934
0.8% (absolute) lower than the hexahydrate standard. A fourth
sample, dissolved in water, evaporated to dryness, and baked
for about 10 minutes, gave a very turbid suspension in ethanol,
and was therefore unsuitable.
The formation of the sparingly soluble basic salt when the
monohydrate is heated above 100” C. precludes this method of
preparing standards, and indicates that care must be taken, in
analyses, to evaporate just to dryness, avoiding baking of the
residue.
For estimrting water in ethanol by the use of cobalt chloride
reagent, it would be immaterial whether hexahydrate or mono-
hydrate is used, inasmuch as the calibration and determination
are made by the use of the same standard solution.
If desired, the determination of cobalt can be made using
ordinary commercial (about 95%) ethanol instead of absolute
ethanol; in this case the calibration curve, for measurements a t
655 mp, is nearly parallel to the curve of Figure 2, but covers a
cobalt concentration range about ten times as high. Spectro-
photometrically, solutions of cobalt chloride in 95% ethanol
have considerable components of both blue and red; solutions
containing about 5000 p.p.m. (5.00 mg. per ml.) of cobalt are
visually blue, and with decreasing cobalt concentration the
solutions show gradations through bluish purple to reddish
purple, Measured a t 655 mp (the absorption maximum of
blue ethanol solutions), the solutions in 95% ethanol showed con-
siderable deviations from Beer’s law, but in such a way as to
increase the analysis accuracy; at 515 mp (the absorption maxi-
mum of pink dilute aqueous solutions; see Figure 3, curve 8)
the measurements followed Beei’s law, but a calibration curve
based on these measurements is flat and if used for analysis would
give larger relative error.
The specifications of range and accuracy given herein for the
cobalt determination apply to the measurements made against a
blank, using the Coleman Model 10-S spectrophotometer with
1.30-cm. absorption cells. As with other spectrophotometric
methods, for a given wave length and cell thickness the range of
the cobalt determination can be extended upward by measuring
against a standard solution of concentration somen hat lower
than that of the solution measured; the standard is so chosen
that the transmittance ratio is near the optimum-theoretically
37y0, although the analysis accuracy is almost as good at trans-
mittancies from about 20 to 60%. TT’hen a Beckman spectro-
Colorimetric Determination of Capsaicin in
Oleoresin of Capsicum
HORACE NORTH, General Control Laboratory, Dodge & Olcott, Znc., Bayonne, N. J.
A method for the colorimetric determination of capsaicin in oleoresin of cap-
sicum has been developed in which the readily available vanillin is employed
for the standard solution in place of capsaicin.
T HE most important constituent of red pepper is the pungent
principle known as capsaicin, discovered by Thresh (8) in
1876. I n 1898 Micko (6) sholyed that the substance had the
properties of a weak phenol and contained one methoxyl group.
He found also that with an alcoholic solution of platinic chloride
an odor of vanilla was developed on standing. In 1919 the struc-
ture of capsaicin was established by Kelson ( 7 ) ,who showed it to
be the vanillyl amide of isodecenoic acid.
Because the pungency of different varieties of peppers varies
enormously, there has long been a demand for an accurate
ANALYTICAL CHEMISTRY
photometer is used, the range can also be extended upward, and
with some increase in accuracy, by the use of the 0.1 selector
switch for measuring transmittancies below 11% ( 1 ) .
The rather high concentration range of the method for cobalt
is a good illustration of the fact that spectrophotometric methods
of analysis are not necessarily limited to the determination of
small amounts of constituent, but can apply to concentrations
comparable to those used in gravimetric and titrimetric methods
(1, 6 ) .
In comparison with gravimetric and titrimetric methods for
cobalt, the proposed spectrophotometric method is somewhat
more rapid, and gives results of comparable precision and ac-
curacy. The 1-nitroso-2-naphthol method requires filtration,
washing, and ignition to constant weight, all of which are time-
consuming. The nitrite-permanganate method requires 12 to
24 hours’ standing for precipitation of the hexanitritocobaltiate,
followed by filtration, washing, dissolving, and back-titrating.
