IRRI

International Rice Research Institute

Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 1 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document maintained electronically.
Any printed copy of this document is not controlled.





International Rice Research Institute
Crop and Environmental Sciences Division
Analytical Service Laboratory















This document is issued under the authority of



MS. LILIA R. MOLINA
Assistant Manager II


THIS IS A CONTROLLED DOCUMENT




Standard Operating Procedure

AVAILABLE PHOSPHORUS
(Olsen Phosphorus)

QM-AD002-AP12

IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 2 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document maintained electronically.
Any printed copy of this document is not controlled.




SIGNATURE PAGE


The signatures below signify approval and concurrence of the signatories with the contents of this
SOP coupled with the commitment to provide the resources necessary to ensure proper quality
operation of the ASL laboratory.


Issuing Authority:

Name & Title Signature Date (yyyy-mm-dd)

Lilia R. Molina
Laboratory Manager/QM



2011-01-09

Christy S. Daniel
Deputy Quality Manager



2011-01-09





IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 3 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document which was condensed from the original SOP and maintained electronically.
Any printed copy of this document is not controlled.


QM-AD002-AP12

AVAILABLE PHOSPHORUS (Olsen Phosphorus)


1.0 Scope and Application

This method estimates the relative bioavailability of ortho-phosphate (PO
4
-P) in soils by
extraction using alkaline sodium bicarbonate (pH 8.5) solution and determining the P
concentration in the extract colorimetrically. It is applicable to soils that are mildly acidic to
alkaline pH and is based on the method developed by Olsen et al., (1954) to correlate crop
response to fertilizer on calcareous soils.


2.0 Summary

A 2.5 gram scoop of soil and 50 milliliters of 0.5 M sodium bicarbonate (pH 8.5) solution
are shaken for 30 minutes. The mixture is then filtered through Whatman filter paper and
the ortho-phosphate in the filtered extract is determined colorimetrically (at 630 nm in
Technicon AutoAnalyzer II) by reacting it with ammonium molybdate using ascorbic acid
as the reducing agent. Results are reported as parts per million (ppm) phosphorus (P) in the
soil.


3.0 Apparatus and Materials

3.1 Analytical balance, capable up to 0.001 g
3.2 Reciprocal shaker, Everbach Model Cat. No. 6000-26.
3.3 Polyethylene bottles with good fitting tops (125 mL and 1L capacity).
3.4 Polypropylene funnels, 65 mm
3.5 No. 40 Whattman filter paper, 11 cm
3.6 20 ml test tubes
3.7 Dispenser or repipet 50 ml or equivalent (calibrated at 50.00 0.20g)
3.8 Technicon AutoAnalyzer II unit or any other equivalent continuous flow, segmented
stream autoanalyzer system
3.9 Autosampler sample cups
3.10 Volumetric flasks
IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 4 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document which was condensed from the original SOP and maintained electronically.
Any printed copy of this document is not controlled.

3.11 Erlenmeyer flasks

4.0 Reagents and Standards

4.1 0.05 % Polyacrylamide. Weigh 0.25 g polyacrylamide to 450 ml of double RO water
and stir overnight with a magnetic stirrer. Make up to 500 ml (Do not try to add the
double RO water to the polyacrylamide.)

4.2 1N Sodium hydroxide for pH adjustment. Weigh 20 g of sodium hydroxide, dissolve
and dilute with double RO water to 500 ml.


4.3 0.5 M sodium bicarbonate, pH 8.5 with polyacrylamide (extracting solution). Weigh
168 g of sodium bicarbonate and place in a 4L Erlenmeyer flask. Dissolve in about
3,900 ml double RO water. Add 20 ml of 0.05% polyacrylamide. Adjust pH to 8.50
with 1N sodium hydroxide (approximately 60-80ml). Add enough double RO water
to make to 4 L if necessary.

4.4 AutoAnalyzer Reagents:

4.4.1 Wash solution - 0.5 M sodium bicarbonate, pH 8.5 with polyacrylamide
(extracting solution).

4.4.2 2.5 N sulfuric acid - Add slowly 70 ml concentrated sulfuric acid to about
500 ml double RO water in a 1 L volumetric flask. Cool down and dilute to
mark.

