On Some of the Tests for Quinine. 
BY 
Theodore G. Wormeey, M.D. ON SOME OF THE TESTS FOR QUININE. 
Theodore G. WormeEy, M.D. 
The recognition of quinine by chemical tests, when present in 
notable quantity, is usually a matter of great ease. Under certain 
conditions, however, as in its extraction from complex organic mix- 
tures or from the tissues, it may be recovered only in minute quan- 
tity, and its presence may then not be so readily determined, at 
least by purely chemical tests. Among the strictly chemical tests 
for this substance, the thalleioquin reaction as it is termed, is one of 
the most characteristic, and at the same time one of the most delicate. 
This test, however, requires caution in its application, since it may 
give a negative result even in the presence of quinine in consider- 
able quantity. 
I. THALLEIOQUIN TEST. 
This test was first proposed by Prof. M. Andre, of Mentz, in 1835,1 
and consists in the production of a bright emerald green coloration 
when a solution of a salt of quinine is treated with chlorine water, 
followed by the addition of a little aqua ammonia. M. Andre 
observed that this order of the application of the reagents was 
necessary for the production of the green color. 
R. Brands, in 1839,2 more fully examined this reaction and found 
that the production of the green color was dependent upon the 
presence of the substances employed in certain proportions, other- 
wise it did not appear. He found that the chlorine caused a 
1 See this Journal, viii, 208. 
2 See Ibid., xi, 36. 2 
decomposition of the quinine which varied with the amount of 
chlorine employed, and thus determined whether a green precipitate 
would be produced, or the liquid simply acquire a green color or 
become yellow. 
The green precipitate thus produced was found to have a bitter 
taste similar to that of quinine; was insoluble in cold water and 
only sparingly soluble in boiling water; insoluble in ether, but 
readily soluble in alcohol, and readily soluble in diluted acids form- 
ing red solutions, from which it was reprecipitated of a green color 
on neutralizing the solution with ammonia. 
In 1853, A. Vogel1 proposed to modify this test by treating the 
quinine solution after addition of chlorine water, with a solution of 
potassium ferrocyanide and then adding a few drops of ammonia, 
when the mixture would assume a deep red color, the green color 
not appearing. 
Professor Fliickiger, in 1861, confirmed the observation of Vogel 
and found that the same red coloration might be produced by sub- 
stituting potassium ferricyanide for the ferrocyanide. 
More recently, Chas. F. Zeller2 has examined this test in regard 
to the production of a green coloration under the action of chlorine 
and ammonia, and confirmed the observations of Brands, namely: 
that the results were influenced by the relative proportion of the 
substances employed. 
It being thus shown that this test was much influenced by the 
relative proportions of quinine and chlorine present, the following 
investigations were made for the purpose of more definitely deter- 
mining the range within which the green coloration would manifest 
itself in different quantities of the same solution, and in solutions 
of different degrees of dilution. 
The quinine was employed in solution both as sulphate and hydro- 
chloride, the i-iooth solutions being prepared by dissolving, by the 
aid of just sufficient of the diluted acid to effect solution, 1 gramme 
of the pure alkaloid in 100 c.c. of water. The more dilute solutions 
were prepared from the i-iooth solutions, by the required dilution 
with water. 
The chlorine water consisted of a saturated aqueous solution of 
1 This Journal, 1853, 516. 
i See Ibid., 1880, 385. 3 
the washed gas. This solution, for uniformity of results, should be 
freshly prepared and preserved from the action of light. When this 
reagent was used in the form of drops, these were delivered from a 
pipette delivering on an average twenty-five drops per cubic centi- 
metre. The ammonia employed had a density of -950. 
A. 1-1 ooth solution of quinine. 
1. 5 c.c. of this solution (= 50 mgras. quinine) yields with: 
(«) 01 c.c. of chlorine water: a colorless solution which, 
on the addition of a drop of ammonia, yields a copious 
white precipitate (of quinine), and after a little time, the 
mixture acquires a rose-red color; on the addition of a 
second drop of ammonia, the mixture becomes colorless, 
then slowly assumes a greenish hue which becomes well 
marked. 
(b) 0-5 c.c. of chlorine water, followed by a drop of 
ammonia, yields a copious white precipitate, and the mix- 
ture quickly assumes a green color, then becomes dark 
rose-red and finally green. 
