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(From the Department of Medicine, Rush Medical College, the University oj
Chicago, Chicago)
(Received for publication, August 12, 1931)
The usual method for the estimation of potassium in the blood
and the urine depends, as is well known, upon the precipitation of
the element as the cobalti-nitrite and the subsequent estimation
of one of the constituents of the precipitate. The reducing power

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of the precipitate is used by Kramer and Tisdall(l); the formation
of a diazo color by the nitrite is employed by Doisy and Bell (2);
the potassium is titrated as the acid tartrate by Fiske and Litarc-
zek (3); the cobalt is determined as the thiocyanate by Breh and
Gaebler (4).
From a theoretical point of view those methods which determine
directly a stable constituent of the precipitate are preferable to
those which depend upon the activity of the unstable nitrite rad-
ical. The methods of Fiske and Litarczek and of Breh and Gaeb-
ler embody this principle, but each of these has a disadvantage:
that of Fiske and Litarczek involves an ashing and a second-pre-
cipitation, and that of Breh and Gaebler depends upon a foreign
factor (alcohol concentration) as well as upon the factor being esti-
It is the object of this paper to describe the formation of a new
stable, colored compound of cobalt and its application to the micro
estimation of potassium in biological fluids.
Principle-When a solution of sodium ferrocyanide is added to
a mixture of a cobaltous salt and choline hydrochloride in water
* Ernest E. Irons Fellow in Medicine; under the supervision of Dr.
George F. Dick.
t National Research Council Fellow in Medicine; under the supervision
of Dr. Wilber E. Post.
686 Calorimetric Estimation of Potassium

solution, an emerald-green color develops immediately. Prelim-

inary experiments with this color showed that it was sensitive to
minute amounts of cobalt, choline, and ferrocyanide, and that,
in the presence of a given excess of choline and ferrocyanide, it
could be used to determine calorimetrically small variations in the
concentration of cobalt. It was further established that the col-
ored solution might be diluted with water without affecting its
quality, and that it developed to maximum intensity in a few
minutes and maintained this intensity for hours.
Procedure-The precipitation of potassium from blood serum

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is carried out according to the method of Ifiamer and Tisdall.
Into a 15 cc. conical centrifuge tube marked at 6 cc. is pipetted 1
cc. of serum. 2 cc. of Kramer and Tisdall’s sodium cobalti-nitrite
reagent’ are added slowly with constant agitation of the serum.
After 45 minutes, 2 cc. of water are added, the contents mixed, and
the tube centrifuged at moderate speed for 15 minutes.
The tube is then inverted and drained briefly. 1 cc. of water is
run down the side of the tube, the tube centrifuged 5 minutes, and
again inverted and drained. The precipitate is then suspended in
2 cc. of 70 per cent alcohol with the aid of.a stirring rod, the tube
centrifuged 5 minutes, inverted, and drained. The alcohol wash-
ing is repeated once.
After the tube has drained for a few minutes, the precipitate is
suspended in 1 cc. of water with the aid of a stirring rod. Another
cc. of water is added and the tube then immersed in a boiling water
bath for 10 minutes. The contents of the tube are agitated several
times during the heating in the bath. The precipitate will dissolve
to form a clear, colorless solution. A small amount of insoluble
material, probably protein, does not interfere. When the tube
has cooled, 1 cc. of 1 per cent choline hydrochloride and 1 cc. of 2
per cent sodium ferrocyanide are added to the solution and made
up to the 6 cc. mark.
1 Sodium Cobalti-Nitrite Reagent-Solution A: 25 gm. of cobaltous nitrate
crystals are dissolved in 50 cc. of water and to this solution are added 12.5
cc. of glacial acetic acid. Solution B: 120 gm. of sodium nitrite (potassium-
free) are dissolved in 180 cc. of water. This gives a total volume of about
220 cc. To all of Solution A are added 210 cc. of Solution B. An evolution
of nitric oxide gas occurs at once. Air is drawn through the solution until
all the gas has passed off. The reagent (thus prepared it has a pH of 5.7)
is placed in the ice chest and filtered each time before using. It will keep
at least 1 month.
H. R. D. Jacobs and W. S. Hoffman 687

