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INDUSTRIAL A N D ENGINEERING CHEMISTRY
centrations of orthophosphate. In 0.0001 M phosphate the accuracy was of the order of 4 per cent. Alkaline earth phosphates can be titrated by the standard procedure. Calcium in large amounts, iron, and organic anions interfere. Methods are described to eliminate the interference.
Dittrich, C., Z. phys. Chem., 29, 448 (1899). Dworzak, R., and Reich-Rohrwig, W., Z . anal. Chem., 77, 14 (1929).
Gmelins Handbuch der anorganischen Chemie, “Uranium”, 8th ed., pp. 67 ff., Berlin, Verlag Chemie, 1936. Hull, D. E., J . Am. Chem. SOC., 63, 1269 (1941). Kolthoff, I. M., “Massanalyse”, 2nd ed., Vol. 11,p. 269, Berlin, Julius Springer, 1930. Kolthoff, I. M., and Lingane, J. J., Chem. Reus., 24, 1 (1939). Kolthoff, I. M., and Lingane, J. J., “Polarography”, p. 296, New York, Interscience Publishers, 1941. Ibid., pp. 447 ff. Langer, A., J. Phus. Chem., 45, 639 (1941). Lewis, D. T., Andust, 65, 256 (1940). Lewis, D. T., and Davis, V. E.,‘J. Chem. SOC.,1939,285. Neuberger, A., 2.anal. Chem., 116, 1 (1939). Rathje, W., Angew. Chem., 51, 256 (1938). Repman, B. R., Lab. Prakt. U.S. S. R., 16,27 (1941). Singh, B., and Ahmad, G . , J . chim. phys., 34, 351 (1937). Stern, A., IXD.ENO.CHEM.,ANAL. ED., 14, 74 (1942). Uhl. F., Z. anal. Chem., 110, 102 (1937).
Acknowledgment Acknowledgment is made to the Carnegie Corporation of New York for a grant which enabled the authors to carry out the present investigation.
Literature Cited Atanasiu, I. A,, and Velculescu, A. I., Z. anal. Chem., 102, 344
(1)
(1935). (2) Bodfors, S., Svensk Kem. Tid., 37, 296 (1925). (3) Chrbtien, A., and Kraft, J., BUZZ.SOC. chim., (5) 5, 372 (1938). (4) Courtois, G . , Ibid., 33, 1773 (1923).
Quantitative Determination of 2-Methyl-
1,4=naphthoquinone AMEL R. MENOTTI, Chemical Laboratory, American Medical Association, Chicago, 111.
T
HE increased use of 2-methyl-l,4-naphthoquinoneas a
therapeutic agent necessitated the development of a n accurate and convenient method for its quantitative estimation in marketed preparations, The procedure described in this paper has been found reliable for the determination of 2methyl-l,4naphthoquinonein quantities as low as 0.05 mg., and has been employed successfully in the assay of oil solutions and of alcoholic extracts containing the drug. I n a study of the reactions involved in this determination, the 2,4dinitrophenylhydrazone of 2-methyl-1,4-naphthoquinonewas isolated and characterized, and its absorption spectrum examined in both alkaline and neutral solvents.
more stable yellow colored derivative in solution. The yellow solution is then compared with a suitable standard. Results of analysis obtained by the method of Pinder and Singer (3) were found, in this laboratory, to be sufficiently accurate, if the naphthoquinone could first be isolated by alcoholic extraction. The method appeared t o be inadequate when applied to vegetable oil solutions from which the naphthoquinone could not be quantitatively extracted. Partial saponification of the vegetable oil, which occurred on the addition of potassium hydroxide, produced turbid solutions that required considerable treatment to effect clarification. It was observed also that the presence of oil complicated the formation and subsequent hydrolysis of the intermediate, unsta6le purpie reaction product. As an alternative, it was considered that the direct spectrophotometric determination of 2-methyl-l,4naphthoquinonein oil solution, employing the absorption maximum at 3360 A., might provide a convenient method of assay. However, direct spectrophotometric examination (1) was found t o be limited in application to those solutions in which the oil used as a vehicle exhibited low absorption in the desired spectral region. The method was of little value when applied to certain commercial products which contained vegetable oil that showed complete absorption at 3360 A.