(c) i-oc.c. chlorine water and a drop of ammonia: an 
immediate green color, which quickly changes to dark red, 
then to purple or blue. 
2. I c.c. of a 1-1 OOth solution (= 10 mgms. quinine) yields with: 
(a) One drop chlorine water and then a drop of 
ammonia: a copious white precipitate, which quickly 
assumes a green color. 
(b) 0-5 c.c. chlorine water and a drop of ammonia: an 
intense green coloration. 
(c) i-o c.c. chlorine water and one drop of ammonia: 
a bright green coloration, which becomes darker in color 
and finally nearly black. 
3. o-1 c.c. of a i-iooth solution (= 1 mgm. quinine) in a very 
small, narrow test tube, yields with : 
(a) One drop of chlorine water and a drop of ammonia: 
a copious green precipitate. 
{b) Two drops chlorine water and a drop of ammonia: 
a bright green coloration. 
(c) Three drops chlorine water and a drop of ammonia: 
no precipitate, but the mixture immediately acquires a 
bright green color, which quickly darkens. 4 
B. i-ioooth solution of quinipe. 
1. 5 c.c. of the solution (= 5 mgms. quinine) yields with: 
(a) One drop chlorine water and a drop of ammonia: 
a white precipitate (quinine) which quickly assumes a 
greenish hue, slowly changing to well-marked bluish green. 
(1b) o-1 c.c. chlorine water and one drop of ammonia : the 
precipitate quickly acquires a strong, bluish-green color. 
(c) o-5 c.c. chlorine water and a drop of ammonia: an 
intense, emerald-green coloration. 
(d) 10 c.c. chlorine water and one drop of ammonia, no 
precipitate, but a strong yellow coloration, which soon 
changes to deep purple. 
(e) 2 0 c.c. chlorine water and a drop of ammonia, a 
colorless mixture; on further addition of ammonia, a dirty- 
brown coloration may be produced. 
2. 1 c.c. of a i-ioooth quinine solution, yields with one drop of 
ammonia, after addition of: 
(a) One drop chlorine water : a green turbid mixture. 
(ft) o-1 c.c. chlorine water: a bright green coloration. 
(c) o-5 c.c. of the chlorine reagent: a pale green color- 
ation. 
(d) po c.c. chlorine water : a slightly yellow mixture. 
3. O'1 c.c. of a i-ioooth solution, under like conditions, yields 
with: 
(a) One drop chlorine water : a very bright green color- 
ation. 
(b) Two drops of the chlorine reagent: a less intense 
green coloration. 
C. l-5000th solution of quinine. 
I. 5 cc. of the solution = (t mg. quinine) yields with a drop of 
ammonia, after addition of: 
(a) One drop chlorine water : a colorless solution, which, 
after a time, may acquire a greenish hue. 
(b) 0-2 c.c. chlorine water : a fine, emerald green solu- 
tion. 
(c) 0-5 c.c. chlorine water : a well-marked green color- 
ation, quickly discharged on shaking the mixture. 5 
(d) of chlorine reagent: no coloration, but, after 
a time, the mixture assumes a yellow hue. 
2. 1 c.c. of a i-5000th solution, with one drop of ammonia 
after adding: 
(a) One drop chlorine water : a fine, green coloration, 
changing to bluish-green. 
(b) 01 c.c. chlorine water: a pale green coloration. 
(c) 05 c.c chlorine reagent: a light green color, quickly 
changing to a faintly yellow. 
3. o-i c.c. of 1-5000th solution, with one drop ammonia, after 
adding: 
(a) One drop chlorine water : a well-marked green color- 
ation. 
([b) Two drops chlorine water: only a faintly yellowish 
color. 
D. i-iooooth solution of quinine. 
1. 5 c.c. of the solution (= 0-5 mg. quinine) yields with one drop 
of ammonia, after addition of: 
(a) One drop chlorine water: .the mixture slowly 
acquires a greenish hue. 
(b) Two drops chlorine water : a strongly marked green 
coloration. 
(c) 0 2 c.c. of chlorine water : a colorless mixture.* 
2. 1 c.c. of a i-iooooth solution, yields with: 
(a) One drop chlorine water and one drop ammonia : 
a marked green coloration. 
