1324
A N A L Y T I C A L CHEMISTRY
Table I.
Solubility of Nitrous Oxide in Nitrogen Dioxide
Solution Temp., K . 263.0 263.1 263.1 268.0 268.0 273.2 273.0 278.2 278.4 283.0 283.1 283.1
Partial Pressure of K20t hlrn. 620 630 630 570 570 510 510 440 430 370
Purity of
so*.
6:
98 3 99 0 97 8 98 0 97.8 98 4 97 8 98 0 97 8 97.8 97.9 98.0
% Solubility by Weight 1.28 1.33 1.52 1.21 1.27 1.11 1,04 0.81 0.82 0.65 0.59 0.63
350 340
Table 11. Freezing-Point Depressions % NzO
Af Observed
k
0.51 0.70 0.81 1.21 1.52
0.45 0.66 0.74 1.12 1.31
3900 4200 4000 4100 3800
Calculated from Heat of Fusion
k Max. 0.50 0.80 1.20
0.48 0.77 1.15
4230 4230 4230
k iMin. 0.50 0.80 1.20
0.42 0.67 1 .oo
3660 3660 3660
results obtained by saturation a t a slightly lower temperature and subsequent heating to the desired temperature. If any supersaturation occurred, the amount of nitrous oxide dissolved in escess of that required for saturation was less than the experimental error. Consequently, the more rapid method as desciibed was adopted. Table I1 gives the freezing point depression data. From the published values for the heat of fusion of nitrogen dioxide (Z), which ranged from 32.2 to 37.2 cal., the spread of values for the molal freezing point depression constant v a s calculated (Table 11). The resulting curves of freezing point depression are plotted in Figure 3 for compaiison with the curve obtained in this investigation. As the experimental curve falls betmeen the curves calculated from the heat of fusion, it may be assumed that
O M
Figure 3.
ow AI
-
om
I W
I20
1.0
I(*
FREEZING POINT DEPRESSION OK
Freezing Point Depression for Nitrous Oxide in Nitrogen Dioxide
Broken lines show freezing point depression charges calculated from highest and lowest heat of fusion values reported in literature
no compound formation takes place. The average value for the molal freezing point depression constant is 4000. LITERATURE CITED (1)
Oiauque, W. F., and Kemp, J. D., J . Chem. P h y s . , 6, 40-52
(2)
Mellor, J. K., “Comprehensive Treatise on Inorganic and Theoretical Chemistry,” London, Longmans, Green & Co.,
(3)
Wright. R. H., and Rlsass, O., Can. J . Research, 6, 94-102
(1938).
1928.
(1 932).
RECEIVED for review February
9. 1952 Accepted June 9 , 1952. Presented at a Symposium on the Practical Factors Affecting the Application of Kitric Acid and Mixed Oxides of Kitrogen as Liquid Rocket Oxidizers, at the Pentagon, Washington. D. C., October 10, 1951.
Colorimetric Method for Estimation of Digitoxin E. L. PRATT Kinthrop-Steurns Inc., Rensselaer,
F
OR many years pharmacologists used the biologic assay as a means of determining t h e potency of digitalis preparations. MIth the extended use of crystalline digitoxin, demands have arisen for a chemical method to supersede the older assays. One of the careful studies of the subject has been macle by Canback ( 8 ) ,who used the Raymond ( 1 2 ) reaction involving ail alcohol solution of rn-dinitrobenzene and sodium hydroxide. A variation of this method u a s described by Anderson and Cheii (1). h similar method wing sodium dinitrobenzoate % a s suggested by Kedde ( 7 , Y). Other methods were proposed by \\ urren, Hornland, and Green (IO),McChesney and colleagues (IO),and finally by Bell and Krantz ( S X ) , who modified the Baljet (9)reaction. This last assay has been included in the XIVth revision of the E. S. Pharmacopeia (15). Various laboratories which have used the C.S.P. XIV (15) assay for digitoxin have found difficulty with the reproducibility (13) and nonspecificitj- ( 1 7 ) of the method, the instability ( 1 7 ) of the reagent, and
iV. Y.
