Chapter XV: Part II: I.--Definition of Poison (2)

From the nature of the case, we can only distinguish in the poisoned fly dulness or vivacity of movement, loss of power in walking on smooth surfaces, irritation of the integument, disorderly movements of the limbs, protrusion of the fleshy proboscis, and paralysis, whether of legs or wings. My experiments were chiefly made by smearing the extracts or neutral solutions of poisons on the head of the fly. In this way some of it is invariably taken into the system, partly by direct absorption, and partly by the insect’s efforts to free itself from the foreign substance, in which it uses its legs and proboscis. For the symptoms witnessed after the application of saponin, digitalin, and aconitine, the reader is referred to the articles on those substances.

In poisoning by sausages, bad meat, curarine, and in obscure cases generally, in the present state of science, experiments on living animals are absolutely necessary. In this, and in this way only, in very many instances, can the expert prove the presence of zymotic, or show the absence of chemical poison.

The Vivisection Act, however, effectually precludes the use of life-tests in England save in licensed institutions. Hence the “methods” of applying life-tests described in former editions will be omitted.

§ 29. =Effect of poisons on the heart of Cold-blooded Animals.=--The
Vivisection Act does not, however, interfere with the use of certain
living tests, such, for instance, as the testing of the action of
poisons upon the recently extirpated hearts of cold-blooded animals.

The heart of the frog, of the turtle, of the tortoise, and of the
shark will beat regularly for a long time after removal from the
body, if supplied with a regular stream of nutrient fluid. The
fluids used for this purpose are the blood of the herbivora diluted
with common salt solution, or a serum albumin solution, or a 2 per
cent. solution of gum arabic in which red blood corpuscles are
suspended. The simplest apparatus to use is that known as
“Williams’.” Williams’ apparatus consists of two glass bulbs (see
diagram), the one, P, containing nutrient fluid to which a known
quantity of the poison has been added; the other, N, containing the
same fluid but to which no poison has been added; these bulbs are
connected by caoutchouc tubing to a three-way tube, T, and each
piece of caoutchouc tubing has a pressure screw clip, V¹ and V; the
three-way tube is connected with a wider tube containing a valve
float, F, which gives free passage of fluid in one direction only,
that is, in the direction of the arrow; this last wide tube is
connected with a Y piece of tubing, which again is connected with
the aorta of the heart under examination, the other leg of the Y
tube is connected with another wide tube, X, having a float valve,
F²: the float containing a drop of mercury and permitting (like the
float valve F) passage in one direction only of fluid, it is obvious
that if the clip communicating with N is opened and the clip
communicating with P is closed, the normal fluid will circulate
alone through the heart; if, on the other hand, the P clip is open
and the N clip closed, the poisoned blood will alone feed the heart.
It is also clear that by raising or depressing the bulbs, the
circulating fluid can be delivered at any pressure, high or low.
Should a bubble of air get into the tubes, it can be got rid of by
removing the cork at S and bringing the fluid up to the level of the
top of the aperture. The observation is made by first ascertaining
the number and character of the beats when the normal fluid is
circulating, and then afterwards when the normal is replaced by the
poisoned fluid. A simpler but less accurate process is to pith two
frogs, excise their respective hearts, and place the hearts in
watch-glasses containing either serum or a solution of common salt
(strength 0·75 per cent.); to the one heart is now added a solution
of the poison under examination, and the difference in the behaviour
and character of the beats noted.

The phenomena to be specially looked for are the following:--

1. The heart at the height of the poisoning is arrested in diastole.
2. The heart at the height of the poisoning is arrested in systole.

=Arrest in diastole.=--The arrest may be preceded by the
contractions becoming weaker and weaker, or after the so-called
heart peristalsis; or it may be preceded by a condition in which the
auricle shows a different frequency to the ventricle.

The final diastole may be the diastole of paralysis or the diastole
of irritation.

The diastole of irritation is produced by a stimulus of the
inhibitory ganglia, and only occurs after poisoning by the muscarine
group of poisons. This condition may be recognised by the fact that
contraction may be excited by mechanical and electrical stimuli or
by the application of atropine solution; the latter paralyses the
inhibitory nervous centres, and therefore sets the mechanism going
again. The diastole of paralysis is the most frequent form of death.
It may readily be distinguished from the muscarine diastole; for, in
muscarine diastole, the heart is full of blood and larger than
normal; but in the paralytic form the heart is not fully extended,
besides which, although, if normal blood replace that which is
poisoned, the beats may be restored for a short time, the response
is incomplete, and the end is the same; besides which, atropine does
not restore the beats. The diastole of paralysis may depend on
paralysis of the so-called excito-motor ganglia (as with iodal), or
from paralysis of the muscular structure (as with copper).

§ 30. =The effect of poisons on the iris.=--Several poisons affect
the pupil, causing either contraction or dilatation. The most
suitable animal is the cat; the pupil of the cat readily showing
either state.

=Toxic myosis, or toxic contraction of the pupil.=--There are two
forms of toxic myosis, one of which is central in its origin. In
this form, should the poison be applied to the eye itself, no marked
contraction follows; the poison must be swallowed or injected
subcutaneously to produce an effect. The contraction remains until
death.

The contraction in such a case is considered to be due to a
paralysis of the dilatation centre; it is a “_myosis paralytica
centralis_;” the best example of this is the contraction of the
pupil caused by morphine.

In the second case the poison, whether applied direct to the eye or
entering the circulation by subcutaneous injection, contracts the
pupil; the contraction persists if the eye is extirpated, but in all
cases the contraction may be changed into dilatation by the use of
atropine. An example of this kind of myosis is the action of
muscarine. It is dependent on the stimulation of the ends of the
nerves which contract the pupil, especially the ends of the _nervus
oculomotorius_ supplying the sphincter iridis; this form of myosis
is called _myosis spastica periphera_. A variety of this form is the
_myosis spastica muscularis_, depending on stimulation of the musc.
sphincter iridis, seen in poisoning by physostigmine. This causes
strong contraction of the pupil when locally applied; the
contraction is not influenced by small local applications of
atropine, but it may be changed to dilatation by high doses.
Subcutaneous injection of small doses of physostigmine does not
alter the pupil, but large poisonous doses contracts the pupil in a
marked manner.

