A METHOD FOR THE RAPID DETERMINATION OF PHOSPHATE
IN FRESH PLANT TISSUES'
E. M. EMMERT
Little work has been done on the distribution of phosphate phosphorus
in living plants.
The colorimetric method worked out by FISKE and SUB-
BAROW (2) for phosphate in animal substances should be equally good when
applied to an appropriate solution from plant tissues.
showed that the solution of plant tissue must not be made alkaline, since
phosphate was precipitated in alkaline solution.
solutions were obtained by using a weak acid solution and a suitable quan-
tity of powdered charcoal or carbon black.
The procedure and results
Preparation of standard solution and checking of reagents
Make up a standard solution by dissolving 0.1755 gm. potassium
dihydrogen phosphate in a liter of water.
0.2 mg. of phosphate phosphorus.
Add the same amount of acid to it as in
the unknown, and neutralize just as is done in the procedure. A blank
should also be prepared to check the reagents, and one standard should be
treated with 1 per cent. acid and charcoal just as the unknown to test for
absorption of phosphate by the charcoal, since different grades vary in their
absorptive power (2).
If the charcoal being used causes absorption, a grade
should be obtained which does not.
phosphorus it should be digested with 5 per cent. by volume sulphuric acid
for one-half hour and leached with 1 per cent. by volume sulphuric acid
until the leachings give no test for phosphates by the FIsKE and SUBBAROW
method, as directed below.
Triturate thoroughly enough green plant tissue to give 0.1 to 0.3 mg.
phosphate phosphorus (usually 1 gram of tissue), in a mortar, with 5 gm. of
fine "decolorizing charcoal."2
Now add exactly 50 cc. of 1 per cent. by
volume sulphuric acid and mix well with the black paste.
5 minutes and filter.
If not clear and colorless, treatment with more char-
coal will be needed.
The amount of charcoal used in trituration should be
regulated according to the amount required to give a clear solution.
to five grams was found to be sufficient for a one-gram sample.
' The investigation reported in this paper is in connection with a project of the
Kentucky Agricultural Experiment Station and is published by permission of the
2 Decolorizing charcoal from J. T. Baker Chemical Co. ivas used.
Five cc. of this solution contains
If the charcoal contains phosphate
Allow to stand
solution is cleared, add a few drops of phenolplitlaleini to an aliquot (isii-
ally 25 cc.) and bring just to neutrality with dilute sodium hydroxide.
Make the neutral solutions of the unknown, blank, and standard up to
about 70 cc. and add 10 cc. of 2.5 per cent. aminoiiuin molybdate made up
in 5 N sulphuric acid. Mix and add 4 cc. of 1, 2, 4-aminio-naplhtholsulphonic
acid, prepared as directed by FISKE and SUBBAROW (1).
up to 100 cc.
After ten minutes read in a colorimeter and calculate the
phosphate phosphorus in the original plant tissues.
Mix and make
Table I shows excellent duplication.
The duplicates wi-ere taken from
PHOSPIIATE PHOSPHIORUS IN GREEN PLANT TISSC ES
(1-GRA-M SAMIPLES WERE TREATED WITH 5 GRAMIS OF PHOSPIIATE-FREE CHARCOAL AND
1 PER CENT. SU-LPhURIC ACID)
PC r celt.
the same leaf by selecting a large leaf and using tissue between the large
In this way uniform samples were obtained.
samples were from plots which varied greatly in treatment.
large applications of 85 per cent. syrupy phosphoric acid which explains
its high phosphate phosphorus content.
which probably explains the smaller amount of phosphate phosphorus ob-
tained.No. 1 was a fresh compost not comparable with the soils in the
Table II shows conclusively that the charcoal used did not absorb phos-
phate from the acid solutions used.
The different lettuce
No. 17 had
No. 3 was from a plot highl in lime
Choice of reducing agent
TRUOG and MEYER (3) used the metlhod of DENIG1S in wlhich stannous
chloride is employed as a reducing agent.
