V O L U M E 2 3 , NO. 6, J U N E 1 9 5 1
925
flask and diluted to approximately 150 nil. with distilled water. Sixty milliliters of 10 AT potassium hydroxide and 20 ml. of the Rochelle salt solution are mixed together and added as rapidly as possible to the flask, which is immediately connected to a trap and condenser. The condenser adapter extends below the surface of a measured amount (about 25 ml.) of standard 0.1 N hydrochloric acid in the receiver. After distillation of about 150 ml. of the flask contents, the condenser is rinsed, the receiver is removed, and the excess hydrochloric acid is t,itrated with standard base, using methyl purple indicator. When the Rochelle salt solution and potassium hydroxide are added, the formation of the blue copper tartrate complex serves to indicate that the contents are alkaline. Shortly thereafter, some copper(1) oxide will be formed from the reaction with the residual hydrazine. If a large excess of potassium hydroxide is not present, the contents of the flask will foam seriously during the distillation. Blanks should be run using the reagents and ammoniafree hydrazine. A determination requires about 2 hours to run exclusive of cooling time. It was usually found convenient to precipitate the samples and allow them to remain in the ice box overnight.
Table 11. Blank Determinations Series A" Series B Reagents Reagents NIHI.HIGO~ NIIr NZHI.H~SOINE8 Gram MQ. Gram MQ. 1.0000 0.22 1.0000 0.37
1.0000 1.0000 1.0000 Av.
0.24 0.19 0.26
0.23
1.0000 1.0000 1.0000 477.
0.37 0.26 0.30 0.33
aeries C Reagents
NzHi
NHI
Gram
Mg.
0.27 0.28 0.27 0.32 Av. 0.28
0.4422 0.3919 0.4187 0,3154
Series D Reagents NaH4 NHI Gram Mg. 0.3443 0.16 0.3895 0.24 0.3229 0.08 0.4115 0.28 A v . 0.19
Seriea refer to different lots of reagents.
are shown i n Table 11. If the blanks are higher than 0.5 mg. of ammonia, it is well to select new reagents. ACKNOWLEDGMENT
The author wishes to express her thanks to D. S. Villara, rvsearch associate, for assistance with the statistical analysis of the data obtained and to E. St. Clair Gantz, under whose supervision this work was completed. This paper is published with the permission of L. T. E. Thonipson, technical director of the U. S. Kava1 Ordnance Test Station.
RESULTS
Table I shows the results of the determination of known amounts of ammonia in synthetic samples. The first series of samples was made from weighed amounts of hydrazine sulfate with ammonia added in the form of a standard solution. The second series was made from weighed amounts of commercial anhydrous hydrazine and a standard solution of ammonia. From Table I, the least significant difference for a mean of two determinations is calculated to be +0.03%. Results of the blank determinations for several lots of reagents
LITERATURE CITED
(1) Bray, R. C., and Cuy, E. J., J . Am. Chem. SOC.,46,858 (1924).
(2) Browne, 8 . W., and Shetteily, F. F.,Ibid., 31,783 (1909). (3) Curtius, T., and Schrader, F., J. prakt. Chem., 50, 311 (1894). (4) Fuchs, W., and Nissel, F., B o . , 60B,209 (1927). (5) Milligan, L. H., J . Phys. Chem., 28, 544 (1924). (6) Penneman, R. A,, and Audrieth, L. F.. ANAL. CHEM.,20, 105s
(1948). RECEIVIDD July 24, 1950. Presented before the Division of Analytical CBeMIcaL SOCIETY, Chemistry a t the 118th Meeting of the AMERICAN Chicago, Ill.
