T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 49, No. 2
Copyright © 1968 by The Williams & Wilkins Co.
Printed in U.S.A.
COULOMETRIC DETERMINATION OF ARSENIC IN URINE
ROBERT K. SIMON, P H . D . , GARY D. CHRISTIAN, P H . D . , AND
WILLIAM C. PURDY, P H . D .
Department of Chemistry, University of Maryland, College Park, Maryland 20742
Arsenic and its compounds are used for
many industrial as well as medical purposes.6, 2S The clinical and toxicologic symptoms of arsenic poisoning have been summarized by Brooks and Alyea.6 The potential clanger of exposure to or ingestion of
arsenic compounds, or both, in industry or
medicine, is significant. In addition, accidental occurrence of arsenic poisoning in the
home, especially in children, appears to be on
the increase.
Urine is the sample usually taken for
arsenic analysis for evidence of harmful exposure because of its larger volume in comparison to blood samples. Under normal
conditions the human body contains traces
of arsenic and some arsenic will be excreted
in urine. The maximal allowable concentration of arsenic in urine is considered to be
1 mg. per liter of urine21 (evidence of harmful
exposure to arsenic will exceed this level).
Toxicologists have determined the levels of
arsenic found in individuals not exposed to
abnormal amounts of arsenic. 2,19
This study was undertaken as a continuation of coulometric methods for determining
toxicologic levels of metals in biologic samples.11 The iodometric titration of small
amounts of arsenic(V) is also reported. The
advantages of coulometric titrations have
been described.10
Myers and Swift23 titrated arsenic(III)
with electrically generated bromine, and
Biswas and Dey3 titrated arsenic(III) with
Received February 18, 1967.
Supported in part under contract No. DA-49193-MD-2593, of the Office of the Surgeon General
of the U. S. Army.
A portion of this material is from the Ph.D.
dissertation of Dr. Simon, whose present address
is E. I. du Pont de Nemours & Co., Stine Laboratory, Newark, Delaware 19711. Dr. Christian's
present address is Division of Biochemistry,
Walter Reed Army Institute of Research, Washington, D. C. 20012.
electrically generated iodine. Bruckenstein
and Johnson0 titrated 10~3 to 10~7 M
arsenic standards coulometrically with bromine, and Purely and associates24 titrated
7 ng. of arsenic at 100 na. of generating current. Christian8 titrated as little as 9 ng. of
arsenic (3 X 10_1J M), using 5 pa. of generating current and a sensitive amperometric
end point; accuracy was ± 4 % .
MATEKIALS AND METHODS
Constant current coulometric titrations
were performed with a ChrisFeld Microcoulometric Quantalyzer or the equivalent.
The titration cell was similar to the one previously described.12 The endpoint of titrations was detected biamperometrically by
impressing 200 mv. between two platinum
foil electrodes with a Sargent Polarograph,
model XV; the amperometric current was
recorded on the polarograph.9 A dead-stop
technic was used to locate the endpoint (see
below, under Titration).
Digestions were carried out in 100-ml.
Pyrex Kjeldahl flasks on a hot plate. Solvent
extractions were performed in 60-ml. separately funnels with Ultramax (Teflon) stopcocks.
Measurements of pH were made with a
Leeds and Northrup pH meter, No. 7405.
Reagents for Method
Arsenic-free acids (assay 5 X 10~7 %).
Concentrated sulfuric, nitric, perchloric, and
hydrochloric acids.
Digestion reagent. Dissolve 2 Gm. of
Na 2 Mo04-2H 2 o'in 40 ml. of water. Add
50 ml. of concentrated sulfuric acid, allow
solution to cool, and then add 10 ml. of 70%
perchloric acid.
Cuprous chloride solution (2 N). Dissolve
10 Gm. of cuprous chloride powder in 500
ml. of concentrated HC1. Store in glassstoppered bottles containing strips of electrolytic copper foil. Allow to stand for 24 hr. to
207
208
SIMON ET
reduce copper(II) to copper (I). The solution
is initially dark and will turn light yellow or
colorless when the copper(II) is reduced.