The electrodeposition method requires fuming down with sulfuric
acid, and an electrolysis time of 2 hours. In contrast, the pro-
posed colorimetric method, although requiring evaporation of
the solution just to dryness, is very rapid from that point on to
the measurement of the desired constituent.
LITERATURE CITED
(1) Ayres, G. H., ANAL.CHEM.,21, 652 (1949).
(2) Brode, W. R., 2.physik. Chem., A187,211 (1940).
(3) Kolthoff, I. M., and Sandell, E. B., “Textbook of Quantitative
Inorganic Analysis,” rev. ed., p. 603, New York, Macmillan
Co., 1946.
(4) Lundell, G. E. F., Hoffman, J. I., and Bright, H. A,, “Chemical
Analy5is of Iron and Steel,” pp. 339-42, New York, John Wiley
& Sons, 1931.
( 5 ) hlellor, J. W., “Comprehensive Treatise on Inorganic and
Theoretical Chemistry,” Vol. XIV, New York, Longmans,
Gieen and Co., 1935.
(6) Ringbom, A., 2 . anal. Chem., 115, 332 (1939).
(7) Toporescu, E., Compt. rend., 192, 280 (1931).
(8) Treadwell, F. P., and Hall, W. T., “Analytical Chemistry,”
9th English ed., Vol. 11, p. 197, New York, John Wiley &
Sons, 1942.
(9) Winkler, C., J . prakt. Chem., [ l ]91, 209 (1864).
RECEIVED Kovember 8, 1948. Condensed f r o m a thesis submitted by Betty
Vining Glanville t o the faculty of the Graduate School of the University of
Texas in partial fulfillment of the requirements of the degree of master of arts,
August 1948.
method for the determination of capsaicin content. The organo-
leptic method formerly official in the United States Pharma.
copoeia and later in the Sational Formulary has now been dis-
carded entirely. Tice (9) brought out a colorimetric method
based on Fodor’s ( 3 ) reaction in which capsaicin gives a blue
color with vanadium oxytrichloride. -4study of this method by
Hayden and Jordan ( 5 ) showed that the results were unreliable.
However, some of Tice’s recommendations relative to the isolatio~
of capsaicin, modified to meet the requirements of an analytical
procedure, have been incorporated in the method described here.
 V O L U M E 21, NO. 8, A U G U S T 1 9 4 9
Folin and Denis (5)devised a solution of phosphotungstic-
phosphomolybdic acid which gives a blue color with phenols, and
applied this reagent to the determination of vanillin ( 4 )in vanilla
extracts. This is now an official method ( 1 ) of the Association of
Official Agricultural Chemists.
A colorimetric method of analysis of capsaicin using the pure
drug as a standard would be unsatisfactory, as the preparation
of pure capsaicin is a difficult, tedious, and very unpleasant task.
This isolation of pure capsaicin has been circumvented by the dis-
covery that vanillin, which like capsaicin contains a phenolic
hydroxy grouR in the same relative position, serves just as well as
capsaicin for the standard solution. The molecular weight of
vanillin is 152, while that of capsaicin is 305. For practical pur-
poses the latter may be considered double the former, so that 5
ml. of a solution containing 0.5 mg. of vanillin are equivalent to
1.0 mg. of capsaicin. Although this relationship is assumed, we
have in hand a practical means for comparing the pungencies of
different oleoresins of capsicum.
Before applying the colorimetric test, however, it is necessary
to isolate the capsaicin present in the sample to be tested, in a
sufficient degree of purity, in order to eliminate other substances of
' separates a t this point be carefully excluded from the filtrate.
a phenolic nature which also give a blue color with the phospho-
tungstic-phosphomolybdic acid reagent. I t is believed that the
number of steps necessary to accomplish this purpose has been
reduced to the minimum possible under the circumstances.
Duplicate results obtained by the application of this method are
in excellent agreement, as evidenced by the following table:
Sample S o . % Capsaicin
1 ration.