the proximity of the maxima of the picrate reagent and the picrate-digitoxin complex ( 7 ) . A method t h a t includes action a t room temperature, simplicity, reproducibility by different operators, and specificity for digitoxin in the presence of similar compounds, especially gitoxin, might well be considered as approaching the ideal. The method described herein satisfies the first three of the above desiderata and produces approximately 50% as much color for gitoxin as i 3 produced by a n equimolar amount of digitoxin. About 85% as much color is given by digoxin, but the latter is not usually found with digitoxin. This method, chosen after the experiments described below, is based on the combination of 3,5-dinitrobenzoic acid and benzyltrimethylammonium hydroxide, reagents which were recently proposed by Tansey and Cross ( 1 4 ) for the assajof certain ketosteroids. With digitoxin the resultant color iz a bluish-red u-ith a maximum at 5?0 mp. Canback (8) has discussed a reaction mechanism for the
V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2
1325
This work was undertaken to obtain a more satisfactory and easily applied method for the assay of digitoxin than has been found in the literature. The reagents, 3,s-dinitrobenzoic acid and benzyltrimethylammonium hydroxide, react w-ith digitoxin in dilute ethj-l alcohol to produce a bluish-red colored solution which has a maximum absorbancy at 550 mfi. The procedure is also applicable in the estimation of digitoxin extracted from tablets. Analytical data are both simply obtained and highly reproducible. These factors enhance the quality control of an extremely toxic substance.
chromophore obtained with na-dinitrobenzene and strong alkali in his comprehensive study of this subject. I n the light of Mulliken's ( 1 1 ) recent article on "Molecular Compounds and Their Spectra," the mechanism of chromophore formation betn-een digitoxin and a polynitroaromatic compound would best be described as a loose combination between a x base, the @-substituted butenolide group of digitoxin, and a T acid, the nitroaromatic. This combination cannot be represented diagrammatically without oversimplification of a. very complex resonance system. The chromophore resulting from t'he combination of the p-substituted butenolide group and a given K arid is primarily influenced by the concentration of hydroxyl ion. The concentration must be such t h a t t h e combination of the x acid and x base can be initiated and promot'ed at a faster rate than the system can be destroyed by hydrolysis effects. The selection of the base, benzyltrimethylammonium hydroxide, and the T acid, 3,5-dinitrobenzoic acid, has been made primarily from the results of rate curves obtained a t room temperature. Similar studies have been made with various comhinationa of the bases and acids bed: Bases Potassium hydroxide (207, in 477, ethyl alcohol)
H Acids m-Dinitrobenzene
Benayltrimethylammoniuni hydroxide (40% in water)
3,5-Dinitrobenzoic acid
Pyridine (reagent grade)
nz-Xitrocinnamic acid p-Xitrocinnamic acid
Benzyltrimethylammoniuni
hydroxide (35% in rnethanol) Choline (50% in water) Diethylamine Diethanolamine Tetraethanolammoniurn hydroxide (40% in water 1 Tetraethylammonium hydroxide (10% in water) Ammonia 28% (reagent) Tetra-n-propylammonium hydroxide (10% in waterj
bidity will develop when the alcohol concentration exceeds 80 volunie 70. A solution of approximately 60 volume Yo ethyl alcohol was chosen as the working solvent. The factor of specificity is of importance. Curves I and 11 of Figure 2 represent the spectra of digitoxin and gitoxigenin. Each point represents a separate color reactiori. The molecular extinction value is calculated from the absorbancy a t maximum intensity. This value is reached approximately 160 seconds after the addition of the base to the digitoxin-3,5-dinitrobenzoic, acid solution in ethyl alcohol. Figure 1 represents the molecular estinction versus time curves for digitoxin, digoxin, and gitoxigenin. The rate curves for digitoxin and digoxin both reach a maximum at approximately the same time. Digoxin gives a molecular extinction equal to 85y0 of t h a t given by digitoxin. Gitoxigenin reaches its maximum about 60 seconds after the addition of the base and then begins to decrease in sbsorbancsy. At 160 seconds, t>heapproxi-
3,5-Dinitrosalicylic acid
Colored reaction products are not obtained with digitoxin and t h e various bases when the aromatic nitro compound used is 3,sdinitrosalicylic acid, m-nitrocinnamic acid, or p-nitrocinnamir acid. ttz-Dinitrobenzene combines with digitoxin in the prebence of t h e bases listed, t o give a chromophore t h a t displays a negative rate curve. \Vith digitoxin and 3,5-dinitrobenzoic acid, no colored reaction product is obtained with pyridine, diethanolamine, or ammonia. Diethylamine (without digitoxin) reacts with 3,5-dinitrobenzoic acid to give a red chromophore. 411 the other quaternary bases \I it h digitoxin and 3,5-dinitrobenzoic acid give chromophore foimation of different degrees of intensity. The reaction uhen carried out with 20% potassium hydroxide in 47% ethyl alcohol and 3,5-dinitrobenzoic acid produces, in the presence of digitoxin, a negative rate curve. Choline compares favorably a ith benayltrimethylammonium hydroxide, the rate curve3 of R hich with various glycosides are shown in Figure 1. The physical nature of choline-namely. high viscosity in 50% solution-makes it less desirable to use. The concentration of alcohol is not critical, however, a tur-
$1
I
I
Figure 1.