The solution can be cleared in 1 hr. by
stirring with a magnetic bar in the presence
of copper shot. Store portions of the clear
solution in 50-ml. glass-stoppered test tubes,
lined with copper-foil strips. If the solution
becomes dark again, it can be cleared by
stirring in the presence of copper shot.
Buffer, pH 7.0. Dissolve 78.5 Gm. of
anhydrous disodium phosphate and 47.1
Gm. of anhydrous monopotassium phosphate in 1 liter of water.
Buffer, pH 9.0. Dissolve 95.0 Gm. of
anhydrous disodium phosphate in 1 liter of
water.
Generating electrolyte. Dissolve 16.6 Gm.
of potassium iodide and 0.1 Gm. of sodium
carbonate in 1 liter of the pH 7.0 buffer.
Benzene, 99.5%, thiophene free.
Additional Reagents for Study of Method
Bromine generating reagent. Sodium bromide 0.2 M in 1 M sulfuric acid.
Toluene, sp. gr. 0.865, thiophene free, was
purified as follows: 1 liter was stirred with
100 ml. of concentrated H2SO4 for 30 min.
at 15 C. The acid layer was removed and the
process was repeated until the sulfuric acid
layer was pale yellow. The toluene was
shaken twice with water, once with 10%
sodium carbonate, and then again with
water, dried over anhydrous calcium chloride, and filtered. The filtrate was distilled
and the distillate, boiling at 100 to 111 C.,
was collected. The toluene was then saturated with HC1 gas.
Arsenic{III) standard. Primary standard
(Baker's 99.9S %) As 2 0 3 was dried for 2 hr.
at 105 C. The requisite amount was dissolved in 20 ml. of water with 2 Gm. of
sodium hydroxide, neutralized with 25 ml.
of 1 M sulfuric acid, and diluted to 1 liter
with water. Standards containing less than
100 jug. per ml. were prepared by dilution of
a 1.000 mg. per ml. stock solution. All
arsenic(III) standards were checked coulometrically with generated iodine.
ArseniciV) standard. Baker's analyzed
As 2 0 6 (99.0%) was dried for 2 hr. at 105 C.
The requisite amount was dissolved and
diluted to 1 liter as explained above for
AL.
Vol. 49
As 2 0 3 . By iodometry, 50 mg. of the AS2O5
were standardized volumetrically against
standard thiosulfate; the assay gave 97.70 ±
0.57% AS2O5. Coulometric standardization
was carried out as follows: 1 ml. of sample
and 0.1 Gm. of KI were added, the flask was
stoppered immediately, and the reduction
was allowed to proceed for 5 min. Thiosulfate solution 1 ml. and 20.0 ml. of 1 M disodium phosphate were added, in that order.
The excess thiosulfate was titrated coulometrically with iodine at 9.650 ma. (sample
titer). The final pH was 2.5 to 3.0. The ratio
of thiosulfate to arsenic(V) was about 2:1
/uEq. A thiosulfate titer was determined by
titration of all reagents and thiosulfate
(minus sample) under the same conditions.
The thiosulfate titer minus the sample titer
gave the microequivalents of arsenic (V) in
the sample; 10 ,uEq. of the above AS2O5 gave
an assay of 97.66 ± 0.89%. Samples containing less than 10 /uEq. of arsenic(V) could
not be determined by this method because
of significant air oxidation of iodide. Smaller
samples can probably be titrated by removing oxygen from solutions.
Iron(II) solution, 0.005 M. Prepared from
ferrous ammonium sulfate.
Iron{III) solution, 0.05 M. Prepared from
ferric nitrate hexahydrate.
Lead(II) solution, 0.025 M. Prepared from
lead nitrate.
Thallium(III)
solution, 0.0005 M. Prepared from thallic fluoride.
Mercury(I) solution, 0.001 M. Prepared
from mercurous nitrate, acidified with
nitric acid.
Mercury(II) solution, 0.01 M. Prepared
from mercuric nitrate.