=Toxic mydriasis, or toxic dilatation of the pupil.=--The following
varieties are to be noticed:--

1. Toxic doses taken by the mouth or given by subcutaneous injection
give rise to strong dilatation; this vanishes before death, giving
place to moderate contraction. This form is due to stimulation of
the dilatation centre, later passing into paralysis. An example is
found in the action of aconite.

2. After subcutaneous or local application, a dilatation neutralised
by physostigmine in moderate doses. This is characteristic of
β-tetrahydronaphthylamine.

3. After subcutaneous injection, or if applied locally in very small
doses, dilatation occurs persisting to death. Large doses of
physostigmine neutralise the dilatation, but it is not influenced by
muscarine or pilocarpine: this form is characteristic of atropine,
and it has been called _mydriasis paralytica periphera_.

The heart at the height of the poisoning stops in systole.

=2. Arrest in systole.=--The systole preceding the arrest is far
stronger than normal, the ventricle often contracting up into a
little lump. Contraction of this kind is specially to be seen in
poisoning by digitalis. In poisoning by digitalis the ventricle is
arrested before the auricle; in muscarine poisoning the auricle
stops before the ventricle. If the reservoir of Williams’ apparatus
is raised so as to increase the pressure within the ventricle the
beat may be restored for a time, to again cease.

A frog’s heart under the influence of any poison may be finally
divided into pieces so as to ascertain if any parts still contract;
the significance of this is, that the particular ganglion supplying
that portion of the heart has not been affected: the chief ganglia
to be looked for are Remak’s, on the boundary of the sinus and
auricle; Ludwig’s, on the auricle and the septum of the auricle;
Bidder’s, on the atrioventricular border, especially in the valves;
and Dogiel’s ganglion, between the muscular fibres. According to
Dogiel, poisons acting like muscarine affect every portion of the
heart, and atropine restores the contractile power of every portion.

VI.--General Method of Procedure in Searching for Poison.

§ 31. Mineral substances, or liquids containing only inorganic matters, can cause no possible difficulty to any one who is practised in analytical investigation; but the substances which exercise the skill of the expert are organic fluids or solids.

The first thing to be done is to note accurately the manner in which the samples have been packed, whether the seals have been tampered with, whether the vessels or wrappers themselves are likely to have contaminated the articles sent; and then to make a very careful observation of the appearance, smell, colour, and reaction of the matters, not forgetting to take the weight, if solid--the volume, if liquid. All these are obvious precautions, requiring no particular directions.

If the object of research is the stomach and its contents, the contents should be carefully transferred to a tall conical glass; the organ cut open, spread out on a sheet of glass, and examined minutely by a lens, picking out any suspicious-looking substance for closer observation. The mucous membrane should now be well cleansed by the aid of a wash-bottle, and if there is any necessity for destroying the stomach, it may be essential in important cases to have it photographed. The washings having been added to the contents of the stomach, the sediment is separated and submitted to inspection, for it must be remembered that, irrespective of the discovery of poison, a knowledge of the nature of the food last eaten by the deceased may be of extreme value.

If the death has really taken place from disease, and not from poison, or if it has been caused by poison, and yet no definite hint of the particular poison can be obtained either by the symptoms or by the attendant circumstances, the analyst has the difficult task of endeavouring to initiate a process of analysis which will be likely to discover any poison in the animal, vegetable, or mineral kingdom. For this purpose I have devised the following process, which differs from those that have hitherto been published mainly in the prominence given to operations in a high vacuum, and the utilisation of biological experiment as a matter of routine. Taking one of the most difficult cases that can occur--viz., one in which a small quantity only of an organic solid or fluid is available--the best method of procedure is the following:--

A small portion is reserved and examined microscopically, and, if thought desirable, submitted to various “cultivation” experiments. The greater portion is at once examined for volatile matters, and having been placed in a strong flask, and, if neutral or alkaline, feebly acidulated with tartaric acid, connected with a second or receiving flask by glass tubing and caoutchouc corks. The caoutchouc cork of the receiving flask has a double perforation, so as to be able, by a second bit of angle tubing, to be connected with the mercury-pump described in the author’s work on “Foods,” the figure of which is here repeated (see the accompanying figure). With a good water-pump having a sufficient length of fall-tube, a vacuum may be also obtained that for practical purposes is as efficient as one caused by mercury; if the fall-tube delivers outside the laboratory over a drain, no offensive odour is experienced when dealing with putrid, stinking liquids. A vacuum having been obtained, and the receiving-flask surrounded with ice, a distillate for preliminary testing may be generally got without the action of any external heat; but if this is too slow, the flask containing the substances or liquid under examination may be gently heated by a water-bath--water, volatile oils, a variety of volatile substances, such as prussic acid, hydrochloric acid, phosphorus, &c., if present, will distil over. It will be well to free in this way the substance, as much as possible, from volatile matters and water. When no more will come over, the distillate may be carefully examined by redistillation and the various appropriate tests.

The next step is to dry the sample thoroughly. This is best effected also in a vacuum by the use of the same apparatus, only this time the receiving-flask is to be half filled with strong sulphuric acid. By now applying very gentle heat to the first flask, and cooling the sulphuric acid receiver, even such substances as the liver in twenty-four hours may be obtained dry enough to powder.

Having by these means obtained a nearly dry friable mass, it is reduced to a coarse powder, and extracted with petroleum ether; the extraction may be effected either in a special apparatus (as, for example, in a large “Soxhlet”), or in a beaker placed in the “Ether recovery apparatus” (see fig.), which is adapted to an upright condenser. The petroleum extract is evaporated and leaves the fatty matter, possibly contaminated by traces of any alkaloid which the substance may have contained; for, although most alkaloids are insoluble in petroleum ether, yet they are taken up in small quantities by oils and fats, and are extracted with the fat by petroleum ether. It is hence necessary always to examine the petroleum extract by shaking it up with water, slightly acidulated with sulphuric acid, which will extract from the fat any trace of alkaloid, and will permit the discovery of such alkaloids by the ordinary “group reagents.”