(b) Two drops chlorine water and a drop of ammonia : 
the mixture may present a greenish hue, which quickly 
changes to yellow. 
From o-i c.c. of a i-iooooth quinine solution, a greenish colora- 
tion may be obtained by employing a drop of a much-diluted solu- 
tion of chlorine. 
From 5 c.c. of a i-20000th solution of quinine, no green colora- 
tion was obtained, even on employing a diluted solution of 
chlorine. 
In 1872, Prof. Fliickiger1 proposed to substitute bromine for 
chlorine in this test, and stated that its reaction was much more 
xNeues Jahr. f. Pharm., 1872. 139. 6 
delicate than that of chlorine, since under it a green coloration 
might be obtained from a i-20000th solution of quinine, whereas, 
chlorine had its limit in about a i-5000th solution. 
In the following examinations a saturated aqueous solution of 
bromine was employed, it being prepared by agitating excess of 
bromine with water, and, after subsidence, decanting the clear, 
highly colored solution. The reagent should be freshly prepared, 
since it may, within twenty-four hours, especially if exposed to 
light, undergo a marked change. A saturated aqueous solution of 
the reagent contains practically i per cent, by volume, or 3 per cent, 
by weight of bromine. 
A. i-iooth solution of quinine. 
1. 5 c.c. of the solution yields with : 
(«) One drop of bromine water : a copious yellow pre- 
cipitate which quickly dissolves to a colorless solution, 
the addition of a drop of ammonia causes a white precipi- 
tate (quinine), which, after a time, acquires a bluish hue, 
then a bluish green color. 
(<$) Three drops bromine water then one drop ammonia, 
yield a precipitate which soon assumes a greenish blue color. 
(<;) 0-5 c.c. bromine water followed by one drop ammonia : 
. the mixture quickly becomes bluish green, which increases 
its intensity. 
(d) i-o c.c. bromine water causes a slightly yellow color- 
ation ; the addition of oi c.c. ammonia produces a white 
precipitate which quickly assumes a bright green color, 
which may change to purple. 
The exact coloration produced in the above solutions depends 
somewhat upon the manner in which the reagents mix with the 
quinine solution. The best results are obtained by dropping the 
reagents into the quinine solution without agitation. 
2. i c.c. of the quinine solution yields with one drop of ammonia 
after addition of: 
(#) One drop bromine water : a white precipitate which 
soon assumes a green color. 
(d) 0'2 c.c. bromine water: quickly a bright green solution. 
(c) 05 c.c. bromine water: a purple precipitate, which, 
after a time, assumes a strong green color. 7 
3. o-i c.c. of a i-iooth quinine solution, yields with one drop of 
bromine water and a drop of ammonia, a white precipitate which 
quickly assumes a green color. 
1. 5 c.c. of the solution yields with one drop of ammonia, after 
adding : 
(a) One drop bromine water: a white precipitate which 
soon assumes a bluish-green color. 
(b) o* 1 c.c. bromine : an immediate bright green color- 
ation. 
(c) 0-3 cc. bromine water alone causes a yellow colora- 
tion, which on addition of the ammonia, is changed to 
deep purple, and this may slowly change to very dark 
green. 
2. 1 c.c. of the quinine solution with one drop of the bromine 
water and one drop of ammonia will yield a bright green coloration. 
Under the action of a slightly larger quantity of the bromine re- 
agent, only a purple color will appear, or the mixture will remain 
colorless. 
3. 01 c.c. of the quinine solution with a minute drop of the brom- 
ine water and one drop of ammonia, will yield a green coloration ; 
but if a full drop of the bromine reagent be employed, a colorless 
mixture will result. With a diluted solution of the bromine water 
(1:4), a fine green coloration may be obtained. 
B. i-ioooth solution of quinine. 
1. 5 c.c. of the solution with one or two drops of the bromine 
water and one drop ammonia, yields a good green coloration. With 
a slightly larger amount of the bromine reagent, the mixture 
remains colorless. 
2. i c.c. of the quinine solution with one drop of bromine water 
and one drop ammonia, will after a time acquire a green coloration. 
If two drops of the bromine water be employed, the mixture remains 
permanently colorless. 
3. o-i c.c. yields with a drop of the bromine reagent, a deep 
yellow color, which is quickly discharged to a permanently colorless 
solution by a drop of ammonia. Under the action of a drop of 
diluted bromine water (1:9), a bright green coloration may bv 
obtained. 