Novelli (2) described a sensitive color test for 2-methyl-1,4naphthoquinone and related substances which forms the basis for the method finally adopted in this laboratory for the quantitative estimation of 2-methyl-l,4naphthoquinone. The procedure detailed below depends on ( a ) formation of the 2,4dinitrophenylhydrazone of 2-methyl-l,4-naphthoquinone,(b) interaction of this dinitrophenylhydrazone with ethanolic ammonia to yield a green t o blue-green colored solution, and ( c ) comparison of the intensity of this color with that produced in a control with known quantities of the naphthoquinone. The 2,4-dinitrophenylhydrazoneof 2-methyl-1,4-naphthoquinone in alcoholic ammonia solutions exhibits a bright blue color. The Preen color described bv Novelli (2) was found to
a
i-
80 -
I L
- 0.4 ii) 2.4-DINIIROPHENYLHYDRAZONE Of 2-METHYL -I.4-NAPHTHOOUINONE (2) 24-OINITROPHENYLHYDRAZINE
-
-0.3
c k
-
I‘
8 c
LE*
240-
-0.2
R
cp
%
s
,
IO
2103
7000
FIGURE
6000
ANGSTROMS
5000
4000
1. LIQHT ABSORPTION OF SOLVTIONS OF THE 2,4-DINITROPHENYLHYDRAZONE OF 2-h’fETHYL-1,4-NAPHTHOQUlNONEAND OF 2,4-DINITROPHENYL HYDRAZINE
may
41Y
nNnLiiiCinL LUIIIUN
13, 134'
A saturated solution of 2.4dinitroohenvlhvdrmine in 2 N hvdrorhloric acid mns added h HI, t t h s n d i f sdution of 2-rnethil1,4nsphthoquinooe; the volution was nnrmcd at C O O to 70' C. ior a few minutes nnd then slowly cooled. I.pon recrystallization f r u m hot chloroform rhe crvitalline nrodiwt v i e l k l brizht orange needles and clusters (Ffgure Z), 'which sGhlimed wcen heated above 200" C. to form long orange needles, and melted with decomposition at 299' C. Analysis for nitrogen gave the following results: Calaulated for CnHuOrN4352.30) N. 15.92 Found N, 16.0
FIGURE2. ~,~-DINII%OPHE~LHYDRAZONE OB 2-METHYL-1,4 NAPHTHOQUINONE
( X 100)
quently the solutions compared colorimetrically were always green to blue-green. Identical shades of green could be obtained in the known and unknown test solutions by controlling the amount of the unknown samule taken for a determine tion Cunres representing the light absorption of the 2,4-dinitrophenylhydrazone of 2-methyl-l,4naphthoquinoneand of 2,4dinitrophenylhydrazine are shown in Figure 1. The data for these curves were obtained with a Cenco-Sheard spectrophotelometer. The 2,4dinitrophenylhydrazone was dissolved in a mixture of one part ethanol and one part concentrated ammonium hydroxide (specific gravity 0.90). The solution of 2,4dinitrophenylhydrazine was prepared by dissolving 2.5 mg. of this substance in 5.0 ml. of 2 N hydrochloric acid contained in a 1oO-ml. volumetric flask, adding 10 ml. of ethanol followed by 10 ml. of a mixture of one part ethanol to one part concentrated ammonium hydroxide, and finally filling to the mark with 95 per cent ethanol. Molecular absorption coefficients were plotted for curve 1, Figure 1, representing the absorption of the dinitrophenylhydraeone, which has an absorption maximum at 635 mp. The curve representing the absorption of 2,4dinitrophenylhydraeine was tbeo plotted to illustrate the relative absorptions of the two components in the solution as used for quantitative determinations, From the curves it may be noted that 2,4dinitrophenylhydraeiue, under the conditions of the determination, possesses no conflicting absorption in the region where the dinitrophenylhydrazone exhibits an absorption maximum. It is suggested that measurements with a spectrophotometer, adjusted for operation at 635 mp, would increase the accuracy of the method. The 2,4dhitrophenylhydrazone of Zmethyl-l,4naphthoquinone was readily obtained according to the following procedure:
