V O L U M E 23, NO. 7, J U L Y 1 9 5 1
1035
to subsequent manipulations. Cool somewhat and add 25 ml. (buret) of the sulfuric-tartaric acid mixture. Cover, heat to boiling, and boil for a minute or two until the sample is in solution. Cool slightly, wash down the cover glass with a small amount of water, and add 50 ml. (buret or pipet) of the caustic mixture. Add 2 ml. of 0.5y0a-benzoinoxime solution and transfer t o a separatory funnel. Extract with chloroform and measure the quantity of copper, according to the procedure of Dunleavy, IT'iberley, and Harley ( 1 ). In Cast Irons. It is advisable to allow more time for the solution of the cast iron sample than for steel. Cast irons may be dissolved in a mixture of 20 nd. of 1 to 1 nitric acid solution and 1.5 ml. (buret) of boric acid solution. Complete the analysis according to the procedure for steel.
4VALYTlCAL R E S U L T S
\z(i11
Four of the National B u k a u of Standard Steels used h y Dunleavy e t al. ( 1 ) were available to determine how this modification compares with the original method. The results obtained with these samples and with Bureau of Standards cast iron 4g are given in Table I. LlTERATURE C l T E D
(1) Dunleavy, R. A , . Kiberley, CHEM., 22, 170 (1950).
5. E., and Harley, d . H.,
A s . 4 ~ .
RECEIVED J u l y 18. 1950
Detection of Nitrogen in Organic Compounds LiWRENCE E. BROWN
AND
CARROLL L. HOFFPSUIK
Southern Regional Research Laboratory, iYew Orleans, Lu.
YEIT and sensitive test for ammonia, which makes use of the
-4 -fluorescence exhibited by the chelate complex of zinc with 8-cluinolinol, has recently been described by Velluz and Pesez ( 7 ) At low p H the complex dissociates and shows no fluorescence, but a solution or sensitized paper made from the complex, after exposure t o ammonia, emits an intense greenish-yellow fluoresence under ultraviolet light. I n adapting this test to the detection of nitrogen in organic compounds Velluz and Pesez (8) recommend a sample size of 1 mg., and use a lengthy digestion and distillation procedure. A more sensitive and reliable technique for converting nitrogen in organic compounds to ammonia is the pyrolysis of the sample in the presence of calcium oxide (1-4, 6). A number of reagents have been suggested for the detection of the ammonia released in the pyrolj sis, including litmus, silver-manganese sulfate, mercurous nitrate, and Kessler's reagent. ?;one of these combines both the convenience and sensitivity of the 8-quinolinol-zinc complex. By adapting the fluorescence method of detecting ammonia to the calcium oxide test, a rapid and eimple test for nitrogen on a microgram scale has been developed. APPARATUS
A source of ultraviolet light, such as a General Electric PurpleS bulb or a KO.360BL fluorescent tube, is required. T h e use of a view box ( 5 ) is convenient but is not essential. T h e combustion apparatus consists of a hard-glass test tube (10 X 75 mm.) equipped with a KO. 0000 one-hole stopper in which is mounted a 5.5-cm. length of 5-mm. tubing. T h e test tube is mounted through a hole in an asbestos board, EO t h a t the lower part can be heated with a microburner. REAGEllTS
Calcium Oxide. Satisfactory calcium oxide can be prepared by igniting analytical reagent grade calcium oxalate for 1 hour a t 950" C. in a platinum dish. Test Paper. A neutral, fairly thick, absorbent filter paper, such as S. & S. 598 or 589 Green Ribbon, cut into 3 X 70 mm. strips. Test Solution. About 0.5 gram of crystalline zinc sulfate heptahydrate and 0.50 gram of 8-quinolinol are dissolved in 4 ml. of 1 X sulfuric acid. While stirring, 60 ml. of 0.005 Asodium hydroxide are slowly added, followed by 35 m]. of water. T h e solution should have a p H of 3.0 * 0.05 and is relatively stable. T h e p H of the solution may drop to 2.8 or 2.9 after several weeks, but this results only in a slightly louer sensitivity. PROCEDURE
Place a few micrograms of the sample material in the bottom of the test tube. Solutions should be evaporated to dryness. Add about 0.1 gram of calcium oxide by means of a funnel t o prevent the lime from adhering to the walls of the tube. T a p the tube t o compact the lime and place it in the combustion apparatus so t h a t it extends 2 to 3 cm. below the asbestos board.
