ANALYTICAL CHEMISTRY
422
Calculate the absorptivity as follom:
ACKNOWLEDGMENT
a = AL
0.9c where A , is the absorbance of the iodine-sample blend minus the absorbance of the iodine blend, c is the concentration of the sample in the final sample-iso-octane blend in grams per liter, and 0.9 is the correction factor necessary to adjust the final concentration to reflect the dilution caused by adding the iodine stock solution. Calculate the weight per cent of sulfide sulfur as follow:
100 X a 44 where 44 is the average absorptivity for sulfide sulfur.
Ft. % ' sulfide sulfur
=
The author wishes to express his appreciation to the Humble Oil and Refining Co. for permission to publish the material included in this article. LITER4TURE CITED
(1) Ball, J. S., I?.S.Bur. Mines, Rept. Invest. 3591 (1941). (2) Benesi, H. A., and Hildebrand, J. H., J . Am. Chem. Soc., 71, 2703 (1949).
~
RECEIVED for review -4ugust 7, 1932. Accepted November 17, 1952.
Reduction of Phosphomolybdic Acid by Compounds Possessing Conjugated Double Bonds SHLOMO BURSTEIN' Hormone Research Laboratory, Hebrew University-Hadassah Medical School, Jerusalem, Israel, and Worcester Foundation for Experimental Biology, Shrewsbury, Mass. The reaction of a specially prepared phosphomolybdic acid reagent w-ith compounds possessing conjugated double bond systems is described. The ability of carotenoid and other conjugated unsaturated compounds to reduce the phosphomolybdic acid reagent to molybdenum blue is increased with the number of the conjugated double bonds present in the molecule. Certain observations in the reduction of phosphomolybdic acid by steroids possessing conjugated systems are described and discussed.
T
H E phosphomolybdic acid reagent of Folin and TVu (6) was originally described in 1920 for the quantitative determination of blood glucose and other reducing sugars. In this method, the glucose is oxidized by alkaline copper tartrate and the cuprous copper formed is estimated colorimetrically by the molybdenum blue produced by reacting with the Folin-Wu reagent. The Folin-7Tu reagent has been extensively used in analytical chemistry. I n 1926 Campbell (3)applied the Folin-Wu reagent for the quantitative determination of dihydroxyacetone, x-hich reduces the reagent to molybdenum blue directly, while the reduction from glucose was only 1/180 as great. Heard and Sobel ( 7 ) in 1946 used the Folin-Wu reagent in a medium of glacial acetic acid for the quantitative determination of corticosteroid. which contain the reducing a-ketolic side chain. While using the method of Heard and Sobel, with a slightly modified Folin-Wu reagent, for the determination of urinary corticosteroids the author observed that carotenoids and other conjugated substances reduce the reagent ( 2 ) . KO previous comparative study of the reaction of phosphomolybdic acid with compounds possessing conjugated double bonds has been reported. A drop test for vitamin A and carotene using phosphomolybdic acid has been described by Levinson and Kushko ( 8 ) , and a spot test using molybdenum phosphotungstic acid by Bezssonoff (1). Linolenic acid which contains four conjugateddouble bonds reacts xvith areenotungstic acid (9). Heard and Sobel ( 7 ) described the ieaction of the Folin-JVu reagent n i t h steroids possessing the a,@-unsaturatedketonic system. The purpose of the present paper is to report on observations found in the ability of carotenoids and other conjugated substances to cause reduction of a specially prepared phosphomolybdic acid reagent to molybdenum blue. 1 Present address, Worcester Foundation for Experimental Shrewsbury, Mass.