The crystalline dinitrophenylhydraeone was very slightly soluble in neutral solvents but dissolved readily on the addition of ammonia or other alkaline reagents with the production of a bright blue color (spectrum, Figure 1). The enhanced solubility and the change in color from orange to blue produced by the addition of bases was attributed to the formation of the aoi-salt of the nitro groups in the dinitrophenylhydraeine nucleus, with the consequent production of an 0- or p quinone configuration coupled to the 2-methyl-1,4-naphthoquinone structure. By careful neutralization of the blue alkaline solutions, orange crystals of the dinitrophenylhydrazone could be recovered. The pnitrophenylbydrazone of Zmethyl-1,4-naphthoquinone was also prepared and found to exhibit properties similar t o those of the 2,4-dinitrophenylhydraaone. However, the instability of p-nitrophenylhydrazie as a reagent prevented its use in quantitative determinations. 2,4,6Trinitrophenylhydrazine was found too insoluble in hydrochloric acid solutions to be used as a reagent. I n Table I a comparison is s b o m of the results obtained on assay of marketed specimens of 2-methyl-1,4-naphthoquinone dissolved in vegetable oil by direct spectrophotometric measurement ( 1 ) and by the method described below. Further controlled experiments indicated that 1 mg. of 2methyl-1,4-naphthoquinonedissolved in ethanol could be determined with an accuracy of ~3 per cent by this method, employing a visual colorimeter; when the naphthoquinone was dissolved in vegetable oil the accuracy was about *5 per cent. Chlorobutanol was found to have no effect on the accuracy of the determination.
Bpeoimen NO.
A
B
C D E F
2-Methyl-1.4-naphtho9uinone Content Found Direct Dinitrophen lhydraaone speotrophotometrio rnetxod method Mdml. Mdml. 0.93 0.94 1.90 1.96 0.82 0.77
.o.m
0.75 1.00
0.94
2.06
2.02
Difference Mdml. 0.01 0.08 0.05 0.05 0.06
0.04
Method
in use. Dinitrophenylhydraeine reagent. Dissolve 50 mg. of reagent in 20 ml. of 2 N hydrochloric quality 2,4dinitrophenylhydr~~zine acid. Ethanolic ammonia solution. Mix one part of 95 per cent ethanol with one part of concentrated ammonium hydroxide (specific gravity 0.90). Transfer a sample (oil solution or alcoholic exPROCEOURE. tract) calculated t o contain about 0.5 mg. ai 2-methyl-1, 4naphthoquinone t o a 50-ml. volumetric 0mk. Place 1.0 ml. of the standard alcoholic solution of 2-methyl-l,4-naphthoquinonein a
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INDUSTRIAL AND ENGINEERING CHEMISTRY
second 50-ml. volumetric flask and adjust the volume in both flasks t o 5 ml. with ethanol. If the unknown contains oil, add an equivalent amount of the same kind of oil, free from naphthoquinone, to the flask containing the standard solution and shake both flasks thoroughly. Add 1.0 ml. of the 2,4dinitrophenylhydrazine reagent t o each flask and place both flasks in a water bath maintained at 70" t o 75" C. for 15 minutes. If oil solutions are being assayed, the flasks must be shaken vigorously once every 3 minutes t o ensure complete reaction; otherwise, only occasional shakingis necessary. At the end of the heating period, cool the flasks to room temperature by immersion in a water bath and add to each flask 5.0 ml. of the ethanolic ammonia solution. Shake the flask thoroughly, fill t o the mark with 95 per cent ethanol, and compare the solutions in a colorimeter. When oil is present in the flasks, allow the solutions to stand for 15 minutes in order that the oil may separate, and use the supernatant liquid for the determination. Note. 2-Methyl-I,4naphthoquinone is decomposed by prolonged exposure to light; consequently, it is best to carry out the determination in subdued light.
