A TITRIMETRIC METHOD FOR THE QUANTITATIVE

ESTIMATION OF LEAD IN BIOLOGICAL MATERIALS

BY M. K. HORWITT* AND GEORGE R. COWGILL
(From the Laboratory of Physiological Chemistry, Yale University School of
Medicine, New Haven)

(Received for publication, March 31, 1937)

The announcement by Fischer in 1929 (1) of the remarkable

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affinity of dithizone (diphenylthiocarbazone) solutions for lead
has stimulated many laboratories to search for practical micro-
methods that could be applied to the determination of lead in
biological materials. The theory and the application of such
methods have been adequately described by Wilkins, Willoughby,
Kraemer, and Smith (2), Ross and Lucas (3), and more recently
by Clifford and Wichmann (4) and will not be treated further
here.
The authors have had the opportunity to apply the various
dithizone methods to a large variety of materials. Most of the
published techniques have been critically investigated and this
paper will present what is in our opinion the most satisfactory
extraction procedure together with a new titration that eliminates
the necessity of investing in expensive photometric equipment.
Interference by Bismuth and Tin-The red color produced by the
reaction between solutions of dithizone in chloroform and those
of a heavy metal in alkaline cyanide solution is not entirely specific
for lead (5). Bismuth and stannous tin react with dithizone in a
similar fashion and must be eliminated at some point in the
analytical procedure. Bismuth may be present in biological speci-
mens as a result of previous medication and stannous tin is not
an uncommon constituent of the normal diet.
Fischer and Leopoldi (6), Winter et al. (7), and Tompsett and
ilnderson (8) have made use of the fact that bismuth may be
separated from lead by extracting the dithizone mixture with
* Lead Research Fellow.
553
554 Lead in Biological Materials

solutions of alkaline cyanide. Clifford and Wichmann (4) have

objected to this procedure on the grounds that the alkaline cya-
nide extracted some of the lead along with the bismuth. Our
investigations have shown us that the amount of lead extracted
by washing with potassium cyanide solution varied not only with
the concentration of the dithizone present but also with the con-
centration of the potassium cyanide used. Thus the 1 per cent
solutions of potassium cyanide caused an appreciable loss of lead
(5 to 10 per cent), but if a 0.5 per cent solution is used, the loss
of lead is negligible, if the extraction is properly conducted.

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Where comparatively large amounts of bismuth are present (25
times the quantity of lead or more), it is advisable to use the
procedure proposed by Willoughby and associates (9) and extract
the weakly acidified solution with an excess of dithizone. We
suggest, however, that the aqueous solution containing the lead
and bismuth be adjusted to pH between 3.0 and 3.5, instead of
pH 2 as recommended by Willoughby et al., in order to obtain a
more satisfactory separation. Since bismuth will react with
dithizone to give a brown color to the chloroform, a large excess
of bismuth is easy to notice.
It is not possible to make an efficient separation of stannous
tin from lead by extracting an acidified mixture with dithizone,
because the optimum pH for the reaction between stannous tin
and dithizone is close to neutrality. Fortunately, stannic tin
does not react with dithizone, and since the process of ashing con-
verts the stannous tin to stannic tin, the former does not inter-
fere. Small amounts of stannous tin can be separated from a
chloroform solution of lead dithizonate by shaking with 0.5 per
cent potassium cyanide. Any traces of tin which have not been
oxidized or which have reverted to the stannous state are removed
from the mixture by the cyanide solution.
Interference by Calcium Phosphate-The presence of large
amounts of calcium phosphate, as in the analyses of bones, pre-
sents a special problem, if one wishes to avoid using a sulfide
precipitation. Winter and collaborators (7) have suggested a
preliminary separation of the lead phosphate which they claim
precipitates at a lower pH than calcium phosphate. Similarly,
in the analyses of urine, Ross and Lucas (3) precipitate the calcium
as the oxalate at pH 4.5, a procedure which entrains the lead and
leaves most of the phosphate in solution.
 M. K. Horwitt and G. R. Cowgill 555

It has long been known that citrates exert a solvent action on

the phosphates of calcium (10). In 1881, Terreil (11) showed
that 7 gm. of calcium phosphate were dissolved by a solution of
100 gm. of citric acid which had been neutralized with ammonia.
In analytical work it is not practical to use such large amounts of
citric acid. Large concentrations of citric acid not only increase
the specific gravity of the aqueous solution, thus slowing the
chloroform separations, but also reduce the affinity of dithizone
for lead so that large excesses of dithizone must be used to insure
complete extraction. These disadvantages may be avoided,

