AN IMPROVED MICROMETHOD OF PHOSPHATE

DETERMINATION
BY BASIL C. SOYENKOFF
(From the Department of Chemistry, New York University College of Dentistry,
New York, New York)

(Received for publication, April 28, 1952)

A highly sensitive method of color development, permitting the colori-

metric estimation of 0.02 to 0.2 mg. of phosphorus per liter, was described
in a previous communication (1). The effects of acids and other inter-
fering substances were the main limitations of the method.

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In the improved method, a readily soluble dye (2-p-dimethylamino-
styrylquinoline ethosulfate) has been substituted for quinaldine red, which
had to be brought into solution by prolonged heating. Interference by
acids, etc., has been reduced and the method adapted to rapid analyses
with routine equipment.
Compared with the Fiske-Subbarow method (a), the sensitivity is about
15 times greater, and the change in readings with time is much smaller.
On the other hand, the molybdate must be more accurately measured
and interference by proteins is likely to be larger than in the molybdenum
blue methods.
In the determination of inorganic phosphate in blood serum, the im-
proved method gave 2 per cent lower values than by the amidol-molyb-
denum blue method. Applications of the method to H&K)4 digests (wet
ashed samples) are being studied.

Preparation of Dye
The dye used in the original method, quinaldine red, is the ethiodide
of 2-p-dimethylaminostyrylquinoline. A more soluble dye was obtained
by substituting sulfate for the iodide to form an ethosulfate of the styryl-
quinoline.
In the preparation, equimolar amounts of quinaldine and diethyl sulfate
gave quinaldine ethosulfate. The styryl dye was formed by condensation
of the ethosulfate 1vit.h p-dimethylaminobenzaldehyde. Finally, the free
ester linkage was hydrolyzed with CH&OOH, as follows.
To 26 ml. of diethyl sulfate (redistilled in vacua at about 100”) were
added 28.5 ml. of quinaldine (dried, then redistilled in va.cuo at about
130”). The mixture was heated under a reflux on a steam bath for 3 hours
and, while still warm, was dissolved in 80 ml. of (commercial) absolute
ethanol.
221
222 MICROMETHOD FOR PHOSPHATE

Meanwhile, 30 gm. of p-dimethylaminobenzaldehyde were mixed with

100 ml. of Cellosolve and 0.3 ml. of morpholine in a 250 ml. wide mouth
flask. A boiling stone was added, and the mixture brought to 120” on a
hot-plate in a hood.
The ethanolic solution was added dropwise during 1.5 hours, the contents
of the flask being kept at 120-125”. After an additional half-hour at
125130”, the contents were poured, with mixing, into 250 ml. of toluene
and placed in a refrigerat,or for 10 hours.
The crystals were filtered off, washed with toluene, and dissolved in 25
ml. of warm CH&OOH plus 3 ml. of water. The mixture was heated under
a reflux on a steam bath for 1 hour, 45 ml. of pyridine were added, and the

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heating continued for 1 hour. The digest was cooled, mixed with 200 ml.
of toluene, and refrigerated for 2 hours. The crystals were dissolved in
45 ml. of acetylene tetrachloride at 120” and reprecipitated by adding 200
ml. of toluene and refrigerating for 10 hours.
The dye was filtered off, washed with toluene, and dried in vucuo at
100’ for 2 hours. The yield was 34 gm.
The N and S content corresponded to the formula C$rH24N208, in
which 1 molecule of 2-p-dimethylaminostyrylquinoline is combined with
1 molecule of ethylsulfuric acid and 1 molecule of water. The dye gained
about 5 per cent in weight on exposure to air.