TinfJI) solution, 0M5 M. Prepared from
stannous chloride dissolved in 1 M hydrochloric acid.
Antimony {111) solution, 0.0005 M. Prepared from Sb 2 0 3 dissolved in sodium hydroxide in the presence of sodium tartrate
and neutralized with H 2 S0 4 .
Procedure
Digestion. Add 10.0 ml. of urine, 1.0 ml.
of nitric acid, 2.0 ml. of the digestion reagent, and 3 or 4 glass beads to a 100-ml.
Kjeldahl flask. Digest in a stone fume hood
at full heat until the solution boils vigor-
Feb. 1968
COULOMETRIC DETERMINATION OF ARSENIC
ously, red fumes appear and evolve, the
digest turns clear, and white fumes evolve
for 1 to 2 min. Digestion time is 15 to 20 min.
Extraction. Transfer the cooled digest to
a separately funnel, using a total of 10 ml.
of concentrated HC1 to transfer. Add 5 ml.
of cuprous chloride reagent and 15 ml. of
benzene and shake the mixture for a few
seconds. Allow the layers to separate and
draw off the aqueous (bottom) layer into a
second separately funnel. Add 15 ml. of
benzene to the second funnel and extract as
before. Draw off the aqueous layer and discard. Add the benzene layer to that in the
first funnel. Wash the combined benzene extracts with three 4-ml. portions of hydrochloric acid to remove traces of yellow
cuprous chloride solution. Back-extract the
arsenic(III) three times with 15-, 10-, and
10-ml. portions of pH 9.0 buffer, adding the
combined aqueous extracts to the titration
eel] (50-ml. beaker). The final pH is 6.5 to
7.0.
Titration. Add 10.0 ml. of generating
electrolyte to the titration cell containing
the extracted sample. Add the pH 7.0 or 9.0
buffer to the isolated cathode compartment.
Titrate the sample with electrogenerated
iodine, using a current of 0.965 ma. (0.01
juEq. per sec.) and a polarograph sensitivity
of 0.01 na./mm. The dead-stop endpoint is
performed by setting the pen of the polarograph at the 50-mm. mark by the displacement knob. The titration is continued until
the indicator current (pen) reaches the 100mm. deflection point (0.05-;ua.) indicator
current. The indicator current will remain
small until the endpoint is reached.
Blank. The blank (duplicate) is run in the
same manner as the sample, with extraction
used as the starting step. The digestion step
does not contribute to the blank. In order to
cleanse the electrodes before each series of
titrations, a generating reagent blank is run
and discarded.
Calculation
The coulometer reads out directly in
microequivalents.
Mg. As/liter of urine
_ (/JEq. sample — ;uEq. blank) X 37.46
ml. sample
209
Or, since 1 sec. is equivalent to 3.746 X
10 -4 mg. As at 0.965 ma.,
Mg. As/liter of urine
_ (sec. sample — sec. blank) X 0.3746
ml. sample
RESULTS AND DISCUSSION
Several factors were considered in selecting the proposed method for the determination of arsenic in urine: sensitivity, selectivity, precision, accuracy, speed, and convenience. Toxicologic studies require a lower
limit of detection, at least 1 ng. As per ml.
of urine. The proposed method consists of
three parts: wet acid digestion of the sample
with a molybdenum (VI) catalyst, selective
extraction of AsCl3 with benzene, and
coulometric titration of the arsenic(III) with
iodine at pH 6.5 to 7.0. The proposed procedure is sensitive to 0.5 ng. As per ml. of
urine with a 10-ml. sample.
The extraction procedure is based upon
the work of Hanke18 and Fischer and associates,15 with a number of modifications.
Arsenic(V) is rapidly reduced with cuprous
chloride before extraction and the arsenic is
back-extracted into phosphate buffer, pH
9.0, instead of water, in order to give a final
pH suitable for titration with iodine.
A series of experiments was undertaken to
determine the effect of different procedures
and parameters on the proposed method.