The precipitation of potassium from ashed or inorganic solutions

is made from 2 cc. of the unknown solution with 1 cc. of the
reagent. The first washing with water after centrifugation is
omitted, and the precipitate washed twice with 70 per cent alcohol
as outlined for serum. The remainder of the procedure is the
The colored solution thus formed is compared with a standard
colored solution made from 1 cc. of standard cobalt nitrate (or
sulfate), 1 cc. of 1 per cent choline hydrochloride, and 1 cc. of 2 per
cent sodium ferrocyanide made up to 6 cc. with water. The

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standard cobalt solution is made from either the nitrate or the sul-
fate: 0.6701 gm. of Co(N0&.6H20 or 0.6469 gm. of CoS04.7Hz0
made up to 1000 cc. with water. These solutions must be stand-
ardized because these cobalt salts are hygroscopic. The stand-
ardization is made against the cobalt contained in the precipitates
of potassium from a standard potassium sulfate solution contain-
ing 0.4011 gm. of KzSO~ per liter (equivalent to 18 mg. of .K per
100 cc.).

This method embodies the theoretical advantage of determining
directly a stable constituent of the potassium precipitate. The
procedure is relatively simple. The use of a permanent standard
permits of more accurate comparisons between values obtained
at different times, and serves as a check upon the efficiency of the
precipitating procedure when this is in question.
There are only two accurate measurements to be made in each
det.ermination: that of measuring 1 cc. of the serum to be used
and that of diluting the colored solution to the 6 cc. mark. All
of the manipulations are carried out in the original tube so that
there is no loss from transfers.
Washing the precipitate in alcohol has the advantage that it is
thoroughly freed from the precipitating agent without danger of
loss, since it is insoluble in alcohol. The alcohol also makes the
precipitate cohere well so that the supernatant fluid may be
poured off and the tube drained upside down. It was found that
there was a large amount of cobalt in the first water washing, a
small amount in the first alcohol washing, a trace in the second
alcohol washing, and none in the combined third alcohol washings
688 Calorimetric Estimation of Potassium

from eight tubes in a test experiment. When the precipitates

are washed with water according to the method of Kramer and
Tisdall, even the fourth washings contain perceptible amounts of
cobalt, which must come either from the residual precipitating
reagent or from solution of the precipit.ate itself.
The quantity of cobaltous salt to be used in the preparation of
the standard cobalt solution is calculated from the formula of the
precipitate, &NaCo(NO& .H*O, in which the amount of potas-
sium is to be 0.18 mg. (equivalent to that in 1 cc. of solution con-
taining 18 mg. per 100 cc.). Cobaltous nitrate and sulfate rapidly

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absorb water during exposure to air, so that weighing is not accu-
rate, and standardization against a known potassium solution is
necessary. Once made, the cobalt solution keeps indefinit,ely.
If the sulfate is used for the standard cobalt solution, it may also
be standardized by determining the sulfate in the solution.
The choline solution is made from choline hydrochloride crystals.
A stock solution of 10 per cent, from which the 1 per cent solution
is made by dilution, may be kept in the refrigerator. Dry crystals
may be conveniently prepared by precipitating an alcoholic solu-
tion with ether.
The sodium ferrocyanide is made in 2 per cent solution from
NalFe(CN)6. 10HzO. It is preserved in a cool, dark place. When
it becomes very yellow or when it develops a red precipitate, it
should be discarded. Of course, potassium ferrocyanide may be
used instead.
The quantities of choline and ferrocyanide used in this method
are sufficient for any amounts of cobalt encountered in potassium
determinations. In developing the color, the choline solution
should be added to the cobalt solution first, and then the ferro-
cyanide. If the ferrocyanide is added to the cobalt solution
before the choline, the color develops much more slowly, instead
of immediately, as in the given procedure.
The object in adding 1 cc. of water as the first washing after the
centrifugation is to remove as much of the protein-containing fluid
as possible before washing the precipitate with alcohol. The
presence of too much coagulated protein makes disintegration of
the precipitate somewhat more difficult.
If the precipitate is dissolved as outlined above, solution will be
rapid and complete. The small particles which sometimes remain
H. R. D. Jacobs and W. S. Hoffman 689

are probably protein, but they give up their salt readily and
become translucent flakes which cause no difficulty. They settle
Determinations of Potassium in Known Potassium Solutions

