165

HOUTIl AFltlC.\:-; JOliR~AL OF' DCIEI\CE,

Vol. XLI, pp. 16:j·171, .
February, 1945.

NIE'l'.-\LLIC l'dISONS; .\ SYSTEM OF ANALYSIS

BY

Dr. P. A. E. KAMERMAN,
(ioL'I'I'IlIllGnt Chemical Laboratories, Johannesburg.

nead 3rd July, 1944.

J w;t as the great organic group of chemistry is treate([

much wore systematically than its relation inorganic chemistry,
in the same way the illvestigation of poisoning by organic sub·
stances can ue said to have received more logical treatment than
t.hat meted out to cases of inorganic poisoning. The latter'are on
a very loose basis, most text books treating the problem more as
if in cases of inorganic poisoning the identity of the poison con·
cerned is known to the analyst before he starts his investigation.
The main reason for this attitude when dealing with metallic
and metalloid poisons is the fact, that once the organic tissues
have been destroyed, the finfll solution is considered to be a
simple solution of metals and that therefore these can be deter·
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mined by the usual text book methods. 1'he amount of

material available for toxicological analysis, however, is of neces·
sity very 'limited, and when one adds the further disadvantage
that the metal responsible for the poisoning "'ill be present only
in little more than traces, it is obvious that this vie\"point is
incorrect.
When dealing with organic poisons, no analogy with ordinary
organic mixtures is possible, and it was a very much felt need,
which led Stas in 1850, to evolve for the first time a metho(l
of analysis, which later became famous as the Stas·Otto method,
and which renders possible the detection of a very large rangc
of organic poisons.
No such general system exists when one has to deal with caSt·,.,
of metallic poisoning. This attitude must be deplored and as ill
addition the old accepted systems of separation as well as the
newer methods of microchemistry are inadequate, it has become
necessary to treat the question of metallic poisoning im'estiga-
tion from a new standpoint, adapting the most suitable of the
old and. of the new processes.
In the last few .vears newer methods of separation have beell
evolved, but these have not rome up to their early promises.
One of these newer methods was the use of a chloroform solution
of diphenyl thiocarbazone (dithizone for short), w11 ich has the
property of extracting a series of metals from acid or alkalin('
solutions.
 166 SOUTH AFHICA:\ JOLll:\.\L OF SClE:\CE.

Wickmann (1939 :66) showed that although the s'eparation

of different metals was largely governed by the pH of the
solution, no clean-cut separation was possible due to overlapping.
In spite of this drawback, however, the dithizone method of
separation and determination can be used to a limited .extent
and does play an important part in toxicological investigatioll,
although in a narrower sphere than was originally hoped for.
In devising a system of analysis, the great consideration
is the number of 'metallic poisons which must be taken into
account. Although their number is large, practical experience
has shown that under South African conditions it is limited to
fourteen. I may mention here that thel'e is a great deal of
difference in the types of poison mostly used in various countries.
Whilst in South Africa arsenic and cantharidin are the most
common poisons employed, in India, for example, arsenic and
opium lead the way. It is obvious that such characteristics
must be considered in the drawing up of any system of toxico-
logical investigation. In South Africa one has to d~al also to
a very large extent with cases of native poisoning, and as any
substance possessing emetic and purgative properties is much
prized by the native as medicine, it is not surprising to find
included in the native herbalist's outfit metallic salts like alum,
copper sulphate and potassium bichromate, the two latter having
the added attraction of colour.
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For practical purposes we can therefore divide the poisonous