Dip a strip of t.est paper about 1 cm. down into the test solution and insert it into the tubing in t,he stopper until it extends within 2 to 3 mm. of the lower end. Bend over the upper end of the strip to hold it in position. Examine the paper under ultraviolet light to establish the absence of fluorescence. Place the stopper in the test tube so that, the end of the test' strip is about 1.5 cm. above the asbestos board. Heat the calcium oxide with a microburner from the top down until all the calcium oxide has been heated to redness. Remove the stopper and again examine the test paper under ultraviolet light. A greenish-yellow fluorescence extending upward from the end is evidence of nitrogen in the sample. DISCUSSION
Using satisfactory reagent and freshly cleaned apparatus, ;I blank test on a sample containing no nitrogen will be completely negative. The sensitivity of the test varies wit,h the nitrogen compound present-from less than 1 microgram for sulfanilic: acid to about 3 microgranis for methy-l o,i-ange, corresponding to about 0.05 to 0.30 microgram of nitrogen. If the type of nitrogen compound present is known, an indication of whether it is present in large or small amounts can be obtained by observing the area of the fluorescent spot on the test paper, or, alternatively, by using an 8-quinolinol-zinc reagent, the sensitivity of which has been reduced by the addition of excess acid. If preferred, the test may be applied t,o samples weighing 0.5 t,o 1.0 mg. by substituting water for the sodium hydroxide in making the test solution, PO that the p H is about 2.5, and pyrolyzing the sample in a 12 X 75 mm. test tube, using 0.5 gram of calcium oxide. Positive tests have been obtained with one or more nitrogen compounds of the following types: amino, amide, nitro, nitroso, nitrile, oxime, azo, ring, semicarbazone, and organic nitrate. Because the fluorescence is dependent on p H , positive teste are obtained with volatile bases other than ammonia. Hence, the test does not fail with compounds, such as trimethylamine oxide, which yield a volatile base other than ammonia on incomplete pyrolysis. KO nitrogen-containing compounds have been encountered which gave a negative test. Nonnitrogenous compounds including those containing phosphorus, arsenic, antimony, and bismuth all gave negative tests. Compounds t h a t are naturally fluorescent, such as anthracene, should be burned carefully t o avoid volatilizing unchanged material onto the test strip. A poor combustion of such a sample is, however, readily detected by the different color of the fluorescence and the fact that the fluorescence is diffused over the inner tube instead of being only on the test strip. The use of manganese dioxide with the calcium oxide as recommended by Feigl ( 4 ) is unnecessary; pyro compounds, if formed, do not give false positive tests in this procedure. Furthermore, its use was found to reduce the sensitivity of the test, probably through oxidation of part of the ammonia. The use of zinc,
ANALYTICAL CHEMISTRY
1036 powder mixed with the lime as suggested by Bennett et ai. (1) gave a false positive test with triphenyl phosphate. Copper powder in the calcium oxide as suggested by Emich and Schneider (S,6 ) offers no advantage, since compounds containing nitrogen linked to oxygen give positive tests \T ith lime alone. LITERATURE CITED
(1) Bennett, E. L., Gould, C. K., Jr., Swift, E. H., and Siemann, Carl, ANAL.CHEM.,19, 1035 (1947). (2) Cheronis, X . D., and Entrikiri, J. B., “Semimicro Qualitative Organic Analysis,” Sew Tork, Thomas Y . Crowell Co., 1947.
(3) Emich, Friedrich, and Schneider, Frank, “Microchemical Laboratory hlanual,” New York, John Wiley & Sons, 1932. (4) Feigl, Fritz, “Qualitative Analysis by Spot Tests,” Xew York, Elscvier Publishing Co., 1946. ( 5 ) Fletcher, M , H., White, C. E., and Sheftel, M. S., IND.ENG. CHEM.,ANAL.ED.,18, 179 (1946). (6) Schneider, Frank, “Qualitative Organic Microanalysis,” Xew Tork, John Wiley 8; Sons, 1946. (7) Velluz, L., and Pesez, >I., Ann. p h a r m . franc., 4, 10 (1946). (8) Ihid., p. 12. RECEIVED September 25, 19.50.
Colorimetric Assay of Diacyl Amides J. B. POLYA
AND
P. L. TARDEW
Chemistry Department, University of Tasmania, Hobart, Australia
IPRIANN and Tutt’le (5) have developed a procedure for assaying acyl phosphates by converting the organic acyl radicals t o hydroxamic acids, which give intense coloration with ferric chloride. Because purrly organic anhydrides or acyl chlorides are not likely t’o occur under biological conditions, and esters or amides do not react under the experimental conditions, the method is claimed t o be specific for acyl phosphates in biological materials. It is possible, however, that diacylamides of the general formula RCO.KR‘.COR” may occur in biological systems. The Lipmann-Tuttle assay was applied t o a number of representative compounds of this class. Dibenzamide ( 1), formyl benzamide ( 4 ) ,diacet’amide (IO),and other diacyl amides with R’ = H (8) were prepared by previously described methods. Other diacyl amides were prepared by the acylation of the appro. priate amines or amides with anhydrides in the presence of acyl halides or thionyl chloride in catalytic amounts ( 3 ) . Ot,her reagents were commercial products of analytical quality. The reagent solutions were those recommended by Lipmann and Tuttle. They were prepared fresh and used in the quantities and order as suggested by the originators of the method. The colors were compared with a Hilger Spekker absorptiometer using 1-cm. cells, Ilford 608 gelatin filters, and a mixture of the reagents only as reference. This gave convenient readings for the assay of succinic anhydride or acet’yl phosphate in concentrations ranging from 0.025 to 0.100 M with an error of +4%.