K taken T K found T K taken K found

WI. w7. m!J. w7. WI.
0.10 0.1043 0.0991 0.16 0.1666

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0.10 0.1064 0.0999 0.16 0.1600
0.11 0.1100 0.1098 0.18 0.1786
0.11 0.11* 0.1091 0.18 0.1830
0.12 0.1204 0.12* 0.20 0.20*
0.12 0.1204 0.1207 0.20 0.2012
0.14 0.1433 0.1476 0.24 0.2420
0.14 0.1386 0.1363 0.24 0.2400
- -
* The solution against which the other solutions of the series were com-

Determinations of Potassium in Human Blood Sera*

- T Potsssium
Case No. Authors’
method method ashing
_- _.
m7. mg. mg. ?w. m7.
per cent per cent per cent pzent per cent per cent
19.9 17.0 19.7 6 18.6 16.6 17.1
19.9 18.5 19.9 18.7
18.4 19.6 7 19.4 19.4
18.2 19.7 19.0
21.1 8 21.9 19.9
21.3 21.9
21.4 19.9 9 22.3 20.2 21.7
21.4 22.1 20.7
21.3 20.2 10 20.6 19.8 20.0
21.3 20.9
- - - -
* Ten consecutive cases.

out after the colored solution is formed and remain in the tube
when the solution is poured into the calorimeter cup.
690 Calorimetric Estimation of Potassium

Color comparisons are best made by daylight. The colors are

easily matched in any good calorimeter.
Tables I to IV present the results of determinations carried
out by this method upon several types of solutions. Tables III
and IV show that known amounts of potassium added to urine
and serum can be determined accurately.
Determinations of Potassium in Urine with Added Potassium

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K in urine K added Total K K found

mp. per cent mg. per cent mg. per cent mg. per cent
178.1 24.0 202.1 204.1
178.1 48.0 226.1 226.2
178.1 72.0 250.1 257.6
178.1 96.0 274.1 277.4
The known amounts of potassium were added to measured portions of
the urine, the mixtures then ashed, and the potassium determined in an
aliquot of the solutions of the ashes. Two cobalt standards, one equivalent
to 18 mg. of K per 100 cc., and the other to 36 mg. of K per 100 cc., were used.

Determinations of Potassium in Serum with Added Potassium
Solution A

K in serum K added Total K K found K in serum K added Total K K found

~- --
fw. WI. ma. ml.
peR!ePcntpm cent peznt per cent p2Lt peznt pm cent per cent
18.6 1.8 20.4 19.9 22.2 1.8 24.0 24.0
18.6 3.6 22.2 22.2 22.2 1.8 24.0 23.8
18.6 5.4 24.0 24.1 22.2 3.6 25.8 25.9
18.6 7.2 25.8 25.9 22.2 5.4 27.6 27.5
22.2 7.2 29.4 29.8

Ammonium salts interfere in this determination because am-

monia also forms a very insoluble cobalti-nitrite precipitate.
For this reason glassware should be cleaned shortly before the
precipitation is made, and, for this reason also, fluids, such as
urine, which contains appreciable amounts of ammonia, must
first be ashed.
In serum potassium determinations it must be remembered that
H. R. D. Jacobs and W. S. Hoffman 691
the red corpuscles contain relatively much more potassium than
the serum, and that potassium values will be incorrect in hemo-
globin-tinged serum and in serum which has been in contact
with the clot for a long time. It is best to collect the blood in
perfectly dry (preferably baked) glassware, allow it to clot in an
incubator for 15 minutes, and then centrifuge it, and separate the
serum at once.

1. The formation of a hitherto unmentioned colored solution

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from cobalt, choline, and ferrocyanide is described.
2. A method is outlined for the determination of potassium in
blood, urine, and ash solutions by means of this color.
3. Data on determinations of potassium in blood sera, in urine,
and in inorganic solution are presented.

1. Kramer, B., and Tisdall, F. F., J. Biol. Chem., 48, 339 (1921).
2. See Briggs, A. P., J. Biol. Chem., 67,351 (1923).
3. Fiske, C. H., and Litarczek, G., J. Biol. Chem., 87, p. xvi (1926).
4. Breh, F., and Gaebler, 0. H., J. Biol. Chem., 87, 81 (1930).