metals into two groups.
A. Those which are commonly encountered in toxicological
practice an(l are more or less toxic.
B. Those which we know to be toxic to a higher or lowel'
degree but which have nevel', as far as we know, figured as
a poison under South African ·conditions ..
In the first group we have aluminium, antimony, arsenic,
barium, bismuth, chromium, copper, iron, lead, manganese,
mercury, thallium, tin and zinc. Although bismuth is not
considered to be of importance as a poison, its presence must
always be expected due to its extensive use as a constituent of
the powders and mi~tures given in cases of stomach disorders.
'fin figures occasionally in undesirable quantities in canned .food
products, although seldom in fatal amounts. Zinc is included
because in spite of its low degree of toxicity, poisoning by means
of the concentrated zinc chloride solutions known to plumbel's
as "killed spirits" has occurred.
Under the second group all the other metals like cadmium:
gold, osmium, silver and so forth, are placed. Although used
to some extent we have never encountered them in our investi-
gations and, therefore, it was decided to leave them out of any
general system, which should be based, after all, on practical
considerations and not on theoretical possibilities.
 ;'IIETALLIC l'UI~U:\i:i: A~ALYi:ili:i. 167
Duch a s.) stem iti herc,yith presented. The tirst step in
the chemical illYestigatioll of acute poisoning is the routine one
of applying the Reinsch test to the minced organs, preferably
the lIver. If the copper strip thus obtained shows a darkening,
or "silvering," the sublimate obtained by heating it ill a small
. fiat, hard, glass tube is examined under a microscope. Shiny
globules show the presence of mercury, whereas arsenic is
indicated by the formation of the characteristic octahedral or
tetrahedral crystals of arsenious oxide. Although most text
books state that the sublimate from antimony is amorphous,
1 have known it to take a crystalline shape very similat to that
obtained from arsenic.
By passing a stream of sulphuretted hydrogen through the
slightly heated tube, the presence of antimony can be confirme(l
by the formation of an orange colour, whilst arsenic shows up
yellow. When the black deposit extends to the platinum wire:
as well, bismuth must be expected.

I-REIXSClI TEST I)OSlTIYE-A. MERCURY PRESE)iT.

If the preliminary test has shown the presence of mercury,

it is necessary to use a special method for the destruction of
the organic tissues, as mercury salts are too volatile for the usual
method~ of destruction. The method described by Winkler
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(1938, :220), consisting of depositing the mercury on zinc powder

and its subsequent determination in an acid solution by means
of a carbon tetrachloride solution of dithjzone, has been found
to be very satisfactory. Since copper and bismuth, when
present in appreciable quantities, interfere, it is necessary to
eliminate these metals. By the addition of eithel' potassium
iodide or sodium thiosulphate to the solution, bismuth and
mercury complexes are formed, which are extracted by the
dithizone, whilst copper is unaffected. Bismuth can be removed
by allowing the original dilute acid solution to stand overnight,
by which most of the bismuth is deposited as the basic salt.

B. MERCURY NOT PRESENT.

If mercury is absent, the organic matter can be efficiently
destroyed by digesting the organs in a Kjeldahl flask by mean"
of boiling sulphuric and nitric acids. The solution thus obtained
is transferred to a standard 100 m!. flask and made up to volume.
Rarilllll
As the barium will have been converted to the insoluble
barium sulphate, this metal must" be looked for in the deposit
formed during the digestion of the organic matter, and which
under om conditions consi"ts mostly of silica. This deposit must
be fused with a mixture of potassium and sodium carbonates,
after which the barium can be determined by any of the common
methods. .
 168 SOUTH AFlUC.-\.:\ ,TOUR:\AL OF SCIENCE.

Arsenic.
If arsenic is present it is determined by the method of
Green (1918, 539). This' method still remains the simplest of
[lll those suggested for the determination of small amounts of
arsenic, and with a slight modification, including the use of a
more dilute iodine solution, we have been able to determine
amounts of arsenic of the magnitude of 5 micrograms arseniou"
oxide, which is usually sufficient for the estimation of arsenie
in hair. It may be mentioned here that for the purpose of
determining the amount of arsenic lvhen only very small amount"
of hair and nails are available, we have modified the Gutzeit
method by making use of a solution of lead acetate instead of
the common solid devices like lead acetate paper, and are now
;tule to determine arsenic with a fair amount of accuracy when
present in amounts as small as 0,1 microgram .
. Intimol/y.
Antimony is determined by the method of Bamford (1934,
101), which depends 011 the measurement of the intensity of
the orange colour produced by mealls of sulphuretted hydrogell
in the presence of a protective colloid like gum acacia.
J3iSl1t1lth.
All accurate Illdhod of determining bismuth is that of
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.\Iahr (1933, 161), which makes use of the thiourea-bismuth

reaction.
II-REI:\SCH TEST KEGA'l'lYE,
A. Reaction with dithizone in allwline solution positiYe,
_\ fresh aliquot of the solution' is 110W taken, and after the
addition of citric acid to the extent of 30 per cent., it is made
alkaline to a pH of 9 by means of dilute ammonia. The solutioll
is now shaken out with successive quantities of a chloroform
>,olution of dithizone until no colour change takes place. Thii'i
extract may contaill bisllluth, lead, thallium, tin or zinc.