l'i,l
I
4-4
I
BO
I
I
20 /do 7-//YE,SECO NOS
I
2w
I
I
244
Rate Curves of Digitoxin, Digoxin, and Gitoxigenin
-
10
D . D / G / TO X / N
- G'G/MX/GfN/N
7
Px
5
z5 8-
$6-
E l2$ 9-'
+ k
c 2, %
$ r ?
I
Figure 2.
I
360
I
I
I
I
I
I
440 480 W AV6d €NG 7 , Mu
CUU
I
I
SZO
I
I
560
Rate Curves of Digitoxin and Gitoxigenin
ANALYTICAL CHEMISTRY
1326 mate maximum absorption time for digitoxin, gitoxigenin contributes 5+lyOas much color as digitoxin on a molar basis. Gitoxin is a common impurity in digitoxin, For this reason it is highly desirable to use a method of assay which will minimize the response due to gitosin when this is present.
Determine the maximum absorbancy of the unknom-n solution in the same manner as outlined for the standard. The per cent digitoxin is obtained as follows:
% ' digitoxin
=
maximum absorbancy of unknown maximurn absorbancy of standard
x
100
For the purpose of evaluating the reproducibility of the foregoing procedure in the hands of different operators, the data i,eported in Table I were obtained on various digitoxin samples. DIGITOXIN TABLETS
.I study of the many methods proposed for the extraction of digitoxin from tablets has shoan these to be either laborious or inefficient or both. Described belon- is a procedure of extraction which, v-hen used with t.he niet'hod for analysis of digitoxin outlined above, has proved satisfactory in the assay of digitoxin tablets (Table JI). CdNCfNTRAA/QN(MG//Z Mf1
Figure 3.
EXTRACTION PROCEDURE
Absorbancy of Digitoxin
Figure 3 shows the conformation to Beer's law of the 3,5dinitrobenzoic acid-benzyltrimethylammonium hydroxide digitoxin procedure. It also gives the range of conrentration of digitoxin over which this method may he used. RE4GEhTS
Ethyl alcohol, 9570. Ethyl alcohol, 47% (1 part of ethyl alcohol-1 part of distilled mater). Benzyltrimethylammonium hydroxide, 40 % aqueous, Eaqtman Kodak. 3,5-Dinitrobenzoic acid, Eastman Kodak i 1% In ethx 1 alcohol). PROCEDURE
Weigh 15 0 mg. of U.S.P. reference standard dlgitoxin and transfer t o a 100-ml. volumetric flask. Dissolve the standard in 9570 ethyl alcohol and make to volume. Prepare an ethyl alcohol solution of the digitoxin sample to be tested as directed for the reference standard. Transfer 3.0 ml. (0.45 mg.) of the standard and unknoan solutions, respectively, t o each of t n o glass-stoppered test tubes. T o each tube add 5.0 ml. of 47Y0 ethyl alcohol and 2.0 ml. of the 3,5-dinitrobenzoic acid solution. Prepare a blank by transferring 3.0 ml of 9570 ethyl alcohol, 5.0 ml. of 47% ethyl alcohol, and 2.0 ml. of the 3,5-dinitrobenzoic w i d solution to a separate glass-stoppered tube. Add 2.0 ml. of the benzyltrimethylammonium hydroxide to the blank tube, then stopper. Mix contents by shaking three or four times and transfer t o a 1-cm. cell or cuvette. With blank solution in light path, set the instrument ( A direct reading spectrophotometer of the Model B Beckman type is excellent for use in this test. However, any spectrophotometer or colorimeter with a narrow band filter having a maximum transmittance at 550 mp should be suitable for use) a t 0.0 absorbancy a t 550 mp. iZdd 2.0 ml. of the benzyltrimethylammonium hydroxide to the standard and treat as outlined above for the blank. Place mixed solution in instrument and determme the maximum absorbancy of this solution.
Table I.