The over-all effects of digestion, extraction,
and titration on the recovery of arsenic were
studied separately. These results are discussed below.
Coulometric titration of arsenic(III). A
quantity of 10 /ig. of arsenic corresponds to
about 0.27 jiEq. Since it was desired to develop a procedure that could be used to
analyze as little as this amount of arsenic,
the accuracy and precision of the coulometric
titration of arsenic (III) standards at approximately this level and higher were determined. Data for iodine titrations of
0.1654 to 31.63 MEq- of arsenic are summarized in Table 1. These standards were
used for the arsenic determinations in urine.
The time of the titration was read to ± 0 . 1
sec. The precision was relatively about 1 %,
210
SIMON ET
TABLE 1
COULOMETMC
TITRATION
OF
ARSENIC(III)
STANDARDS*
Taken
Found
pEq.
liEq.
0.1054
0.3208
1.002
1.205
2.004
31.63
0.1634
0.3218
1.003
1.273
2.610
31.37
Nt
R.S.D.t
%
%
15
18
16
15
15
17
1.3
1.1
0.90
0.72
0.93
0.41
-1.2
0.31
0.10
0.61
0.46
-0.75
Error
* Iodine titrant, pH 7.0, 0.9650 ma.
t Number of samples.
% Relative standard deviation.
and the accuracy ranged from —1.2 to
+0.61%.
Iodine as titrant gave biamperometric
titration curves very close to the theoretical
one in the arsenic determination; the current
remained very small until the endpoint was
reached and then increased rapidly as a
result of excess iodine. Occasionally a slight
negative drift in the baseline was observed
with actual samples (extracted standards or
arsenic in urine digests); this behavior did
not affect the precision or accuracy. Bromine
titrant, however, frequently exhibited curious indicator electrode phenomena. Initial
residual currents of up to 0.5 no,, were sometimes observed. A series of five samples
would give two good titration curves, followed by three curves that exhibited current
reversals before the endpoint. The current
reversals usually increased in magnitude
with succeeding titrations; however, the precision of the dead-stop endpoint was surprisingly unaffected. Various treatments
such as washing the electrodes with 6 M
nitric acid and then with water every 4 to 5
runs helped somewhat; however, the problem returned with the next set of runs. New
electrodes did not exhibit this phenomenon
for several weeks. Overnight cleaning with
chromic acid cleaning solution eliminated
the problem for about 40 consecutive runs.
Heating the electrodes to red heat did not
remove the problem. Commercial platinum
electrodes were more trouble-free than were
homemade foil electrodes. Reversal of the
polarity usually decreased the current re-
AL.
Vol. 49
versal significantly, only to have it return
after a few series of runs. Decreasing the
applied voltage (and increasing the sensitivity) from 200 to 40 mv. decreased the
reversal to a small value. If the electrodes
were rinsed with water immediately after
titration, the current problem was minimized. When electrodes were allowed to
soak in solutions containing arsenic for 1 day
or more, the current reversal was pronounced. The accumulated data seem to
indicate that the problem is one of adsorption on the electrode surface.
Iodine was selected as the titrant because
the current reversal problem was minimized
and because the titration blank was smaller
(see below) than it was with bromine, a
stronger oxidant.
Reagent blanks. The absolute value and
reproducibility of the reagent blank, if any,
were important factors in the proposed
method for arsenic in urine. A precisely determined reagent blank with a magnitude
almost twice as small as the lower sensitivity limit of arsenic (10 Mg-) was desired. A
number of different reagents were tested for
use in extraction and titration. Table 2 lists
10 different reagents used in the extraction
and titration. Iodine titrant, as expected,
gave lower blank values than bromine.
Benzene and carbon tetrachloride gave significantly lower blanks than "purified"
toluene. Benzene that was purified in a
manner similar to that used for toluene did
not give significantly lower blanks. Cuprous
chloride, when washed thoroughly from the
organic layer with concentrated hydrochloric acid, had only a slight effect on the
total blank. Aqueous solutions of disodium
phosphate were preferable to other salt solutions (trisodium phosphate and sodium acetate) for the aqueous extraction procedure.
