THE

DETERMINATION
OF ZINC
BIOLOGICAL
MATERIAL

BY MELVILLE
(From

SAHYUN

the Biochemical

(Received

AND

ROLLAND

Research
Laboratory,
Company,
Detroit)
for publication,

September

F.
Frederick

IN
FELDKAMP
Stearns

and

4, 1936)

Several investigators have already reported on the occurrence

of zinc in the pancreas, Lutz (l), Delezenne (2), and recently
Fisher and Scott (3). In the estimation of zinc in biological
material, ashing was employed. This is a tedious procedure and
there is always the possibility of the volatilization of zinc due to
overheating. Moreover, since zinc along with other metals such
as magnesium, copper, iron, cobalt, nickel, etc., is present in small
amounts in the tissues, its separation renders the gravimetric procedure quite hazardous. Of course, the quantitative estimation
of zinc by spectrographic analysis can be performed fairly accurately, but such apparatus is not always available and it requires
an experienced operator.
In 1929 and later in 1933, Lang (4) published a method for the
estimation of zinc in aqueous solutions by treating the slightly
acidified solution of zinc with an excess of potassium ferricyanide
and potassium iodide, whereby iodine is liberated and titrated
against a standard solution of thiosulfate. Lang found that 1 cc.
of 0.1 N sodium thiosulfate is equivalent to 10 mg. of zinc. Hibbard (5) confirmed Langs findings and adopted the method for the
estimation of zinc in soil and plants. The reaction involved was
expressed as follows :
2KsFe(CN)G

+ 2KI

+ 3ZnS04

= K2Zns(Fe(CN)&

+ 3K2SOa

+ I:!

In this investigation we made use of the principle involved in

the above equation for the estimation of zinc in proteins and in
tissues, and determined the zinc content of the pancreas of various
species.
555

556

Zn in Biological

Material

Reagents0.5 per cent starch.

10 per cent potassium iodide. Afresh solution is prepared daily.
0.001 N sodium thiosulfate. This is prepared daily from a stock
solution of 0.1 N sodium thiosulfate, and standardized against a
standard solution of zinc containing 0.1 mg. per 1 cc.
Zinc standard. 1 gm. of pure zinc is dissolved in concentrated
hydrochloric acid and diluted to 1 liter. 1 cc. contains 1 mg. of
zinc and requires 10 cc. of 0.001 N sodium thiosulfate.
Potassium ferricyanide.
A fresh 1 per cent solution is prepared
daily.
Special phosphate. To 50 cc. of C.P. orthophosphoric acid
(HBPO.J sufficient 10 per cent potassium hydroxide is added to
bring the reaction to about pH 3.0. This is diluted to 1 liter.
Special ammonium acetate. Approximately 2 M ammonium
acetate buffer, with a pH of about 6.0, is prepared.
Method

250 gm. of ground fresh pancreas are treated with 3 volumes of 4

per cent trichloroacetic acid, thoroughly stirred for at least 1 hour,
allowed to stand overnight, and filtered through a hardened filter
paper. The aqueous trichloroacetic acid filtrate contains all the
zinc in solution. Samples (such as 200 cc. or as may be desired)
of this filtrate are transferred to 250 cc. capacity centrifuge bottles;
concentrated ammonium hydroxide is added until the reaction is
alkaline to litmus, whereupon 5 cc. of the special ammonium acetate buffer are added, stirred, and followed by 1 or 2 cc. of concentrated acetic acid. The mixture is saturated with hydrogen sulfide, allowed to stand overnight, and centrifuged. The precipitate
is next dissolved in about 1 or 2 cc. of hot 5 N hydrochloric acid
and the solution is quantitatively
transferred to a 50 or 100 cc.
Pyrex flask. The liquid is evaporated to dryness over an asbestoscovered hot-plate until the excess of hydrochloric acid is almost
completely driven off. While the flask is still warm, 5 cc. of 0.02
N sulfuric acid are slowly introduced along the sides of the flask,
followed by 5 to 10 cc. of distilled water and 1 cc. of the special
phosphate reagent. The liquid is boiled for 1 or 2 minutes, removed from the hot-plate, and allowed to cool to room temperature. It is now ready for titration.