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however, if proper adjustments are made between the volume of
solution and the amounts of calcium phosphate and citrate used.
Thus it was found that the lead in an aliquot equivalent to not
more than 1.5 gm. of dry bone could readily be extracted from
350 cc. of solution at pH 8.0 containing 15 gm. of sodium citrate.
A “wet digestion” with sulfuric, nitric, and perchloric acids was
used during our preliminary work on lead methods, but was soon
abandoned because of the large amounts of lead introduced by
this acid mixture. Furthermore, the use of sulfuric acid com-
plicated the analysis of materials high in calcium. Better results
were obtained when “dry ashing” at a temperature below 500’
was used. In order to facilitate the preliminary charring of the
biological materials, an overhead heater was devised by Nims and
Horwitt (12), which greatly shortened the time required for an
analysis. By application of radiant heat from above, a sample
can be dehydrated and charred without danger of foaming or
spattering. The resulting product may be placed directly into
a muffle furnace at 475”.
A source of error in the analysis of lead may be found in the
type of dish used for ashing when the quantities of ash are small.
When 5 cc. portions of a lead nitrate solution containing 0.01 mg.
of lead per cc. were evaporated in a porcelain dish and heated at
450’ for 2 hours, less than 80 per cent of the original lead was
recovered by extraction with hot 20 per cent hydrochloric acid.
Two extractions with alkaline citrate and two more with 10 per
cent potassium cyanide accounted for another 15 per cent of the
original lead. When the same experiment was repeated with
silica dishes, the hydrochloric acid alone extracted 97 per cent or
more of the added lead.
Interference by Iron-Dithizone solutions are oxidized by ferric
556 Lead in Biological Materials

iron in the presence of cyanide and special precautions must be

taken when blood or other materials containing much iron are
analyzed. Wilkins and associates (2) resorted to a preliminary
dithizone extraction in which the partial destruction of this re-
agent was not important. Tompsett and Anderson (8) also used
a preliminary extraction, except that in their case sodium di-
ethyldithiocarbamate was used instead of dithizone. A simple
procedure was suggested by Fischer and Leopoldi (13) who add
hydroxylamine hydrochloride to prevent the oxidation of dithi-
zone. This treatment is effective if the amounts of iron present

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are small.
Since ferric iron in the concentrations encountered does not
oxidize dithizone in the absence of cyanide, Wichmann et al. (14)
have suggested that less cyanide be used. Similarly, Cheftel and
Pigeaud (15) reduced the amount of cyanide used and claimed
that iron did not interfere with the extraction of lead under these
conditions. In our own laboratory the best results were ob-
tained when both hydroxylamine hydrochloride and reduced
amounts of potassium cyanide were employed; this procedure
permitted the analysis of larger amounts of blood and gave good
results.
Principle of Titration-The method described below differs
from other dithizone techniques in that it is not necessary to
standardize the dithizone solution. The final titration is carried
out .direetly with a dilute lead solution, thus eliminating the neces-
sity for special precautions in the handling of the dithizone. The
lead is separated from a given solution by means of dithizone and
the resulting lead-dithizone complex is then isolated. The latter
is freed of lead by washing with acid. The chloroform solution
of dithizone remaining is mixed with some dilute cyanide solution
which removes most of the dithizone from the chloroform, im-
parting a brown color to the aqueous layer. A lead solution is
added from a burette to this mixture until all the dithizone has
been reconverted to lead dithizonate, as indicated by (1) the dis-
appearance of the brown color in the aqueous layer, and (2) the
absence of a red color when the aqueous layer is mixed with chloro-
form and additional lead solution.
Apparatus-All glassware should, whenever possible, be made
of Pyrex glass. Silica dishes are recommended for the ashing
 M. K. Horwitt and G. R. Cowgill 557

procedures. The cleansing of the separatory funnels before each

analysis is especially important. Washing with hot dilute nitric
acid followed by a thorough rinsing with water redistilled from an
all-Pyrex still is usually sufficient for the separatory funnels, but
the dishes in which materials have been ashed should be cleansed
with the aid of a warm solution of alkali as well.
An overhead heater of some sort is recommended for preparing
biological samples (12). Such a device not only shortens the
time required to complete a determination but also affords protec-
tion from laboratory dust during the diminished period of hand-

