A NOTE ON A CLOSED TITRATION FLASK FOR USE IN THE

BROMOMETRIC DETERMINATION OF MAGNESIUM

WITH 8-HYDROXYQUINOLINE
APPLICATION TO THE ESTIMATION OF MAGNESIUM IN
TISSUES AND URINE
BY DAVID M. GREENBERG, CARL ANDERSON, AND
ELMA V. TUFTS
(From the Division of Biochemistry, University of California Medical
School, Berkeley)

(Received for publication, July 29, 1935)

While the bromometric method of determining magnesium with

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S-hydroxyquinoline is based on stoichiometric principles, the
relatively high volatility of bromine may cause analytical difficul-
ties if the bromination is carried out in an open vessel. When
small quantities of magnesium are to be estimated, the loss of
bromine by volatilization may lead to very large errors. In the
method of Greenberg and Mackey (1) for the determination of
magnesium in blood, this error was minimized by definitely timing
the bromination reaction and by running parallel blank titrations
under the same experimental conditions with each analytical
series. In the hands of a careful analyst, this procedure yields
accurate results.
However, to avoid this rather difficult step, we have devised a
simple means of preparing a closed titration flask which practically
eliminates the possibility of loss of bromine by volatilization. A
diagram of the flask is shown in Fig. 1. It consists of a ‘250 ml.
capacity suction flask regularly used in the method of Greenberg
and Mackey which is stoppered with a long stemmed glassfunnel
(6 inch stem). The funnel is fitted into a thoroughly paraffined
cork stopper.
In the side arm of the suction flask, as is indicated by the arrow
in Fig. 1, there is introduced a bit of glass wool soaked in 20 per
cent potassium iodide solution to trap the bromine from the air
561
562 Mg Determination in Tissues and Urine

which is displaced when liquids are introduced into the flask

through the funnel.
To carry out an analysis, the magnesium precipitate is filtered
and washed as described by Grccnbcrg and R/lackey. The precipi-
tate is then dissolved with strong hydrochloric acid and washed
into the suction flask with water. With a pipette, 1 ml. of 50 per
cent potassium bromide is introduced into the flask. Now a fun-
nel-cork unit is fitted into the mouth of the flask so that the lower
end of the stem of the funnel is below the level of the liquid in the
flask and a glass wool plug soaked with potassium iodide ispushed
into the side arm with a small glass rod.

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FIG. 1. Closed titration flask for bromination

Then 5 ml. of standard 0.005 M potassium bromate are added

through the funnel from an accurately calibrated pipette and
washed down with distilled water. The flask is gently shaken to
mix the contents thoroughly and then allowed to stand for about 10
minutes to permit the bromination of the hydroxyquinoline. At
the end of this time, 1 ml. of 20 per cent potassium iodide solution
is added to the flask through the funnel and washed down with a
small quantity of water. The contents of the Aask are again mixed
and set aside for about 20 minutes to insure the complete reaction
of the bromine with the iodide. The funnel is then removed and
washed off with water to remove any adhering iodine. The glass
 Greenberg, Anderson, and Tufts

wool plug is pushed into the flask with a small glass rod and the
rod and side arm are rinsed off with a stream of water. The iodine
which has been formed is now titrated with 0.02 N sodium thio-
sulfate in the usual manner, starch being used as the indicator.

Estimation of Magnesium in Tissues and Urine

The major problems involved in the analysis of tissues are in the
methods of ashing and the means of removing iron. After consid-
erable experimentation on our part with dry and wet ashing
methods, it was concluded that dry ashing in an electric muffle was
the most satisfactory procedure. For the removal of iron, Alten,

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Weiland, and Kurmies (2) have suggested that this element be
precipitated with hydroxyquinoline at pH 5.0. At this pH value
the magnesium remains in solution. Arnoux (3) suggests remov-
ing the iron oxyquinolate after it is precipitated along with the
magnesium by washing with chloroform in which the iron but not
magnesium oxyquinolate is appreciably soluble. In our hands
the method of Alten, Weiland, and Kurmies proved the more
satisfactory. However, if a trace of iron oxyquinolate is carried
down with the magnesium (this can be detected by the black
coloration of the precipitate), it can be removed by washing the
precipitate with chloroform.
Procedure for Tissues-Dissect out the tissue and place it imme-
diately into a tared, stoppered weighing tube or bottle to prevent
evaporation. After the tissue in the tube has been weighed, it is
t,ransferred to a silica crucible and dried in an oven at about 100’.
It is then ashed in the same crucible at the temperature of about
500” in an electric muffle furnace. The subsequent treatment
depends upon the magnesium content of the tissue sample and
whether or not it is desired to estimate other substances besides
magnesium. The lower limit of magnesium in the aliquot taken
for analysis after iron and calcium have been removed is about
0.02 mg. in a volume not greater than 10 ml.; the upper limit
about 0.2 mg. The optimum amount for the analysis is between
0.05 and 0.1 mg. of magnesium.
In samples in which there is a plentiful amount of magnesium,
the ash is dissolved in 1 N HCl and is transferred to a volumetric
flask of suitable size which is then filled to volume. A suitable
aliquot portion of this solution is pipetted into a 25 ml. volumetric
564 Mg Determination in Tissues and Urine