C. i-5QOOth solution of quinine. D. i-iooooth solution of quinine. 
1. 5 c.c. of the solution with : 
(#) One drop bromine water yields a colorless solution 
which on addition of a drop of ammonia quickly assumes 
a green color; this becomes deep bright green, which 
remains unchanged for many hours. 
('b) Two drops of bromine water causes a yellow color, 
which is immediately discharged by a drop of ammonia, 
and the mixture remains colorless. 
2. i c.c. of the quinine solution fails to yield a green coloration 
unless the bromine reagent be diluted. 
3. From 01 c.c. of the quinine solution, no green coloration was 
obtained, even with the diluted bromine reagent. 
E. i-2000Oth quinine solution. 
5 c.c. of this solution yields no green coloration with the undi- 
luted bromine reagent; but under a dilution of 1:2 a well-marked 
green color may be obtained. 
From the foregoing it is obvious that the production of a green 
color under the action of this test depends upon the presence of the 
quinine and bromine in proportion contained within rather narrow 
limits; otherwise the green coloration will not manifest itself. 
Should it be desired to apply this test to only a small and limited 
quantity of a suspected solution, it would be necessary, or at least 
advisable, to first ascertain under what conditions a similar volume 
of solution of quinine of known strength would give a positive reac- 
tion with a given quantity of the bromine reagent, after proper dilu- 
tion of the latter if necessary. 
If an aqueous solution of chlorine, instead of bromine, be employed 
in the thalleioquin test, a positive reaction will manifest itself 
through a greater range than when bromine is employed, the result 
being less readily affected by excess or deficiency of the reagent. 
In regard to the production of a red coloration, by treating the 
quinine solution, after addition of bromine or chlorine, and before 
adding the ammonia, with potassium ferrocyanide solution, as advised 
by Vogel, the results were less satisfactory than the test without the 
use of the potassium salt. With solutions containing more than 
-1000th of the alkaloid, a green or red or other coloration may be 9 
developed, the result depending upon the relative proportions of 
the reagents employed. 
On treating 5 c.c. of a i-ioooth quinine solution with one drop of 
bromine water followed by one drop of potassium ferrocyanide solu- 
tion (1:12) and a drop of ammonia, the result is about the same as 
without the presence of the potassium salt; that is, a white precipi- 
tate soon becoming green is produced. But, if under these condi- 
tions o-2 c.c. of the potassium solution be employed, a portion of the 
mixture may be red and a portion green in color. 
5 c.c. of a r-50ooth solution of the alkaloid under the action of 
one drop each of the reagents as above, yields a fine green colora- 
tion. If, however, to this quantity of the quinine solution one drop 
of bromine water be added and then 0-5 c.c. of the ferrocyanide solu- 
tion, the mixture, without the addition of ammonia, immediately 
assumes a deep red color. If a drop of ammonia be now added, 
the red color quickly changes to a beautiful purple. 
5 c.c. of a i-iooooth quinine solution with one drop each of the 
reagents, yields a fine red-purple coloration which soon changes to 
green. 
5 c.c. of a i-20000th solution, under like conditions, yields a fine 
red coloration, which quickly fades to a light yellow color. 
2. HERAPATHITE TEST. 
This test was first proposed by Dr. Herapath, in 1852, and con- 
sists in the formation of quinine iodosulphate or Herapathite, as it 
has been termed. This compound may be obtained by treating a 
solution of quinine in a mixture of strong acetic acid and alcohol, 
with an alcoholic solution of iodine. In a little time the iodosuL 
phate separates out in the form of characteristic plates and rosette 
groups of crystals. By reflected light, the crystals are of a dark 
green color; under transmitted light they are dichroic and strongly 
polarize light. 
The reagents may be prepared as follows : (a) Thirty volumes 
of strong acetic acid are mixed with ten volumes of strong alcohol 
and one volume of diluted sulphuric acid (1:10). (b) One part of 
iodine is dissolved in about twenty parts by weight of alcohol. 