The substance now being freed for the most part from water and from fat, is digested in the cold with absolute alcohol for some hours; the alcohol is filtered off, and allowed to evaporate spontaneously, or, if speed is an object, it may be distilled _in vacuo_. The treatment is next with hot alcohol of 90 per cent., and, after filtering, the dry residue is exhausted with ether. The ether and alcohol, having been driven off, leave extracts which may be dissolved in water and tested, both chemically and biologically, for alkaloids, glucosides, and organic acids. It must also be remembered that there are a few metallic compounds (as, for example, corrosive sublimate) which are soluble in alcohol and ethereal solvents, and must not be overlooked.

The residue, after being thus acted upon successively by petroleum, by alcohol, and by ether, is both water-free and fat-free, and also devoid of all organic poisonous bases and principles, and it only remains to treat it for metals. For this purpose, it is placed in a retort, and distilled once or twice to dryness with a known quantity of strong, pure hydrochloric acid.

If arsenic, in the form of arsenious acid, were present, it would distil over as a trichloride, and be detected in the distillate; by raising the heat, the organic matter is carbonised, and most of it destroyed. The distillate is saturated with hydric sulphide, and any precipitate separated and examined. The residue in the retort will contain the fixed metals, such as zinc, copper, lead, &c. It is treated with dilute hydrochloric acid, filtered, the filtrate saturated with SH₂ and any precipitate collected. The filtrate is now treated with sufficient sodic acetate to replace the hydric chloride, again saturated with SH₂ and any precipitate collected and tested for _zinc_, _nickel_, and _cobalt_. By this treatment, viz.:--

1. Distillation in a vacuum at a low temperature,
2. Collecting the volatile products,
3. Dehydrating the organic substances,
4. Dissolving out from the dry mass fatty matters and alkaloids,
glucosides, &c., by ethereal and alcoholic solvents,
5. Destroying organic matter and searching for metals,

--a very fair and complete analysis may be made from a small amount of material. The process is, however, somewhat faulty in reference to phosphorus, and also to oxalic acid and the oxalates; these poisons, if suspected, should be specially searched for in the manner to be more particularly described in the sections treating of them. In most cases, there is sufficient material to allow of division into three parts--one for organic poisons generally, one for inorganic, and a third for reserve in case of accident. When such is the case, although, for organic principles, the process of vacuum distillation just described still holds good, it will be very much the most convenient way not to use that portion for metals, but to operate on the portion reserved for the inorganic poisons as follows by destruction of the organic matter.

The destruction of organic matter through simple distillation by means of pure hydrochloric acid is at least equal to that by sulphuric acid, chlorate of potash, and the carbonisation methods. The object of the chemist not being to dissolve every fragment of cellular tissue, muscle, and tendon, but simply all mineral ingredients, the less organic matter which goes into solution the better. That hydrochloric acid would fail to dissolve sulphate of baryta and sulphate of lead, and that sulphide of arsenic is also almost insoluble in the acid, is no objection to the process recommended, for it is always open to the analyst to treat the residue specially for these substances. The sulphides precipitated by hydric sulphide from an acid solution are--arsenic, antimony, tin, cadmium, lead, bismuth, mercury, copper, and silver. Those not precipitated are--iron, manganese, zinc, nickel, and cobalt.

As a rule, one poison alone is present; so that if there should be a sulphide, it will belong only exceptionally to more than one metal.

The colour of the precipitate from hydric sulphide is either yellowish or black. The yellow and orange precipitates are sulphur, sulphides of arsenic, antimony, tin, and cadmium. In pure solutions they may be almost distinguished by their different hues, but in solutions contaminated by a little organic matter the colours may not be distinctive. The sulphide of arsenic is of a pale yellow colour; and if the very improbable circumstance should happen that arsenic, antimony, and cadmium occur in the same solution, the sulphide of arsenic may be first separated by ammonia, and the sulphide of antimony by sulphide of sodium, leaving cadmic sulphide insoluble in both processes.

The black precipitates are--lead, bismuth, mercury, copper, and silver. The black sulphide is freed from arsenic, if present, by ammonia, and digested with dilute nitric acid, which will dissolve all the sulphides, save those of mercury and tin, so that if a complete solution is obtained (sulphur flocks excepted), it is evident that both these substances are absent. The presence of copper is betrayed by the blue colour of the nitric acid solution, and through its special reactions; lead, by the deep yellow precipitate which falls by the addition of chromate of potash and acetate of soda to the solution; bismuth, through a white precipitate on dilution with water. If the nitric acid leaves a black insoluble residue, this is probably sulphide of mercury, and should be treated with concentrated hydrochloric acid to separate flocks of sulphur, evaporated to dryness, again dissolved, and tested for mercury by iodide of potassium, copper foil, &c., as described in the article on _Mercury_. Zinc, nickel, and cobalt are likewise tested for in the filtrate as described in the respective articles on these metals.

AUTENRIETH’S GENERAL PROCESS.

§ 32. A general method of procedure has been published by W.
Autenrieth.[43]

[43] _Kurze Anleitung zur Auffindung der Gifte_, Freiburg, 1892.

He divides poisonous substances, for the purposes of separation and
detection, into three classes:--

I. Poisons capable of distillation from an acid aqueous solution.
II. Organic substances which are not capable of distillation from
acid solutions.
III. Metallic poisons.

Where possible, the fluid or solids submitted to the research are
divided into four equal parts, one of the parts to be kept in
reserve in case of accident or as a control; one of the remaining
three parts to be distilled; a second to be investigated for organic
substances; and a third for metals. After the extraction of organic
substances from part No. II. the residue may be added to No. III.
for the purpose of search after metals; and, if the total quantity
is small, the whole of the process may be conducted without
division.