Discussion Because the excess reagent contributes to the final color, the same volume of dinitrophenylhydrazine reagent solution must be added to the unknown and to the standard when the color comparison is performed visually. I n practice, if the developed color of the unknown solution is found to vary more than 10 per cent from the standard, a second determination is made with an adjusted volume of the unknown sample. Consistently accurate results may be obtained in this manner. Ammonia was found t o be the most satisfactory alkaline reagent for the development of the blue color. Strong alkalies, such as sodium or potassium hydroxides in small amounts, led to extensive decomposition of the excess dinitrophenylhydrazine and to the production of colored derivatives, which interfere with color comparison. It was observed that the addition of concentrated ammonium hydroxide solution produced some decomposition of the
excess dinitrophenylhydrazine as a result of high local concentrations of alkali at the point of addition. This difficulty was overcome by the use of less alkaline alcoholic ammonia solution to develop the color. A final concentration of 0.6 N ammonium hydroxide was found necessary to effect complete conversion of the alcohol-insoluble 2,44initrophenylhydrazone to the blue alcohol-soluble compound.
Summary
A method for the quantitative estimation of 2-methyl-1,4naphthoquinone and related substances, applicable t o oil solutions or alcoholic extracts, depends upon the interaction of 2,Pdinitrophenylhydrazine with 2-methyl-l,4-naphthoquinone and the subsequent production of a blue to bluegreen color by the addition of alcoholic ammonia. The 2,4dinitrophenylhydrazone of Z-methy1-1,4-naphthoquinone has been isolated and characterized, and the absorption spectra of the 2,4dinitrophenylhydrazone of 2-methyll14-naphthoquinone and of 2,Pdinitrophenylhydrazine dissolved in alcoholic ammonia have been determined. Acknowledgment The data for the absorption curves reproduced in Figure 1 were obtained through the courtesy of Thorfen R. Hogness, in the laboratories of Spectroscopic Biological Investigations, University of Chicago, with the assistance of Richard Abrams. The author wishes to express his appreciation to A. E. Sidwell, Jr., of this laboratory, for suggestions and criticisms during the course of this work.
Literature Cited (1) Kreider, H. R., unpublished data. ( 2 ) Novelli, A., Science, 93,358 (1941). (3) Pinder, J., and Singer,J., AnaZyst, 65,7 (1940).
Colorimetric Determination of Low Concentrations of Sodium Nitrate in Sodium Nitrite WILLIAM SEAMAN, A. R. NORTON, W. J. MADER, AND J. J. HUGONET Calco Chemical Division, American Cyanamid Company, Bound Brook, N. J.
A
METHOD was needed for the determination of sodium nitrate in sodium nitrite samples in concentrations of about 1 per cent or less. I n order to obtain fairly precise values, those methods were excluded from consideration which depend upon the determination of nitrates by the difference in the total nitrogen value and the nitrite value, because the errors would all be thrown upon the minor constituent. Of the direct methods which have been reported for nitrate, the colorimetric ones seemed most promising. These fall into two classes. The first class comprises tests such as those with diphenylamine and with ferrous sulfate which are not specific for nitrates but are also given by nitrites. The use of such tests would require the preliminary removal of nitrites. In order to avoid the necessity for doing this, attention was turned to the second class-namely, tests which have been reported as being specific for nitrates and not subject to interference by nitrites. This claim has been made for two methods in particular; one by Wolf and Heymann (11) involves the formation of a color by means of 2,4-diamino-6-hydroxypyrimidinein the presence of sulfuric acid; the other by Pesez (7, 8) depends upon nitrating nitrobenzene to dinitrobenzene by means of the nitrate in sulfuric
acid and reacting the product according to Janowski (2) with acetone and alkali to produce a color which is a measure of the nitrate present. The authors of both methods claimed that nitrites do not interfere. The present authors have been unable to confirm their claims in this respect; on the contrary, they find that nitrites do interfere. Even recrystallized sodium nitrite gives a variable intensity of color. The reason for this is of fundamental importance in considering claims for the specific!ty of any method for nitrates in the presence of nitrites. Whenever it is necessary to carry out such an analysis by treatment of the sample, which still contains nitrite, in the presence of a considerable concentration of strong acid, then the analysis cannot be reliable, because some of the free nitrous acid which is formed will decompose to form nitric acid, presumably according to the well-known reaction: 3HN02 +HNOs 2N0 H20
+
+
When analyzing samples consisting almost entirely of sodium nitrite, the amount of nitrate which is formed is sufficient to vitiate the analysis. Since no method was available which could be applied
