Revised
ITR-18
GRAVIMETRIC CARRIER SEPARATION OF PHOSPHORUS-32
IN WATER SAMPLES
by
N. A. Talvitie
Technical Services
Southwestern Radiological Health Laboratory
Department of Health, Education, and Welfare
Public Health Service
Consumer Protection and Environmental Health Service
May 7, 1969
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INTRODUCTION AND OBJECTIVES
The objective of this study was to provide a readiness
capability at the Southwestern Radiological Health Labora-
tory for the determination of phosphorus-32 in environmental
samples. This was to be accomplished by evaluation of methods
and continuing participation in the Columbia River Cross-
check program of the Analytical Quality Control Service of
the Northeastern Radiological Health Laboratory. Inasmuch as
phosphorus-32 determinations are not conducted routinely at
the laboratory, a basic method adaptable to all types of
environmental samples was selected for study. This method is
one which generally is used not only for the determination of
phosphorus-32 but also for the gravimetric determination of
total phosphorus. The chemical yield is quantitative which
is of importance in analysis of bone and other phosphate-
containing samples. The study was limited to a few analytical
runs to corrent any problem areas and to coordinate the separa-
tion with requirements of the beta counting systems. The
procedure is given in detailed laboratory manual style.
Time estimate for one analyst conducting a set of 1 to
4 determinations in water samples is 1 man-day with results
reported at the end of the second day. Time estimate for two
analysts conducting a set of 1 to 4 determinations in other
types of environmental samples is 1-1/2 man-days with results
reported at the end of the third day.
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GRAVIMETRIC CARRIER SEPARATION OF PHOSPHORUS-32
IN WATER SAMPLES
Principle of Method;
This separation of phosphorus-32 in a form suitable for counting
in a low-level beta counter is based on classical precipitation methods
having quantitative yields. Total phosphorus can be determined simul-
taneously when the amount in the analyzed aliquot is greater than a few
milligrams. The method has direct application to potable and saline
water and, following suitable ashing and dissolution steps, is adaptable
to sewage, sediments, and biological samples.
Phosphorus-32 in water is concentrated by precipitation as ferric
phosphate in the presence of phosphorus carrier, separated from the iron
by precipitation as ammonium phosphomolybdate and separated from the
molybdenum by double precipitation as magnesium ammonium phosphate..
*
Reagents :
Iron carrier, 0.2M Fe, 1 ml = 11.17 mg Fe. Dissolve 27.03 g of
FeCl3-6H20 in 1.2M HC1 and dilute to 500 ml with 1.2M HC1.
Phosphorus carrier, 1 ml = 10 mg P. Dry KH2POL, at 105°C in an
oven and cool in a desiccator. Dissolve 4.3937 g in water and dilute
to 100 ml.
Molybdate reagent. Add 150 ml of water and 60 ml of concentrated
NH^OH to 58.5 g of 85% molybdic acid. Warm gently, stir until dissolved
and dilute to one liter.
Magnesia mixture. Dissolve 50 g of MgCl2-6H20 and 100 g of NHUC1
in 500 ml of water. Add five drops of 0.1% methyl orange and concen-
trated NH^OH until yellow and then add one milliliter in excess. Let
stand overnight and filter. Make just acid with concentrated HC1 and
dilute to one liter.
*
See appendix A
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Ammonium nitrate, 2% w/v and 50% w/v
Nitric acid, 4M (1:3) and 0.3M (1:50)
Ammonium hydroxide, 1. 5M (1:9) and 0.75M (1:20)
Hydrochloric acid, 1.2M (1:9)
Citric acid
Potassium permanganate
Sulfurous acid, 8.9% w/w
Apparatus:
Fisher filtrator
Millipore Pyrex filter holder
47-mm diameter 0.45-micron pore-size membrane filters
Procedure:
A. Ferric Phosphate Precipitation
1. Transfer a 1-liter aliquot of water to a 1500-ml beaker and add
2 ml of 10 mg P/ml phosphorus carrier and 50 ml of concentrated
nitric acid. Heat to the boiling point and add saturated potassium
permanganate solution drop by drop until a pink color persists.