To apply the test, a drop of the quinine solution is evaporated to 
dryness, and the residue treated with a drop of the first-mentioned 
solution; a minute drop, or sufficient to color the liquid brownish- 10 
yellow, of the alcoholic solution of iodine is then added. Very 
soon portions of the mixture will present a dark green deposit, 
which when examined by a moderate power (75 diam.) of the 
microscope will be found to consist of the crystals in question. The 
formation of these crystals is perfectly characteristic of quinine. 
The residue from one drop of a i-iooth solution of quinine, in 
the form of sulphate, when treated with a drop of the acetic acid 
mixture and then sufficient of the iodine solution to impart a strong 
color, will yield innumerable crystals of the iodosulphate compound. 
A 1-500th solution residue will generally yield an abundant 
deposit of the crystals. 
The residue from a drop of a i-ioooth solution, if simply mois- 
tened with the acetic acid liquid and then with a minute drop of 
the iodine solution, may yield satisfactory results; but it requires a 
nice adjustment of the reagents to obtain satisfactory results from 
this quantity of the alkaloid. 
3. FLUORESCENCE. 
One of the most striking properties and at the same time the 
most delicate reaction of quinine at present known, is the 
fluorescence of solutions of its oxysalts, especially the sulphate. 
Normal solutions of the hydrochloride, hydrobromide, hydriodide 
and hydrocyanide present little or no fluorescence. A solution of 
the hydrochloride containing a limited excess of hydrochloric acid, 
may, as pointed out by Prof. R. A. Witthaus,1 present, a well 
marked or even strong fluorescence, especially in dilute solutions; 
but this is permanently destroyed by a slightly larger quantity of 
the free acid. 
* When in solution as quinine normal acid sulphate, without excess 
of acid, and examined by ordinary reflected light in volumes of 50 
to 100 c.c. the blue fluorescence is very intense in the i-iooth, 
i-ioooth, and i-iooooth solutions, and very well marked in a 
i-5oc>ooth solution; but it is not apparent in a i-ioooooth solution, 
and is only faintly marked even under a cone of condensed sunlight. 
If 1 c.c. of the acid sulphate solution, placed in a small test tube, 
be examined it will present as follows: 
(1) 1-1 ooth solution, a very strong blue fluorescence in reflected 
light, which becomes intense in condensed sunlight. 
1 Researches Loomis Laboratory, 1892, 91. 11 
(2) i-ioooth solution, a strongly marked fluorescence in reflected 
light; only feebly marked in direct sunlight; but intense in a cone 
of sunlight. 
(3) i-iooooth solution, a just perceptible fluorescence in reflected 
light; appears colorless in sunlight; intensely fluorescent in a cone 
of sunlight. 
(4) i-50000th solution exhibits no marked fluorescence in either 
reflected or in direct sunlight, and only a faint fluorescence under a 
cone of sunlight. On the addition of a drop of dilute sulphuric 
acid, the solution presents a quite well-marked fluorescence in con- 
densed sunlight. 
(5) i-ioooooth solution in condensed sunlight presents only a 
faint fluorescence; but on addition of a drop dilute sulphuric acid, 
the fluorescence is well marked. 
The last-mentioned degree of dilution is by no means the limit of 
the fluorescence of quinine solutions, under certain conditions. 
Kerne, rby a specially constructed instrument, termed by him the 
fluorescope, was able to obtain the fluorescence beautifully marked 
in a solution of the alkaloid under a dilution of two million times. 
It should be borne in mind that the fluorescence of quinine solu- 
tions, even of the sulphate, may be entirely prevented by the pre- 
sence of chlorides, bromides and iodides, and the free acids of these 
salts. If to 1 c.c. of a i-iooth solution of quinine sulphate, which 
is strongly fluorescent, one drop of hydrochloric acid or of a solution 
of sodium chloride (1 : 10), be added, the fluorescence is imme- 
diately and wholly destroyed, and is not reproduced on the addition 
of even 0 5 c.c. of dilute sulphuric acid. The interference of bromine 
and iodine is as prompt and complete as that of chlorine. 
According to the recent observations of MM. Sestini and Cam- 
pani1 the fluorescence of sulphuric acid solutions of quinine, especi- 
ally when dilute, may also be concealed by the presence of phenace- 
tine. According to these observers, this substance may also greatly 
interfere with the normal reaction of quinine with both chlorine 
and bromine in the thalleioquin test. 
