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934

0.8% (absolute) lower than t he hexahydrate standard. A fourthsample, dissolved in water, evaporated to dryness, and bakedfor 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 es timrting 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 ins tead of absolute

ethanol; in this case the calibration curve, for measurements at

655 mp, is nearly parallel t o 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 at 655 mp (the absorption maximum of

blue ethanol solutions), he solutions in 95 % ethanol showed con-siderable deviations from Beer’s law, but in such a way as to

increase the analysis accuracy; at 51 5 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 fo r 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-

A N A L Y T I C A L C H E M I S T R Y

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 fo r measuring transmittancies below 11% (1) .

The rather high concentration range of the method for cobal t

is a good illustration of t he fact tha t 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 t he hexanitr itocobaltiate,

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 tothe measurement of th e desired constituent .

LITER ATUR E C ITED

(1) Ayres,G. H., ANAL.CHEM., 1, 652 (1949).(2) Brode, W. R., 2. hy s i k . Chem., A187, 11 (1940).(3) Kolthoff, I. M., and Sandell, E. B., “Textbook of Quantitative

Inorganic Analysis,” rev. ed., p. 603, New York, MacmillanCo. , 1946.

(4) Lundell, G . E. F., Hoffman, J. I., and Bright, H. A , , “ChemicalAnaly5is of Iron and Steel,” pp . 339-42, Ne w York, John Wiley& Sons, 1931.

( 5 ) hlellor, J. W., “Comprehensive Treatise on Inorganic andTheoretical Chemistry,” Vol. XIV, New York, Longmans,Gieen and Co., 1935.

(6) Ringbom,A. , 2 . anal . Chem. , 115, 332 (1939).(7 ) Toporescu, E. , Comp t . 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 . prak t . Chem . , [ l ] 1, 209 (1864).

RECEIVEDovember 8, 1948. Condensed fr om a hesis submitted by Betty

Vining Glanville t o the faculty of the Graduat e School of the University ofTexas in partial fulfillment of the requirements of t he degree of master of arts ,

August 1948.

Color imetr ic Determinat ion of Capsaicin in

Oleoresin of CapsicumHORACE NORTH, General Control Laboratory, Dodge & Olcott, Znc., Bayonne, N. .

A me thod for th e colorimetric dete rm inat ion of capsaicin in oleoresin of cap-sicum has been developed in which the readily available vanillin is employed

fo r th e stan dard so lution in place of capsaicin.

HE most important constituent of red pepper is the pungentT rinciple known as capsaicin, discovered by Thresh (8 ) in

1876. In 1898 Micko (6 ) sholyed that the substance had th e

properties of a weak phenol and contained one methoxyl group.

He found also th at 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

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. -4 tudy of thi s method by

Hayden and Jordan ( 5 ) howed that the results were unreliable.

However, some of Tice’s recommendations relative to the is ol at io ~

of capsaicin, modified to meet the requirements of an analytical

procedure, have been incorporated in the method described here.

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V O L U M E 21 , NO. 8, A U G U S T 1 9 4 9

Folin and Denis (5) evised a solution of phosphotungstic-

phosphomolybdic acid which gives a blue color with phenols, and

applied this reagent t o th e determination of vanillin ( 4 ) n 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

dru g as a standard would be unsatisfactory, as the preparation

of pure capsaicin is a difficult, tedious, and very unpleasant task.

Th is isolation of pure capsaicin has been circumvented by t he 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 th at 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 pur ity, in order to eliminate other substances of

a phenolic nature which also give a blue color with the phospho-

tungstic-phosphomolybdic acid reagent. I t is believed that the

number of s teps necessary t o 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

935

123

The substitu tion 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 st ate and in

which other substances of su itable composition are so obtainable

and can be used for the preparation of the standard solutions.

A N A L Y T IC A L P R OC E D U R E

Special Reagents. Ultrasene. This is purified, deodorizedkerosene much more suitable for analytical work than keroseneitself. Kerosene can be used, if it is treated wi th sulfuric acid andredistilled

Acetone, 607, by volume.Phosphotungstic-Phosphomolybdic Acid. To 100 grams of

pure sodium tungstate and 20 grams of phosphomolybdic acid(free from nitra tes and ammonium salts) add 100 grams of sirupyphosphoric 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 amoun t of pure molybdic acid may be substi tuted for thephosphomolybdic acid.

