through a small cation exchange column in the hydrogen form to remove the excess ammonia and convert the ammonium sulfate to the acid. The sulfate is then titrated in 80% alcohol with 0.01M barium perchlorate, using thorin as the indicator ( 3 ) . PROCEDURE
Keigh 0.2- to 0.3-gram samples into the macro peroxide fusion cup and add 0.5 gram of powdered sucrose, 0.5 gram of potassium perchlorate, and 12 grams of sodium peroxide. Mix thoroughly with a tiny spatula or \\-ire. Place 3 grams of sodium peroxide on top of the mixture. Assemble, screw tight, and ignite by placing the bomb over the sharp blue tip of a Bunsen flame for 90 seconds. Quench the reaction by immersing in cold distilled water. Open the cup and dissolve the fused contents by laying the cup in a 400-ml. beaker containing sufficient water to cover the cup. Cover with a watch glass and heat to near boiling. \\-lien the melt has dissolved, rinse the cup with mater and remove. K i t h cover glass in place, add sufficient concentrated hydrochloric acid (28 to 30
ml.) to make the solution distinctly acidic. After allowing the carbon dioxide to escape, transfer the solution (with filtration if necessary) to a 500-ml. volumetric flask. Dilute to volume and pipet 50- or 100-ml. portions into 150ml. beakers. Dilute to approximately 125 ml. Pass the solution through the alumina column in the perchlorate form a t the rate of 2 drops per second. Rinse the beaker with a little water and pass this through the column. Rinse the column with 10 ml. of water added in two portions, then place the original 150-ml. beaker beneath the column. Elute the sulfate by passing through the column 5 ml. of 1M ammonia and 20 nil. of 0.1M ammonia added in four portions. Finally, rinse with 10 ml. of water. Pass the eluate through the cation exchange column, and collect in a 100-ml. volumetric flask. Dilute to volume. To IO-ml. portions, add 40 ml. of ethanol and titrate with 0.01M barium perchlorate to the first permanent pink color using a drop each of thorin and methylene blue. The above procedure was applied to the determination of sulfur in a wide variety of organic compounds. The experimental results tabulated in Table VI are the averages of two or more
analyses. The average deviation from the mean was approximately *0.03%. Some of the compounds were very hygroscopic; consequently, it was necessary to establish their purity on the basis of their neutralization equivalent. The separation and titration methods described may also be applied in the determination of sulfur following oxidation of the sample by the Carius procedure, or with nitric and perchloric acids. In the Carius method the alumina column separation may be eliminated if the excess nitric acid is removed by careful evaporation. LITERATURE CITED
( 1 ) Dean, J. A , , ASAL. CHEM.23, 1096 f19.51l \ - - - - ,
(2) Fritz, J. S., Freeland, M. Q., Ibid., 26, 1593 (1954). ( 3 ) Fritz, J. S., Yamamura, S. S., Ibid., 27, 1461 (1955). ( 4 ) Nydahl, F., Ibid., 26,580 (1954). ( 5 ) Nydahl, F., Gustafsson, L., Acta Chem. Scand. 7, 143 (1953).
RECEIVEDfor review March 14, 1956. Accepted October 19, 1956. Contribution from Ames Laboratory, U. S. Atomic Energy Commission.
Color Reaction between 17-Ketosteroids and 3,5Dinitrobenzoic Acid SASSON COHEN and ASHER KALUSZYNER Medical Research laboratories, Medical Corps, Israel Defence Forces, Israel
b A number of dinitro compounds were investigated as substitutes for m-dinitrobenzene in the determination of 17ketosteroids with the view to reducing the interference from urinary chromogens. 3,5-Dinitrobenzoic acid, under controlled conditions, reacts with the more important urinary 17-ketosteroids, giving a color with maximum absorbance a t 550 mp. Interference from urinary chromogens is negligible. When applied to urine extracts, the method gives results 18% lower than the Nathanson-Wilson method, and 8% higher than the Callow method. The method may b e adapted, with advantage, to the quantitative determination of urinary 17-ketosteroids.