Spectrophotometric Determination of Dihydrostreptomycin DOROTHY J. HISCOX Laboratory of Hygiene, Department of National Health and Welfare, Ottawa, Canada
TREPTOMYCIN may be determined spectrophotometrically by measuring the maltol ( 4 ) formed by alkaline hydrolysis in the visual ( 1 , b) or ultraviolet (6) range of the spectrum. In this laboratory it is estimated by measuring the absorbance a t 275 and 290 mp of a solution of streptomycin, 1 N in sodium hydroxide, \vhich has been heated for 3 minutes a t 100" C. and immediately neutralized (unpublished). Dihydrostreptomycin does not give the maltol reaction ( 3 ) . I n the ultraviolet region it, shows only end absorption even after prolonged treatment with alkali. When streptomycin is heated in acid solution, its ultraviolet spectrogram, as determined using the Beckman DU quartz spectrophotometer, shows absorption maxima a t 245 and 315 mp with a minimum a t 285 mp. When dihydrostreptomycin is treated similarly, a single absorption maximum appears a t 265 mp. The extent of these maxima depends upon the normality of th(3 acid and the heating time employed. On refluxing solutions of streptomycin sulfate or the calcium chloride complex which are nornit31 in sulfuric acid, the maxima show their greatest absorbance after 30 minutes' treatment. On mor(>prolonged heat.ing the maxiniuni :it 315 mp decreases rapidly, while that at 245 mp broadens but does not decrease significantly. When a similar solution of dihydrostreptomycin is refluxed, the maximum at 265 mp requires 1.5to 2 hours to reach it? greatest value. .ifter 30 minutes' refluxing another absorption peak begins to appear a t 220 mp. Its position shifts to longer wave lengths and its absorbance increases as the refluxing time is lengthened. After 2 hours its position is a t 227.5 mp and it absorbance is greater than that of the 265 inp maximum.
To determine if use could be made of the maximum a t 265 m,u for the quantitative estimation of dihydrostreptomycin with the mode of heating changed from refluxing on a sand bath to heating in boiling water, solutions in which the concentration of sulfuric acid varied from 0.25 to 5 S were heated from 30 minutes to 2 hours. K h e n a heating time of 2 hours was used for a solution of dihydrostreptomycin in 0.25 A' sulfuric acid, the absorbance wad as great as in a 1 N solution refluxed for 2 hours. The seeondarj absorption peak did not appear under these conditions. Figure 1 shows the absorption spectra of solutions of streptomycin sulfate and dihydrostreptomycin in 0.25 N sulfuric acid which have been heated in boiling water for 2 hours. It was necessary to dilute the solutions before taking readings in the Beckman spectrophotometer. The concentration of acid in the final solutions was 0.06 Ar, I t is evident that thr quantitative estimation of dihydrostreptomycxiri is possible. For the tlrtermination of dihydrostreptomycin the following procedure is uqed in this laboratory: In a 25 X 200 mm. test tube place an aqueous aliquot contairiing 1 to 3 mg. of dihydrostreptomycin. Make to a volume of 3 ml. with water. Add 3 ml. of 0.5 N sulfuric acid. Insert a foilwrapped rubber stopper through which a 12-inch (30-cm.) piece of glass tubing has been passed to serve as an air condenser. Heat the tube in boiling water for 2 hours. Cool. Transfer the contents to a 23-ml. volumetric flask and make to volume with water. Measure the absorbance of the solution a t 265 and 380 mp. The difference in absorbance is proportional to the dihydrostreptomycin present.
ANALYTICAL CHEMISTRY
'924
With seventy-five aliquots containing 0.25 to 4 mg. of dihydrostreptomycin a regression line was established using this procedure. The equation of the line was Y = 104.011847 X - 0.4236 where X is the difference in absorbance and Y the micrograms of dihydrostreptomycin in 1 ml. of the h a 1 solution. The correlation coefficient was +0.9998 and the standard error of estimation 10.97.
Y,
, WAVELENGTH
Figure 1. Spectrograms of Streptomycin Sulfate and Dihydrostreptomycin Heated in 0.25 N HzSO4 for 2 hours in boiling water Dihydrostreptomycin. 30 micrograms/ml. 0.06
NHkKh
Dihydrostreptomycin. 90 micrograms/ml. 0.06
NHYSO~
Streptomycin sulfate.