Biology,
MhTERIALS AND METHODS
Molybdic acid, sodium tungstate, phosphoric acid, glacial acetic acid. Baker analyzed reagent. Solutions of the compounds to be studied in glacial acetic acid were kept a t 0" to 5' C. Phosphomolybdic acid reagent. The phosphomolybdic acid was prepared according to Folin and Wu (6), omitting, however, the tungstic acid. The original Folin-\Vu reagent, which was used by Heard and Sobel, is actually phosphomolybdotungstic acid and is referred to here as such. The color reaction was carried out in glacial acetic acid solution. according to Heard and Sobel ( 7 ) . Bn aliquot of the solution n as placed in a 10 X 1 cm. test tube and diluted lvith glacial acetic acid to 1 ml. One milliliter of the phosphomolybdic acid reagent was then added and the tube was heated in a boiling water bath for 1 hour. After cooling in tap water, the reaction mixture was diluted to 10 ml. with a mixture of phosphomolybdic acid reagent and glacial acetic acid (1 to 1). The optical density of the molybdenum blue was measured in a 1-cm. wide cuvette with a Coleman Universal Model 14 spectrophotometer a t 650 mp. The blank consisted of 1 ml. of glacial acetic acid and 1 ml. of phosphomolybdic acid reagent; it TT as heated, diluted, and run in the same manner as above. RESULTS AND DISCUSSION
Reduction of Phosphomolybdic Acid by Carotenoids and Other Open Chain Conjugated Substances. The reduction of phosphomolybdic acid by carotenoid and other open chain conjugated substances is given in Table I. Column 3 shows the optical densities of the molybdenum blue obtained for 1 micromole of compound. I n column 4 are given the optical densities calculated for one conjugated double bond by dividing the molar optical density by the number of conjugated double bonds in the molccule. A4general relationship between the number of the conjugated double bonds in the molecule and the molar optical deniity could not be found. Hovever, as can be seen from the data piesented in Table I, the molar optical density of the molybdenum
V O L U M E 25, N O , 3, M A R C H 1 9 5 3
423
Table 1. Optical Density of Molybdenum Blue Obtained with 1 hlicromole of Compound by Phosphomolybdic iicid Reaction Compound
No. of Conj. Double Bonds
Log Ia/I
Log Io/I for 1 Conj. Double Bond 0.55 0.48
@-Carotene 11 6 lla Cryptoxanthin 11 5.33a 5.89a 0.59 a-Carotene l? Titarnin A 1.48" 0.30 1,8-Diuhen~loctatetraene 4 3.3v 0.84 B-Ionone 3 0.28 0.09 Citral 2 0.41 0.20 1,4-(ol,@-Dinaphthyl)huta2 0.24 0.12 diene l-Phenyl,4(-9-phenanthryl)butadiene 2 0.18 0.09 1,4-Dighenylbutadiene 2 0.11 0.05 a-Ionone 2 0.02 0.01 Sorbic acid 2 0.0 0.0 a Obtained b y extrapolation. All compounds studied gave linear relationship between optical density and concentration.
blue obt:iiiied Trith vitamin A, a-carotene, 8-carotene, and cryptoxanthin increases Tvith the number of conjugated double bonds. The increase in the optical density of the molybdenum blue formed n-ith the number of the conjugated double bonds is greater in the diarylpolyenes studied. 1,s-Diphenyloctatetraene has an optical density per conjugated double bond which is about 17 times greater than that of 1,4-diphenylbutadiene. By using the phosphomolybdic acid reaction for the two isomers, p-ionone and tu-ionone, it is possible to determine the degree of conjugation. 8-Ionone, which contains three double bonds forniirig a conjugated system (C=C-C=C-C=O), gives a molar density 10 times greater t,han that of a-ionone which also contains three double bonds, only two of which are conjugated. The reaction could serve as a qualitative test to distinguish between the tn-o compounds. CXtral, with a two conjugated double bond system (C==CCH=O), has a greater molar optical density than a-ionone, possibly because of the higher reducing properties of the a,Purisaturated aldehydic system. S o t all the conjugated compounds tested reduce the reagent. Sorbic acid, for instance, which contains two conjugated double bonds (CH,CH=CIi-CH=CH-COOH], does not reduce the reagent even at a concentration as high as 500 micrograms. Reduction of Phosphomolybdic Acid by Steroids Possessing Conjugated Double Bonds. The optical densities of the molybdenum blur obtained with 1 micromole of steroid are given in Table 11. The A4-3-keto conjugated system of A4-androstene-3one gives an optical density of 0.07. Introduction of a keto group in the 17-position of the steroid molecule seems to lower the reducing pon-er of the A4-3-keto system (A4-androstene-3,17tlione), whereas a keto group at carbon 11 increases the optical density (adrenosterone). An acetyl side chain in position 17 (loci: not change the optical density (progesterone). KO significant rhange is observed in A"cholestene-3-one and in 3-ketoetiochoienic acid methyl ester. Introduction of a keto group in poeition 6 lowers the optical density of bhe 44-3-keto system (A4cholpsteii~-:'s,G-dioiie:t. A 8-hydrosyl group at carbon 17 does not :rlter the optical density (testosterone). A 17a-hydroxyl, boxever, increases the optical density approximately tv-ofold (epitwtosteronr). A still larger increase of the optical density of the A4-3-keto system is observed in methyl- and vinyltestosterone. The A1-8-keto system gives a very low optical density ( A 1 nndrostene-3. li-dione) as does the Al6-20-keto system (A'6pregriene-3p-ol-20-one acetate). The very low optical density shown by the A6-7-keto system in 7-ketorholesterol is sigiiificantly increased in the A3,5-diene-i-keto system ( A3s5cholest'adiene-7-one), which constitutes a con;lugated triene syst ~ n i . -411 extremely Ion- optical density for 7-ketocholesterol has :ilso been obtained by Heard and Sobel ( 7 ) using the Folin-Ku reagent. Thc optical density of the molybdenum blue produced by the
carbon-carbon diene system in the steroid molecule seems to depend upon the position of the double bonds in the rings. The homoannular A517-diene system (7-dehydrocholesterol acetate and A6s7-androstadiene-3P.l7p-diol) gives a much higher optical density than the A4-3-keto system. The homannular A2s4cholestadiene, however, gives a much lower optical density. The latter observation may be due to the rearrangement into a A3z5heteroannular diene which the homoannular Az,4-diene readily undergoes in an acid medium (4). The heteroannular diene systems studied showed a loir- optical density. The systems A7f9*(l1)diene and A1p4,6-trienehave a reducing power of the same order of magnitude as that of the A4-3-ketosystem. The isolated double bond does not react with phosphomolybdic acid under the experimental conditions described. S o reaction was observed with A7-cholestenol or with cholesterol. Heard and Sobel using the Folin-Wu reagent obtained a much higher optical density for the A4-3-keto system than was obtained in the present investigation where the phosphomolybdic acid reagent was used. That a change in the composition of the phosphomolybdic acid reagent may change its selectivity of reacwho prepared the tion rras shown by Folin and Denis in 1912 (6), uric acid and the phenol reagents.