The property of fluorescence is possessed, although in a less 
degree, by some of the other cinchona alkaloids; and Dr. Bence Jones, 
of London, has described a substance normally present in the body, 
which has a similar property, and named by him animal quinoidine. 
1 Jour. Chem. Soc., Abs., May, 1892, 665. 12 
Beside these substances, certain vegetable principles and extracts 
and the hydrocarbon oils present fluorescent properties. 
4. TEST OF TASTE. 
The intensely bitter taste of quinine and its salts may serve as a 
test of its presence. Of the ordinary preparations of quinine, the 
tannate is the least and the free alkaloid next least bitter; the nor- 
mal sulphate is less bitter than the bisulphate, hydrobromide or 
hydrochloride.1 
The taste of the acid sulphate is very distinct and strongly 
marked in a single drop of a i-iooooth solution; and is still dis- 
tinct in the same quantity of a i-2C>oooth solution ; but, according to 
several persons, is not perceived in a drop of a i-50000th solution. 
In the very elaborate investigations of Dr. G. Kerner2 on the 
absorption and elimination of quinine, he found that when taken 
into the stomach in 0-5 gram (7-5 grains) doses, it appeared in the 
urine in fifteen minutes thereafter ; and that one-half of the total 
quantity taken was thus eliminated in six hours; and one-fourth in 
the following six hours; but that a trace might still be present in 
the urine at the end of seventy-eight hours. 
Of the quinine thus eliminated Dr. Kerner found that a large por- 
tion had undergone a material change, in that while it still possessed 
fluorescent properties, responded to the reaction with chlorine and 
ammonia, and with chlorine and potassium ferrocvanide, it no 
longer had a bitter taste, it now being tasteless. By experiments 
he found that under the limited oxydation of quinine by potassium 
permanganate, a substance was produced which was tasteless and 
corresponded in every respect to this eliminated quinine. On analy- 
sis this oxydation product was found to be dihydroxyl quinine, hav- 
ing the composition C20H2iN2O2, 2OH. From these results Dr. 
Kerner concluded that it was under this form that the alkaloid, in 
part at least, appeared in its tasteless condition in the urine. 
If then, in the absorption of quinine or under oxydation, this 
change to the tasteless variety may take place, it is easy to under- 
stand that in the extraction of the alkaloid from the urine or the 
tissues, this important corroborative test might fail, even when the 
chemical tests indicated its presence. At present we have little or 
1 Prescott Organic Analysis, p. 127. 
2Archiv fur Physiologie, ii, 1869, pp. 200-243; iii, 1870, 93-165 13 
no experience in regard to the recovery of quinine from the body, 
long periods after death. 
That the alkaloids in general may undergo a slow and gradual change 
in the presence of decomposing animal matter seems to be fully 
established by the investigations of Profs. Buchner, Gorup-Besanez 
and others1, as well as our own observations, in the case of strych- 
nine, which, under the conditions stated, after a time, loses its prop- 
erty of responding to the color and certain other chemical tests, but 
still retains its bitter taste and the property of producing tetanic 
convulsions in frogs. 
So also, Dr. Pellacani2 on mixing a definite quantity of curarine 
with a given quantity of fresh blood and allowing the mixture to 
putrify under favorable conditions of temperature, found after some 
months that the alkaloid no longer responded to the physiological 
test, although it still retained its chemical properties, except with 
the sulphuric acid test. 
From our own experience we are strongly led to believe that 
when morphine and strychnine are taken in moderate toxic quantity, 
that portion of the alkaloid which is carried to the tissues by the 
ordinary process of absorption, entirely loses its property of crystal- 
lizing, even when present in quantity sufficient to promptly respond 
to chemical tests. 
When the morphine or strychnine is taken in excessive quantity, 
a portion seems to be distributed to the organs by simple exosmotic 
diffusion, and this may readily be recovered in the crystalline state. 
In a case in which 1-68 grams (about 26 grains) of strychnine were 
administered subcutaneously to a dog during a period of four hours, 
in divided doses, so as to keep the animal paralyzed, 56 mgs. of well- 
crystallized strychnine was recovered from the urine, 26 mgs. from 
the liver, and 14 mgs. from the blood. 
University of Pennsylvania, 
Medical Department. 
1 Ann. d' Hyg., 1881, 385. 
3 Rivista Sper. Med. Legal, xiii, 2, p. 237.