Dissolve 0.1 gram of vanil lin insufficient 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 smallbeaker and transfer to a 125-m1. Squibb separatory funnel bysolution in 20 ml. of ultrasene, using the ultrasene in portions.Dissolve 1.0 gram of sodium chloride in 80 ml. of 60% acetone (byvolume) and n as h out the beaker Lvith 20 ml. of this solution, inportions, transferring the washings to the separatory funnel.Shake the funnel sufficiently to keep the liquids well mixed andcontinue 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 thelower layer into a 125-ml. Squibb separato ry funni.1 and continuethe extraction of the solution of oleoresin in like manner, using thebalance of the acetone solution in 20-ml. portions. To the com-bined extractions add 5 ml. of ultrasene and shake gently for a fewminutes Let stand 1 hour to separate. Draw off the still hazylower layer into a 100-nil. volumetric flask containing 0.5 gram ofFilter-Cel, cork the flask, and shake 0.5 hour in a machine.

Standard T'anillin Solution.

Make up to the mark with acetone, mix thoroughly, andfilter through a dry double filter. The filtrate should be per-fectly clear. .

Pipe t 50 ml. of the clear filtr ate into a 250-ml. beaker marked at20 ml. and evaporate on top of a steam bath (no t directly over thesteam) at a temperature not over 65" C. , using a small thermom-eter a s a stirr ing rod, until th e volume of liquid is reduced to 20 ml.By this treatment the acetone is removed from the solution andthe crude capsaicin separa tes as an oily sediment. Solutions ofcapsaicin should be heated as little a s possible and a t as lo w a tem-perature as possible.

Cool the liquid to room temperature, add 10 ml. of 0.5 Nsodium hydroxide, and stir until the oily sedimen't has dissolved.Pour the solution into a 250-ml. Squibb separatory funnel, andwash the beaker with two further 5-ml. portions of 0.5 N sodiumhydroxide and finally with two 5-ml. portions of water, pouring thewashings into the separatory funnel. Now add to the funnel 5.0grams of sodium hica rbona te and 150 ml. of petroleum ether,shake moderately 15 minutes, and let stand unt il the layers sepa-rate sharply (overnight, if necessary). The amount of petroleumether is sufficient for 1.0 gram of a normal oleoresin. In specialcases i t may be necessary to use a larger quant ity of solvent.

Draw off and reject the lower layer and carefully filter theupper layer into a clean 250-ml. Squibb separatory funnel, wash-ing the separatory funnel and the filter with small portions ofpetroleum ether. It is essential th at the yellow substance whichseparates at this point be carefully excluded from the filtrate.Shake the petroleum ether solution with 10 ml. of 0.5 N sodiumhydroxide, add 10drops of 957, ethy l alcohol, and without furthershaking let stand until the layers separate sharply. Filter the

lower layer into a 50-ml. volumetric flask and extract the petro-leum ether furthe r with three IO-ml. portions of wa ter, passing th eextract ions successively through the filter into the flask. Fill upthe flask with water to the 50-ml. mark and mix thoroughly. Thissolution should be nearly colorless. The concentration remainsthe same as the 50 ml. of clear filtrate originally taken for evapo-ration.

Pipet 5 ml. of the solution into a 50-ml. volumetric flask andinto another 50-ml. volumetric flask pipet 5 ml. of standard vanil-lin solution. To each flask add from a pipet 5 ml. of the ph05photungstic-phosphomolybdic acfd reagent, allowing it to flowdown the neck of t he flask in such a way a s to wash downthe solution t ha t may be on the sides of t he flask. RIix con-tents of flasks by rotating and after 5 minutes dilute contents to50 ml. with saturated sodium carbonate solution. Mix thor-oughly by inverting the flasks several times and shaking and thenplace the flasks in a shaking machine until 30 minutes haveelapsed since the phosphotungstic-phosphomolybdic acid reagentwas first added to the solutions. This thorough shaking is neces-