On the basis of the data in Table 2, the following reagents were preferred: benzene as
the organic extractant, anhydrous disodium
phosphate as the aqueous back-extractant,
and iodine titrant. In addition to the lower
blank, benzene was chosen as the organic
solvent because of its commercial availability
in a pure form; it has a specific gravity
(0.879) less than that of water, so that it was
the upper phase in solvent extractions, and
Feb. 1968
211
COULOMETRIC DETERMINATION OF ARSENIC
TABLE 2
REAGENT BLANKS*
Reagent
Amount Titrant Reducing Matter
ml.
Benzene
Benzene, purified
Benzene, purified
ecu
CuCl (0.05 M)
CuCl (0.05 M ) , acid
washed
CuCl (2 M ) , acid
washed
Cone. HC1
0.1 M K I
Sodium acetate (1 M)
20
20
20
20
5-20
5-20
0.2 M N a B r , 1 M
H2SO4
2 M Na2HPOi
0.2 M N a 2 H P 0 4 - 7 H 2 0
0.15MNa3PO4-12
HjO
Toluene
Toluene, purified
5
»Eq.
Br2
Br 2
I2
Br 2
Br2
Br 2
0.11
0.09
0.004
0.0s
ca. 0.2-0.5
<0.05
h
0.008
Br 2
Is
Br 2
I2
Br 2
0.0
<0.01
ca. 0.5
0.21
<0.02
25
I2
Br 2
I2
I2
0.036
0.038
0.038
>2.8
20
20
Br 2
Br 2
1-2
0.3-0.35
1-3
10
25
5
30
25
* Bromine titrant, pll 0.5; iodine titrant, pH
7.0, 0.9650 ma.
it could be used for the quantitative extraction of microgram amounts of arsenic.
Table 3 is a compilation of data on the
total procedural blank with iodine titrant.
The total (digestion, extraction, and titration) blank was 0.024 ^Eq. For a 10-ml.
urine sample containing 1 ng. of As per ml.,
this constituted a sample-to-blank ratio of
about 10:1. The relative reproducibility of
the blank determination for 20 runs was
2.S%. It was thus possible to determine
arsenic accurately at the lower sensitivity
limit. The digestion reagent and digested
urine did not cause a measurable blank.
I t should be noted that the listed blanks
are true blanks, that is, the tiEq. of iodine
required to reach the break in the titration
curve. In actual practice, a dead-stop
method is used, and an additional 0.03 or
0.04 fiEq. of iodine is required to deflect the
recorder pen by 50 mm. This, however, is
not a contribution to the blank and is very
reproducible.
Hydrochloric acid concentration. The effect
of the concentration of hydrochloric acid on
the extraction efficiency of arsenic(III) is
shown in Figure 1; 1 /uEq. of arsenic standard was extracted and then titrated. The
final pH of the extract-electrolyte solution
ranged from 7.7 to 6.6 for 2 to 11.3 M hydrochloric acid; this variation in pH did not
affect the titration. A hydrochloric acid concentration equal to or greater than 10 M was
necessary to recover 9 5 % or more of the
arsenic. This is in agreement with the work
of Fischer and associates,15 in which arsenic
was extracted into carbon tetrachloride.
Number of extractions and pH in backextraction. Table 4 is a tabulation of data
concerning the number of organic and aqueous (back) extractions and the pH of the
aqueous extract. It was concluded that at
least three extractions, both organic and
aqueous, were required to yield 95% recovery of up to 50 /^Eq. of As. There was no
significant difference in the recovery of the
TABLE 3
R E A G E N T B L A N K S OF PROCEDUHE*
Blank
Extraction
Extraction
Digestion and extraction
Digestion and extraction
Total (including generating electrolyte)
* Iodine titrant, pH 6.7 ± 0.2, 0.9650 ma.
f Number of determinations.
J Relative standard deviation.
Conditions
No CuCl
CuCl
No urine
Urine
Urine
Reducing Matter
Nt
u£«.