U
extracts, prepared by the usual methods for the determination of 17-ketosteroids, contain a nonketonic fraction that possesses a RIXE
significant absorption in the region of nonketonic material by adsorption on 520 mp when treated with m-dinitrocharcoal ( 9 ) . benzene, the reagent of the Zimmermann reaction (19, 20). The use of Girard’s reagent (6, 15) seems to be the most effective means for the removal of the interfering nonketonic fraction, but it does not lend itself to rapid routine work. In the Callow modification (W), a correction may be applied for the interfering chromogens (4, 5 ) ; however, the instability of the ethanolic potassium hydroxide solution used in this method is a major drawback. The Nathanson-Kilson modification (11, 18) of the Holtorff-Koch method ( 7 ) is perhaps the most suitable for routine work, but no valid correction for the 1 2 3 L 5 6 1. DINlTROSEU201C ACID urinary chromogens can be applied (4). Other methods for the elimination of Figure 1. Effect of concentration of interference depend either on the ex- 3,fi-dinitrobenzoic acid (curve A) and traction of the color with an immiscible of potassium hydroxide (curve E ) on color intensity solvent (10, 17) or on the remoyal of the VOL. 29, NO. 1 , JANUARY 1 9 5 7
161
While adhering to the general procedure of the Holtorff-Koch method which excels because of its simplicity, the authors have investigated a number of aromatic dinitro compounds as possible substitutes for m-dinitrobenzene, in the hope of finding a color system less susceptible to interference by the nonketonic fraction. Both 3,5-dinitroanisole and 3,bdinitrophenetole were found to react with androsterone in alkaline medium, giving a rose color reaction that changed very rapidly to purple and brown. Changes in the conditions under which the reaction was carried out failed t o stop the reaction in its first stages and thus made it useless for analytical purposes. 3,5-Dinitrosalicylic acid, used for the microdetermination of reducing sugars (lS),was found to be inadequate, as its potassium salt is intensely yellow and very sparingly soluble in alcohol. 2,4-Dinitro compounds such as 2,4dinitrotoluene and 2,4-dinitroanisole are obviously unsuitable, as they gire, by themselves, intense color reactions with alcoholic alkali.
O5
r
values and the interference of the nonketonic fraction can be kept very low. The absorption maxima (550 mp) and the extinction coefficients are practically identical for all four compounds. Under the same conditions, estrone gives a similar but weaker color and acetophenone an intense brown-red reaction; slight to insignificant color reactions are given by testosterone, progesterone, estradiol, cholestanone, cyclohexanone, cyclopentanone, and ethyl acetoacetate. KOcolor developed when cortisone, benzophenone, stilbestrol, or cholesterol was tested. The evaluation of the reaction with 3,5-dinitrobenzoic acid for the analytical determination of 17-ketosteroids is exemplified for the case of dehydroepiandrost erone . EXPERIMENTAL
Reagents and Apparatus. Absolute methanol. Methanol solutions of 25, 50, and 75 volume yoin distilled water. Dehydroepiandrosterone acetate (Organon) solutions in methanol in concentrations equivalent to 0.20 to 2.00 mg. of dehydroepiandrosterone per rnl. 3,5Dinitrobenzoic acid (Eastman) solutions in methanol, .from 1 t o 6%.
tion in ethyl alcohol, equivalent to 1.00 mg. of dehydroepiandrosterone per ml. Ethanolic potassium hydroxide solution, 2.50 rt 0.02N. 70% aqueous ethyl alcohol. Leitz Rouy Photrometer. Beckman Model B spectrophotometer. General Method. Mix 0.2 ml. of methanolic dehydroepiandrosterone solution, 0.2 ml. of 3,Ei-dinitrobenzoic acid solution, and 0.2 ml. of aqueous potassium hydroxide solution, all measured from 0.2-ml. pipets, in a 10ml. volumetric flask and leave the mixture a t a definite temperature for a knom-n period of time in the dark. At the end of this period, dilute the mixture with the required concentration of methanol, n-ith shaking, to the 10-ml. mark, and measure the intensity of the resulting color in the Photrometer, using filter Yo. 550, after the lapse of 3 to 4 minutes. Blanks have been performed in every experiment by replacing the dehydroepiandrosterone solution with 0.2 ml. of methanol. Every point represents the average of two experiments.
G'5r1 a.