2 Pipet 5 ml. of the solution into a 50-ml. volumetric flask and
3 into another 50-ml. volumetric flask pipet 5 ml. of standard vanil-
The substitution of vanillin for capsaicin in the present analyti-
cal procedure suggests that similar procedures may be applicable
to some other colorimetric determinations in which the substances
to be determined are not readily obtainable in a pure state and in
which other substances of suitable composition are so obtainable
and can be used for the preparation of the standard solutions.
ANALYTICAL PROCEDURE
Special Reagents. Ultrasene. This is purified, deodorized
kerosene much more suitable for analytical work than kerosene
itself. Kerosene can be used, if it is treated with sulfuric acid and
redistilled
Acetone, 607, by volume.
Phosphotungstic-Phosphomolybdic Acid. To 100 grams of
pure sodium tungstate and 20 grams of phosphomolybdic acid
(free from nitrates and ammonium salts) add 100 grams of sirupy
phosphoric acid (containing 857, H3POI) and 700 ml. of water;
boil over a free flame for 1.5 to 2 hours; then cool, filter if neces-
sary, and make up with water to a volume of 1 liter. An equiva-
lent amount of pure molybdic acid may be substituted for the
phosphomolybdic acid.
Standard T'anillin Solution. Dissolve 0.1 gram of vanillin in
sufficient distilled water to make 1000 ml. This solution must be
freshly prepared each day.
Procedure. JTeigh 1.0 gram of oleoresin red pepper in a small
beaker and transfer to a 125-m1. Squibb separatory funnel by
solution in 20 ml. of ultrasene, using the ultrasene in portions.
Dissolve 1.0 gram of sodium chloride in 80 ml. of 60% acetone (by
volume) and nash out the beaker Lvith 20 ml. of this solution, in
portions, transferring the washings to the separatory funnel.
Shake the funnel sufficiently to keep the liquids well mixed and
continue this gentle shaking for about 5 minutes. On standing,
the mixture wparates within 2 or 3 minutes into two sharply de-
fined layers but the lower layer is always cloudy. Draw the
lower layer into a 125-ml. Squibb separatory funni.1 and continue
the extraction of the solution of oleoresin in like manner, using the
balance of the acetone solution in 20-ml. portions. T o the com-
bined extractions add 5 ml. of ultrasene and shake gently for a few
minutes Let stand 1 hour to separate. Draw off the still hazy
lower layer into a 100-nil. volumetric flask containing 0.5 gram of
Filter-Cel, cork the flask, and shake 0.5 hour in a machine.
935
Make up to the mark with acetone, mix thoroughly, and
filter through a dry double filter. The filtrate should be per-
fectly clear. .
Pipet 50 ml. of the clear filtrate into a 250-ml. beaker marked at
20 ml. and evaporate on top of a steam bath (not directly over the
steam) a t a temperature not over 65" C., using a small thermom-
eter as a stirring rod, until the volume of liquid is reduced to 20 ml.
By this treatment the acetone is removed from the solution and
the crude capsaicin separates as an oily sediment. Solutions of
capsaicin should be heated as little as possible and a t as low a tem-
perature as possible.
Cool the liquid to room temperature, add 10 ml. of 0.5 N
sodium hydroxide, and stir until the oily sedimen't has dissolved.
Pour the solution into a 250-ml. Squibb separatory funnel, and
wash the beaker with two further 5-ml. portions of 0.5 N sodium
hydroxide and finally with two 5-ml. portions of water, pouring the
washings into the separatory funnel. Now add to the funnel 5.0
grams of sodium hicarbonate and 150 ml. of petroleum ether,
shake moderately 15 minutes, and let stand until the layers sepa-
rate sharply (overnight, if necessary). The amount of petroleum
ether is sufficient for 1.0 gram of a normal oleoresin. I n special
cases it may be necessary to use a larger quantity of solvent.