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ling. The muffle furnace used should preferably be equipped with
a stainless steel sleeve to protect the samples from contamination
by the brittle parts of the oven.
Reagents-The water should be distilled from an all-Pyrex
still and all reagents should be stored in Pyrex containers.
1. Potassium cyanide solution. 10 gm. in 100 cc. prepared
daily.
2. Hydrochloric acid, 20 per cent. Concentrated acid (sp.
gr. 1.19) mixed with an equal volume of water and the mixture
distilled from the all-Pyrex still.
3. Hydrochloric acid, 0.5 per cent. Prepared by diluting 25
cc. of Reagent 2 to 1 liter.
4. Chloroform, U.S.P.
5. Hydroxylamine hydrochloride solution, 25 per cent.
6. Dithizone solution. Dissolve 40 mg. of diphenylthiocar-
bazone in 400 cc. of chloroform and filter into a 500 cc. Pyrex
separatory funnel. Add 50 cc. of water containing 2 cc. of 25
per cent hydroxylamine hydrochloride solution and shake. Keep
in a cool dark place and withdraw the chloroform solution as
needed. The acid aqueous layer not only prevents the oxidation
of the dithizone but also extracts any lead which might be pres-
ent. Further purification was not found necessary for the titri-
metric method to be described below.
7. Potassium cyanide solution, 0.5 per cent. Prepared daily
by diluting 25 cc. of Reagent 1 to 500 cc. It is important that
this solution be lead-free. To insure this, place 100 cc. of Re-
agent 1 in a separatory funnel and extract with 2 cc. of chloroform
containing 2 drops of dithizone solution. If a pink color appears
in the chloroform layer, withdraw it and repeat the extraction
558 Lead in Biological Materials

until the chloroform layer is colorless. The slight excess of dithi-

zone which remains in the 10 per cent potassium cyanide is not
significant, since the amounts which remain after dilution to form
the.0.5 per cent solution are not detectable.
8. Nitric acid. Redistil the concentrated reagent.
9. Ammonium hydroxide. Distil the concentrated reagent
into cold redistilled water.
10. Standard lead solutions. Dissolve 1.599 gm. of recrystal-
lized lead nitrate (or the equivalent of lead acetate) with the aid of
1 cc. of nitric acid and dilute to 100 cc. This solution which con-

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tains 10 mg. of lead per cc. is quite stable. By diluting 10 cc.
of this solution to 100 cc. and then in turn diluting 10 cc. of the
latter to 1 liter, a solution containing 0.01 mg. of lead per cc. is
prepared. This is stable in Pyrex glass containers for at least
5 days.
11. Sodium citrate, 20 per cent. To 800 cc. of this solution add
8 cc. of 10 per cent potassium cyanide and extract in a 1 liter
separatory funnel with 15 cc. portions of dithizone solution until
the citrate mixture is free of lead. Wash twice with 25 cc. por-
tions of chloroform, acidify with 4 cc. of 20 per cent hydrochloric
acid, and complete the extraction of the excess dithizone with 20
cc. portions of chloroform.

Procedure
Blood-A 10 cc. sample in a 50 cc. silica evaporating dish is
dried and completely charred beneath a radiant heater. This is
accomplished in approximately 45 minutes. Transfer to a muffle
adjusted to a temperature of about 475”. After 2 hours, remove
from the muffle, moisten with 2 cc. of nitric acid, and place the
dish beneath the radiant heater until the reaction has ceased and
the material is free of excess acid. This requires approximately
30 minut,es. Return to the muffle for about half an hour to com-
plete the oxidation,
Place the dish on a hot-plate, carefully add 15 cc. of 20 per
cent hydrochloric acid, and heat until the ash is dissolved. Wash
the contents into a 125 cc. separatory funnel with about 20 cc.
of hot water. Add 10 cc. of 20 per cent sodium citrate and 3 cc.
of ammonium hydroxide to the silica dish, mix, and transfer to
the separatory funnel with enough water to make a total volume
 M. K. Horwitt and G. R. Cowgill

of about 75 cc. Cool, add 1 cc. of hydroxylamine hydrochloride,

1 drop of phenol red, and bring to pH 8.0 with ammonium hydrox-
ide delivered from a Pyrex burette. Cool, add drop by drop,
shaking between additions, 0.5 cc. of 10 per cent potassium
cyanide, and immediately extract with 0.5 cc. of dithizone solu-
tion and 4 cc. of chloroform. If, after shaking, the chloroform
layer does not contain a noticeable excess of uncombined dithizone,
add 0.2 cc. portions of dithizone solution, shaking between addi-
tions, until the green excess becomes evident. Remove the chlo-
roform phase to another separatory funnel and repeat the extrac-