0.012
0.020
0.026
0.019
0.024
10
10
10
10
20
R.S.D4
%
2.8
5.0
4.9
3.9
2.8
212
SIMON ET
Vol. 49
AL.
12
significantly with benzene were undesirable
since they would have reacted with generated iodine. Reductants which required
heating to effect reduction, remove their
excess, or eliminate undesirable products
of the reaction weie avoided because of
the volatility of AsCl3. Table 5 lists data on
a series of reductants studied under a variety
of conditions. Arsenic(V) has reportedly
been reduced with boiling ammonium iodide
in 4 M hydrochloric acid,1 hydrazinium
sulfate,14 and sodium sulfite,20 among others;
however, in these experiments, only cuprous
chloride in concentrated hydrochloric acid
F I G . 1. Effect of hydrochloric acid concentration of extraction efficiency of arsenic ( I I I ) into
benzene.
E F F E C T OF N U M B E R OF EXTRACTIONS AND P H OF
0 1
2 3 4 5
6 7 8 9
10
MOLARITY HYDROCHLORIC ACID
II
TABLE 4
AQUEOUS BACK EXTRACTION
aqueous extraction between pH 3.3 and 7.3.
Below pH 3, recovery was not complete.
Oxidation state of arsenic. While arsenical!) was extracted satisfactorily, less
than 0.1 % of arsenic(V) was extracted. After
digestion, all arsenic is in the pentavalent
state (see below). In order to determine the
total arsenic in urine, the arsenic(V) must
first be reduced to the tripositive state.
Studies were run to determine the best reducing agent under the experimental conditions.
The most desirable reductant would be one
which rapidly and stoichiometrically reduced
arsenic (V) in concentrated hydrochloric acid
(during the extraction) without interfering
side reactions. Reductants that extracted
N*
Mt
1
1
1
2
2
2
1
1
2
2§
311
311
pH Aqueous
Extract
As (III)
Taken
liEq.
%
7.3
3.3
6.5J
6.5
6.5
6.5
1.002
1.002
1.002
50.00
10.00
10.00
95.3
96.7
90.3
90.0
8S.0
94.5
As Recovered
* N u m b e r of extractions with 20.0 ml. of benzene.
| Number of extractions with 15.0 ml. of p H 9.0
buffer.
X Water as the back-extractant.
§ Portions of 15 + 10 ml.
If Portions of 15 + 10 + 10 ml.
TABLE 5
REDUCTION OF A R S E N I C ( V ) *
Reductant
CuCl (2 N)
N2HV2HC1
NH 2 OHHCl
Iodide
30% H 2 0 2
K,Fe(CN)o
Na2SO,
Na&Os
SnCl2
Reduction
Conditions
Reduction
to As (III)
Cone. HC1
Cone. HC1
1 MHC1
1 M HC1
pH7
Hot cone. HC1
6 M HC1
1 M HC1, hot
1 MHC1
1 M HC1
%
95 ± 5
53
7
Unknown
0
0
Unknown
30
22
13
Undesirable
Interferences
Asl3 precipitate and I 2
Fe(CN) 6 4_ extracts
Black metallic As formed
* Solutions of 0.1 M of the reductant used unless otherwise specified; 5-min. reduction time.
Feb. 1968
COULOMETRIC DETERMINATION OF ARSENIC
TABLE 6
RECOVERY OF AHSENIC AFTER R E D U C T I O N WITH
C U C L REAGENT
As(V) Taken
N*
Recovery
R.S.D.f
%
liliq.
1.000
10.00
50.00
6
6
5
94.9
94.7
96.2
Total
17
95.3
%
3.8
2.7
2.4
* No. of determinations.
f Relative standard deviation.
TABLE 7
RECOVERY O F ARSENIC STANDARDS*
Sample
As
Nf
V
V
III, V (ca.