I. SUBSTANCES SEPARATED BY DISTILLATION.

The substances are placed in a capacious flask, diluted if necessary
with water to the consistence of a thin soup, and tartaric acid
added to distinct acid reaction, and distilled.

In this way phosphorus, prussic acid, carbolic acid, chloroform,
chloral hydrate, nitrobenzol, aniline,[44] and alcohol may be
separated and identified by the reactions given in the sections of
this work describing those substances.

[44] Aniline is a weak base, so that, although a solution be acid, some of the aniline distils over on heating.

II. ORGANIC POISONS NOT VOLATILE IN ACID SOLUTION.

Part No. II. is mixed with double its volume of absolute alcohol,
tartaric acid added to distinct acid reaction and placed in a flask
connected with an inverted Liebig’s condenser; it is then warmed for
15 to 20 minutes on the water-bath. After cooling, the mixture is
filtered, the residue well washed with alcohol and evaporated to a
thin syrup in a porcelain dish over the water-bath. The dish is then
allowed to cool and digested with 100 c.c. of water; fat and
resinous matters separate, the watery solution is filtered through
Swedish paper previously moistened: if the fluid filtrate is clear
it may be at once shaken up with ether, but if not clear, and
especially if it is more or less slimy, it is evaporated again on
the water-bath to the consistence of an extract: the extract treated
with 60 to 80 c.c. of absolute alcohol (which precipitates mucus and
dextrin-like substances), the alcohol evaporated off and the residue
taken up with from 60 to 80 c.c. of distilled water; it is then
shaken up with ether, as in Dragendorff’s process, and such
substances as digitalin, picric acid, salicylic acid, antipyrin and
others separated in this way and identified.

After this treatment with ether, and the separation of the ether
extract, the watery solution is strongly alkalised with caustic soda
and shaken up again with ether, which dissolves almost every
alkaloid save morphine and apomorphine; the ethereal extract is
separated and any alkaloid left identified by suitable tests.

The aqueous solution, now deprived of substances soluble in ether
both from acid and from solutions made alkaline by soda, is now
investigated for morphine and apomorphine; the apomorphine being
separated by first acidifying a portion of the alkaline solution
with hydrochloric acid, then alkalising with ammonia and shaking out
with ether. The morphine is separated from the same solution by
shaking out with warm chloroform.[45]

[45] Hot amyl alcohol would be better (see “Morphine”).

III. METALS.

The substances are placed in a porcelain dish and diluted with a
sufficient quantity of water to form a thin soup and 20 to 30 c.c.
of pure hydrochloric acid added; the dish is placed on the
water-bath and 2 grms. of potassic chlorate added. The contents are
stirred from time to time, and successive quantities of potassic
chlorate are again added, until the contents are coloured yellow.
The heating is continued, with, if necessary, the addition of more
acid, until all smell of chlorine has ceased. If there is
considerable excess of acid, this is to be evaporated away by
diluting with a little water and continuing to heat on the
water-bath. The dish with its contents is cooled, a little water
added, and the fluid is then filtered.

The metals remaining on the filter are:--

Silver chloride,
Lead sulphate,
Barium sulphate;

in the filtrate will be all the other metals.

The filtrate is put in a flask and heated to from 60 to 80 degrees
and submitted to a slow stream of hydric sulphide gas; when the
fluid is saturated with the gas, the flask is securely corked and
allowed to rest for twelve hours; at the end of that time the fluid
is filtered and the filter washed with water saturated with hydric
sulphide.

The still moist sulphides remaining on the filter are treated with
yellow ammonium sulphide containing some free ammonia and washed
with sulphide of ammonium water. Now remaining on the filter, if
present at all, will be:--

Mercury sulphide,
Lead sulphide,
Copper sulphide,
Cadmium sulphide;

in the filtrate may be:--

Arsenic sulphide,
Antimony sulphide,
Tin sulphide,

and there may also be a small portion of copper sulphide, because
the latter is somewhat soluble in a considerable quantity of
ammonium sulphide.

The filtrate from the original hydric sulphide precipitate will
contain, if present, the sulphides of zinc and chromium in solution.

INVESTIGATION OF THE SULPHIDES SOLUBLE IN AMMONIUM SULPHIDE, VIZ., ARSENIC, ANTIMONY, TIN.

The ammonium sulphide solution is evaporated to dryness in a
porcelain dish, strong nitric acid added and again dried. To this
residue a little strong caustic soda solution is added, and then it
is intimately mixed with three times its weight of a mixture
composed of 2 of potassic nitrate to 1 of dry sodium hydrate. This
is now cast, bit by bit, into a red-hot porcelain crucible. The
whole is heated until it has melted into a colourless fluid.

Presuming the original mass contained arsenic, antimony, and tin,
the melt contains sodic arseniate, sodic pyro-antimonate, sodic
stannate, and tin oxide; it may also contain a trace of copper
oxide.

The melt is cooled, dissolved in a little water, and sodium
bicarbonate added so as to change any caustic soda remaining into
carbonate, and to decompose the small amount of sodic stannate; the
liquid is then filtered.

The filtrate will contain the arsenic as sodic arseniate; while on
the filter there will be pyro-antimonate of soda, tin oxide, and,
possibly, a little copper oxide.

The recognition of these substances now is not difficult (see the
separate articles on _Antimony_, _Tin_, _Zinc_, _Arsenic_,
_Copper_).

INVESTIGATION OF THE SULPHIDES INSOLUBLE IN SULPHIDE OF AMMONIUM, VIZ., MERCURY, LEAD, COPPER, CADMIUM.

If the precipitate is contaminated with organic matter, it is
treated with hydrochloric acid and potassic chlorate in the manner
already described, p. 51.