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tion of the aqueous phase twice with 0.2 cc. portions of dithizone
in 2 cc. of chloroform. To the combined chloroform solutions
add an amount of 0.5 per cent potassium cyanide equal to 1.5
times the volume of the chloroform solution and shake for 10
seconds. Withdraw the chloroform layer to another separatory
funnel and wash the aqueous cyanide solution with 1 cc. of chloro-
form. Combine the chloroform solutions and again extract
with 1.5 volumes of 0.5 per cent potassium cyanide solution.
Any lead which may have dissolved in the aqueous phase is re-
moved by extraction with 2 cc. of chloroform.
The extraction with cyanide solution described above removes
the uncombined dithizone unless a very large excess has been used,
in which case the extraction with 0.5 per cent potassium cyanide
is continued until the absence of color in the aqueous layer indi-
cates that the dithizone excess has been removed. The lead is
separated from the red dithizone complex by shaking for 15
seconds with 2 volumes of 0.5 per cent hydrochloric acid. With-
draw the green chloroform layer and then extract the acid aqueous
solution with 1 cc. of chloroform to recover the last traces of dithi-
zone. Combine the chloroform fractions.
Titration-Add to the dithizone solution 0.5 volume of 0.5 per
cent potassium cyanide and shake. Most of the dithizone goes
into the aqueous layer, giving that mixture a brown color. Add
the standard lead solution (0.01 mg. per cc.) from a burette a drop
at a time, shaking between additions, until only a very faint color
remains in the aqueous phase. ‘This is evidence that practically
all of the dithizone has combined with lead and gone into the
chloroform layer. Discard the red chloroform phase and wash
the aqueous layer with chloroform, 2 cc. at a time, until the chloro-
 Lead in Biological Materials

form layer remains colorless after shaking. (In order to prevent

any loss of uncombined dithizone the color in the cyanide solu-
tion should not be greater than that color which 0.2 cc. of Re-
agent 6 will impart to 10 cc. of 0.5 per cent potassium cyanide
solution.) Add a drop or two of the lead solution and shake for
5 seconds. Withdraw the pink chloroform solution and continue
the extraction with 2 cc. portions of chloroform plus a drop or two
of lead solution until further addition of lead gives no pink color
to the chloroform solution after shaking. The end-point is a slight
pink in the chloroform solution; extraction with 1 more drop

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results in a colorless solution. In order to facilitate the titration,
a solution of the lead-dithizone complex containing a small amount
(1 or 2 drops) of the lead solution in 2 cc. of chloroform is kept
for comparison. When the color obtained after an addition of
lead solution is less than that given by 1 drop of lead, the end-
point has been attained. It is suggested that the beginner add
2 drops (about 0.0006 mg.) at a time until his eyes become accus-
tomed to the change.
Urine-Measure 200 cc. of urine into a 250 cc. silica dish and
ash as described above for blood, except that 5 cc. of nitric acid
should be used instead of 2 cc.
Because the ash of urine is sometimes difficult to dissolve,
moisten it with 15 cc. of 20 per cent hydrochloric acid and heat
until almost dry. Transfer the contents to a 500 cc. separatory
funnel with the aid of an additional 15 cc. of hydrochloric acid,
50 cc. of 20 per cent sodium citrate, and 5 cc. of ammonium hydrox-
ide. ,Make to a volume of 250 cc. and cool. Add 1 drop of
phenol red and slowly bring to pH 8.0 with ammonium hydroxide.
Add 3 cc. of 10 per cent potassium cyanide; extract with an excess
of dithizone using 0.5 cc. portions in 3 cc. of chloroform and pro-
ceed as with blood beginning with “TO the combined chloroform
solutions add. . .”
Bone-Place a known amount of dried bone in a silica dish
and heat in a muffle at 475” for 2 hours. Remove, add an amount
of nitric acid equivalent to 3 cc. for each 1.5 gm. of dried bone, and
evaporate to dryness beneath the radiant heater. Return to the
muffle for about 3 hour.
Dissolve the contents of the silica dish, using 15 cc. of 20 per
cent hydrochloric acid for each 1.5 gm. of dried bone, and transfer
 M. K. Horwitt and G.‘R. Cowgill

an aliquot containing not more than 1.5 gm. to a 500 cc. Pyrex
separatory funnel. Add 75 cc. of sodium citrate solution, 2 drops
of phenol red, and enough water to make a volume of about 350
cc. Add ammonium hydroxide 1 drop at a time, shaking and
cooling during the addition, until pH 8.0 has been attained. Add
slowly 5 cc. of 10 per cent potassium cyanide and extract the clear
solution with an excess of dithizone. Add 3 cc. of dithizone solu-
tion and 3 cc. of chloroform, and shake for 30 seconds. If the
chloroform layer is not purple, add 1 cc. of dithizone solution at
a time until the purple color remains after shaking, indicating that