Continue heating near the boiling point for 1 hour or longer
to break down organic matter and to hydrolyze polyphosphates.
2. If a dark brown precipitate of manganese dioxide has appeared
at the end of the digestion period, dissolve it by adding 8.9%
sulfurous acid a drop at a time. Otherwise add only enough
sulfurous acid to decolorize the permanganate.
3. Cool to room temperature, add 10 ml of 0.2M Fe carrier solution
and adjust initially to about pH 2 with concentrated ammonium
hydroxide and then to pH 5.0 with 1.5>1 ammonium hydroxide.
4. Heat and keep near the boiling point for 20 to 30 minutes
and then cover and set aside to cool until the precipitate of
ferric phosphate and ferric hydroxide has settled.
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5. Decant the supernatant liquid through a 0.45-micron membrane
filter and transfer the precipitate and remaining liquid to
a centrifuge tube. Rinse the beaker with 2% ammonium nitrate
and add the rinses to the centrifuge tube. Centrifuge,
resuspend the precipitate in 2% ammonium nitrate and centri-
fuge again. Wash the small amount of precipitate on the
filter with 2% ammonium nitrate.
6. Dissolve any precipitate adhering to the 1500-ml beaker with
30 ml of 4M nitric acid and pour the solution into the centri-
fuge tube. When the precipitate has dissolved, pour the solution
into the filter assembly and collect the filtrate in a 250-ml
beaker. Use another 30 ml of 4M nitric acid to successively
rinse the beaker, centrifuge tube and filter. The total,, volume
of 4^1 nitric acid used must equal 60 ml. Use water for additional
rinses to give a total filtrate volume of 75 to 85 ml. More
water may be used but the solution must then be evaporated to
reduce the volume to 85 ml.
B. Ammonium Phosphomolybdate Precipitation
1. Heat the solution to the boiling point, remove from the hot
plate and immediately add 15 ml of 50% ammonium nitrate
solution. The temperature at this point will approximate the
required 80 C. Immediately add 50 ml of molybdate reagent
slowly from a pipette with constant stirring. Continue stirring
for several minutes and then set aside to cool. After 30 minutes,
set the beaker in an ice bath to cool for an additional 30 minutes
or allow to stand at room temperature for 1 hour to overnight.
2. Filter by vacuum on a 0.45-micron membrane filter and wash the
beaker, funnel and precipitate with 0.3M nitric acid to remove
soluble ferric salts.
3. Dissolve the precipitate from the beaker, funnel, and filter with
1.5M ammonium hydroxide - 0.01M ammonium citrate solution and
collect the filtrate in a 150-ml beaker. Continue washing with
1.5M ammonium hydroxide - 0.01M ammonium citrate until 80 ml of
filtrate has been collected.
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C. Magnesium Ammonium Phosphate Precipitation
1. Heat the solution to the boiling point and remove from the
hot plate. Fill a 15-ml pipette with magnesia mixture and
add drop by drop to the solution with continuous stirring
until a slight turbidity develops. Interrupt the addition of
reagent for 1 or 2 minutes but continue to stir and then
slowly add the remainder. Cover the beaker and cool for 30
minutes in an ice bath or from 1 hour to overnight at room
temperature.
2. Filter on a 0.45-micron membrane filter and wash the beaker,
filter and precipitate with 0.75M ammonium hydroxide.
3. Replace the beaker containing the filtrate with the 150-ml
beaker used for the precipitation and dissolve the preci-
pitate from the filter with 1. 2N[ hydrochloric acid. Con- ,
tinue washing with 1.2M hydrochloric acid until 50 ml of <
filtrate has been collected and then wash with water until
the filtrate volume is 80 ml.