Afterwards it is once more saturated with hydric sulphide, the
precipitate is collected on a filter, well washed, and the sulphides
treated with moderately concentrated nitric acid (1 vol. nitric
acid, 2 vols. water). The sulphides are best treated with this
solvent on the filter; all the sulphides mentioned, save mercury
sulphide, dissolve and pass into the filtrate. This mercury sulphide
may be dissolved by nitro-muriatic acid, the solution evaporated to
dryness, the residue dissolved in water acidified with hydrochloric
acid and tested for mercury (see “Mercury”).

The filtrate containing, it may be, nitrates of lead, copper and
cadmium is evaporated nearly to dryness and taken up in a very
little water. The lead is separated as sulphate by the addition of
dilute sulphuric acid.

The filtered solution, freed from lead, is treated with ammonia to
alkaline reaction; if copper be present, a blue colour is produced,
and this may be confirmed by other tests (see “Copper”). To detect
cadmium in the presence of copper, potassic cyanide is added to the
blue liquid until complete decolorisation, and the liquid treated
with SH₂; if cadmium be present, it is thrown down as a yellow
sulphide, while potassic cupro-cyanide remains in solution.

SEARCH FOR ZINC AND CHROMIUM.

The filtrate from the hydric sulphide precipitate is divided into
two parts; the one half is used in the search for zinc, the other
half is used for chromium.

=Search for Zinc.=--The liquid is alkalised with ammonia and then
ammonium sulphide is added. There will always be a precipitate of a
dark colour; the precipitate will contain earthy phosphates, iron
and, in some cases, manganese. The liquid with the precipitate is
treated with acetic acid to strong acid reaction and allowed to
stand for several hours. The portion of the precipitate remaining
undissolved is collected on a filter, washed, dried and heated to
redness in a porcelain crucible. The residue thus heated is cooled
and dissolved in a little dilute sulphuric acid. To the acid
solution ammonia is added, and any precipitate formed is treated
with acetic acid; should the precipitate not completely dissolve,
phosphate of iron is present; this is filtered off, and if SH₂ be
added to the filtrate, white zinc sulphide will come down (see
“Zinc”).

=Search for Chromium.=--The second part of the SH₂ filtrate is
evaporated to a thin extract, mixed with double its weight of sodic
nitrate, dried and cast, little by little, into a red-hot porcelain
crucible. When the whole is fully melted, the crucible is removed
from the flame, cooled, and the mass dissolved in water and
filtered. Any chromium present will now be in solution in the easily
recognised form of potassic chromate (see “Chromium”).

INVESTIGATION OF THE RESIDUE (p. 52) AFTER THE TREATMENT OF THE ORIGINAL SUBSTANCE WITH HYDROCHLORIC ACID AND POTASSIC CHLORATE FOR PRESENCE OF SILVER CHLORIDE, LEAD AND BARIUM SULPHATES.

The residue is dried and intimately mixed with three times its
weight of a mixture containing 2 parts of sodic nitrate and 1 part
of sodium hydrate, This is added, little by little, into a red-hot
porcelain crucible. The melted mass is cooled, dissolved in a little
water, a current of CO₂ passed through the solution to convert any
caustic soda into carbonate, and the solution boiled. The result
will be an insoluble portion consisting of carbonates of lead and
baryta, and of metallic silver. The mixture is filtered; the
insoluble residue on the filter is warmed for some time with dilute
nitric acid; the solution of nitrates of silver, lead and barium are
concentrated on the water-bath nearly to dryness so as to get rid of
any excess of acid, and the nitrates dissolved in water; then the
silver is precipitated by hydrochloric acid, the lead by SH₂, and
the barium by sulphuric acid.

VII.--The Spectroscope as an aid to the Identification of certain Poisons.

§ 33. The spectra of many of the metals, of phosphine, of arsine and of several other inorganic substances are characteristic and easily obtained.

It is, however, from the employment of the _micro-spectroscope_ that the toxicologist is likely to get most assistance.

Oscar Brasch[46] has within the last few years studied spectroscopy in relation to the alkaloids and organic poisons. Some of these, when mixed with Froehde’s reagent, or with sulphuric acid, or with sulphuric acid and potassic dichromate, or with nitric acid, give characteristic colours, and the resulting solutions, when examined by a spectroscope, for the most part show absorption bands; these bands may, occasionally, assist materially in the identification of a poison. By far the best apparatus is a micro-spectroscope of the Sorby and Browning type, to which is added an apparatus for measuring the position on a scale of the lines and bands. Seibert and Kraft of Wetzlar make an excellent instrument, in which a small bright triangle is projected on the spectrum; this can be moved by a screw, so that the apex may be brought exactly in the centre of any line or band, and its position read on an outside scale. The first thing to be done with such an instrument is to determine the position on the scale of the chief Fraunhofer lines or of the more characteristic lines of the alkalies and alkaline earths,[47] the wave lengths of which are accurately known. If, now, the scale divisions are set out as abscissæ, and the wave lengths in millionths of a millimetre are made the ordinates of a diagram, and an equable curve plotted out, as fully explained in the author’s work on “Foods,” it is easy to convert the numbers on the scale into wave lengths, and so make the readings applicable to any spectroscope. For the purpose of graphical illustration the curve method is convenient, and is adopted in the preceding diagrams, all taken from Oscar Brasch’s monograph. Where the curve is highest there the absorption band is thickest; where the curve is lowest there the band is weak. The fluid to be examined is simply placed in a watch-glass, the watch-glass resting on the microscope stand.

[46] _Ueber Verwendbarkeit der Spectroscopie zur Unterscheidung der Farbenreactionen der Gifte im Interesse der forensischen Chemie_, Dorpat, 1890.

[47] The alkalies and earths used for this purpose, with their wave lengths, are as follows: KCl, a line in the red λ 770, in the violet λ 404. Lithium chloride, red line, 670·5; sodium chloride, yellow, 589; strontium chloride, line in the blue, 461. It is also useful to measure the green line of thallium chloride = 535.

NOTES TO CURVES INDICATING ABSORPTION BANDS.