4.4.3 Acid molybdate - Dissolve 4.3 g ammonium molybdate tetrahydrate
(NH
4
)
6
Mo
7
O
24
.4H
2
O) in about 200 ml of double RO water in a 1-liter
volumetric flask. Add carefully 52 ml concentrated sulfuric acid. Make to
volume with double RO water and shake well to mix.

4.4.4 1 %(w/v) Ascorbic acid solution (with SDS). Prepare this solution fresh each
time before use. Dissolve 5g of ascorbic acid in 500 ml of double RO water.
Add 10 -15 drops of 5% w/v sodium dodecyl sulfate (SDS) as a wetting
agent to improve the autoanalyzer bubble pattern.

4.5 Stock solution A (2000 ppm P) - Dry reagent grade potassium dihydrogen
phosphate (KH
2
PO
4
) at 105
O
C for 2 hr. Cool in a dessicator. Accurately weigh
out 4.394 g KH
2
PO
4
, dissolve in double RO water and dilute to 500 ml. Shake
well to mix.

4.6 Intermediate standard solution B (100 ppm P) - Pipet 5.00 ml of the stock
solution A containing 2000 ppm P and dilute to 100 ml with the extracting
solution (0.5 M sodium bicarbonate, pH 8.5 with polyacrylamide). Shake well to
mix.

IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 5 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document which was condensed from the original SOP and maintained electronically.
Any printed copy of this document is not controlled.

4.7 Working standard solutions (0.5, 1, 2, 3, 4 and 5 ppm P)- Pipet 0.5, 1, 2, 3, 4, and
5 ml of the intermediate standard solution B containing 100 ppm P and dilute to
100 ml with the extracting solution (0.5 M sodium bicarbonate, pH 8.5 with
polyacrylamide).


Concn. of Working Std (ppm P) Intermediate Standard Soln, B (ml) Final Volume (ml)
0.5 0.5 100
1 1 100
2 2 100
3 3 100
4 4 100
5 5 100

4.8 Calibration blank extracting solution (0.5 M sodium bicarbonate, pH 8.5 with
polyacrylamide).

4.9 QC standard check, 3 ppm P Prepared as in 4.7.

4.10 Reference Material

Soil In-house Reference Material (SIRM-Coarse), Control No. 251.xxx



5.0 Sample Preparation, Handling and Storage

See QM-AD036-MP24 - Plant and soil sample preparation procedures for the appropriate
sample preparation, handling, and storage.


6.0 Procedure

6.1 Sample Extraction

6.1.1 Weigh 2.50 g of 2 mm air-dried soil sample and place in a clean dry 125 ml
polyethylene bottle. Run 2 blanks in the same way. Weigh also reference
materials and sample repeats.
Note: Perform moisture determination by weighing another test portion according to
QM-AD002-AP26 (Moisture Determination of Soil Samples) and incorporate results
in the calculation of P when results are reported in oven-dry basis.

6.1.2 Add 50 ml of the extracting solution (0.5 M sodium bicarbonate, pH 8.5 with
polyacrylamide) from a dispenser.
6.1.3 Place bottles in a reciprocating shaker and shake for 30 min.
IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 6 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document which was condensed from the original SOP and maintained electronically.
Any printed copy of this document is not controlled.

6.1.4 Filter through no. 40 Whattman filter paper (11 cm) into a clean, dry 20 ml
test tube.
6.1.5 Determine available phosphorus in the clear filtrate by colorimetry using
Technicon II Autoanalyzer.

6.2 Colorimetric Determination of Phosphorus Using the Technicon AutoAnalyzer II
Refer to the Technicon AutoAnalyzer II Operations Manual for a complete and
detailed operating procedure for the autoanalyzer. When determining phosphorus,
ensure that the Olsen Phosphorus manifold is set-up for analysis (Refer to Figure 1).