Reagent Solutions
Distilled water and Pyrex glassware were used.
Phosphate standards. Stock phosphate (100 mg. of P per liter) con-
tained 0.4390 gm. of KHzPOd and 10 ml. of 10 N H$Od per liter. Stand-
ards containing 0.05 to 0.5 mg. of P per liter were prepared from the stock
plus 5 ml. of 10 N H$04 per liter.
Stock dye solution. 0.1 per cent in water. Stable for half a year in
the dark.
Reagent dye solution (volume ,900 ml.). To 1.22 gm. of benzoic acid (re-
agent grade) and 1.23 gm. of nicotinic acid (Eastman Kodak) were added
94 ml. of water, followed by 2.84 gm. of NHdHCO, (reagent grade). The
contents were stirred until dissolved, then 1.18 gm. of succinic acid (re-
agent grade) mere added, and excess CO* liberated by vigorous stirring.
Next were added 10 ml. of the stock dye, 2 ml. of freshly prepared 1 per
cent gum arabic (select grade), and 88 ml. of water, with mixing after
each addition.
The pH of the solution was 5.60 to 5.64. The reagent was stored in
glass-stoppered bottles and kept out of direct sunlight. Solutions older
than 2 weeks were not studied extensively. The most important change
on aging appears to be in the H&O4 tolerance range (see below).
 B. C. SOYENKOFF 223

Molybdate-Sulfate-To a freshly prepared solution of 8.85 gm. of am-

monium molybdate (reagent grade, 81.4 per cent of MoOa) in about half a
liter of water were added 270 ml. of 10 N HzS04, and the solut,ion was made
up to 1 liter with water.

Procedure

Preparation of Samples---The method is mainly intended for solutions

of ash or calcified tissues in H&30+ for CCl,COOH fihrates and H&304
digests diluted to contain 0.025 to 0.125 N acid. HCIO, digests are not
suitable.
The blood serum samples used in this study were deproteinized with

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9 volumes of 10 per cent CCl&OOH and the filtrates diluted ten times with
water.
Development of Color-Into a dry or well drained Klett micro tube
were measured 2 ml. of the sample and 2 ml. of the dye solution; then 1
ml. of the molybdate was added from a transfer pipette, with stirring.
The color intensity was read after 10 minutes. The inside of the tube was
then rinsed with H&304, flushed with an upward jet of water, and the tube
set to drain in an inclined position.
The above operations required 25 minutes for three samples, one set
of tubes draining on the rack while the other three were in use. The
time could be shortened with the aid of autopipettes, etc., since the color
readings showed little change after 5 minutes.
The molybdate should be accurately measured, and the time of out-
flow of the pipette should not be less than 20 seconds, if maximum ac-
curacy is desired. The dye can be added from a burette, with about 2 per
cent accuracy (see under the sources of error).
The tendency of the dye to deposit on glass, which was troublesome in
the old method, was not observed here.
Reading of Color; Calibration Curve-The absorption curves, traced in a
Hardy photometer, were closely similar to those obtained by the quinaldine
red method (l), with the maxima centered near 510 rnl.r.
The samples in the Klett tubes were read in a Klett-Summerson color-
imeter equipped with a Corning No. 4010 filter of 4 mm. thickness. A
large reading lens was substituted for the plastic magnifier supplied with
the instrument. To reduce parallax, a mark was made on the pointer
window, and the eye so placed that the mark fell on the dividing line.
With the above filter, whose transmission maximum was 25 per cent,
at 525 rnp, and the half width 60 mp, sufficient pointer response was ob-
tained for readings to 0.1 division.
As seen from Table I, the readings deviated appreciably from propor-
tionality to the phosphate concentrations. Over the range of 0.04 to 0.2
224 MICROMETHOD FOR PHOSPHATE

mg. per liter, the deviations did not exceed 5 per cent for a concentration
ratio of 1:2.
Below 0.08 mg. of P per liter, better proportionality was obtained by
subtracting the reagent blank from the readings. The blank was smaller
and the 0.02 mg. per liter reading was larger than in the quinaldine red
method. Above 0.3 mg. per liter, the readings showed large deviations
from proportionality.

Estimates of Precision and Sources of Error

Drift-Readings of 0.02 mg. of P per liter increased by 0.6 per cent
between 5 and 10 minutes after mixing, and by an additional 0.6 per cent

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during the 10 minutes following.

TABLE I
Deviations from Proportionality
k = Klett readings at 23”; A, percentage deviations of the total readings from
proportionality; 6, percentage deviations of readings corrected for the reagent
blank.