O4
I
-
-
-
-
=
=
:
Figure 2. Effect of incubation time (curve A ) and temperature (curve B) on final color intensity
ANALYTICAL CHEMISTRY
15
20
25
30
O4
Figure 4. Changes in the color intensity subsequent to dilution
t 450
600
550
600
WAVE LENGTH, y
3,5-Dinitrobenzoic acid is known to give a color reaction with acetone (12) and with creatinine (1, 3, 8) in the presence of alkali, and with 17-ketosteroids in the presence of benzyltrimethylammonium hydroxide ( I @ , the absorption maxima lying a t 570, 510, and 530 mp, respectively. I n the presence of aqueous alkali and under suitable conditions, 3,5-dinitrobenzoic acid was found to give a brilliant mauve color reaction with androsterone, epiandrosterone (3p-hydroxy-17-androsterone), etiocholanolone, and dehydroepiandrosterone (3p-hydroxy-5-androsten17-one). By carrying out the reaction in aqueous methanol instead of the conventional ethyl alcohol, both thc 1)Imk
(0
MHUTES
4Q4
162
BLANK
5
ALANK
O.'t
1
Figure 3. Effect of composition of diluent on final color A.
B. C. D.
E.
Absolute methanol 75% Methanol 50% Methanol 2 5 % methanol Distilled water, to 10 ml.
Potassium hydroxide solutions in distilled water, 1 to ION (accuracy within *0.2%)* .4bsolute ethyl alcohol. m-Dinitrobenzene in absolute ethyl alcohol, 2%. This compound had been purified according to Callow ( 2 ) . De'- yclroepiandrosterone acetate solu-
A.
0.2 ml. of 5N potassium hydroxide; incubation at 25' C. for 60 minutes then dilution with 75% methanol to 10 ml.
8. C. D. E.
Dilution with absolute methanol lncubction period for 30 minutes EN potassium hydroxide used 3N pofassium hydroxide used
Effect of Concentration of 3,s-Dinitrobenzoic Acid. Add quantities of 0.2 ml. of 3,5-dinitrobenzoic acid solution from 1 t o 6% t o 0.2 ml. of dehydroepiandrosterone solution (1 mg. per mi.) and 0.2 ml. of 5N potassium hydroxide. Incubate the mixture a t 25" C. for 60 minutes. Then dilute with 75% methanol and measure the intensity of the resulting color in the Photrometer. There is a gradual increase in intensity up to a concentration of 4y0,above which the intensity remains a t the same level (Figure 1). Effect of Concentration of Potas-
sium Hydroxide. Add 0.2 ml. of dehydroepiandrosterone solution (1 mg. per ml.) to 0.2 ml. of 4% dinitrobenzoic acid solution and 0.2 ml. of concentrations of potassium hydroxide from 1 to 10N. Apply the same procedure as above. Below 7 N , the alkali concentration is a critical factor in determining the color intensity. However, it is not practical to work with solutions stronger than 5N, for a t such high concentrations some urine extracts tend to discolor (Figure 1).
Belolv 50% methanol, the presence of water has a marked hypsochromic effect on the color and causes a sharp decrease in its intensity. When the diluent is distilled water, the maxiniiim absorbance is shifted to 510 mp (Figure 3). Maximum intensity is obtained when the diluent is absolute methanol; but fading is very rapid in such a solution (Figure 4), so that the presence of water seems to constitute a stabilizing factor. Changes in Color Intensity Subsequent to Dilution. Maximum color intensity is reached only 3 minutes after dilution of the reaction mixture. It remains at this level for about 2 more minutes and then falls a t a steady rate. The magnitude of the rate of decrease appears to depend on the duration of the incubation period prior to dilution, the composition of the diluent, and the alkali concentration in the mixture. Incubation periods of less than 60 minutes and higher alcohol concentrations in the diluent increase it greatly, increasing alkali concentration only slightly (Figure 4). Calibration Curve at Optimum Conditions. A calibration curve was constructed by treating 0.2 ml. of dehydroepiandrosterone solutions of graded concentrations with 0.2 ml. of 4% dinitrobenzoic acid solution and 0.2 ml. of 5-V potassium hydroxide a t 25OC. for 60 minutes, then diluting with 75% methanol and measuring the color intensity against a blank within 3 to 5 minutes after the dilution step. Beer's law is followed in the range of 0.040 to 0.300 mg. The absorbance for larger quantities tends to be lower than the extrapolation of the curve would indicate. Application to Urine Extracts. Neutral, crude urine extracts were pre-
Effect of Incubation Period. This was investigated by keeping for 15, 30. 45, 60, 90, and 105 minutes the mixture of 0.2 ml. of dehydroepiandrosterone solution (1 mg. per ml.), 0.2 nil. of 49;', dinitrobenzoic acid solution, and 0.2 ml. of 5Ai potassium hydroxide a t 25'. The further procedure followed was as aboye. Above 60 minute.. . prolonged incubation periods do not contribute to any significant increase i n the color intensity (Figure 2 ) . Effect of Incubation Temperature. Allow mixtures of 0.2 ml. of dehydroepiandrosterone solution (1 mg. per ml.), 0.2 ml. of 4% dinitrobenzoic acid solution, and 0.2 ml. of 5 N potassium hydroxide to react in water baths for 60 minutes a t temperatures of 15", 20°, 25", 30". and 35" 0.5" C. Maximum color intensity is attained a t 30". Howerer, a t this temperature some urine estrscts tend to discolor, so that the pIactica1 working temperature is 25' C. (Figure 2 ) . Effect of Alcohol Concentration in Diluent. The mixture of 0.2 ml. of dehydroepiandrosterone solution (1 mg. per ml.), 0.2 ml. of 4% dinitrobenzoic acid solution, and 0.2 ml. of 5 N potassium hydroxide, which had stayed for 60 minutes a t 25' C., was diluted with distilled mater, 2 5 . 50, 75Cr,, and absolute methanol.
