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138 MIDDLETON AND STUCKEY: THE PREPARATIOIG OF BIOLOGICAL [Vol. 79

The Preparation of Biological Material for the

Determination of Trace Metals
Part 11. A Method for the Destruction of Organic
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Matter in Biological Material

(Presented at the meeting of the Society on Wednesday, October 7 t h , 1953)
BY G. MIDDLETON AKD R. E. STUCKEY
A method for the destruction of organic matter is described; it is
capable of dealing with a very wide range of organic products of biological
origin. Nitric acid is the only reagent used in quantity, and the mineral
constituents are obtained in acid solution with but small quantities of other
reagents. The maximum temperature attained does not exceed 350” C.
The method is specially advantageous for treating large amounts of animal
tissues and proteins.

A REVIEW of the various methods that have been proposed and of the criticisms and objections
raised against them (see Part I)l shows that, although many variations of dry ashing and
sulphuric acid wet combustion have been tried,, not one of them has been found generally
acceptable. Methods of destruction with chlorine oxy-acids are unpromising, while those
in which perchloric acid is used are potentially dangerous. Hence we are left with the nitric
acid method as the most promising for further development.

PRINCIPLES
In methods involving the use of both sulphuric and nitric acids, as well as in methods
in which nitric acid is used alone, the oxidation results mainly from the action of nitric acid,
but the mixed-acid methods are subject to two major disadvantages: the production of
resistant compounds, in particular sulphonic acids, and the fact that most of the nitric acid
is lost by volatjlisation before the temperature is sufficiently high for appreciable oxidation
to take place. Even if it were possible to overcome the first objection by using some inert
solvent in place of sulphuric acid, the second would remain.
The Carius method depends on the action of nitric acid a t a high temperature, and a
sealed tube is used to retain the acid. I n the other nitric acid methods described (Part I,
p. 538 et seq.) the organic matter is dispersed in nitric acid, and the residue from the evaporation
of this solution usually catches fire when a certain stage is reached, leaving a white ash.
In practice these methods are generally inconvenient-in one procedure it is necessary to
use a flask immersed in a bath of fusible metal a t 300” C, and in another an acid-resistant
cast-iron vessel must be used.
It is, however, possible and practicable to destroy a great variety of animal and vegetable
tissues and products, in considerable quantity, by a simple procedure without incandescence
and without exceeding a temperature of about 350°C. The procedure was first applied
in the determination of cobalt in liver tissue, a material that can never be completely destroyed
by any combination of sulphuric, nitric and perchloric acids, and which also is troublesome
and difficult to ash in the dry way at a low temperature.
On heating liver with diluted nitric acid, it is fully dispersed in a short time with evolution
of a comparatively small quantity of oxides of nitrogen; this reaction produces a yellow
solution that contains a fair amount of suspended matter representing the fat originally
present. On further heating, this solution evaporates or boils until, at a certain concentration,
itbbecomes syrupy and begins to evolve a gas, at first colourless, later brown. On further
heating the mass blackens suddenly and leaves a black “char.” If this residue is again
treated with nitric acid, brown fumes are evlolved, the mixture swells into a froth and
ultimately leaves a hard black residue. Successive repetitions of this treatment result in
the production of a white residue that does not. char on heating.
The procedure as described above was found to be a very simple and satisfactory method
of “wet ashing” liver tissue without removal of the fat, and was also effective in destroying
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March, 1964] M A T E R I A L F O R THE I)ETERMINATION OF TRACE M E T A L S 1 39