sary in order to precipitate the sodium phosphate completely andpreven t the filtra te from becoming hazy while the solution is beingread in the colorimeter. Filter the solutions through dry doublefilters and compare the blue colors of the clear solutions withoutdelay 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 th is labora-

tory it is customary for two observers to read the color and their

results uniformly agree within one or two ten ths of th e color-

imeter scale. It is essential that the blue solutions be perfectly

clear. Ordinarily the standard blue color is set at 20, but if the

test solution is pale it may be necessary to set t he 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. Th e 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 th e spice, 5 to 10

grams of t he ground material a re extracted with acetone or

ether in a Soshlet extraction apparatus and the extract is tested as

above described.

A CKN OW L E D GM E S T

Acknowledgments are due to V. H. Fischer, vice president of

Dodge & Olcott, Inc., who assigned this problem for study, to

Herman Wachs, director of research, who supervised the prepara-

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936 A N A L Y T I C A L C H E M I S T R Y

tion of this material for publication, and to Thaddeus Ptaszynski,

Philip Catanzaro, and Thomas hledwick who performed a large

par t of t he experimental work.

(3 ) Folin and Denis, J . Bid. Chem., 12 , 239 ( 1912) .

(4)(5 ) Hayden and Jordan, J . Am . Pharm. Assoc., 30, 107 ( 1941) .(6) Micko,2. Vahr. Genussm., 1 , 8 1 8 ( 1 8 9 8 ) ; 2, 4 1 1 ( 1 8 9 9 ) .

and Denis, J . Ind. Chem.*4, 670 ( 1912) .

L I T E R A T U R E C I T E D

( 7 ) Nelson, J . A m . Chem. Soc., 41 , 1115 ( 1919) .(8 ) Thresh, Pharm. J . Trans . ( 3 ) ,7 , 21, 259 , 473 ( 1876- 77) ; 8, 18 7

/ 1 Q77-7Qj, L U , I I Y,.

(1 ) Aasoc. Offic. Agr. Chemista, “Official and Tentative Methods of

(2 ) Fodor, 2. Cntersuch. Lebensm., 6 1 , 9 4 ( 1 9 3 1 ) .

(9 ) ~ i ~ ~ ,~ ., pharm., 1 0 5 , 3 2 0 ( 1 9 3 3 ) .

RECEIVEDOctobrr 9, 1948.

Analysis,” 6t h ed., p. 366, 1945 .

Rapid Ident i f icat ion o f Manganese Dioxide, OresG L E N N A. M A R S H ’ AND HUGH J. McDON..ILD2

Illinois Institu te 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 com mon Leclanchk t> e of

dry cell. Ores are comm only tested for their quality

by constructing an actual cell and making suitable

measurements o n it s current capacity and shelf life.

Because such tests are time-consuming, a rapid

S A dry cell of the common Leclanche type, a depolarizer orI 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 analyt ical 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 character istics 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 bu t also shelf life, is highly desirable from the standpoint

of batte ry makers and ore suppliers. al though these character-

istics have not been studied, the work described here indicates

that it may be possible to establish rapidly the depolarizing

characteristics of an ore as measured by the initial capacity of t he

(Bell.THE P U L S E P OL A R I Z E R

The method described here involved use of the pulse polarizer,

developed during 1947. The instrument employs a system of

electronic circuits t o 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 at 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 (%,$).

E X P E R I M E N T A L T E C HN I QU E

A technique has been developed which permits rapid study of

the depolarization characteristics of manganese dioxide ores. -4

1 Present address, The Pure 011Company, Research and Development

2 Prpsent address, Loyola C-nirersity, Str i tch School of Medicine, Chicago,Laboratories, Northfield, Ill.

Ill.

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. In the top of the cylinder an elec-

troly te is added, in which an “ine rt” 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 appara tus, 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 tha t 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 attr ibuted

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

M E C H A N I S MIC A L O M E L

HALF- CELLfnbill I I

A M P L I F I E R

LPLATINUM

Figure 1. Experimental Setup

I