Table 1.
Assaying Increasing Aliquots of Neutral, Crude Urine Extracts
Volume
Taken, $11. 0.05
0.10 0.15 0.20
17-Ketosteroid Found, Mg./Ml. of Extract B C D
A
0.48 0.49 0.49 0.46
0.43 0.48 0.46 0.43
Table 11.
Urine Sample
y-17-KS Present
A
48 69 49 74 48
B
C
D
71 46 66
0.46 0.48 0.49 0.47
Av .
0.41 0.46 0.44 0.43
0.4;i 0.48 0.47 0.45
Recovery Experiments
Dehydroepiandrosterone Added Recovered 50 50 50 50
50 50
50 50
Recovery,
%
46 44 47 43 47 42 46 43
Av.
92 88 94 86 94 84 92 86 89.5
'
n,
WAVELENGTH, mp.
Figure 5. A. 6. C.
D.
Absorption curves
0.20 mg. of dehydroepiondrosterone Crude, neutral urine extract Nonketonic fraction b y present method Same nonketonic froction by NathansonWilson method
pared according t o Robbie and Gibson (13). The removal of nonketonic fractions by means of Girard's reagent was carried out as directed by Talbot and coworkers (16). Each extract was conveniently divided into two equal parts, one made up with ethyl alcohol, and the other with methanol, to such a concentration that 1 ml. of the final solution was equivalent to 25 ml. of the original urine. Except Then otherwise stated, 0.2 ml. of extract was taken for the assay and treated as in the procedure described under "calibration curve." The absorption spectra of the nonketonic fraction were determined for a number of samples by the present method and by the Nathanson-Wilson modification of the Holtorff-Koch method. Typical results are shown in Figure 5. Increasing aliquots of the same neutral, crude urine extract were assayed by the present method (Table I). Recovery tests were performed (Table 11). The 17-ketosteroids content of several extracts was determined by the present method, the NathansonWilson modification of the HoltorffKoch method, and the Callow modification of Zimmermann's method (Table 111). In the first two methods, the measured absorbance of the sample was corrected by subtracting the sum of the reagent blank and the sample blank from the observed absorbance. In the third method, use was made of the Gibson and Evelyn correction formula (5:. RESULTS AND DISCUSSION
The extinction coefficient of the more important neutral 17-ketosteroids by the present method does not match with that obtained by the Nathanson-Wilson VOL. 2 9 , NO. 1, JANUARY 1957
163
Table 111. Assaying Neutral, Crude Urine Extracts by Different Methods
Urine Sample il B C
D E
Av.
17-Ketosteroid Found, lIg./lll. Extract A B C 0.43 0.46 0.47 0.43 0.37
0.49 0.53 0.55 0.51 0.47
0.40 0.43 0.43 0.41 0.34
0.43
0.51
0.40
-4.By present method. B. By Nathanson-Wilson method. C. By Callow method, using correction.
method, but still some 8% higher than those obtained by Callow's method, using a correction. Though Beer's law is followed fairly well by crystalline dehydroepiandrosterone acetate, there is a lack of linearity between absorbance and concentration for neutral urine extracts or in recovery experiments. I n order to obtain comparable results, therefore, measurements were made in the transmittancy range of 40 to 60%. *4s in the case of m-dinitrobenzene, the reaction must be carried out under strictly standardized conditions, but gives reproducible and accurate results. ACKNOWLEDGMENT
modification of the Holtorff-Koch method; their ratio is roughly 1 to 2 . On the other hand, the color due to the nonketonic fraction is, in the former method, much lower than in the latter. The observed absorbance via the present method, therefore, would appear to represent the true content of 17-ketosteroids more accurately. Values obtained for neutral extracts by the present method are about 18% lower than those obtained by the Sathanson-Wlson
148.