M. Sahyun and R. F. Feldkamp

557

Titration of Zinc-l
cc. of 0.5 per cent starch and 1 cc. of 10 per
cent potassium iodide are added. The mixture should,. at this
stage, be colorless.
However, if after 1 minute iodine is liberated
owing to traces of copper or some other ions, 0.001 N sodium thiosulfate is added 1 drop at a time until the solution is clear. Then
1 cc. of 1 per cent potassium ferricyanide
is introduced.
The
The iodine
mixture is stirred and allowed to stand for 1 minute.
liberated is titrated against the standard 0.001 N sodium thiosulfate until the addition of 1 drop of this reagent does not cause any
change in the yellowish
color at the point of contact with the
liquid.
1 cc. of 0.001 N sodium thiosulfate is equal to 0.1 mg. of
zinc.
EXPERIMENTAL

Experiment ~--TO a known amount of zinc in solution (in 15 cc.

centrifuge tubes) sufficient trichloroacetic
acid is added to bring
about a concentration
of 3 to 4 per cent. The acidity is neutralized with 5 N ammonium hydroxide and 1 cc. of the special ammonium acetate is added, followed by 1 or 2 cc. of 2 N acetic acid.
The mixture is saturated with hydrogen sulfide and the tubes
allowed to stand overnight and then centrifuged.
The clear supernatant liquid is decanted and the precipitate is dissolved in 0.5
cc. of 5 N hydrochloric
acid. The contents of the tubes are transferred quantitatively
(with hot distilled water) into a 50 cc. or 100
cc. Pyrex flask.
The liquid is carefully evaporated to dryness over
an asbestos-covered
hot-plate; 5 cc. of 0.02 N sulfuric acid are introduced, along with 1 cc. of special phosphate reagent, and the
The liquid is
sides of the flask washed with distilled water.
allowed to boil for 1 or 2 minutes, removed from the hot-plate, and
cooled to room temperature.
Zinc is next titrated as described
in the method.
The results of this experiment
are found in
Table I.
Determination

of Zinc in Presence of Iron

In the determination
of zinc by the method described in this
paper, the removal of iron is not essential, as it is in the gravimetric
1 For the quantitative
precipitation
of zinc with hydrogen
sulfide
there
are two prerequisites:
(a) The solution
must
contain
an excess of ammonium
sulfide
and (b) the acidity
of the solution
must be well controlled.
For this purpose
ammonium
acetate
and acetic acid are added.

558

Zn in Biological

Material

method.
It was observed in this laboratory
that in solutions
containing a mixture of zinc and iron in various proportions,
even
up to 5 mg. of iron to every 0.1 or 0.2 mg. of zinc (a ratio of 50 or
25 to 1) the addition of the acid phosphate reagent inhibits the
reaction of iron with ferricyanide and potassium iodide.
This is
due to the formation of the insoluble iron phosphate in slightly
acid solution.
The zinc can then be quantitatively
estimated in
the manner described.
It was further observed that when iron
phosphates are absent, or present in moderate amounts, the zinc
reaction proceeds fairly rapidly (within 1 minute) after the addiI

TABLE

Estimation
of Zinc
Added
to Solution
of Approximately
Trichloroacetic
Acid, and to Concentrated
Insulin
A-8
Trichloroacetic
Acid

Experiment

No.

T-

Zinc

Amount

added

rots1

amount

9.

0.10
0.25
0.32
0.60
1.04

2
3
4
5
A-l
A-2
A-3
A-4

J$ Per Cent
in .J Per Cent

(Control)
4.25
7.12
9.50

w?.

found

Total amount
found
less zinc in sample

?w.

0.10

0.255
0.31
0.59
1.09
0.68
5.10
7.55
10.01

4.32
6.87
9.33

tion of the last reagent.

However,
in the presence of large
amounts of iron phosphate, as in the proportions mentioned above,
the liberation of iodine is definitely retarded, necessitating
a 3
minute wait before titration.
Experiment
Z-It
was observed that by using the well known
method of precipitating proteins with 3 to 4 per cent trichloroacetic
acid, the zinc associated with the proteins remains in the filtrate.
In order to determine whether or not the zinc is quantitatively
recovered the following experiments were performed.
To each of three 100 mg. samples of dry insulin powder, 20 units
per 1 mg., zinc was added and the samples were dissolved in dilute

M. Sahyun and R. F. Feldkamp

hydrochloric
acid. To each 10 cc. of insulin protein in solution
5 cc. of 10 per cent trichloroacetic
acid were added. This quantity
was found sufficient for the complete precipitation
of the insulin
protein.
See Table I for the results.
A solution of crystalline
insulin prepared in this laboratory,
containing a known amount of zinc, was used for this demonstration. The zinc content of this material had been determined
spectrographically
and was shown to be in the neighborhood
of
0.9 mg. per 1000 units.
The zinc content of this crystalline
II

TABLE

Estimation

of Zinc

in Samples

of Crystalline

Technique

Material
Lot

Insulin,

B-2
B-3

B-4
B-5
B-6

Ashing
<
L(

as sulfate

<
<
,

Trichloracetic
extraction

<
Digestion

<

acic
(I

1657-I

used

No.

Zinc
Per sample

units
B-l

Lots

and 3499-I
found
Per
1000 units

m7.

ml.