7,ill('.
The zmc can be recovered from tlle diUlizone extract by
shaking it out with a solution of potassium cyanide, which
removes the zinc but leaves the other four metals comuined with
the dithizone. As it is rarely necessary to estimate traces of
zinc, any of the stnnd[lrd methods can be employed far, its
determin a tion.
If zinc is known to ue absent, the potassium cyanide solutioll
e:m be added directly to the alkaline solution, but in such event
it is nec'essary to gum-d against the possibility of oxidation of
the dithizone by the ferric iron present in the viscera, as' the
ferric;yanide formed destroys the reaction capacity of the dithi-
zone. This can be done most suitably through the addition of
a few crystals of hydroxylamine hydrorhloride to the solution
 ~mT_\LLlC 1'01S0:\S: A:\ALYSLS_

prior to the shakmg ouL This has an added beneficial effect,

as it stabilises the type of dithizone complex formed. A red
(:olour then indicates the presence of bismuth, lead, thallium or
tin, and it therefo~:e becomes necessary to separate these meta/,;.
The first step in this is to shake out the dithizone extract witll
<1 buffer solution of pH 0·4 _which. according to Bambach nIl(]
Burkey (1943, 904), removes all the lead. The author has fOUlHl
that this treatment also removes any thallium and some traces
of tin. The latter cnn be removed again by shaking out the
buffer solutio.p. with more dithiwne solution. The cOlnplek
separation of the lead amI thallium on tbe one side, an(l of the
bismuth and tin on tIle other has thus bern affected.

1_('(ul and Thalli1lm.

The buffer extract is now made nlkalille and re-extractetl
with dithizone. ~\ftel" the removal by distillatio-n of the bulk
of the chloroform, the organic matter is destroyed by means of
boiling sulphuric nnd llitric acids, which lea yes a residue of lead
:md thallium sulphates .. After the addition of an equal volume
of alcohol to the diluted solution, the lead sulpbate is filtere(l
o.ff through a micro filter, which affords a complete separation
between the lead and the thallium salts. ('fhis separation is
based on the unpu blishetl method of ;;eparation in the deter-
mination of lead by Copeman.)
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Thallium can then be determined in the filtrate by DlE-;III"

of a standardised dithizone solution.
Lead is determined in the sallIe way, after the illsolllbk
lead suI ph nte has been redissolved by the use of all ammOll iUlll
"alt such as ammoniulll citrate.

Til!.
If it is desired to determine till, \,11is cm] be achievecl bv
the use of any of the standard methods. Its separation fro~l
bismuth can be effected by making lise of the fact_ that whilst
the tin sulphides arc soluble in yellow ammonium sulphide, thnt
c;>f bismuth is insoluble.

B_ Henction with dit11izone in albdilll' ;;oluiion lll'g;\ti\-e_

c\nothcr aliquot is no\v boiled with a few- drops of llitril~
acid, in order to oxidise all the iron present, and is then made
alkaline \yith ammonia. Should a strong blue colo11r dcoyelop,
wpper in appreciable amOlI)lts may he expectefl .

.Copper.
Although it is rare to have to cll'termine copper in toxico-
logical investigatiOlv, its estimation is easily effected by the
method of Haddock and Evers (1932, 495) using the reaction
between ropper and -di-ethyl-c1i-thiocal·bamate.
 17U • SOt:TH AFRIC.\:-I JOlJR:-IAL' OF SCIE:-ICE.

The addition of ammonia to the solution made from viscera

always causes the formation of a precipitate which consists
mainly of the phosphates of iron, calcium and magnesium. This
masks the presence of chromium and aluminium, and for further
tests it is necessary to re-dissolve it in dilute sulphuric acid,
after washing it thoroughly with a 2 per cent solution of
ammonium sulphate until free from chloride. I should mention
here that if bisllluth or tin are also pre.sent these should be
removed either through precipitation with sulphuretted hydrogell
Dr by extraction: with dithizone as described above.