The authors wish to thank Ernst D. Bergmann for valuable adrice in preparing this report. LITERATURE CITED
(1) Benedict, S. R., Behre, J. A., J . B i d , Chem. 114, 515 (1936). (2) Callow, N. H., Callow, R. K., Emmens, C. IT.,Biochem. J . 32, 1312 (1938). (3) Carr, J. J., A s - 4 ~ .CHEM.25, 1859 (1953).
(4) Engstrom, W. W., Mason, H. L., Endocrinology 33, 229 (1943). (5) Gibson, J. G., 11, Evelyn, K. .4., J . Clin. Invest. 17, 153 (1938). (6) Girard, A., Sandulesco, G., Helv. Chim. Acta 19, 1104 (1936). (7) Holtorff, A. F., Koch, F. C., J . Bzol. Chem. 135, 377 (1940). (8) Langley, W. D., Evans, If., Ibid., 115,333 (1936). (9) Lombardo, ll. E., Viscelli, T. rl., Mittelman, A., Hudson, P. B., Ibid., 212, 353 (1955). (10) Lfasuda, bl., Thuline, H. C., J . Clzn. Endocrinol. and Xetabolism 13, 581 (1953). (11) Nathanson, I. T., Wilson, H., Endocrinology 33, 189 (1943). (12) Porter, C. C., ANAL.CHEW27, SO5 (1955). (13) Robbie, TV. A , Gibson, R. B., J . Clin.Endocrznol. 3, 200 (1943). (14) Sumner, J. B., J . B i d . Chem. 47, 5 ( 1921) . (15) Talbot, X. B., Butler, A. X., Maclachlan, E., Zbid., 132, 595 (1940). (16) Tasney, R. P., Cross, J. hl., J . A m . Pharm. Assoc. 39, 660 (1950). (17) Werbin, H., Ong, S., ANAL.CHEM.26, 762 (1954). (18) Wilson, H., Nathanson, I. T., Endocrinology 37, 208 (1945). (19) Zimmermann, W., Hoppe-Seyler's 2.physiol. Chem. 233, 257 (1935). (20) Zbid., 245, 47 (1936).
RECEIVED for review March 17, 1955. ACcepted July 16, 1956.
Dirubidium Uranyl Tetranitrate, Rb,UO,(NO,),
EUGENE STARITZKY and DONALD 1. WALKER', The Universify of California, los Alamos Scientific laborafory, los Alamos, N. M.
'
of dirubidium uranyl tetranitrate have been prepared by allowing an aqueous solution containing rubidium nitrate and uranyl nitrate in the molar ratio 5 to 1 to evaporate a t room temperature with agitation. RTSTALS
CRYSTAL MORPHOLOGY System and Class. Alonoclinic, prismatic. Axial Elements. a:b:c = 0.8224:l: 1.627; fl = 108" 11'.
1 Present address, Department of Chemistry, University of Colorado, Boulder, Colo.
164
ANALYTICAL CHEMISTRY
Crystal Habit. Tabular { i O Z ) with prominent { i l l } and less prominent { 100 1, { O O l ) , { O l l ] , and (1111. Polar Angles. (lOO)A(011) = 71'49'; (001) A (102) = 53' 40'; ( i i i ) A ( i i i )= 102' 16'; (001) A (011) = 5i' 12' (calcd. 57" 6'); (001) A (111) = 56' 32' (calcd. 56" 24'); (001) A ( i i i ) = 81' 5' (calcd. 81' 4').
x-R.4~DIFFRACTION D~TA Space Group. P2Jc ( G ) . Cell Dimensions. ao = 6.47 -4.;bo = 7.90 *4.;co = 12.84 A.; p = 108.2'; ao:bo:co = 0.819:1:1.625.
Formula Units per Cell. 2; volume per formula unit 312 A.3.
Figure 1. Orthographic projections of crystal of dirubidium uranyl tetranitrate on (102) and parallel to b