flask and sufficient potassium oxalate,-usually 2 ml. of a 4.5 per

cent solution are added to precipitate the calcium. Then a few
drops each of glacial acetic acid and brom-cresol green or other
suitable indicator are added and the pH is adjusted to about 5.5
with 10 per cent sodium hydroxide. At this stage 2.5 ml. of the
hydroxyquinoline solution are introduced to throw down the iron
and the flask is filled to the graduation mark. If a considerable
amount of iron is present, a black precipitate will form at once.
However, if the amount of iron is quite small, no precipitate will
appear, but the solution will turn dark. In such a case 2 or 3
drops of 1 per cent ferric chloride are added to supply a sufficient

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TABLE I
Test of Hydroxyquinoline Method for Determination of Magnesium in
Tissues

Magnesium

Deter- Test
Test material
mined material
Theo- As oxy-
retical aa pxy- quino- MgNA&P04
qmno- late
late

mg. per mg. per 100

100 gnz. gm.
Artificial salt solution.. . . . 0.040 0.042 Muscle.. 21.2 21.0
“ ‘I “ . . . . . . 0.030 0.028 Liver.. . . 15.8 16.3
Aliquot of whole rat. . . . . . . 0.113 Wholerat. 31.2 30.7
“ ‘I ‘I “ +0.0516
mg. Mg. . . . . . . . . . . . . . . . . . . 0.164f 0.165 1 I

amount to induce the complete precipitation of the iron. After

being allowed to stand for at least 2 hours, the contents of the
flask are filtered through an ashlessfilter paper and an aliquot of
the filtrate containing between about 0.05 to 0.1 mg. of magnesium
is taken for analysis. The procedure from here on is the sameas
has been described by Greenberg and Mackey for blood.
If the sample to be analyzed contains but little magnesium, the
ash is dissolved in 1 N HCI and is directly transferred to a 10 ml.
volumetric flask, a total volume of about 6 ml. of acid and wash
water being used for this purpose. The precipitation of the cal-
cium and iron is carried out in the same way as in the larger volu-
metric flask with a proportionally calculated amount of the rea-
 Greenberg, Anderson, and Tufts 565

gent. After the calcium and iron precipitate are filtered out, an
aliquot of as much as 8 ml. may be obtained for the magnesium
analysis.
Procedure for Urine-If no protein is present, 10 ml. of urine
are pipetted into a 50 ml. volumetric flask, brom-cresol green or
other suitable indicator is added, and the pH is adjusted between
about 5 and 5.5 with 10 per cent sodium hydroxide or acetic acid
as is required. There are now added 5 ml. of 4.5 per cent potas-
sium oxalate and sufficient water to fill the flask. If the urine
contains protein, 20 ml. of 10 per cent trichloroacetic acid are first
added to the 10 ml. of urine to precipitate the protein. The acid of

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the protein-free filtrate is neutralized to the required pH in the man-
ner just described and potassium oxalate added. After standing
for 2 hours the calcium oxalate precipitate is filtered off. The
procedure from here on also is the same as for blood. With
normal urines, a 10 ml. aliquot of the filtered solution contains an
amount of magnesium suitable for the analysis. If the magnesium
level is suspected of being high, the aliquot used is correspondingly
reduced. A few representative results obtained on the test
materials used to check the method are given in Table I.

BIBLIOGRAPHY

1. Greenberg, D. M., and Mackey, M. A., J. Biol. Chem., 96,419 (1932).

2. Alten, F., Weiland, H., and Kurmies, B., Angew. Chem., 46,697 (1933).
3. Arnoux, M., Compt. rend. Sot. biol., 116,436 (1934).
 A NOTE ON A CLOSED TITRATION
FLASK FOR USE IN THE
BROMOMETRIC DETERMINATION OF
MAGNESIUM WITH
8-HYDROXYQUINOLINE:
APPLICATION TO THE ESTIMATION
OF MAGNESIUM IN TISSUES AND
URINE
David M. Greenberg, Carl Anderson and Elma
V. Tufts

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J. Biol. Chem. 1935, 111:561-565.

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