D. Reprecipitation of Magnesium Ammonium Phosphate
1. Add 2 ml of magnesia mixture to the solution at room temperature.
Fill a 15-ml pipette with concentrated ammonium hydroxide and
add dropwise while stirring until a turbidity appears. Interrupt
the addition for 1 to 2 minutes but continue to stir and then
slowly add the remainder of the 15-ml of ammonium hydroxide.
Cover the beaker and cool for 30 minutes in an ice bath or
from 1 hour to overnight at room temperature.
2. Filter on a tared 0.45-micron membrane filter. Loosen the
adhering precipitate from the beaker with a policeman. Trans-
fer and wash the precipitate with 0.75M ammonium hydroxide.
3. Dry at room temperature. The precipitate loses water of hy-
dration if heated above 35
P = 158.5 mg MgNHi+POl+-6H20
dration if heated above 35 C. Weigh to obtain yield. 20 mg
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E. Standardization
Pipette into a 150-ml beaker a volume of calibrated phos-
phorus-32 standard containing about 1000 dpm. Add 2 ml of
10 mg P/ral phosphorus carrier solution and dilute to 50 ml
with 1.2M hydrochloric acid. Cover and heat for 30 minutes
to assure carrier exchange.
Cool to room temperature, dilute to 80 ml with water and
proceed as described in Section D "Reprecipitation of
Magnesium Ammonium Phosphate" except use 5 ml of magnesia
mixture.
F. Calculations
1. Yield
Y = Fractional chemical yield = (observed weight in milli-
grams) /I 58. 5 mg
Standardization
,, Ns-N,
E cpm/dpm = b
A x D x Y x V
s
where:
Ns = Count rate of standard sample in counts per minute
Njj = Counter background in counts per minute
As = Activity of calibrated phosphorus-32 solution at
time of calibration in dpm/ml
D = Fractional decay correction factor for tj-tg
where tg = time of calibration and t^ = midpoint of
counting interval
Y = Fractional chemical yield
V = Volume of calibrated phosphorus-32 solution in
milliliters
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3. Sample
pCi P/liter = — —° " • —
YxDxEx2.22xV
N -N,
s b
where:
NS = Count rate of sample in counts per minute
Njj = Counter background in counts per minute
Y = Fractional chemical yield
D = Fractional decay factor for tj-tQ where tp = time
sample collected and tj = midpoint of counting interval
E = Counter fractional efficiency factor
V = Volume of aliquot in liters
G. Results and Discussion
Chemical yield is 100 percent reproducible to about ± 1%. Two runs
with 1 liter aliquots of tap water gave 157.3 and 159.8 mg. No yield
correction is made except for accidental mechanical loss of precipitate.
The magnesium ammonium phosphate precipitate does not adhere too well to
membrane filters. For routine determinations the final precipitate may
be centrifuged and transferred to a tared planchet with alcohol. All
operations following the initial precipitation of ferric phosphate can
be conducted in centrifuge bottles but with decreased decontamination
from other radionuclides and increased chance of mechanical losses of
phosphorus. The magnesium ammonium phosphate may be ignited in a muffle
furnace to magnesium pyrophosphate. When mounted on an inert support,
heating to 500 C for 15 minutes is sufficient. Stainless steel planchets
discolor at this temperature but do not undergo a detectable change in
weight. When adapted to phosphate-containing materials such as bone, ,
carrier phosphorus need not be added but a curve of counter efficiency
versus weight of precipitate is required.
H. References
1. W. F. Hillebrand, G. E. F. Lundell, H. A. Bright, and
J. I. Hoffman, Applied Inorganic Analysis, John Wiley,
New York, 1953.
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2. H. H. Willard and H. Dlehl, Advanced Qaaat lr.-<-ive Analysis,
D. Van Nostrand Co., Inc., New York, 1943.
3. W. T. Mullins and G. W. Leddicotte, The Radiochemistry of
Phosphorus, National Academy of Sciences, Nuclear Science
Series, NAS-NS-3056, 1962.
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