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a large excess of dithizone has been used. Good results are ob-
tained in the presence of large quantities of citrate, if a 100 per
cent excess of dithizone is used at this stage. Withdraw the
chloroform layer to a 125 cc. separatory funnel and extract the
aqueous mixture three times with 1 cc. of dithizone solution plus
2 cc. of chloroform, 0.5 cc. of dithizone plus 2 cc. of chloroform,
and 3 cc. of chloroform, respectively. Combine the chloroform
solutions and extract once with 2 volumes and twice with equi-
volumes of 0.5 per cent potassium cyanide. Remove the lead
from the lead-dithizone complex with 2 volumes of 0.5 per cent
hydrochloric acid and titrate the resulting dithizone solution as
described under blood beginning, “Add to the dithizone solution
0.5 volume. . .”
Blank Determination-A separate blank is run for each type
of material analyzed. It is also important that an amount of
lead approximately equivalent to that which one is likely to ob-
tain from the material be added to the reagents when the blank
is estimated in order that the blank determination may serve
as a daily check on the technique of the analyst. This permits
the use of as much dithizone as in the regular procedure and may
bring out errors of manipulation that might not otherwise be
caught. Thus, if 0.02 mg. of lead is added to the reagents and the
final titration shows that 0.0223 mg. was present, the blank for
reagents for a determination between 0.01 mg. and 0.03 mg. of
lead would be 0.0023 mg.

Notes on Procedure
1. A good grade of white Vaseline is used for stop-cocks.
2. Care must be taken during the separation of the liquid
562 Lead in Biological Materials

phases that no drops of the chloroform layer remain on the surface

of the aqueous layer or adhere to the sides of the funnel.
3. If a precipitate should appear in the alkalinized solution of
the ash just prior to the extraction of the lead with dithizone,
redissolve the suspension with hydrochloric acid and add more
sodium citrate before again alkalinizing.
4. It is sometimes more satisfactory to extract the aqueous
layer with more chloroform rather than wait for the last traces of
chloroform to separate out.
5. A small chloroform trap should always be maintained in the

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separatory funnel, rather than to attempt a complete separation.
EXPERIMENTAL

Accuracy of Fundamental Titration-Inasmuch as the final

measurement in this method involves a determination of an un-
known quantity of dithizone in terms of its combination with lead,
it is necessary to prove the accuracy of such a titration. This
was done as follows:
Measured quantities of a dilute solution of dithizone were trans-
ferred to a separatory funnel, diluted with chloroform to 15 cc.,
mixed with 10 cc. of 0.5 per cent potassium cyanide, and titrated
with lead nitrate solution. The average time for each titration
was approximately 5 minutes. The results as given in Table I
indicate that the sensitivity of the titration is about 0.0002 mg.
Accuracy of Method Applied to Biological Materials-Repre-
sentative results for the recovery of different amounts of lead
added to blood, urine, and bones are given in Table II. The
amounts added correspond to the lead which may be found in
normal and pathological states. The accuracy of the method for
blood is about 10 per cent, for urine and bones about 3 per cent.
A large number of foods and diets have been analyzed for lead
during the past year, and we find it preferable not to use a definite
procedure in these cases because of the varying amounts of iron
and calcium in different products. Instead, the method is modi-
fied to suit the material. Thus, animal rations which may be high
in calcium are treated by the technique described under bone.
Should the material contain a considerable amount of iron, care
is taken to use some hydroxylamine hydrochloride and a minimum
of the 10 per cent potassium cyanide solution before extraction.
 M. K. Horwitt and G. R. Cowgill

The analyst is safe, whatever procedure is used, provided a blank

determination accomplished in a similar manner gives a good
recovery.
TABLE I
Titration of Dithizone with Lead Nitrate

Dithirone PbNO, (0.01 III&!. Per cc.) Lead nitrate used for each cc.
of dithizone

cc. cc. cc.

