ANALYTICAL CHEMISTRY
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 monohydrate 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. Spectrophotometrically, 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 considerable deviations from Beer’s law, but in such a way as to increase the analysis accuracy; at 515 mp (the absorption maximum 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 transmittancies from about 20 to 60%. TT’hen a Beckman spectro-
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 accuracy. The 1-nitroso-2-naphthol method requires filtration, washing, and ignition to constant weight, all of which are timeconsuming. 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 proposed 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
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). (1)
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.
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 capsicum 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 structure 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
method for the determination of capsaicin content. The organoleptic method formerly official in the United States Pharma. copoeia and later in the Sational Formulary has now been discarded 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.
935
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 phosphotungsticphosphomolybdic 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 discovery 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 purposes 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 a phenolic nature which also give a blue color with the phosphotungstic-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 . 1 2 3
% Capsaicin
The substitution of vanillin for capsaicin in the present analytical 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 necessary, and make up with water to a volume of 1 liter. An equivalent 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 defined 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 combined 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.
Make up to the mark with acetone, mix thoroughly, and filter through a dry double filter. The filtrate should be perfectly 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 thermometer 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 temperature 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 separate 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, washing the separatory funnel and the filter with small portions of petroleum ether. It is essential that the yellow substance which separates a t this point be carefully excluded from the filtrate. 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 petroleum 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 evaporation. Pipet 5 ml. of the solution into a 50-ml. volumetric flask and into another 50-ml. volumetric flask pipet 5 ml. of standard vanillin 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 contents of flasks by rotating and after 5 minutes dilute contents to 50 ml. with saturated sodium carbonate solution. Mix thoroughly 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 necessary 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 difference between the standard solution and the test solution because then the traces of color carried through from the oleoresin have a greater influence on the total color. This does not interfere in any way with the usefulness of the method. In this laboratory it is customary for two observers to read the color and their results uniformly agree within one or two tenths of the colorimeter 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 before 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-
ANALYTICAL CHEMISTRY
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
(3) (4) (5) (6) (7) (8)
Folin and Denis, J . Bid. Chem., 12, 239 (1912). and Denis, J . Ind. Chem.*4, 670 (1912). Hayden and Jordan, J . Am. Pharm. Assoc., 30, 107 (1941). Micko, 2..Vahr. Genussm., 1 , 818 (1898); 2, 411 (1899). Nelson, J . Am. Chem. Soc., 41, 1115 (1919). Thresh, Pharm. J . Trans. ( 3 ) , 7, 21, 259, 473 (1876-77); 8, 187 / 1 Q77-7Qj I Y,. ,LU, I
(1) Aasoc. Offic. Agr. Chemista, “Official and Tentative Methods of Analysis,” 6th ed., p. 366, 1945. (2) Fodor, 2. Cntersuch. Lebensm., 61, 94 (1931).
(9) ~
i A ~ J ,. pharm., ~ , 105, 320 (1933).
RECEIVEDOctobrr 9, 1948.
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 depolarizing 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 providing 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-consuming to construct. Rapid evaluation of the characteristics of manganese 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 characteristics 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 interval. 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 potential is amplified by means of an electronic direct current voltage amplifier, and then recorded. The pulse polarizer has been surcessfully employed in the field of corrosion research (%,$).
method for testing ores would be of great interest. Through the use of the pulse polarizer, it was possible 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 electrolyte 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 obtained 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
f
CALOMEL HALF- CELL
n
bill I I
EXPERIMENTAL TECHNIQUE
A technique has been developed which permits rapid study of the depolarization characteristics of manganese dioxide ores. -4 Present address, The Pure 011 Company, Research and Development Laboratories, Northfield, Ill. 2 Prpsent address, Loyola C-nirersity, Stritch School of Medicine, Chicago,
AMPLIFIER
1
Ill.
LPLATINUM
Figure 1.
Experimental Setup