Their tests, however, show that
EMMERT: DETERMINATION OF PHOSPHATE
RECOVERY OF PHOSPHATE IN ACID SOLUTIONS FILTERED FROM PHOSPHATE-FREE CHARCOAL
(TREATED WITH THE SAME REAGENTS AS WERE USED ON THE PLANT TISSUE)
the range of acidity and concentration of molybdate in which accurate re-
sults are obtained, is very narrow.
gives a color in the absence of phosphate; and if it is too strong, the blue
color fails to form even if phosphate is present.
tain a uniform solution of stannous chloride, a layer of mineral oil being
recommended to preserve
The solution of
sulphonic acid will keep at least a month without any special treatment.
It does not have such a narrow range of acidity in which it is accurate and
never causes a color to form when phosphorus is not present, as long as the
solution is acid and cold.
On boiling, however, a blue color is produced
without phosphorus. Trials with different degrees of acidity, from neutral
to very acid, in the cold, produced no color from molybdate alone.
nous chloride acts on molybdate in the cold even if phosphate is not present,
unless a certain acidity is maintained, and small increases in temperature
may cause a color even if the acidity is right.
Another advantage of the amino-naphtholsulphonic acid reagent is that
it is not so sensitive to interference by the presence of ferric iron as stan-
nous chloride. TROUG and AIEYER (3) found that the limit with stannous
chloride was 2 ppm. of ferric iron.
30 ppm. of ferric iron may be present in the solution with 1, 2, 4-amino-
naphtholsulphonic acid without interfering.
It is true that stannous chloride brings the color faster than the
amino-naphtholsulphonic acid reagent, but as long as the standard and
unknowns are treated alike as to time and reagents accurate results are
obtained as shown by tables I and II.
factor in the plant solutions, since silica would not be put into solution by
the weak acid used, even if the plant contained a significant amount.
If the acidity is weak the molybdate
It is also difficult to main-
1, 2, 4-amino-naphthol-
Table III shows that as much as
Interference by silica- was not a
EFFECT OF FERRIC IRON ON THE BLUE PHOSPHATE COLOR
(FERRIC SULPHATE WAS USED)
NOTES ON COLOR
Blue matched well
Blue, slight cloudiness
Slight green, quite cloudy
Titanium and arsenates cause errors if present (3), but w-ould not likely
be present in significant amounts in plant extracts.
also found that nitrate, if present at the rate of 200 ppm. of nitrogen,
caused a fading of the color.
However, it would be highly improbable that
a one-gram sample of any plant in 100 cc. would cause a conicentration of
200 ppm. of nitrate.
1. A method of determining phosphate phosphorus in fresh plant tissue
The tissue is extracted with dilute sulphuric acid and cleared
with phosphate-free powdered charcoal or carbon black.
FISKE and SUBBAROW is used on the cleared extract.
2. Data are presented which show good duplicates on tomato, tobacco
and lettuce leaves.
3. No adsorption of phosphate by the charcoal used could be detected.
4. Preference is given to 1, 2, 4-amino-naphtholsulphonic acid over stan-
nous chloride as a reducing agent, because:
(1) The reagent keeps better.
(2) It does not require as narrow a range of acidity for accuracy.
(3) A blue color does not develop in the absence of phosphate at any
degree of acidity used in these experiments.
(4) Larger quantities of iron may be present in the tissue extract with-
out causing interference.
UNIVERSITY OF KENTUCKY,
TRUOG and MEYER (3)
The method of
EMMERT: DETERMINATION OF PHOSPHATE
1. FISKE, C. H., and SUBBAROW, Y.
2. HARPER, H. J.
Ind. Eng. Chem. 16: 180-183.
3. TRUOG, E., and MEYER, A. H.
method for phosphorus and arsenic.
(Analytical edition) 1: 136-139.
The colorimetric determination of
Jour. Biol. Chem. 63: 375-400.
The accurate determination of nitrates in soils.
Improvements in the Deniges colorimetric
Jour. Ind. Eng. Chem.