934 0.8% (absolute) lo wer than t he 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 theref ore unsuitable. Th e formation o f 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 timrtin g water in ethanol by th e use o f cobalt chloride reagent, it would be immaterial whether hexahydrate or mono- hyd rate is used, inasmuch as th e calibration and determi nation are made by the use o f th e same standa rd solution. I f desired, the determination of cobalt can be made using ordinary commercial (about 95%) ethanol ins tead o f absolute ethanol; in this case the calibration curve, for measurements at 655 mp, is ne arly parallel t o th e curve o f Figure 2, but covers a cobalt concentration range about ten times as high. Spectro- photometrically, solutions of cobalt chloride in 95% ethanol have consider able compone nts o f b oth blue and red ; solutions containing about 5000 p.p.m. (5.00 mg. per ml.) o f 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 measuremen ts is flat and i f used for analysi s would give larger relative error. Th e speci fications of range and accuracy given herein fo r the cobalt determina tion 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 th e cobalt determination can be extended upward by measuring against a standa rd solution o f concentration somen hat lower than that of the sol ution measured; the standa rd i s 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- ANALY TICAL CHEMISTRY photometer is used, the range can also be extended upward, and with some incre ase in accuracy, by th e use of th e 0.1 selector switch fo r measuring tran smittan cies belo w 11 % (1). Th e rather high concentration range of the method for cobalt is a go od i llustration o f t he 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, th e proposed spectrophotometric method is somewhat more rapid , and gives results of comparab le precision and ac- curacy. Th e 1-nit roso-2-na phthol method requires filtrat ion, washing, and ignition to constant weight, all of which are time- consuming. The nitrite- perman ganate method requires 1 2 to 24 hours’ standing for precipitation o f t he hexanitr itocob altiate, 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 t o the measurement of th e d esired constituent . LITERATURE CITED (1) Ayres, G. H., ANAL. CHEM., 1, 652 (1949). (2) Brode, W. R., 2. hysik. Chem., A187, 11 (1940). (3) Kolthoff, I. M., and Sandell, E. B., “Textbook o f Quantitative Inorganic Analysis,” rev. ed., p. 603, N ew York, Macmillan Co., 1946. (4) Lundell, G. E. F ., Hof fma n, J. I., and Bright, H. A , , “Chemical Analy5is o f Iron and Steel,” pp . 339-42, Ne w 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 ] 1, 209 (1864). RECEIVED ovember 8, 1 9 48 . Condensed from a hesis 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 o f Capsaicin in Oleoresin o f Capsicum HORACE NORTH, General Control Laboratory, Dodge & Olcott, Znc., Bayonne, N. . A method for the colorimetric determination of capsaicin in oleoresin of cap- sicum has been developed in which the readily available vanillin is employed fo r the standard solution in place of capsaicin. HE mos t important constituent of red pepper is the pungent T rinciple known as capsaicin, discovered by Thresh (8 ) in 1876. In 1898 Micko (6) sholyed that the substance had the properties of a weak phenol and contained one methoxyl group. He found also th at with an alcoholic solutio n o f platinic chloride an odor o f vanilla was de velope d on standing. In 1919 th e struc- tu re o f capsaicin was established by Kelson (7), who showed it to be th e vanillyl ami de o f isodecenoic acid. Because the pungency o f different varieties peppers varies enormously, there has long been a demand for an accurate method for the determination o f capsa icin content. The organo- leptic method formerly official in the United States Pharma. copoeia and later in the Sational Formulary has now been dis- carded enti rely. Tice (9) broug ht out a co lorime tric method based on Fodor’s ( 3 ) reaction in which capsaicin gives a blue color with vanadium oxytrichloride. - 4 tud y o f thi s method by Hayden and Jordan ( 5 ) howed that the results were unreliable. However, some o f Tice’s recommendations relative to the is ol at io ~ of capsaicin, modified to meet th e requirements of an analytical procedure, have been incorporated in th e method described here .
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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