1. Strychnine, treated with sulphuric acid and potassic dichromate
(violet).
2. Brucine, treated with potassic nitrate and sulphuric acid (clear
red).
3. Quebrachine, treated with vanadium sulphate (dark blue).
4. Quinine, Vogel’s reaction (red).
5. Caffein, Murexid reaction (violet-red).
6. Dephinoidin, Froehde’s reagent (cherry-red).
7. Veratrine, treated with sulphuric acid (straw-yellow).
8. „ „ „ (cherry-red).
9. „ „ „ (carmine-red).
10. Veratrine, Furfurol reaction (blue-violet).
11. Sabadillin, treated with sulphuric acid (red).
12. Veratroidine, „ „ (brown-red).
13. Jervine, Furfurol reaction (blue).
14. Sabadine, „ „ (blue).
15. Sabadine, treated with sulphuric acid (cherry-red).
16. Physostigmine, „ „ (grass-green).
17. Morphine, treated with Froehde’s reagent and sugar (dark-green).
18. Narcotine, treated with a mixture of sulphuric acid and nitric
acid (30 drops of sulphuric to 1 drop of nitric), (red).
19. Codeine, treated with Froehde’s reagent and sugar (dark violet).
20. Papaverine, treated with Froehde’s reagent (green-blue).
21. Sanguinarin, „ „ (violet-red).
22. Chelidonin, „ sulphate of vanadium (dark green).
23. Solanin, „ sulphuric acid and allowed to stand 4
hours (brown-red).
24. Digitalin, „ Erdmann’s reagent (red).
25. Aniline, „ sulphuric acid and potassic dichromate
(blue).]

The wave lengths corresponding to the numbers on the scale in the diagram are as follows:--

W.L.
0 732
1 656
2 589·2
3 549·8
4 510·2
5 480·0
6 458
7 438

Examination of Blood, or of Blood-Stains.

§ 34. Spots, supposed to be blood--whether on linen, walls, or weapons--should, in any important case, be photographed before any chemical or microscopical examination is undertaken. Blood-spots, according to the nature of the material to which they are adherent, have certain naked eye peculiarities--_e.g._, blood on fabrics, if dry, has at first a clear carmine-red colour, and part of it soaks into the tissue. If, however, the tissue has been worn some time, or was originally soiled, either from perspiration, grease, or filth, the colour may not be obvious or very distinguishable from other stains; nevertheless, the stains always impart a certain stiffness, as from starch, to the tissue. If the blood has fallen on such substances as wood or metal, the spot is black, has a bright glistening surface, and, if observed by a lens, exhibits radiating fissures and a sort of pattern, which, according to some, is peculiar to each species; so that a skilled observer might identify occasionally, from the pattern alone, the animal whence the blood was derived. The blood is dry and brittle, and can often be detached, or a splinter of it, as it were, obtained. The edges of the splinter, if submitted to transmitted light, are observed to be red. Blood upon iron is frequently very intimately adherent; this is specially the case if the stain is upon rusty iron, for hæmatin forms a compound with iron oxide. Blood may also have to be recovered from water in which soiled articles have been washed, or from walls, or from the soil, &c. In such cases the spot is scraped off from walls, plaster, or masonry, with as little of the foreign matters as may be. It is also possible to obtain the colouring-matter of blood from its solution in water, and present it for farther examination in a concentrated form, by the use of certain precipitating agents (see p. 61).

In the following scheme for the examination of blood-stains, it is presumed that only a few spots of blood, or, in any case, a small quantity, is at the analyst’s disposal.

(1) The dried spot is submitted to the action of a cold saturated solution of borax. This medium (recommended by Dragendorff)[48] does certainly dissolve out of linen and cloth blood-colouring matter with great facility. The best way to steep the spots in the solution is to scrape the spot off the fabric, and to digest it in about a cubic centimetre of the borax solution, which must not exceed 40°; the coloured solution may be placed in a little glass cell, with parallel walls, ·5 centimetre broad, and ·1 deep, and submitted to spectroscopic examination, either by the ordinary spectroscope or by the micro-spectroscope; if the latter is used, a very minute quantity can be examined, even a single drop. In order to interpret the results of this examination properly, it will be necessary to be intimately acquainted with the spectroscopic appearances of both ancient and fresh blood.

[48] _Untersuchungen von Blutspuren_ in Maschka’s _Handbuch_, Bd. i. Halfband 2.