Draw off and reject the lower layer and carefully filter the
upper layer into a clean 250-ml. Squibb separatory funnel, wash-
ing the separatory funnel and the filter with small portions of
petroleum ether. It is essential that the yellow substance which
Shake the petroleum ether solution with 10 ml. of 0.5 N sodium
hydroxide, add 10 drops of 957, ethyl alcohol, and without further
shaking let stand until the layers separate sharply. Filter the
lower layer into a 50-ml. volumetric flask and extract the petro-
leum ether further with three IO-ml. portions of water, passing the
extractions successively through the filter into the flask. Fill up
the flask with water to the 50-ml. mark and mix thoroughly. This
solution should be nearly colorless. The concentration remains
the same as the 50 ml. of clear filtrate originally taken for evapo-
lin solution. To each flask add from a pipet 5 ml. of the p h 0 5
photungstic-phosphomolybdic acfd reagent, allowing it to flow
down the neck of the flask in such a way as to wash down
the solution that may be on the sides of the flask. RIix con-
tents of flasks by rotating and after 5 minutes dilute contents to
50 ml. with saturated sodium carbonate solution. Mix thor-
oughly by inverting the flasks several times and shaking and then
place the flasks in a shaking machine until 30 minutes have
elapsed since the phosphotungstic-phosphomolybdic acid reagent
was first added to the solutions. This thorough shaking is neces-
sary in order to precipitate the sodium phosphate completely and
prevent the filtrate from becoming hazy while the solution is being
read in the colorimeter. Filter the solutions through dry double
filters and compare the blue colors of the clear solutions without
delay in a colorimeter.
With samples poor in capsaicin, there may be a slight hue dif-
ference between the standard solution and the test solution be-
cause then the traces of color carried through from the oleoresin
have a greater influence on the total color. This does not inter-
fere in any way with the usefulness of the method. In this labora-
tory it is customary for two observers to read the color and their
results uniformly agree within one or two tenths of the color-
imeter scale. It is essential that the blue solutions be perfectly
clear. Ordinarily the standard blue color is set a t 20, but if the
test solution is pale it may be necessary to set the standard a t 10
or even 5. After a reading is made, the positions of the cups
should be reversed and another reading made. The average of
these two readings is used for the calculation. The zero points on
the colorimeter should be checked and corrected if necessary be-
fore the instrument is used.
If it is a question of determining capsaicin in the spice, 5 to 10
grams of the ground material are extracted with acetone or
ether in a Soshlet extraction apparatus and the extract is tested as
above described.
ACKNOW LEDGM E S T
Acknowledgments are due to V. H. Fischer, vice president of
Dodge & Olcott, Inc., who assigned this problem for study, t o
Herman Wachs, director of research, who supervised the prepara-
936
tion of this material for publication, and to Thaddeus Ptaszynski,
Philip Catanzaro, and Thomas hledwick who performed a large
part of the experimental work.
LITERATURE CITED
(1) Aasoc. Offic. Agr. Chemista, “Official and Tentative Methods of
Analysis,” 6th ed., p. 366, 1945.
(2) Fodor, 2. Cntersuch. Lebensm., 61, 94 (1931).
Rapid Identification of Manganese Dioxide, Ores
GLENN A. MARSH’ AND HUGH J. McDON..ILD2
Illinois Institute of Technology, Chicago, I l l .
Alanganese dioxide ores obtained from different
geographic locations and as a result of different
methods of preparation differ in their depolarizing
ability when used in the common Leclanchk t >pe of
dry cell. Ores are commonly tested for their quality
by constructing an actual cell and making suitable
measurements on its current capacity and shelf life.
Because such tests are time-consuming, a rapid
I S A dry cell of the common Leclanche type, a depolarizer or
oxidizing agent is used to provide the cathodic reaction.
Manganese dioxide is ordinarily used for this purpose, but the de-
polarizing characteristics of different commercial batches vary
considerably. Ordinary chemical and x-ray analytical method?
have not been used extensively in the evaluation of manganese
dioxide ores. The method presented here shows promise of pro-
viding a rapid and reliable means of identifying good and poor
battery depolarizer ores.