the organic part of the fat extracted from liver and other animal materials; but when the
method was applied to a number of other substances (e.g., blood, milk, gelatin or bone),
it was found that the "char" often ignited in the nitric acid vapour. This did not occur
with liver, egg or casein. Although the deflagration was not violent, high temperatures
were obviously reached in the mass so that there was danger of loss of trace metals. For-
tunately it was found possible to keep the reaction under control, and to obviate the danger
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of deflagration, by the addition of a little sulphuric acid in the first evaporation. I t should
be empliasised that this sulphuric acid is added to decrease the reactivity of the "char"
and that its purpose is quite different from that of the large quantity of sulphuric acid used
in the nitric - sulphuric acid procedure with the intention of breaking down the organic
matter, assisting the action of the oxidising acids and raising the temperature of the mixture.
The added acid is in fact all lost before the end of the procedure.
Although this method was originally devised to deal with animal tissues that are
particularly resistant to ordinary wet-oxidation methods, it was found subsequently that
it could be applied also to practically all organic materials of biological origin (see Table I,
p. 141).
MECHANISMOF THE METHOD
Some information concerning the mechanism of the method can be gained by simplifying
the conditions, and it was found possible to destroy the organic matter of liver by either
of two methods: (1) continuous heating in nitric acid vapour at 260" C ; or (2) alternate
evaporation with nitric acid at a low temperature in an open vessel, and heating the residue
(without nitric acid) at a temperature of 300"to 350" C on a hot-plate. Both of these processes
come into play under the conditions used in the proposed method; the residue prepared from
the nitric acid solution of the organic matter is intermittently and alternately treated with
nitric acid and heated in a large covered beaker on a hot-plate at a temperature of about
320" C. The temperature gradient of the solid frothy mass on the bottom of the beaker is
very great, and favourable conditions for the reaction occur only locally and temporarily.
On the addition of fresh nitric acid, the partly oxidised residue is dissolved and re-distributed
on the bottom of the beaker. The number of repetitions required depends inter alia on the
nature of the material in question, and on the quantity taken or the relation of this quantity
to the surface area of the bottom of the beaker. In general, from four to ten repetitions of
the procedure are sufficient to give a white ash free from organic matter (see Table I).
When the sample consists of animal tissues, this final residue is not completely soluble
in dilute acids, but contains a proportion of insoluble matter that is apparently a meta-
phosphate. In order to get the whole of the residue into solution, it may either be digested
with sodium hydroxide before acid is added, or be evaporated with dilute sulphuric acid
until fumes are evolved.
ADVANTAGESOF THE PROPOSED METHOD
The proposed method offers considerable advantages over others that have been put
forward. In the first place the temperature of the material at no time exceeds about 350" C,
so that the possibility of loss of trace elements (except mercury) by volatilisation is greatly
reduced. The time taken depends largely on the amount and nature of the organic matter,
more especially as in practice there is a limit to the surface area of the bottom of the vessel
that can be used. I n general, the organic matter in 5 g of dried material can be completely
destroyed within 2 hours, and little attention is required during this time; larger quantities
may require longer. It should be remembered that all the other methods commonly used
are liable to become tedious and troublesome when applied to large quantities , especially
of animal tissues, and that with this method the only attention required during this period
is the intermittent addition of fresh acid.
Compared with the nitric - sulphuric acid procedure, the proposed procedure is effective
with a number of materials for which the former fails to effect complete destruction of the
organic matter (e.g., with liver). Further, the classical method of wet combustion requires a
large quantity of sulphuric acid, of which a great deal is lost in the course of the procedure,
and the residue must generally be neutralised, as removal of any considerable quantity of
sulphuric acid by evaporation is impracticable. Hence the trace elements must be determined
in the presence of very large quantities of sodium sulphate, and a blank determination must
be made on the acids and on the alkali used. For this blank determination an amount of
sulphuric and nitric acids equal to the amount used for the wet combustion should be
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140 MIDDLETON AND STUCKEY: THE PREPARATION OF BIOLOGICAL [Vol. 79

