chase (fine, montmoril lonitic , mesic A q u i cA r g iu d o l l ) a n d E u d o r a silty clay loam (coarse-silty, mixed,mesic F l u v e n t i c H a p l u d o l ) . T h e n a t iv e prairie soils weresilty a n d cher t y clay loams of the Benfield-Florence com-plex (Be nfie l d soils a re f ine , mixed, mesic U d i c Argius-tolls, Florence soils are clayey-skeletal,montmoril lonit ic,mesic U d i c Argiustolls) a n d t h e Tully silty c lay loam (fine,mixed, mesic Pachic Argiustoll). Table 1 gives selectedproperties of these soils a s d etermin ed b y s t a n d a r d m e t h o d sfor th is region ( D a h n k e , 1980).

I sobuta n ol s a t u r a t e d w i t h w a t e r (65 mL w a t e r L-

1

iso-b u t a n o l ) a n d aqueous solutions satu rate d w i t h i s o b u t a n o l(65 mL i sobut anol L-

1w a t e r , 50 mL i sobut anol L-

10.5

M H2SO4, or 35 mL i sobut anol L-1

2.3 M H2SO4) wereused for all reagents involved in th e l iq u id extractions. M u -

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N O T E S 763

SOIL SA M PLE

d)

HCOa E X T R A C T

(2)

TOTAL P P|

TOTA L P -P|.P0

(5)

ISOBUTA NOL EXTR A CTION

WI TH A CID M OLYBDA TE

ISOBUTA NOL PHA SE AQ U EO U S PHA SE

Pi PO

(8)

ISOBUTA NOL EXTR A CTION

WI THO U T A CID M OLYBDA TE

ISOBUTA NOL PHA SE A QUEOUS PHA SE

N°P

T O T A L P Pj

TOTA L P - P j = P0

Fig. 1. The sequence of analyses used toevaluate 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 f u n n e l fol low ed by 5 mL of a c id if ied mo-l ybdate , 10 mL of isobutanol saturated with deionized water,an d 1 0 mL of d eion ized w a ter sa tura ted w ith isobuta n ol .Th e sepa ra tory f u n n e l w a s sh a ken for 2 m in a n d a l low edto set t le . In t h i s process, m o l y b d e n u m complexed w i t h Pf

ions, and the phosphomolybdate complex was extracted intoth e isobuta n ol ph a se. A f t e r ph a se sepa ra t ion w a s c omplete ,th e more d en se a queous ph a se w a s d ra in ed from the bottomof the f u n n e l a n d saved fo r fu rt he r a n a ly sis . T h e isobuta n olp h a s e w a s w a s h e d b y s h a k i n g for 1 min w i t h 10 mL of 0.5M

H2SO4 sa tura ted w ith isobuta n ol a n d th e a queous ph a sed i s c a r d e d .T o d etermin e Pj in the isobuta n ol , 15 mL o f r e d u c i n g

a g en t w ith out moly bd a te w a s a d d ed to th e isobuta n ol ph a se ,shake n for 1 min, a l lowed to settle, a n d t h e colorless, aqueous

for 2 min , a l low ed to se t t le , a n d th e a queous l a y er sa vedfor P j and total P analysis. T h e Pj in both a queous a n disobuta n ol ph a ses w a s d etermin ed color i met r i ca l ly as de-scribed above.

Tota l P in th e a queous a n d isobuta n ol ph a ses w a s d eter-m i n e d after HCIO4-HNO3 digestion. A 5-mL a l iquot w a sa d d ed to a d ig est ion tube a lon g w ith 0.25-mL of sa tura tedMgCl2 (167 g MgCl2-6H2O i n 100 mL of d eion ized w a ter)to prevent P loss d urin g a c id d ig est ion (Brookes a n d Fowl-son, 1981). O n e mill i l i ter o f 6 M HNO3 and 8 mL of 1:1

HNC>3/rICl O 4 w ere a d d ed , th e sa mples w ere d ig ested in ablock digester, a n d total P w a s d etermin ed . Org a n ic P w a sc a lc u la ted a s th e d ifferen c e betw een tota l P a n d Pj .

T h e Pj in the original HCO3 extra c t w a s d etermin ed c ol -orimetric a l ly (Olsen et al., 1954). Tota l P w a s d etermin ed

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764 SOIL SCI. S O C . A M . J., VOL. 56, M A Y - J U N E 1992

Table 2. Recovery of inorganic (P ,) and organic P (P 0) fromH CO 3 s o l u t i o n s .