The depolarizing characteristics of manganese dioxide ores are
ordinarily evaluated on the basis of dry cell tests, carried out ill
special test cells, which are rather complicated and time-consum-
ing to construct. Rapid evaluation of the characteristics of man-
ganese dioxide ores, from the standpoint of not only immediate
capacity but also shelf life, is highly desirable from the standpoint
of battery makers and ore suppliers. although these character-
istics have not been studied, the work described here indicates
that i t may be possible to establish rapidly the depolarizing
characteristics of an ore as measured by the initial capacity of the
(Bell.
THE PULSE POLARIZER
The method described here involved use of the pulse polarizer,
developed during 1947. The instrument employs a system of
electronic circuits to polarize an electrode over a brief time inter-
val. The polarization and depolarization a t the surface of the
electrode are recorded continuously on a high-speed strip chart
(Brown Instruments Division, Minneapolis-Honeywell Regulator
Co., Philadelphia, Pa.; single record; full scale, 5 mv.) and
show up as a curve which is distinctive for each set of conditions.
The polarizing circuit consists of a condenser (125 mfd.) which is
charged to 310 volts. Discharge of this condenser brings about
the polarization a t the electrode surface. The polarization poten-
tial is amplified by means of an electronic direct current voltage
amplifier, and then recorded. The pulse polarizer has been sur-
cessfully employed in the field of corrosion research (%,$).
EXPERIMENTAL TECHNIQUE
A technique has been developed which permits rapid study of
the depolarization characteristics of manganese dioxide ores. -4
1Present address, The Pure 011 Company, Research and Development
Laboratories, Northfield, Ill.
2 Prpsent address, Loyola C-nirersity, Stritch School of Medicine, Chicago,
Ill.
ANALYTICAL CHEMISTRY
(3) Folin and Denis, J . Bid. Chem., 12, 239 (1912).
(4) and Denis, J . Ind. Chem.*4, 670 (1912).
(5) Hayden and Jordan, J . Am. Pharm. Assoc., 30, 107 (1941).
(6) Micko, 2..Vahr. Genussm., 1 , 818 (1898); 2, 411 (1899).
(7) Nelson, J . Am. Chem. Soc., 41, 1115 (1919).
(8) Thresh, Pharm. J . Trans. ( 3 ) , 7, 21, 259, 473 (1876-77); 8, 187
/ 1 Q77-7Qj
,LU, I I Y,.
(9) ~ i A ~ J ,. pharm.,
~ , 105, 320 (1933).
RECEIVEDOctobrr 9, 1948.
method for testing ores would be of great interest.
Through the use of the pulse polarizer, it was pos-
sible to differentiate in a few minutes between the
poor and good ores in a set of samples. The ores
were rated independently on the basis of test cells,
and the comparisons between predicted and actual
depolarizing ability were, in the majority of cases,
found to be good.
platinum cylinder 0.5 inch in diaiiieter is filled with ore and
tamped until tightly packed. I n the top of the cylinder an elec-
trolyte is added, in which an “inert” electrode is immersed. This
electrode, which merely completes the electrical circuit, is a fine
iron wire. A thin calomel half-cell is then lorered into contact
with the ore surface. The experimental setup is shoivn i n
Figure 1.
Either the cathodic or anodic polarization may be studied with
the apparatus, but the cathodic polarization is of interest in this
parti ular case. By cathodic polarizat’ion is meant the change in
the electrode potential of the manganese dioxide when electrons
are forced into it from the external circuit.
The voltage applied is constant for each pulse, and the time
during which the ore surface is polarized is between 0.05 and 0.1
second. Experiments showed that the curves obtained with
platium alone are long but very narrow, radically unlike those ob-
tained with the ores. On this basis, the results must be attributed
almost entirely to the depolarizing characteristics of the ore.
The electrical discharge obtained from the pulse polarizer is
standardized to the extent that curves obtained are, for the most
part, reproducible in minute detail, and curves obtained with a
ELECTRICAL
I
MECHANISM
I
CALOMEL
n
f HALF- CELL
bill I I
AMPLIFIER
LPLATINUM
Figure 1. Experimental Setup