P in mg. per liter of processed sample

Age of dye solution Values of
0 0.02 0.04 I 0.08 i 0.20 0.40
,- I--.- ‘-

1 day 7.3 28.6 51.4 1 97.6 / 256.7 420.4

+14.5 +4.9 I 0 +4.8 -16.1
-6.1 -2.5 0 +9.4 -9.3
2 wks. 7.5 28.8 53.1 101 .o 254.6 432.7
+12.5 +3.3 0 +1.6 -16.8
-9.8 -2.6 0 +5.4 -10.0
__.__

Readings of 0.1 to 0.2 mg. per liter declined by 0.1 to 0.2 per cent be-
tween 5 and 20 minutes after mixing.
l’emperature Coe&ientsThe following estimates are based on tem-
perature readings of the sample made immediately after the mixing. For
0.02 mg. of P per liter, +0.3 per cent between 20-25”; for 0.1 to 0.2 mg.
per liter, +0.6 per cent between 2CL25”; for 0.1 to 0.2 mg. per liter, i-O.5
per cent between 25-30”.
Amounts and Rate of Addition of ReagentsThe following estimates are
based on Klett readings of processed samples containing 0.1 mg. of P
per liter.
A 10 per cent increase in the volume of the dye solution raised the
readings by 0.7 per cent. A 10 per cent decrease raised the readings by
0.2 per cent.
A 5 per cent increase in the volume of the molybdate lowered the read-
 B. C. SOYENKOFF 225

ings by 4.0 per cent. A 5 per cent decrease raised the readings by 5.6
per cent.
When the time of outflow of the molybdate was increased from 26 to
55 seconds, the Klett readings increased by 2.2 per cent.
Estimates of Pretition-The estimates in the accompanying tabulation
are each based on ten replicate samples, read at 25-26”: P is in mg. per
liter; a is the mean, in Klett divisions; d is the average deviation, expressed
in per cent of the mean; V is the coefficient of variation.

P a d V
____~- ~

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Samples in micro tube read after 10 min.. 0.02 29.7 0.47 0.64
“ “ 10 ml. “ “ “ 5 “ 0.04 57.5 0.40 0.49
“ “ micro “ “ “ 10 “ 0.26 255.1 0.71 0.80

H2S04 Tolerance Range; Efects of Strong Acids-The strong effect of pH,

observed in the quinaldine red method, was modified or even reversed
by the precipitating action of some anions on the dye. In the revised
method, the specific effects of benzoate, nicotinate, and succinate have
been combined to produce a tolerance range, within which the H&O4
concentration of the sample can be varied between 0.025 and 0.125 N
without changing the color reading by more than 1 per cent. As seen
from Table II, the tolerance range is decreased when the age of the reagent
dye solution exceeds 2 weeks. Similar findings have been obtained with
several dye solutions, made from two preparations of the dye.

TABLE II
H&O, Tolerance Range
P = 0.1 mg. per liter of the sample-reagent mixture; k is the Klett reading at 25”;
A is the percentage difference from the reading of the sample which contained 0.05
N HzSO,.
-
Normality of H&h in untreated sample
Age of dye Values of
s&t ion
0.025 0.050 0.075 .I00 0.125

1 day 129.6 128.5 12i.4 127.6 128.9

+0.8 0 -0.8 -0.7 +0.3
2 wks. 129.3 129.9 129.5 / 130.6 130.1
+0.5 I 0 -0.3 +0.5 +0.3
2 mos. 129.0 128.9 129.2 131.1 135.3
+0.1 0 +0.2 +1.7 +4.9

Specific effect of CCLCOOH was not observod in the improved method.

The P standards containing CCl&OOH gave slightly lower color readings
226 MICROMETHOD FOR PHOSPHATE

than the standards in 0.5 N HpSO,, as might be expected from the ob-
served pH difference (see “Inorganic phosphate content of serum”).
HClO, increased the readings and produced upward drifts.
Other Interfering Substances--Except for serum albumin, the substances
listed in Table III increased the color readings. The effects of citrate
and ferric ion were reversed at higher concentrations.
The tolerance ratios of silicate and ferric ion compare favorably with
those reported for the Fiske-Subbarow method (2), while the interference
by citrate is probably stronger than in the latter method (3).
Compared with the original quinaldine red method, the effects of citrate,
of proteins, and of gum arabic (used in the dye solution) have been reduced