Table 11. Optical Densities of Molybdenum Blue Obtained with 1 Micromole of Steroids Possessing Conjugated Double Bonds by Phosphomolybdic Acid Reaction Compound Log Io/I Ad-Androstene-3-one A4-Androstene-3,17-dione Adrenosterone Progesterone A4-Cholestene-3-one A.'-3-Ketoetiocholenic acid methyl ester A.'-Cholestene-3,6-dione Testosterone Enitestosterone Eihyltestosterone Vinyltestosterone Al-Androstene-3,17-dione A~6-Preanene-38-0l-20-oneacetate 7-Ketocholesterol AW-Cholestadien-7-one 7-Dehydrocholesterol acetate Abi-Androstadiene-3@,17j3-diol diacetate A21 4-Cholestadiene h i t s : 11-.4ndrostadiene-38,17j3-diol A h 4~6-.4ndrostatriene-17-01 AWholestenol
0.07 0.045 0.18 0.068 0.09 0 . 10 0.02 0,062 0.14 0 31
0 29 0 008
0 0 0.012 0.06 0.33 0.26 0.04 0.05 0.06 0.00
Table 111. Optical Densities of Molybdenum Blue Obtained with 300 Micrograms of Compound by Phosphomolybdic Acid and Phosphomolybdotungstic Acid Reactions Compound Progesterone Testosterone A4-Androstene-3,l'l-dione A1-Androstene-3,17-d1one
Phosphomolybdic Phosphornolybdotungstic Acid Acid 0 068 0 18 0 062 0 15 0 054 0 13 0 0 0 0
A comparison of the Folin-Wu reagent and the phosphomolyhdie acid reagent using testosterone, A4- and Al-androstene-3,lidione, and progesterone is shown in Table 111. Phosphomolybdotungstic acid gives an optical density approximately 2 to 3 times greater than t h a t obtained with phosphomolybdic acid. The color with phosphomolybdotungstic acid did not reach its maximum intensity in 1 hour of heating, but continued to increase even in the cold. Under the same conditions, the color obtained with the phosphomolybdic acid reagent was much more stable. The phosphomolybdotungstic acid reagent appears to react more strongly with the A4-3-keto system than the phosphomolybdic acid reagent. No difference was, however, observed for the A'-3keto system where no reaction was obtained (Table 111). ACKNOWLEDGMENT
The author is grateful to Bernhard Zondek and Felix Bergman of the Hebrew miversity-HadiLssah Medical School for their en-
ANALYTICAL CHEMISTRY
424
couragenient and valuable advice during the course of this inyestigation, and to Paul Karrer of the University of Zurich, for generously supplying the carotenoids.
Folin, O., and Denis, TI'., J . Biol. Chon., 12, 239 (1912). Folin, O., and Wu, H.. Ibid., 41,367 (1920). ( 7 ) Heard, R. D. H., and Sobel, H., Ihid., 165, 687 (1946). ( 8 ) Levinson, AI. S., and Kushko, 1'. M.,Lab. Prakt. (C.S.S.R.), (5) (6)
1938, SO.6, 17-19.
LITERATURE CITED
(1) (2) (3) (4)
Bezssonoff, K,,Bull. SOC. chim. b i d , 11, 294 (1929). Burstein, S., Harejuah, 41,KO.8 (Oct. 16, 1951). Campbell, W. R., J . Bid. Chem., 67,59-69 (1926). Fieser, L. F., and Fieser, AI., "Satural Products Related to Phenanthrene," Xew T o r k , Reinhold Publishing Corp., 1949.