§ 35. =Spectroscopic Appearances of Blood.=--If defibrinated blood[49] be diluted with water until it contains about ·01 per cent. of oxyhæmoglobin, and be examined by a spectroscope, the layer of liquid being 1 centimetre thick, a single absorption band between the wave lengths 583 and 575 is observed, and, under favourable circumstances, there is also to be seen a very weak band from 550 to 532. With solutions so dilute as this, there is no absorption at either the violet or the red end of the spectrum. A solution containing ·09 per cent. of oxyhæmoglobin shows very little absorption in the red end, but the violet end is dark up to about the wave length 428. Two absorption bands may now be distinctly seen. A solution containing ·37 per cent. of oxyhæmoglobin shows absorption of the red end to about W.L. 720; the violet is entirely, the blue partly, absorbed to about 453. The bands are considerably broader, but the centre of the bands occupies the same relative position. A solution containing as much as ·8 per cent. of oxyhæmoglobin is very dark; the two bands have amalgamated, the red end of the spectrum is absorbed nearly up to Fraunhofer’s line a; the green is just visible between W.L. 498 and 518. Venous blood, or arterial blood, which has been treated with reducing agents, such, for example, as an alkaline sulphide, gives the spectrum of reduced hæmoglobin. If the solution is equivalent to about ·2 per cent., a single broad band, with the edges very little defined, is seen to occupy the space between W.L. 595 and 538, the band being darkest about 550; both ends of the spectrum are more absorbed than by a solution of oxyhæmoglobin of the same strength. In the blood of persons or animals poisoned with hydric sulphide--to the spectrum of reduced hæmoglobin, there is added a weak absorption band in the red, with its centre nearly corresponding with the Fraunhofer line C. Blood which has been exposed to carbon oxide has a distinct spectrum, due, it would seem, to a special combination of this gas with hæmoglobin; in other words, instead of oxygen, the oxygen of oxyhæmoglobin has been displaced by carbon oxide, and crystals of carbon oxide-hæmoglobin, isomorphous with those of oxyhæmoglobin, may be obtained by suitable treatment. The spectrum of carbon oxide-hæmoglobin, however, differs so little from that of normal blood, that it is only comparison with the ordinary spectrum, or careful measurements, which will enable any person, not very familiar with the different spectra of blood, to detect it; with careful and painstaking observation the two spectra are seen to be distinct. The difference between the carbon oxide and the normal spectrum essentially consists in a slight moving of the bands nearer to E. According to the measurements of Gamgee, the band α of CO-hæmoglobin has its centre approximately at W.L. 572, and the band β has for its centre W.L. from 534 to 538, according to concentration. If a small quantity of an ammoniacal solution of ferrous tartrate or citrate be added to blood containing carbon oxide, the bands do not wholly fade, but persist more or less distinctly; whereas, if the same solution is added to bright red normal blood, the two bands vanish instantly and coalesce to form the spectrum of reduced hæmoglobin. When either a solution of hæmoglobin or blood is exposed to the air for some time, it loses its bright red colour, becomes brownish-red, and presents an acid reaction. On examining the spectrum, the two bands have become faint, or quite extinct; but there is a new band, the centre of which (according to Gamgee) occupies W.L. 632, but (according to Preyer) 634. In solutions of a certain strength, four bands may be seen, but in a strong solution only one. This change in the spectrum is due to the passing of the hæmoglobin into _methæmoglobin_, which may be considered as an intermediate stage of decomposition, prior to the breaking up of the hæmoglobin into hæmatin and proteids.

[49] In this brief notice of the spectroscopic appearances of the blood, the measurements in wave lengths are, for the most part, after Gamgee.--_Text-Book of Physiological Chemistry_, London, 1880.

A spectrum very similar to that of methæmoglobin is obtained by treating ancient blood-stains with acetic acid--viz., the spectrum of _acid hæmatin_, but the band is nearer to its centre, according to Gamgee, corresponding to W.L. 640 (according to Preyer, 656·6). The portion of the band is a little different in alkaline solution, the centre being about 592. Hæmatin is one of the bodies into which hæmoglobin splits up by the addition of such agents as strong acetic acid, or by the decomposing influence of exposure; the view most generally accepted being that the colouring-matter of the blood is hæmatin in combination with one or more albuminoid bodies. The hæmatin obtained by treating blood with acetic acid may be dissolved out by ether, and the ethereal solution then exhibits a remarkable distinctive spectrum. Hence, in the spectroscopic examination of blood, or solutions of blood, for medico-legal purposes, if the blood is fresh, the spectrum likely to be seen is either that of oxyhæmoglobin or hæmoglobin; but, if the blood-stain is not recent, then the spectrum of either hæmatin or methæmoglobin.

The colouring-matter of cochineal, to which alum, potassic carbonate, and tartrate have been added, gives a spectrum very similar to that of blood (see “Foods,” p. 82); but this is only the case when the solution is fresh. The colour is at once discharged by chlorine, while the colour of blood, although changed in hue, remains. The colouring-matter of certain red feathers, purpurin-sulphuric acid, and a few other reds, have some similarity to either the hæmatin or the hæmoglobin spectrum, but the bands do not strictly coincide; besides, no one would trust to a single test, and none of the colouring-matters other than blood yield hæmatin.

The blood in CO poisoning has also other characteristics. It is of a peculiar florid vermilion colour, a colour that is very persistent, lasting for days and even weeks.

Normal blood mixed with 30 per cent. potash solution forms _greenish_ streaky clots, while blood charged with CO forms red streaky clots.

Normal blood diluted to 50 times its volume of water, and then treated successively with yellow ammonium sulphide in the proportion of 2 to 25 c.c. of blood, followed by three drops of acetic acid, gives a grey colour, while CO blood remains bright red. CO blood shaken with 4 times its volume of lead acetate remains red, but normal blood becomes brown.[50]

[50] M. Rubner, _Arch. Hyg._, x. 397.

Solutions of platinum chloride or zinc chloride give a bright red colour with CO blood; normal blood is coloured brown or very dark brown.

Phospho-molybdic acid or 5 per cent. phenol gives a carmine-coloured precipitate with CO blood, but a reddish-brown precipitate with normal blood (sensitive to 16 per cent.).

A mixture of 2 c.c. of dilute acetic acid and 15 c.c. of 20 per cent. potassic ferrocyanide solution added to 10 c.c. of CO blood produces an intense bright red; normal blood becomes dark brown.

Four parts of CO blood, diluted with 4 parts of water and shaken with 3 vols. of 1 per cent. tannin solution, become at first bright red with a bluish tinge, and remain so persistently. Normal blood, on the other hand, also strikes bright red at first, but with a yellowish tinge; at the end of 1 hour it becomes brownish, and finally in 24 hours grey. This is stated to be delicate enough to detect 0·0023 per cent. in air.

If blood be diluted with 40 times its volume of water, and 5 drops of phenylhydrazin solution be added, CO blood strikes rose-red; normal blood grey-violet.[51]

[51] A. Welzel, _Centr. med. Wiss._, xxvii. 732-734.

Gustave Piotrowski[52] has experimented on the length of time blood retains CO. The blood of dogs poisoned by this agent was kept in flasks, and then the gas pumped out by means of a mercury pump on the following dates:--

[52] _Compt. Rend. Soc. de Biol._, v. 433.