This procedure was not suitable for diacyl amides, because some of them were not sufficiently soluble and even diacetamide gave low density readings with an error of = t 1 8 ~ o .Table I refers t o average values from four measurements each, with probable errors indicated above.
Table I. Optical Density Molar Concentration 0.100 0.075 0.050 0,025
Optical Density Succinic anhydride Diacetamide 1.18 0.13 0.11 0.89 0.59 0.30
0.09 0.06
Better results were obtained by increasing the alkalinity of the reaction mixture and limiting the reaction time t o 2 minutes. ’ I n this modified procedure 1 ml. each of 4 Jf hydroxylamine hydrochloride solution and 3.5 .IT sodium hydroxide are mixed and 1.8 ml. of the rrcommended acetate buffer are added. The solution to be analyzed (2 ml.) followed by another 0.2 ml. of the 3.5 M sodium hydroxide is added in this order and the mixture is allowed t o stand for 2 minutes a t room temperature (18’ to 20”). The subsequent operations are those recommended by Lipmann and Tuttle. Ilford 604 gelatin filters were used in the modified procedure as they were found to give better results than Ilford 608.
Dibenzamide is not sufficiently soluble in water and was used dissolved in 1% sodium hydroxide. Other relatively insoluble diacyl amides like acetyl and propionyl benzamide hydrolyze too rapidly in alkaline solutions. I n such cases the assays had to be restricted to lower concentrations in water only. Although Smethyldiacetamide is very soluble in water, such’solutions hydrolyze rapidly and the assay must be carried out without delay. against +4% in other Even then the assay has an error of =ti% cases, n-ith the exception of acetyl chloroaeetamide, which is assayed v i t h an error of =t22’%. The method is not suitable for the assay of h’-formyl amides, as the color fades within a few seconds. KH20H+ The reaction therefore proceeds as PhCONHCHO PhCONH2 HC( .O) SHOH, in agreement with previous theories (8). Hydantoin, S-methyl-S’-acetvlurea, surcinimide, and acetamidine gave little or no color. hcetamide in a 5% (0.847M) solution gave an intensity of 0 27 * 0.02, corresponding to that given by approuimately 0.0025 Jf solutions of diacyl amides The S-bromo and S-bromoniagnesium derivatives of diacetamide (6) gave the color test which, however, was not suitable for quantitative purposes, owing to the instability of these compounds under the experimental conditions. The same applies to a lesser extent to acetyl chloroacetanlide, but the colorimetric assay of some halogenated diacyl amides could be supplemented by fluorometric or spectrographic methods ( 7 , Q ) . Esters interfere in the presence of excess alkali ( I l ) , although this is not the case in the original method of Lipmann and Tuttle. However, the removal of esters from solutions containing acyl phosphates is difficult or impossible, whereas the separation of esters from most diacyl amides is comparatively simple. Average results of six assays (four assavs only for acetyl chloroacetamide) are shown in Table 11. The errom indicated above refer to concentrations from 0.0025 to 0.0100 M . At 0.0010 M concentration the errors are estimated a t *9 to *loyo. The curves of density versus concentration may be regarded as linear within the stated range of errors and may be used for the rapid determination of small amounts of diacyl amides. The assays may be duplicated with an error of *2% only if succinic anhydride is used in the reference cell as in the original LipmannTuttle procedure. Diacetamide is unsatisfactory as a reference in the original but useful in the modified method. Analytically pure disilver acetyl phosphamide has been prepared by dehydrating and acylating monoammonium phosphate with acetic anhydride in excess in the presence of thionyl or acetyl chloride and precipitating from an aqueous solution with silver nitrate ( 2 ) . Like benzoyl phosphamide ( l a ) , acetyl phosphamide appears to be relatively stable jn aqueous solution. Removal of the silver by thioacetamide resulted in solutions from Khich the original silver compound salt could be regenerated with a loss of 71 to 86%. Such silver-free solutions could not be used for the assay because of a sloa-ly fading green color due to residual thioacetamide in the solution. Fresh extracts of disilver acetyl
+
+