90 microgramslml. 0.06
NHgSO4
Comparison of Methods for Determination of Dihydrostreptomycin
Table I. Sample 54550 53827 54281 55278 50207 50323 50419 50381 55626 5038 5040 5072 50317 50318 50327 55245 55477 51051 51825 50830 50256 5020 55936 50513
Potency, Micrograms/hlg. Bioassay Ultraviolet 702 733 685 701 736 697 690 751 700 696 741 742 668 676 775 760 739 746 766 772 807 798 826 793 811 798 770 792 793 741 736 752 755 73 1 712 733 696 737 764 834 742 696 770 791 756 790 710 697
~ l t ~ ~ Ls ~ . i ~ l ~ t of Bioassay 104.4 97.7 94.7 108.8 100.6 99.9 98.8 98.1 100.9 100.9 98.9 96.1 101.6 97.2 93.4 102.2 103.3 97.1 94.4 91.6 106.6 97.3 95.7 101.9 Mean 99.5
tivrty of the method is less A reading at 235 mp is useful as N check on the identity of the compound. In Table I results by the ultraviolet assay are compared with those by the bioassay on twenty-four commercial samples of dihydrostreptomycin. There is no significant difference between the potencies obtained by the two methods, as the t value is 1.050. The fourth column of the table lists the ultraviolet determinations as per cent of the bioassay. The bioassay figures mere determined by Miss K. Fitzpatrick. LITERATURE CITED
Although one reading must be made a t 265 mg, there is a choice of wave lengths for the second reading; 380 mp was chosen a8 being well within the horizontal part of the curve and yet not too close to the upper limit of the ultraviolet range. Readings may be made a t 235 and 265 mp for the quantitative determination of dihydrostreptomycin, but the difference in absorbance is not as great as with the two wave lengths chosen and the sensi-
(1) Boxer, G. E., Jelinek, V. C., and Leghorn, P. M.,J . B i d . Chem., 169, 153 (1947). (2) Eisenman, W., and Bricker, C. E., ANAL.CHEM.,21, 1507 (1949). (3) Peck. R. L., Hoffhine, C. E., and Folkers, K., J . $m. Chem. soc.. 68, 1390 (1946). (4) Schenk, J. R., and Spielman, hf. A , , Ihid., 67, 2277 (1945). (5) Titus, E., and Fried, J., J . B i d . Chem., 174, 57 (1948).
RECEIVED September 15, 1950
Quantitative Test for Nornicotine LOUIS FEINSTEIN AND EDWARD T . MCCABE Bureau of Entomology and Plant Quarantine, U. S. Department of Agriculture, Beltscille, Md.
NEW quantitative test has been found and developed for the Nicotine fails to give any similar color even when taken in amounts of 80 times that of nornicotine. Anabasine develops a color only 80% as intense as nornicotine Fvhen taken in an amount 80 times that of the nornicot rnr Synthetic and natural nornicotines react quantitatively like cach other. I t 15 as observed that nornicotine produced a very intense violet color when added to an acetone solution of l,3-diketohydrindene if diipopropyl ketone was also present (1). The violet color, howwer, failed to appear with the use of a new batch of diisopropyl ltrtonc~. The old bottle of ketone had a cork stopper and an experirnc,nt was set up to extract a new cork with the neiv ketone. This cork extract caused the violet color to appear with nornicotine and the acetone solution of 1,3-diketohydrindene. Other experiments showed that tannic acid, gallic acid, or p-hydrouytmizoic acid could be used to make the new ketone reactive. The present method employs as reagents, acetone, diisopropyl bctone, p-hydroxybenzoic acid, and 1,3-diketohydrindene. Us-
A Nicotiana alkaloid, nornicotine.
40 42 44 46 40 50 52 54 56 58 60 62 64 66 6 8 7C FILTER NUMBER
Figure 1. Determination of Nornicotine
