Scribd Logo
Upload

Welcome to Scribd!

  • 54 views

    555 Full PDF

    Uploaded by

    Med Aj
    1) The document describes a method for estimating the amount of zinc in biological materials using potassium ferricyanide and potassium iodide. The method involves liberating iodine, which is then titrated with sodium thiosulfate to determine the amount of zinc present. 2) Experiments found that the method accurately determined zinc added to solutions containing proteins and insulin. Even in the presence of large amounts of iron, the method still allowed accurate zinc quantification. 3) Applying the method to pancreas samples from various species and insulin samples, the zinc content was determined with good reproducibility using different sample pre-treatment techniques like ashing, trichloroacetic acid extraction, and acid digestion.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    555 Full PDF

    Uploaded by

    Med Aj
    54 views8 pages
    1) The document describes a method for estimating the amount of zinc in biological materials using potassium ferricyanide and potassium iodide. The method involves liberating iodine, which is then titrated with sodium thiosulfate to determine the amount of zinc present. 2) Experiments found that the method accurately determined zinc added to solutions containing proteins and insulin. Even in the presence of large amounts of iron, the method still allowed accurate zinc quantification. 3) Applying the method to pancreas samples from various species and insulin samples, the zinc content was determined with good reproducibility using different sample pre-treatment techniques like ashing, trichloroacetic acid extraction, and acid digestion.

    Original Title

    555.full.pdf

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    1) The document describes a method for estimating the amount of zinc in biological materials using potassium ferricyanide and potassium iodide. The method involves liberating iodine, which is then titrated with sodium thiosulfate to determine the amount of zinc present. 2) Experiments found that the method accurately determined zinc added to solutions containing proteins and insulin. Even in the presence of large amounts of iron, the method still allowed accurate zinc quantification. 3) Applying the method to pancreas samples from various species and insulin samples, the zinc content was determined with good reproducibility using different sample pre-treatment techniques like ashing, trichloroacetic acid extraction, and acid digestion.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    54 views8 pages

    555 Full PDF

    Uploaded by

    Med Aj
    1) The document describes a method for estimating the amount of zinc in biological materials using potassium ferricyanide and potassium iodide. The method involves liberating iodine, which is then titrated with sodium thiosulfate to determine the amount of zinc present. 2) Experiments found that the method accurately determined zinc added to solutions containing proteins and insulin. Even in the presence of large amounts of iron, the method still allowed accurate zinc quantification. 3) Applying the method to pancreas samples from various species and insulin samples, the zinc content was determined with good reproducibility using different sample pre-treatment techniques like ashing, trichloroacetic acid extraction, and acid digestion.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 8

    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).

    You might also like