AutoAnalyzer Settings:
Filter: 630 nm
Heating bath: 90
O
C

Tube requirements:
Function Color Block
position
Flow rate
ml/min
Wash purple/orange 3U 3.40
sample (from up) purple/purple 4U 2.50
H
2
SO
4
red/red 5L 0.80
acid molybdate purple/purple 3L 2.50
ascorbic acid red/red 6U 0.80
Air black/black 7U 0.32
pull thro' cell red/red 5U 0.80
resample (from debubbler) blue/blue 2L 1.60


6.2.1 Set up autoanalyzer and analyze the samples using the flow diagram shown
in Figure 1.

6.2.2 Transfer adequate amount of the standards and samples into the autosampler
cups and start the sequence run.

6.2.3 After the last sample in the sequence has been taken for colorimetric
analysis, turn OFF the autosampler and timer.
6.2.4 Flush the system with deionized RO water for a minimum of 15 minutes (or
as long as the dead time) before turning off the autoanalyzer.






IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 7 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document which was condensed from the original SOP and maintained electronically.
Any printed copy of this document is not controlled.





































Figure 1. Flow diagram for the determination of available P in sample extracts.



7.0 Quality Control

7.1 At a minimum, an analytical batch (50 samples or less) must contain a method
blank, sample repeat and reference material.

7.2 Unusual situations occurring during sample preparation and analysis must be
documented on the work sheet, and non-conformity/corrective action reports
must be issued if necessary (See QM-AD017-MP08- Non-conformities and
Corrective Actions).
IRRI
International Rice Research Institute
Analytical Service
Laboratory
Document Control No.:
QM-AD002
SOP Code: AP12 Issue Date: 2011-01-09 Revision No.: 03 Page: 8 of 8
Title: Available Phosphorus (Olsen Phosphorus)


This is a controlled document which was condensed from the original SOP and maintained electronically.
Any printed copy of this document is not controlled.

8.0 Calculations

8.1 Calculate soil P (mg/ kg) follows:

Soil P (mg/kg) = [(A x B x C x M )/E]
(oven-dry basis)
where:
A = Sample extract reading (mg l
-1
)
B = Extract volume (ml)
C = Dilution, if performed
M = Moisture correction factor
E = Sample weight (g)


9.0 References

9.1 Blakemore, L. C., Searle, P. L. and B. K. Daly. 1987. Methods for chemical
analysis of soils. NZ Soil Bureau Scientific Reprt 80. Dept. of Scientific and
Industrial Research. Lower Hutt, New Zealand.

9.2 Faithfull, N.T. 2002. Methods in agricultural chemical analysis: a practical
handbook. CABI Publishing. Wallingford, UK

9.3 Kalra, Y.P.; Maynard, D.G. 1991. Methods manual for forest soil and plant
analysis. For. Can., Northwest Reg., North For. Cent., Edmonton, Alberta, Canada.
Inf. Rep. NOR-X-319.

9.4 Olsen, S. R., C. V. Cole, F. S. Watanabe and L. A. Dean. 1954. Estimation of
Available Phosphorus in Soils by Extraction with Sodium Bicarbonate. U. S.
Department of Agriculture Circular No. 939. Banderis, A. D., D. H. Barter and K.
Anderson. Agricultural and Advisor.

9.5 QM-AD036-MP24 - Plant and soil sample preparation. ASL QMS. IRRI-CESD.

9.6 Rayment, G.E. and Higginson, F.R. 1992. Australian Laboratory Handbook of Soil
and Water Chemical Methods. Inkata Press, Melbourne.

9.7 Service, England (ADAS) 1976. The use of Polyacrylamide in the Determination
of Olsens Extractable Phosphate in Soil. J. of Soil Science. 27:71-74. Watanabe,
F. A. and J. R. Olsen. 1965.

9.8 Test of an Ascorbic Acid Method for Determining Phosphorus in water and in
sodium bicarbonate extract from soil. Soil Sci Soc Proc. 28:677-678.

9.9 USDA Soil Survey Laboratory Methods Manual. Version 4.0. November 2004.


(END OF DOCUMENT)