1:1)
III, V (1:1)
Total
R.S.D.}
Error
%
%
%
0.0113
1.204
3.749
9.742
31.35
5.578
19.45
2.664
4 94.9
4 95.0
7 94.7
5 94.0
6 97.1
5 94.8
6 97.2
4 95.2
2.6
2.0
2.7
2.7
3.0
2.8
1.2
3.1
-5.1
-5.0
-5.3
-6.0
-2.9
-5.2
-2.8
-4.8
49.98
5
94.4
1.4
-5.6
46
95.2
lig./ml.
in
in
in
in
in
Recovery
* Sample of 10 ml.
t N o digestion performed before extraction.
t Relative standard deviation.
solution quantitatively reduced arsenic(V)
to arsenic(III) without serious side effects at
room temperature. Cuprous chloride is
easily air-oxidized and thus must be stored
in glass-stoppered containers in the presence
of copper metal. Since small amounts
(droplets) of the aqueous layer will generally
remain in the extraction funnel with the
organic layer, it is necessary to wash with
hydrochloric acid to remove the last traces
of copper(I).
Data on the reduction and extraction of
arsenic(V) standards and mixtures of
arsenic(III) and (V) are given in Tables 6
and 7. From 1 to 50 /iEq. of arsenic(V) were
recovered, with an average of 95.3 ± 0.6%
213
(Table 6). The average recovery in Table 7
for 0.6 to 50 Mg- of arsenic per ml. (10-ml.
sample) was 95.2%. The average precision
(pooled relative standard deviation) was
2.5%. A total of 63 determinations was
made.
Digestion. Preliminary experiments were
designed to test the possible loss of arsenic
during the molybdenum wet acid digestion
procedure. Samples of arsenic(III) and (V)
(1.00 juEq.) were added to 10 ml. of urine
before and after digestion. The digests were
extracted and titrated with iodine. Recoveries of five samples, added before digestion,
averaged 94.1 ± 3.7%. Recoveries of four
samples added after the digestion averaged
93.7 ± 3.7%. These data indicated that
arsenic was not lost by volatilization during
the digestion. The similar recoveries of
arsenic(III) and arsenic(V) indicated that
arsenic was rapidly oxidized to the pentavalent state. Under the conditions of the
digestion, arsenic(V) was not volatilized.
Extractions in the presence and absence of
cuprous chloride, after digestion of 1 juEq.
of arsenic (III), verified that arsenic was in
the pentavalent state after digestion. The
presence of about 0.1 M chloride in urine
did not affect the recovery. Thus, maintenance of strongly oxidizing conditions prevented loss as the volatile AsCl3.
If the digest was not heated for several
minutes after fumes of perchloric acid appeared, residual currents due to oxidation of
the electrolyte were sometimes observed in
the titration. This effect was thought to be
the result of oxidation of the iodide due to
residual traces of nitric acid (cold perchloric
acid is a weak oxidant) and was avoided with
sufficient reflux. The small amount of urine
salts present in the digest did not seriously
affect the extraction procedure.
The employment of a molybdenum(VI)
catalyst in wet acid digestions was described
by Bolin and Stamberg4 in the treatment of
organic compounds before phosphorus determination; a mixture of sulfuric and perchloric acids was used in the digestion.
Cummins and colleagues13 used essentially
the same digestion procedure for determining
selenium in biologic materials. Christian and
co-workers" found that addition of nitric
214
SIMON ET
Vol. 49
AL.
TABLE 8
TABLE 9
RECOVERY OF ARSENIC FROM U R I N E *
SELECTIVITY F O R A R S E N I C
Sample
As
Nt
Recovery
%
%
%
5
5
5
5
5
4
5
4
93.3
94.3
96.9
94.4
95.6
96.5
94.7
95.2
2.4
1.8
1.4
3.7
0.85
5.0
3.2
3.3
-6.7
-5.7
-3.1
-5.6
-4.4
-3.5
-5.3
-4.8
38
95.1
Pg./ml.