evaporated down; this also is a troublesome procedure owing to the creeping of hot sulphuric
acid.
With the proposed procedure, on the other hand, the mineral components are obtained
by themselves with no large quantities of added reagents, which allows the widest possible
choice of further treatment, while at the same time they have not been exposed to a high
temperature, which is sometimes liable to convei-t traces of metals to insoluble compounds.
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A blank experiment can easily be performed after evaporation of the same amount of nitric
acid, and if necessary the nitric acid-the only reagent used in quantity-can be purified
before use by distillation, although nitric acid with low trace-metal content is now generally
available.
It is not suggested that this method is necessarily superior to others for all materials.
The superiority is most evident when the method is applied to nitrogenous animal products
and fatty materials. Cocoa powder, for example, can be ashed easily in the ordinary way,
but it is difficult to carry out this procedure in such a way as to avoid ignition of the material,
and the fat content makes wet combustion troublesome. Further, no attempt has been
made to test its suitability for the determination of particular trace elements, with the
exception of cobalt and lead; it is hoped that details of this work will be published shortly.
The application to the determination of arsenic has not been examined, while for tin it would
be necessary to use special means to get the tin into solution after the completion of the
procedure. It does not appear that the determination of metals such as copper, iron, zinc
manganese or aluminium would offer any difficulty.
METHOD
The procedure is carried out in a suitable vessel of Pyrex glass with a large flat area
of base, and which can be covered by a clock-glass. Lipless beakers, which can be closed
more effectively by a clock-glass, are preferable. For quantities of material equivalent to
about 5 g of dry matter, we have used 1 and 2-litre beakers, but the same size of vessel can
be used for much larger quantities, e.g., 50 g of dried material, although, of course, the
procedure then takes longer.
The hot-plate should be maintained at a temperature of about 310" to 350°C. The
temperature can be measured by physical methods or by placing small quantities of substances
of suitable melting-point on the hot-plate. Among available substances are : potassium
acetate, anhydrous, 292" C ; sodium nitrate, 307" C ; 2-amino-anthraquinone, 310" C ; sodium
acetate, anhydrous, 324" C ; potassium nitrate, 334" C; theobromine, 337" C ; acridone,
364" C. Organic compounds under such conditions often sublime rapidly before melting;
by pressing a small pellet of compound on the hot-plate with a spatula, melting before sub-
limation can usually be observed. The inorganic compounds listed are, however, usually
preferable, although melting points of both organic and inorganic substances in this range
vary considerably with purity and moisture content ; the actual melting point should therefore
be checked before use. If an electrical hot-plate working within this range is not available,
or if it is considered undesirable to operate one under fume-cupboard conditions, a satisfactory
alternative is a thick (&inch) sheet of iron heated over a gas ring or burner.
General directions for carrying out the procedure are as follows.
PROCEDURE-
Place about 5 g of material in the beaker with 10 to 20 ml of water and 5 to 10 ml of
nitric acid, spgr. 1.4, containing 6; per cent. of sulphuric acid, and heat gently until the sample
has dispersed. Evaporate the mixture, adding if necessary a few drops of capryl alcohol to
control frothing, and take care to avoid loss by spurting. Leave uncovered on the hot-plate
until there is little or no further visible change; this stage is generally reached in about
16 minutes. Allow the beaker to cool, add sufficient nitric acid, sp.gr. 1.4, to moisten the
residue, cover with the clock-glass, and leave the beaker on the hot-plate for at least 15minutes
after the residue has become dry. Allow the beaker to cool and repeat the operation as often
as necessary. When the residue is whitish with d.ark patches, use fuming nitric acid in place
of ordinary nitric acid, and continue until a white residue is left.
With some materials, such as animal tissues, in order to dissolve the residue it is necessary
to add about 1 to 3 ml of concentrated sulphuric acid and 20 rnl of water and to heat on the
hot-plate until the residue fumes strongly. Then allow the mixture to cool, and dilute it
with water.
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March, 1954) MATERIAL FOR THE DETERMINATIOS OF TRACE METALS 141

POSSIBLE
MODIFICATIONS IN THE PROCEDURE-
The procedure detailed above is a general one and can be applied directly to most
materials, but where possible it is advisable to carry out a preliminary test with the material
in question, as this may indicate that certain modifications are desirable or advantageous,
as follows-
1. Dilution of the acid in the first stage is used to moderate the reaction and facilitate
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the dispersion of the material. When the material does not react with diluted acid,
there is no point in diluting it.
2. Sulphuric acid is added to the first portion of nitric acid only, to moderate the
reactivity of the “char” and thereby to prevent ignition. It can often be omitted, as
it only slows down the procedure.
3. For substances containing fats and fatty materials, the first heating (uncovered)
is used to remove a large proportion of the volatile matter.
4. Any volatile matter (e.g., from fat) that condenses on the clock-glass should be
removed with cellulose wadding at intervals.
5. The change to fuming acid should be made at the earliest convenient occasion
at which it is possible to do so without causing incandescence; this depends on the

TABLEI
APPLICATIONSOF THE METHOD
Number of repetitions
Size of Total volume
Weight beaker acid, acid, of nitric acid,
Material taken, used, sp.gr. 1.4 fuming sp.gr. 1.4, used,
g litres ml
Animul tissues and products-
Fresh liver . . .. .. 100 2 7 4 85