1657-I
1657-I
2499-I
2499-I
1657-I

1000
1000
1050
1050
1000

0.942
0.932
0.950
0.970
0.924

0.942
0.932
0.905

1657-I
2499-I
2499-I
1657-I
1657-I
2499-I
2499-I

1000
1050
1050
1000
1000
1050
1050

0.914
0.913
0.915
0.891
0.900
0.883
0.950

0.914

0.925
0.924

0.870
0.870
0.891
0.900
0.840
0.905

material was analyzed in duplicate by three different preliminary

procedures.
(1) 1000 units of crystalline insulin were brought to
The ash was
dryness and ashed as sulfates in platinum crucibles.
dissolved in hydrochloric
acid and zinc was estimated.
(2) 1000
units of crystalline
insulin in solution were precipitated
with
trichloroacetic
acid, the filtrate was recovered quantitatively,
and
zinc was estimated.
(3) 1000 unit samples of crystalline insulin
in solution were digested with concentrated
sulfuric acid, potassium sulfate, and nitric acid. The clear digested material was
neutralized
and the process of sulfiding was performed.
The
results of these experiments are shown in Table II.

560

Zn in Biological
III

TABLE

Estimation

of Zinc

in Pancreatic

Material

Tissue

&ith
Zinc,

Experiment

C-l

c-2

added

B-l
B-2

mg.

No.

Material

Beef
I
I
I

Calf

<
I
L

s-2
s-3
H-l
H-2
H-3

found

Sheep
I
I
I
I

Hog
I
I(

I
I

Net amount
ml.

27 06
26.20
27.00
27.00

IV

Found
in Fresh Pancreas
of Beef, Calf,
Extraction
with Trichloroacetic
Acid

C-l

S-l

amount

27.06
26.20
47.00
47.00

c-3

Total

ml.

B-3

c-2

Zinc

per kilo

None
i
20
20

TABLE

of Zinc

Determination

Added

No.
Amount

Amount

and without

Amount
found per
kilo fresh pancreas

m7.
32.4
34.8
43.2
40.6
41.4
42.0
34.9
33.3
38.8
43.2
36.2
36.6
19.9
19.04
24.0
25.0
24.8
24.7
42.9
44.4
35.2
34.6
31.0
28.2

Sheep,

Average

and

Hog.

per kilo
WJ.

33.6
41.9
41.7
34.1
41.0
36.4
19.5
24.5
24.75
43.6
34.9
29.6

M. Sahyun and R. F. Feldkamp

561

500 gm. of well ground pancreas were divided into two equal
parts.
250 gm. were treated with 3 volumes of 4 per cent trichloroacetic acid (final volume was 1 liter), thoroughly
mixed,
The filtrate was labeled
allowed to stand overnight, and filtered.
C-l.
To the other 250 gm., 20 mg. of zinc in solution were added
and the ingredients intimately
mixed; 3 volumes of 4 per cent
trichloroacetic
acid were added (final volume 1 liter), mixed,
allowed to stand overnight, and filtered. This filtrate was labeled
C-2. Zinc determination was then performed on Filtrates C-l and
The results of this experiC-2 according to the method described.
ment are shown in Table III.
Zinc Content of Pancreas
Experiment 3-l
kilo of fresh pancreas of each of the following
species was analyzed for zinc: beef, calf, sheep, and hog. The
pancreas of each species was divided into three lots; each lot was
ground separately, and 250 gm. samples of each were used. To
250 gm. of ground tissues enough 4 per cent trichloroacetic
acid
was added to make up a volume of 1 liter.
The mixture was
thoroughly
stirred for 1 hour, treated with a few cc. of toluene,
and allowed to stand overnight.
The following day each lot was
filtered separately and zinc was determined in accordance with the
method outlined.
The results are shown in Table IV.
Zinc Content of Commercial

Insulin

Experiment ~-AS
a matter of interest samples of different
preparations of insulin found on the market were secured and zinc
was determined by trichloroacetic
acid extraction according to the
method described.
The results are as follows:
Preparation

A
B
C
D

Insulin

per sample

units
800
800
400
800

Zinc

found

per 1000 units

VW.

0.043
0.068
0.094
0.050

562

Zn in Biological

Material

SUMMARY

A method for the microestimation

of zinc in biological materials
is described.
Values for the zinc content of the pancreas of beef, calf, sheep,
and hog are given. These values are somewhat higher than those
reported by Fisher and Scott (3).
Commercial insulin of different sources was observed to contain
small amounts of zinc. These values are not constant but will
vary considerably, one way or another, from batch to batch.
BIBLIOGRAPHY

1.
2.
3.
4.
5.

Lutz,
R. E., J. Ind. Hyg., 8, 177 (1926).
Delezenne,
C., Ann. Inst. Pasteur,
33, 78 (1917).
Fisher,
A. M., and Scott, D. A., Biochem.
J., 29,1055
(1935).
Lang, R., 2. anal. Chem., 79, 161 (1929); 93,21
(1933).
Hibbard,
P. L., Ind. and Eng. Chem., Anal. Ed., 6, a-23 (1934).