(9)
Martin, CT. J., J . Am. Chem. SOC.,58, 364-5 (1936).
.
RECEIYED for review .January 8, 1952. Accepted December 5 . 1952. ported i n part by U. S. Public Health Grant S o . G-3247(C.).
Sup-
Apparatus for Measuring Gas Permeability of Sheet Materials D.4VID WILLIAM BRLBAKER AND KARL KAM3lERIIIEYER Chemical Engineering, State Unicersity of Iowa, Iowa City, Iowa Gas permeabilities of plastic films are of basic importance in many applications. For this reason, a rapid and convenient method for determining permeabilities of sheet materials to various gases is much needed. An apparatus and procedure for use in determining the permeabilities of sheet materials are presented. The operation of this apparatus is based upon the principle of measuring a change in volume at conditions of constant pressure and temperature. Typical permeability data obtained are included. The significance of the apparatus and procedure lies in its general applicability and in the fact that results may be obtained rapidly and with a high degree of accuracy.
T
HAITplastic materials have liecome of paramount iniportance in a great variety of industrial and domestic applications is illustrated by the use of plastics in the form of film, as \vel1 as of coated cloth and paper, in the packaging industry, in the frozen food industry, and in the manufacture of balloons. I t is natural, therefore, that increasing attention has been given by many laboratories to methods of determining vapor and gas permeability values of sheet materials. Despite keen interest, and the fact that many methods have been developed which allow the vapor transmission rate to be quickly determined, there has been no development of a really satisfactory rapid method of making gas permeability measurements. Barrer ( 2 ) , Cartwright (4), Shunian ( 7 ) , van Amerongen ( I ) , and others have described methods for gas permeability determinations, making use of a vacuum system, in which the pressure, volume, temperature, and time are recorded to perniit calculations of the permeation rate. While these methods :ield accurate results and the equipment is not too elaborate, i n general, the time required to obtain results is rather long. Todd (8) developed an instrument in which the test gas, which was held at 1-atmosphere pressure in a closed chamber, permeated through a test specimen into a relatively large evacuated chamber, The decrease in volume of the test gas was read on a scale along the capillary by observing the movement of a short column of liquid which separated the test gas from the outside air. This slug of liquid also maintained the test ga.: in the capillary at atmospheric pressure. The sensitivity of 'Todd's apparatus has been reported (6) to be greater than that of most other available testing methods, but it is subject to considerahlv greater error due to slight changes in atmospheric pressure and temperature, Davis (6) and others have described an apparatus which makes use of the sweep gas principle. I n this method, separate streams of two gases are passed through a cell in which the gases are separated only by the sheet material under test. The pressures and flow rates of the two gases are maintained by suitable control devices and the effluent stream of each gas is collected and ana-
lyzed for its content of the other gas. I n this appratus small leaks will not have a pronounced effect upon the accuracy of the results as in the case of the equipment of Barrer and Todd. However, several difficulties frequently result from the use of the sweep gas and other dynamic t>pes of permeability apparatus (6). Because large quantities of sweep gas are usually required, many problems in gas regulation are encountered. It is not unusual to observe higher values of the permeability at higher rates of gas flow, resulting in doubt as to the actual permeability values. I n an effort to find a rapid and convenient method for determining the permeabilities of sheet materials to various gases, the authors developed a relatively simple and rugged piece of equipment. The apparatus differs from most of the other Currently used equipment in that it does not require a vacuum seal or precise regulation of the gas flow. Leaks on the high preqsure side of the membrane will cause no error in the permeability determinations. Because the low pressure side of the apparatus is essentially at atmospheric pressure, the occurrence of errors due to leaks is rather remote. I n general, the principle employed in thip apparatu. 1" that of measuring the permeated gas rate a t conditiom of constant teniperature and pressure. CONSTRUCTION AND OPERiTIO\
OF 4PPARATUS
The apparatus shown in Figures 1, 2 . and 3 is conctructed chiefly of steel. I n operation, the plastic film is placpd between two rubber gaskets, which are inserted between two steel flanges. These flanges are held together by means of a screw prcss. It has been found that the rate of permeation through the rubber gasket is negligible, as would be expected, as the ratio of area to thickness is small. The film is supported on mch side hy porous stainless steel disks, which are inserted in the face of ench of the steel flanges, and also act as diffusers for the gas. A few sheets of filter paper or glass fiber mats may be placed hetn een the porous steel and the film to protect the membrane. -450-inch manometer or a 100-pound pressure gage, is uqed to measure the pressure difference across the membrane, and a thermometer serves t o measure the temperature of the surrounding air. This tempera-