Reproducibility of Data % "f
Weigh a counted number of not less than 10 digitoxin tablets and reduce them to a fine poR-der. Transfer t o a ground-glassstoppered tube (Figure 4) a portion of the powdered tablets equivalent to 3.75 mg. of digitoxin. Quantitatively transfer 25.0 ml. of 95% ethyl alcohol t o the tube containing the weighed mixture and then stopper. Place the stoppered tube in a shaking machine regulated to oscillate approximately 250 times per minute. Orient this so that the long axis of the tube is parallel t,o the path of oscillation. Permit the shaking operation to continue for 1 hour. Transfer the closed tubes to a centrifuge :tnd operate a t 2000 r.p.m. for 15 minute...
27 mm.
90mm.
18mm.
Transfer 3.0 ml. (0.45 mg. of digitoxin) of the standard and 3.0 ml. of the supernatant extract, respectively, t o each of two glass-stoppered test tubes. Continue procedure as directed for digitoxin above, beginning with ,'To each tube add 5.0 ml. of +li70 et,hyl alcohol. . . '' ACKNOW-LEDGMENT
The samples of the glycosides used in this study were U.S.P. reference standards, Tit'h the exception of those listed in Table I and the sample of gitoxigenin. The gitoxigenin sample waa kindly furnished by E. K. IIcChesney, Sterling-Winthrop Research Institute. The initial supply of benzyItrimeth~-lammoniun~ hydroxide in methanol was very generously furnished hg D. L. Felley, Rohm & Haas Co., Philadelphia. Acknowledgment and thanks are accorded to M. E . Auerbach, Sterling-JVinthrop Research Institute, E. L. Bauer, W i n t h r o p Stearns Inc., and Ilse lIemelsdorff, Xnthrop-Stearns Inc., for their assistance in obtaining the collaborative data tabulated above. Thanks are also extended t o Hugh H. Corbitt, Winthrop-
Table 11. 2
c
I I I I1
C
I1
4
C C
111 I I I11
5
A C C
D
D
1 1
3
1 1 5
Beckman Model B Beckman Model B Beckman Model B Fisher electrophotometer Fisher electrophotorneter
6 8 5
964 84.3 853
4
84 9 )
4
85.2
Lumetron Model 401 Beckman Model D U Beckman Model B Lumetron Model 401
2 3
0.6
4
87.1 87.8 103.5
2
103.5
0.3
0 4 1 1 1 1
i 0.9 0.2
7 mm.
Figure 4. Modified Centrifuge Tube
Results of Tablet Assay
(Data obtained by one operator relative to U.S.P. reference standard digitoxin) yo of Estimated Std Digitoxin, ExtracNO.of Lot Theory, tion Detns. per Theory Denation ?io. hfg./lOO M g . Xo. Extraction round, Av. Subgroup Av. 20 0.2 I 3 99.4 0.50 3 99.3 0.22 0.2 I1 20 0.65 0.40 20 0.2 111 3 98.4 13 0.1 I 3 94.9 0.17 3 93.1 0.12 0.1 I1 13 0.81 1.27 I11 3 96.7 13 0.1
V O L U M E 2 4 , NO. 8, A U G U S T 1 9 5 2
1327
Stearne Inc., and Elmer J. Lawson, Sterling-Winthrop Research Institute, for their helpful criticism and suggestions in the preparation of this paper. LITERATURE CITED
(1) Anderson, R. C.. and Chen. K. K.. J . A m . Pharm. Asaoc.. Sci. Ed., 35,353 (1946) (2) Baljet, H., Schweiz. Apoth.-Ztg., 56, 71. 89 (1918). (3) Bell, F. K.. and Krantz, J. C., Jr., J . A m . Pharm. Assoc., Sci. Ed., 37,297 (1948). (4) Bell, F. K., and Krantz. J. C.. .Jr.. b. Pharmucol. Etptl. Therap., 83.213 (1945). (5) Ibid.;87,198 (1946). (6) Ibid., 88, 14 (1946). (7) Canback, T.. J . Pharm. Phormacol., 1, 201 (1949).