The re-dissolved precipitate should no\v be tested for

aluminium, chromium and iron.
Aluminiuln.
The test for alumiuiulll is the qualitative one devised by
Hammett and Sottery (1925, 142), using the ammonium salt
of aurine tricarboxylic acid. This test can be used quantitatively
as wel~ but for most purposes of toxicological investigations the
method of Monier-Williams (1935, 9), which utilises the reaction
be,tween aluminium and 8-hydroxy-quinoline, can be recom-
mended.
Chromium.
Chromium cun be deterlllined by oxidising it to chromate
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by boiling the solution with sodium peroxide. The chromate,

which is indicated by the formation of an Intense yellow colour,
can then be determined volumetrically, using for the titration
O'01N sodium thiosulphate. If the amount of chromium is very
small, then the colorimetric lllethod based on the colour reaction
with diphenylcarbazide as adapted by yan del' \Yalt and van del'
Merwe (1938, 809) is advisable.
Iron. , .
Iron is a uormal constituent of the organs, especially the
liver, and from a to?,icological viewpoint oilly very large amounts
.can have any significance. Hence the iron can be determined
by any of the common titration methods.
Manganese.
Most of the manganese is precipitated as phosphate by
ammonia, and by treating the re-dissolved precipitate with a
persulphate in the presence of silver nitrate, the manganese is
-easily indicated through the formation of a yiolet colour. For
a quantitative estimation it is advisable to take a new aliquot,
and carry out the persulphate oxidation on the solution direct.
The permanganate formed cttn then be estimated colorimetric ally,

SUlIIMARY.
As under South African conditions, experience has shown
that the number of metals encountered in cases of poisoning is
 ~lETALLIC POISO!\S: A!\ALYSIS. 171

limited to fourteen, a system is herewith presented by which

it is possible to treat the investigation logically.
In this system the Reinsch test is used to indicate tlw
presence of antimony, arsenic, bismuth and mercury; a chloro-
form solution of c1iphenylthiocarbazone to show the presence of
bismuth, lead, thallium, tin and zinc; and ammonia is used to
separate the remaining metals, viz., aluminium, chromium,
copper, iron and manganese, with the exception of barium,
which is determined directly in the deposit formed during
the destruction of the organic matter by means of boiling
sulphuric and nitric acids. The methods of determination fol'
each metal nre indicated.

REFERENCES.
BAllBACH, K. und BURKEY, R. E.: "Micro-determination of lead by
dithizone." Ind. Eng. Chem. (Anal. ed.), 14 :904-907 (1942).
BAMFORD, F.: "Deter~ination of antimony in "iscera and excreta."
Analyst, 59: 101-102 (1934).
COPEMAN, P. V. d. R.: Private communication (1940).
GREEN, H. H. : "The micro-titration of arsenic." 5th and 6th Report
of the Director of Vet. Research; 539 et seq. (1918).
HADDOCK, L. A. and EVERS, N.: "The determination of minute
amounts of copper in the presence of iron and certain other
metals." A 1Ullyst, 57: 341-346 (1932).
JlAMMATT, L. P. and LOTTERY, C. T.: "New reagent for aluminium."
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2010).

Journal Am. Chem. Society, 47: 142-143 (1925).

)fAHR, C.: "Colorimetric determination of bismuth." Analyst, 58:
714 (1933).
~IONIEIi-WILLIAlIIS, C. 'V.: "Aluminium in food." Reports on Public
Health and Medical Subjects No. 78, 9-11 (1935).
V. D. WALT, C. F. and v. D. MERWE, A. J.: "Colorimetric deter-
mination of chromium in plant, ash, soil, water and rocks."
Analyst, 83: 809-811 (1938).
WIOKMAN, H. J.: "Isolation and determinatioIe of traces of metals."
Ind. Eng. Chem. (Anal. Ed.) II: 66-72 (1939).
WINKLER, W. 0.: "Report on mercury." J.A.O.A.C., 21: 220-228
(1938).