III
0.C113
III ' 1.204
3.749
III
9.742
III
50.35
III
1.460
V
3.853
V
10.50
V
Total
R.S.D4
Error
* Urine sample of 10.0 nil.
t N u m b e r of determinations.
t Relative s t a n d a r d deviation.
acid allowed the digestion to proceed more
smoothly and without charring, with subsequent danger of loss of selenium. The removal of organic matter from urine by a
variety of methods has been described.27
Although arsenic samples are conventionally digested without a molybdenum
catalyst 16,21 we found that unless extreme
care was taken, arsenic was lost. In addition,
digestions required 1 hr. instead of 15 to
20 min., and appreciable blanks from urine
were a danger in the absence of a molybdenum
catalyst.
Recovery of arsenic from urine. Recovery of
added arsenic(III) and (V) from 10 ml. of
urine is summarized in Table S. The average
recovery of 0.6 to 50 /ug. of As per ml. of
urine was 95.1%, with an average standard
deviation of 2.7%. Thirty-eight determinations were made. The data in this table yield
several important conclusions: arsenic is reproducibly recovered from urine; there is no
significant difference between extracted
aqueous standards (Tables 6 and 7) and
urine samples with respect to precision and
percentage recovery; arsenic (III) and arsenic(V) are recovered to the same extent;
0.6 to 50 Mg- per ml. are recovered at 95%.
The entire loss of arsenic in the proposed
method occurs in the extraction step, because digestion was shown to cause no loss.
Further evidence to support this conclusion
was obtained by adding arsenic(III) stand-
Element Tested
Taken
fEq.
Sb(III)
Fe(II)
Fe(III)
Pb(II)
Hg(D
Hg(II)
Tl(III)
Sn(II)
1.0
5.0
50.0
50.0
1.0
1.0
1.0
5.0
Extracted
%
<0.1
<0.1
<0.1
Not titrated
0
0
0
0
ards after the extraction step; recovery in
the titration was 100.0 ± 0.50%.
Arsenic(III) and arsenic(V) are quantitatively distinguished by extraction; however,
this does not apply to urine samples with
the present method.
Selectivity. The selectivity of the extraction
for arsenic(III) is shown in Table 9. The
given amount of foreign metal was taken
through the extraction procedure and then
was titrated. Antimony and iron gave less
than 0.1 % extraction. Thallium, tin, and
mercury did not interfere. Lead is not
titrated by iodine. Thus, as a toxicologic
method, the proposed procedure can selectively distinguish arsenic in the presence of
these common heavy metals or those generally present in the body. Fischer and associates15 found that in the extraction of
arsenic(III) from hydrochloric acid into
carbon tetrachloride, only germanium(IV)
could also be extracted; selenium(IV),
arsenic (V), antimony (III) and (V), and
tin(IV) could not be extracted. Using the
same extraction system, Milaev and Voroshima22 and Saito and associates26 extracted
arsenic (III); cadmium, cobalt, vanadium,
silicon, iron, lead, selenium, mercury,
molybdenum, tungstic oxide, and phosphorus pentoxide did not interfere.
Organic arsenic samples were not determined in this work; however, the work of
Gross and Rivers,17 Carey and colleagues,7
and Remington and associates25 suggests
that most organic arsenic compounds are
converted to soluble arsenic during wet acid
digestion. The action of stomach acid would
also help to solubilize ingested organic
arsenic compounds.
Feb. 1968
COULOMETRIC DETERMINATION OF ARSENIC
SUMMARY
A rapid and reproducible method for the
toxicologic determination of arsenic in urine
is described. A 10-ml. sample is digested in a
mixture of sulfuric, perchloric, and nitric
acids containing a molybdenum(VI) catalyst; digestion time is 15 to 20 mi'n. The
arsenic in the digested sample is simultaneously reduced to arsenic(III) with cuprous
chloride and extracted into benzene from
10 M or more hydrochloric acid as AsCl3. I t
is then back-extracted into disodium phosphate, potassium iodide is added, and the
arsenic(III) is titrated with electrogenerated
iodine. Recovery is 95.1 % for 0.6 to 50 ^gper ml. of urine, with a standard deviation
of ± 2 . 7 % (38 determinations). The method
is specific for arsenic.
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