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five to ten times.
TABLE III
Effects of Some Constituents of Biological Samples
The sample-reagent mixtures contained 0.1 mg. per liter; i.e. the phosphate con-
centration was 3.2 X 10-s M and was read 10 minutes after mixing. The tolerance
ratio is the molarity of the substance which produces 0.5 per cent change in the
reading, divided by the molarity of the phosphate.
T&rfLlKX
ratio substance

Ethanol >6000 Lactic acid 70

Urea 3300 NaF 40
NaCl 1000 Fe+++ as FeNHd(SOJ2 26
CaC12 1600 Serum albumin 8*
Mg++ as MgSO, 200 NakliOa 7
140 Citric acid 7

* Calculated from the concentrations in mg. per liter.

Inorganic Phosphate Content of Serum

CCl&OOH filtrates were prepared from ten human blood sera, ob-
tained from nine subjects. Each filtrate was sampled for twelve deter-
minations by the new method and six determinations by the molybdenum
blue method (with amidol as the reducing agent).
Two sets of standards were used in the molybdenum blue method.
The first one contained 0.5 N H&04. The second one contained 8.7 per
cent CCl&OOH to match the pH value of the filtrates. The color read-
ings of the second set were about two divisions higher than those of the
first, but checked closely when corrected for the difference between read-
ings of the untreated samples. The color readings of the serum filtrates
were therefore corrected for their sample blanks. The corrections ranged
from -0.6 to -1.8 per cent.
The P standards for the new method were prepared by lo-fold dilution
of the standards used in the molybdenum blue method. The standards
 B. C. SOYENKOFF 227

containing 0.5 N Ha04 were more alkaline by 0.08 pH unit and gave
0.8 per cent higher color readings than the standards containing 0.87
per cent CCl&OOH, which matched the diluted filtrates in pH value.
The sample blank corrections to readings of the standards and diluted
filtrates were of the order of 0.1 per cent.
The inorganic P content of the sera, determined by the new method
with P standards in 0.87 per cent CCLCOOH as the reference, ranged
from 2.34 to 5.05 mg. per 100 ml. The values by the molybdenum blue
method, corrected for the sample blanks, were 0.2 to 3.7 per cent higher
than the above, the average difference being 2.0 per cent. The uncorrected
molybdenum blue values, with P standards in 0.5 N HaSO4 as the reference,

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were 1.1 to 5.3 per cent (average 3.1 per cent) higher than the values by
the new method.
Thus, the values by the new method are probably too low by about
2 per cent. If the preparation of special P standards for serum is dis-
pensed with, and the standards in 0.05 N HaSO4 are used instead, the ad-
ditional error is about 1 per cent. These errors are not large, compared
with the differences reported between two molybdenum blue methods (4)
which are often used in serum analysis.

The nitrogen determinations used in determining the formula of the

reagent dye were made by Dr. Carl Tiedcke of Teaneck, New Jersey.
Dr. Addison V. Bowman participated in the experiments leading to the
development of the method. The second estimate of precision is from
the readings by Mr. Bernard0 E. Taylor. The blood samples were ob-
tained through the kindness of Dr. Harry Blechman and of the do-
nors. The work was supported by a grant from the United States Public
Health Service.
SUMMARY

The quinaldine red method of phosphate determination has been re-

vised so as to decrease the effects of pH and interfering substances and has
been adapted to routine use. The sensitivity, as before, is about 15
times that of the Fiske-Subbarow method. A precision of 0.5 to 0.8 per
cent is obtained over the range of 0.02 to 0.2 mg. of P per liter. The
method is mainly intended for CCl+ZOOH filtrates, for samples ashed by
ignition and by digestion in HzSOd, and for solutions of calcified tissues.
The accuracy, when applied to serum filtrates, is estimated at 2 per cent.

BIBLIOGRAPHY

1. Soyenkoff, B., J. Biol. Chem., 168, 447 (1947).

2. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66, 382 (1925).
3. Berenblum, I., and Chain, E., Biochem. J., !D, 298 (1938).
4. Looney, J. M., and Dyer, C. G., J. Lab. and Clin. Med., 27,554 (1942).
 AN IMPROVED MICROMETHOD OF
PHOSPHATE DETERMINATION
Basil C. Soyenkoff
J. Biol. Chem. 1952, 198:221-227.

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