Phosphorus recovery

Extractionf P, + P.f P, + P 0#

P partitioningAcid molybdate:

P, in isobutanol (6) 24.2 ±P, in aqueous (7)P0 in aqueous (7)

W ithout acidifiedmolybdate:

Total P in aqueous(11)

Pi in aqueous (12)P0 in aqueous

(11 mi nus 12 )P, in isobutanol (9)P0 in isobutanol (9)

D irect analysis of H C O ,extractsTotal P (3)P I (4)P0 (3 minus 4)

m g P L-

0.40.0

NDft

0.0 9.1 ± 0.1 13.8 ± 0.10.0 0.0 0.0N D N D N D

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 ± 0.10.0 0.0 0.0 0.00.0 0.0 0.0 0.0

24.3 ± 0.3 24.3 ± 0.3 24.2 ± 0.2 24.1 ± 0.124.0 ± 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

t Total P in each case was 24 m g P L ~ * . N u m b e r s in parenthesesrefer tothe steps in Fig. 1.

t P, isK H 2PO 4-P .§ P0 is glycerophosphate-P.11 Original solution contained 9 mg P, L "

1+ 15 mg P0 L ~ ' .

# Original solution contained 1 4 m g P, L-' + 10 mg P0 L-1.

ttN ot determined because molybdate interfered with acid digestion.

Table 3. Recovery o f P in soil extracts by the i sobutanole x t r a c t i o n method.

Soil

E xtract iont C h a s eBenfield-

E ud ora Floren ce T ully

P partitioningA c i d molybdate:

P, in isobutanol (6)P, in aqueous (7)P0 in aqueous(7)

Without acidifiedmolybdate:Total P in aqueous

(11)P, in aqueous (12)P0 in aqueous

(11 minus 12)P, in isobutanol (9)P0 in isobutanol (9)

D irect analysis of HCO,extractsT ota l P (3)P I (4)P0 (3 minus 4)

m g P L -1

37.1 ± 0.60.0

51.7 ±36.3 ±

1.00.6

34.2 ± 0.3 12.2 ± 0.2 7.8 ± 0.10.0 0.0 0.0N D N D N D

41.6 ± 1.0 17.3 ± 0.6 24.3 ± 1.233.8 ± 0.3 12.6 ± 0.2 8.2 ± 0.1

15.4 ± 0.60.00.0

7.8 ± 0.70.00.0

4.7 ± 0.90.00.0

16.1 ± 1.20.00.0

51.0 ± 1.0 41.1 ± 1.0 18.0 ± 0.6 23.8 ± 1.236.3 ± 0.6 34.1 ± 0.3 12.6 ± 0.2 8.1 ± 0.114.7 ± 0.6 7.0 ± 0.7 5.4 ± 0.9 15.8 ± 1.2

t Num b ers in parentheses refer to the steps in Fig. 1 . P, = inorganic P; P0= organic P.

t N ot determined because molybdate interfered with acid digestion.

remain i n t h e aqueous phase because th e molybdateis not present to complex it . Indeed, al l of the P re-m ai n e d i n t h e aqueous phase a n d w a s recovered a s P;

(Table 2).W h e n Gly-P was extracted with acidified molyb-

date, n o Pj w a s detected i n t h e isobutanol (Table 2 ).A s discussed in more detail below, this indicates thatth e Gly-P d i d n o t hydrolyze during th e extraction. I nth e absence of acidified molybdate, all of the Gly-P

Cleavage of organic, phosphoester bonds by HCO3

or H2SO4 to convert P0 to P; would confound the re-sults. This possibility was tested by determining theformation of P; after (i ) incubating P0 in HCO3 orH2SO4 solutions and (ii) extracting solutions of or-

ganophosphates with isobutanol in the presence of aci-dified molybdate. Hydrolysis of the P0 to Pj w a s n o tobserved under a n y o f these conditions for AMP, CMP,Phy, or Gly-P, even after 16 h of incubation. How-

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N O T E S 765

eralization experiments. Although molybdate-catalyzed

hydrolysis of labile P0 compounds has been observed

(Marsh, 1959; Parvin and Smith, 1969), hydrolysis oflabile P0 was not observed for labile P0 compounds

except for ATP a f t e r long exposure periods.

A C K N O W L E D G M E N T S

T h e a u t h o r s t h a n k D r . R . D a v i d J o n e s f or p r e l i m i n a r yr e s e a r c h , a n d Drs. J a n E. J^each a n d C h a r le s W . R i c e f ort h e i r cr i t ica l e val u at io n of t h i s s t u d y . T h i s s t u d y w a s sup-ported i n p a r t b y N a t i o n a l S c ie n c e F o u n d a t i o n LTER pro-g r a m , G r a n t no. BSR-8514 327.