(8)Canback, T.. Suensk Farm. Tid., 54, 201 (1950). (9) Kedde, “Bijdrage tot ket chemisch Onderzock van Digitalispreparaten,” dissertation, Leiden, 1946. (10) McChesney, E. R., et al., J . Am. Pharm. Assoc., Sei. Ed., 37, 364 (1 . 948). . (11) Mulliken, R. S., J. Am. Chem. Soc., 74,811 (1952). (12) Raymond, W. D., Analyst. 63, 478 (1938). (13) Rice, W., Chairman, Contact Committee on Digitoxin, Eli Lilly & Co., Indianapolis, Ind., private communication. (14) Tansey, R . P., and Cross, J. JI., J . A m . Pharm Assoc., Sci. Ed., 39, 660 (1950). (15) United States Pharmacopeia, XIVth ed., p. 180, 1950. (16) Warren, 4.T., Hon-land,F. 0..and Green, L. W., .I. A m . Phnrm. Assoc., Sci. Ed., 37, 186 (1948). (17) Winthrop-Stearns Inc., unpublished reports. RECEIVED for revieiv l l a r c h 6, 1952.
Accepted June 7 1952.
Classifying Butyl Rubber with Respect t o Modulus A Chemical Method L. L. CURRIE Butyl Control Laboratory, Polymer Corp., Ltd., Sarnia, Canada The stress-strain method of determining the modulus of Butyl rubber possesses severe shortcomings when applied to plant control, the most serious being the time lag in reporting reliable results. A chemical method is described which is less time-consuming and at the same time more reproducible. The basic iodine-mercuric acetate method for Butyl unsaturation has been improved by the standardization of reaction conditions, and a correlation between unsaturation and stress strain values in the National Bureau of Standards formula has been developed. This comprehensive study has covered commercial grades of Butyl rubber over an 18-month period. The application of the method to plant control is described, and improved product uniformity is indicated. Reliable results suitable for control purposes are available in 3 to 4 hours.
I
N THE commercial production of Butyl rubber, the deter-
mination of the modulus is of paramount importance. In addition to being the most important product specification, this curing characteristic is of primary concern to both the plant operatoi and the rubber processor. Since the inauguration of the synthetic rubber program, the modulus level has been determined by the stress-strain method of compounding, curing, and testing sample specimens of the finished polymer. In spite of numerous improvements ( 8 ) , the stress-etrain procedure is time-consuming and unreliable. Mill roll and press temperatures, humidity conditions, and chemicals are variables that require constant attention. The most serious shortcoming is the 24-hour time lag between the time of sampling and the reporting of a result. Consequently, stress-strain testing has been of little value in the control of Butyl rubber production. For maximum product uniformity, a rapid reliable method for evaluating the effect of plant variables is required. This paper deals with the development of a chemical method for predicting modulus and its adoption for plant control purposes. This study has been based on the testing of the raw isoprene Butylpolymers listed in Table I, and its application to vulcanized stocks would require further work. Although all the Butyl polymers listed in Table I possess low unsaturation compared to natural rubber, i t has been shown (9) that each possesses specific properties depending on the amount of diolefin present. The properties chiefly affected are rate of vulcanization and the characteristics of the stress-strain curve. In developing a chemical method for predicting the rate of vulcanization, it has been necessary to improve the reproduGibility of the method for determining the unsaturation content of
Butyl rubber and to correlate the unsaturation values with the modulus values as measured by stress-strain testing. HISTORY
Iodine chloride ( 3 , 7 ) has been the principal reagent used for the determination of the unsaturation of Butyl polymers. Rehner (6) developed a satisfactory but time-consuming method based on the limiting viscosity of the polymer solution after degradation by ozone. Gallo, Wiese, and Selson ( 1 ) introduced a procedure involving a reaction between the pol-mer and iodine in the presence of mercuric acetate and trichloroacetic acid. This method is a rapid, reproducible test for determining the relative unsaturation of raw isoprene-isobutylene polymers and with slight modification was used in this study. PROCEDURE
A 5.OC-gram sample of the polymer weighed on an analytical balance is cut into small pieces and placed in 500 to 600 ml. of Table I.
Commercial Butyl Grades
hlooney Range 40-Minute Feed (ML 8 Minutes Modulus Range. Commercial Stock” at 212’ F.) Lb./Sq. Inch Grades B-1. O 40-50 ,.... PB. 100, GRI-R-2 B-2.0 40-50 876-1 125 PB.200, GRI-50 B-3.0 40-50 1125-1375 P B . 300, GRI-Y-15 B-2.5 70-80 1125-1375 PB.301, GRI-18 B-4.0 40-50 1326-1575 P B . 400, GRI-Y-25 a B-1.0 represents a mixture of 99% isobutylene a n d 1% isoprene, B-2.0 represents a mixture of 98% isobutylene and 2% isoprene, eto.