Date. Content of gas in CO.
Jan. 12, 1892, 24·7 per cent.
„ 20, „ 23·5 „
„ 28, „ 22·2 „
Feb. 8, „ 20·3 „
„ 16, „ 15·5 „
„ 26, „ 10·2 „
March 3, „ 6·3 „
„ 14, „ 4·6 „
„ 22, „ 1·2 „

The same dog was buried on the 12th of January, and exhumed on March 28th, and the gas pumped out from some of the blood; this gas gave 11·7 per cent. of CO; hence it is clear that burial preserves CO blood from change to a certain extent.

N. Gréhant[53] treated the poisoned blood of a dog with acetic acid, and found it evolved 14·4 c.c. CO from 100 c.c. of blood.

[53] _Compt. Rend._, cvi. 289.

Stevenson, in one of the cases detailed at p. 67, found the blood in the right auricle to contain 0·03 per cent. by weight of CO.

(2) =Preparation of Hæmatin Crystals=--(Teichmann’s crystals).--A portion of the borax solution is diluted with 5 or 6 parts of water, and one or more drops of a 5 or 6 per cent. solution of zinc acetate added, so long as a brownish-coloured precipitate is thrown down. The precipitate is filtered off by means of a miniature filter, and then removed on to a watch-glass. The precipitate may now be dissolved in 1 or 2 c.c. of acetic acid, and examined by the spectroscope it will show the spectrum of hæmatin. A minute crystal of sodic chloride being then added to the acetic acid solution, it is allowed to evaporate to dryness at the ordinary temperature, and crystals of hæmatin hydrochlorate result. There are other methods of obtaining the crystals. When a drop of fresh blood is simply boiled with glacial acetic acid, on evaporation, prismatic crystals are obtained.

Hæmatin is insoluble in water, alcohol, chloroform, and in cold dilute acetic and hydrochloric acids. It may, however, be dissolved in an alcoholic solution of potassic carbonate, in solutions of the caustic alkalies, and in boiling acetic and hydrochloric acids. Hoppe-Seyler ascribes to the crystals the formula C₆₈H₇₀N₈Fe₂O₁₀2HCl. Thudichum considers that the pure crystals contain no chlorine, and are therefore those of hæmatin. It is the resistance of the hæmatin to decomposition and to ordinary solvents that renders it possible to identify a certain stain to be that of blood, after long periods of time. Dr. Tidy seems to have been able to obtain blood reactions from a stain which was supposed to be 100 years old. The crystals are of a dark-red colour, and present themselves in three forms, of which that of the rhombic prism is the most common (see fig.). But crystals like _b_, having six sides, also occur, and also crystals similar to _c_.

If the spot under examination has been scraped off an iron implement the hæmatin is not so easily extracted, but Dragendorff states that borax solution at 50° dissolves it, and separates it from the iron. Felletar has also extracted blood in combination with iron rust, by means of warm solution of caustic potash, and, after neutralisation with acetic acid, has precipitated the hæmin by means of tannin, and obtained from the tannin precipitate, by means of acetic acid, Teichmann’s crystals. A little of the rust may also be placed in a test tube, powdered ammonium chloride added, also a little strong ammonia, and after a time filtered; a small quantity of the filtrate is placed on a slide with a crystal of sodium chloride and evaporated at a gentle heat, then glacial acetic acid added and allowed to cool; in this way hæmin crystals have been obtained from a crowbar fifty days after having been blood-stained.[54]

[54] _Brit. Med. Journ._, Feb. 17, 1894.

(3) =Guaiacum Test.=--This test depends upon the fact that a solution of hæmoglobin develops a beautiful blue colour, if brought into contact with fresh tincture of guaiacum and peroxide of hydrogen. The simplest way to obtain this reaction is to moisten the suspected stain with distilled water; after allowing sufficient time for the water to dissolve out some of the blood constituents, moisten a bit of filter-paper with the weak solution thus obtained; drop on to the moist space a single drop of tincture of guaiacum which has been prepared by digesting the inner portions of guaiacum resin in alcohol, and which has been already tested on known blood, so as to ascertain that it is really good and efficient for the purpose; and, lastly, a few drops of peroxide of hydrogen. Dragendorff uses his borax solution, and, after a little dilution with water, adds the tincture and then Heunefeld’s turpentine solution, which is composed of equal parts of absolute alcohol, chloroform, and French turpentine, to which one part of acetic acid has been added. The chloroform separates, and, if blood was present, is of a blue colour.

§ 36. To prove by chemical and physical methods that a certain stain is that of blood, is often only one step in the inquiry, the next question being whether the blood is that of man or of animals. The blood-corpuscles of man are larger than those of any domestic animal inhabiting Europe. The diameter of the average red blood-corpuscle is about the 1/126 of a millimetre, or 7·9 µ.[55] The corpuscles of man and of mammals, generally speaking, are round, those of birds and reptiles oval, so that there can be no confusion between man and birds, fishes or reptiles; if the corpuscles are circular in shape the blood will be that of a mammal. By careful measurements, Dr. Richardson, of Pennsylvania, affirms that it is quite possible to distinguish human blood from that of all common animals. He maintains, and it is true, that, by using very high magnifying powers and taking much trouble, an expert can satisfactorily identify human blood, if he has some half-dozen drops of blood from different animals--such as the sheep, goat, horse, dog, cat, &c., all fresh at hand for comparison, and _if the human blood is normal_. However, when we come to the blood of persons suffering from disease, there are changes in the diameter and even the form of the corpuscles which much complicate the matter; while, in blood-stains of any age, the blood-corpuscles, even with the most artfully-contrived solvent, are so distorted in shape that he would be a bold man who should venture on any definite conclusion as to whether the blood was certainly human, more especially if he had to give evidence in a criminal case.

[55] 1/3200 of an inch; the Greek letter µ is the micro-millimetre, or 1000th of a millimetre, ·00003937 inch.

Neumann affirms that the pattern which the fibrin or coagulum of the blood forms is peculiar to each animal, and Dr. Day, of Geelong, has independently confirmed his researches: this very interesting observation perhaps has not received the attention it merits.