Dried liver

Bone
Brain (rabbit)
..
..
..
.. ..
.. . .
..
i
..
50
5
5
10
5
2
2
1
2
1
15

3
3
5
0
3
3
2
120
13
18
20
10 2 2 1 16
.. ..
Shrimps (fresh)
Goose quills . . ,. ..
{. . 50
5
2
2
2
4
2
2
65
27
Mouse (whole) .. .. .. 25 2 7 3 35
Cochineal insects . . .. .. 10 2 4 2 30
Desiccated hog’s stomach . . .. 5 1 9 1 35
Milk . . .. .. .. .. 50 ml 2
Wool fat .. .. .. .. 5 1 6 6 19
Rat faeces . . . . .. .. 10 1 5 3 25
Vegctable tissues and products-
Apple (whole) .. .. .. 30 2 4 3 30
Cocoa powder .. .. .. 10 2 5 2 40
Pepper, white .. .. .. 5 2 2 4 25
Arachis oil . . .. .. .. 10 ml 2 2 4 35
Treacle .. .. .. .. 10 2 6 5 35
Shellac .. .. .. .. 5 1 5 3 20
Balsam of Tolu .. .. .. 12 2 3 6 30
Sundvy-
Asphalt .. .. .. .. 5 2 1 6 16

material being destroyed. If the weaker acid does not appear to be having any
appreciable effect, it is probably safe to change to the fuming acid.
6. AS an alternative to the procedure described above for getting an insoluble
final residue into solution, it may be digested for an hour on the water-bath with 5 per
cent. sodium hydroxide, after which it should dissolve readily on the addition of hydro-
chloric acid. If phosphorus is absent, the final residue may dissolve directly in dilute
acid.
7. The total amount of acid used should be noted so that allowance can be made
for any trace metals in it. The amount used at each stage need only be sufficient to
damp the residue, and therefore decreases continually as the bulk of residue decreases.
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142 MIDDLETON AND STUCKEY [Vol. 79

8. In general, no metallic contamination is to be anticipated from the use of Pyrex-
glass vessels, but silica could be used if desired. It may be necessary to distil the nitric
acid in silica before use in order to remove traces of metals.
APPLICATIONOF THE METHOD
Table I shows the great variety of substances that have been treated successfully, and
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gives an indication of the quantity of acid used and the number of repetitions of the treatment.
As the conditions cannot be exactly standardised, these results only show what has been
achieved. It would, for example, be possible to effect an appreciable reduction in the amount
of acid used in many instances.
In addition to the materials tabulated, the following have been successfully treated :
egg (whole), kipper, dried blood, extract of ox bile, liver fat, casein, gelatin, peptone, keratin,
lecithin, sodium nucleate, beeswax, yeast (dried), cork, wood, curry-powder and rubber.
ADVANTAGES
OF THE PROPOSED METHOD
The advantages claimed for the proposed method are as follows-
The maximum temperature attained does not exceed 330” C.
The mineral constituents are obtained in an acid solution with only small
quantities of added reagents.
Nitric acid is the only reagent used in quantity. This can easily be purified bJ-
distillation if necessary.
A blank determination can easily be made by evaporating an equivalent quantity
of the nitric acid used.
Atmospheric contamination is reduced -to a minimum.
There is no evaporation of sulphuric acid.
The method is especially effective for all animal tissues and proteins, which are
in general the most difficult to deal with by other methods.
The authors wish to thank the Directors of the British Drug Houses Limited, for
permission to publish these results.
REFEREXCE
Middleton, G., and Stuckey, R. E., Analyst, 1053, 78, 532.
1.
THE BRITISHDRUGHOUSESLIMITED
GRAHAMSTREET, LONDON, N.l April 271’h, 1953

ADDENDA:September (1953) issue, p. 536. Add to Part I of the foregoing paper, under the
heading “DIRECT OXIDATION WITH PERCHLOF:IC ACID,” at the end of the paragraph, the
following sentence-
Perchloric acid has been used extensively for the oxidation of organic matter by
Kahane,125who also deals with the associated dangers.126
Ibid., p. 542. Add to the References-
125. Kahane, E., IXeme’Congrks International de Chimie Pure et Appliqu6, Madrid, 1934, 6 , 40.
126. __ , 17eme Congrks de Chimie Industrielle, Paris, 1937, Communications, p. 476c.