1 0.52 0.52
3 1.56 0.52

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3 1.54 0.51
6 3.14 0.52
6 3.13 0.52
10 5.18 0.52
10 5.18 0.52

TABLE II
Recovery of Lead from Blood, Urine, and Bones
- T

Material Lead in ElT0r I tWOVery

-
ml. “8. mg. w. mg. per cent

10 cc. beef blood 0.0016 0 .OOlO 0.0025 0.0009 0 .OOOl

0.0020 0.0036 0.0020 0.0000 1::
0.0030 0.0047 0.0031 0 .OOOl 103
0.0040 0.0060 0.0044 0.0004 110
0.0050 0.0066 0.0050 0.0000 100
200 cc. human urine 0.0060 0.0100 0.0163 0.0103 0.0003 103
0.0200 0.0265 0.0205 0.0005 103
0.0300 0.0358 0.0298 0.0002 99
0 .0400 0.0455 0.0395 0.0005 99
0.0500 0.0560 0.0500 0.0000 100
1.5 gm. bone ash* 0 .O,l28 0.0100 0 .0230 0.0102 0.0002 102
0.0300 0.0429 0.0301 0.0001 100
0.0500 0.0637 0.0509 0.0009 102
0.1000 0.1128 0.1000 0.0000 100
0.1500 0.1618 0.1490 0.0010 99
- - -
* Aliquots of an acid solution of bone ash.

DISCUSSION

The dithizone methods published to date depend upon the

standardization, at some time or other, of the dithizone solution
used. Since the ultimate standard is a lead solution, a method
 Lead in Biological Materials

which does not depend on the concentration of the dithizone and

makes direct use of a lead standard instead has many advantages.
Furthermore, since the dithizone complexes fade on standing, a
method in which use of a 10 cc. burette is substituted for a colorimet-
ric procedure should prove more accurate. Another advantage of
the method is that the amounts of bismuth and tin which are
present in biological specimens are eliminated by the regular pro-
cedure.
Each determination of the blank serves not only as a daily
check on the skill of the operator, but also on the efficacy of the

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method. The short period of heating described is important in
the prevention of loss of lead; therefore, care should be exercised
not to prolong the ashing procedure.
For routine analyses of foods in a control laboratory, it is prob-
ably sufficient to titrate to the disappearance of the brown color
in the potassium cyanide solution.
SUMMARY

A quantitative method for the determination of lead based on

a new titrimetric procedure is described. Results of the applica-
tion of this method to biological materials are reported.
BIBLIOGRAPHY

1. Fischer, H., 2. angew. Chem., 42, 1025 (1929).

2. Wilkins, E. S., Jr., Willoughby, C. E., Kraemer, E. O., and Smith,
F. L., Ind. and Eng. Chem., Anal. Ed., 7, 33 (1935).
3. Ross, J. R., and Lucas, C. C., J. Biol. Chem., 111, 285 (1935).
4. Clifford, P. A., and Wichmann, H. J., J. Assn. Off. Agric. Chem., 19,
130 (1936).
5. Fischer, H., Mikrochemie, 8, 319 (1930).
6. Fischer, H., and Leopoldi, G., Z. angew. Chem., 47, 90 (1934).
7. Winter, 0. B., Robinson, H. M., Lamb, F. W., and Miller, E. J., Ind.
and Eng. Chem., Anal. Ed., 7, 265 (1935).
8. Tompsett, S. L., and Anderson, A. B., Biochem. J., 29, 1851 (1935).
9. Willoughby, C. E., Wilkins, E. S., Jr., and Kraemer, E. O., Ind. and
Eng. Chem., Anal. Ed., 7, 285 (1935).
10. Nicholls, J. R., Analyst, 66, 594 (1931).
11. Terreil, M. A., Bull. Sot. chim., 36, 548 (1881).
12. Nims, F. L., and Horwitt, M. K., Znd. and Eng. Chem., Anal. Ed., 8,
275 (1936).
13. Fischer, H., and Leopoldi, G., Z. ges. exp. Med., 97,819 (1936).
14. Wichmann, H. J., Murray, (3. W., Harris, M., Clifford, P. A., Loughrey,
J. H., and Vorhes, F. A., Jr., J. Assn. 08. Agric. Chem., 17,108 (1934).
15. Cheftel, H., and Pigeaud, M. L., Ann. fuls., 29, 76 (1936).
 A TITRIMETRIC METHOD FOR THE
QUANTITATIVE ESTIMATION OF
LEAD IN BIOLOGICAL MATERIALS
M. K. Horwitt and George R. Cowgill
J. Biol. Chem. 1937, 119:553-564.

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