1034
ANALYTICAL CHEMISTRY
Unidimensional chromatograms, made by the "ascending method," were used in most of these experiments. T h e chroniatograms n-ere developed a t room temperature (20-23" C.) for 6 to 8 hours. Thoroughly washed, dried papers 45 cm. in length and 25 em. in widt.h were used, and the ammonium salts of the acids were applied to a starting line 2.5 cm. from the bottom of the paper, usually in 0.01 ml. of aqueous solution, containing 0.5 t o 1.5 micromoles of each acid. If the fatty acids were added t o the paper as the sodium salts, as in the method of Brown and Hall ( I ) , the rates of migration of the acids with ethyl alcohol-ammonia as the developing solvent were considerably sloyer, and regions of excess alkalinity usually marked the t'racks of the acids. If a solution of the sodium salts was first mised with an equimolar amount of ammonium sulfate and made alkaline with a little free ammonia, and this mixture was applied t o the paper, the rates of migration were then approximately the same as those of the pure ammonium salts alone. .liter application of the salts of the acids, the paper was rolled into a cylinder and pinned together, and the chromatogram was developed. -1 number of developing solvents were used successfully. These included aqueous ethyl alcohol-ammonia solutions, aqueous acetone-ammonia, and aqueous butanol-ammonia. With the first two solvents, which are miscible with water, the Rj values could be altered considerably by varying the percentage of water. In general, the addition of a larger portion of water to the mixture caused the organic acids to travel more rapidly on the chromatogram. This flexibility of such watermiscible solvents may prove useful in separating the ammonium salts of dibasic acids which do not move appreciably in the aqueous ammonia-butanol mixture described by Brown and Hall. .\ simple solvent mixture that proved useful in this work was composed of 100 ml. of 95% ethyl alcohol, to which was added 1 nil. of concentrated ammonium hydroxide. Rr values of a number of acids in this system are shown in Table I. .Ift,er development of the chromatogram, the papers were dried in an oven a t 100" C. for 5 minutes. The spots were then located bj- spraying with a solution of 50 mg. of bromophenol blue in 100 nil. of water, made acid with 200 mg. of citric acid. Because of the buffer capacity of the acid anions, the location of the spots is shown hy the intense blue (alkaline) color of the indicator in
Table I.
Rj Values of Acids in A m m o n i a c a l Ethyl alcohol Solution .Icid Formic Acetic Propionic n-Butyric n-Valeric n-Caproic n-Heptanoic n-Octanoic Vinylacetic n-@-Ketohexanoic
R: 0 0 0 0 0
31 33
44 54 60 0 68 n 72 0 76 0 46 0 53
these regions, while the background is orange-yellow. This method of locating the spots was found to be more satisfactory than the use of bromocresol green made alkaline with a little sodium hydroxide ( I , 4 ) . In experiments in which radioactive fatty acids were to be separated, and it was desired to make radioautographs of the chromatograms, the apers were not sprayed with indicator, but instead were sprayez before drying with a 0.5 Jf solution of potassium monohydrogen phosphate, which fixes the acids on the paper as the potassium salts. Radioautographs of the papers may then be made by conventional techniques. LITERATURE CITED
(1) Brown, F., and Hall. L. P., .Vatwe. 166, 66 (1950).
(2) Elsden. 5.R., Biochem. J . , 40,252 (1946). (3) Fink, K., and Fink, R. AI., Proc. S O C .E.@. Bid. M e d . , 7 0 , 654 (1949). (4) Hiscox, E. R., and Berridge, S . ,J., .Vatwe. 166, 522 (1950). (5) Lugg, J. W. H., and Overell, B. T., d i ~ s t m Z i a nJ. Sei. Research, (A) 1, 98 (1948). RECEIVEDSeptember 5 , 1950. Investigation supported in part by research grants from the American Cancer Society and the Division of Research Grants and Fellowships of t h e National Inititutes of Health, United States Public Health Service.
Modified Photometric Determination of Copper in Ferrous Alloys KENDALL W. N-INCE Kaiser Steel Gorp., Fontana, Calif.
HE photometric determination of copper in ferrous alloys, T a b l e I.
according to the method of Dunleavy, \J-iherley, and Harley
(I), may be modified to obviate the use of a pH meter. Such a modification simplifies the method as an implement for the control analyst. The folloning procedure will produce a solution from which optimum extraction of the copper coniplei with a-benzoinouime is o1,t:iined
D e t e r m i n a t i o n of Steel and Cast Iron
160
20d 21c 55b
Cast iron 4g
1 12.1 12.3 la.1 12.3
12.0
2
True value,
Kiett-Summerson, %
PH B.S. Steel
2
3 0.062
%
1 0.061 0 164 0.056 0 038
0.061 0.161 0.054 0.039
0.164
0,164
11.3
3 12.0 12.1 12.3 12.3
0,040
0.040
12.0
11.4
0 236
0.252
0.238
0.240
11.4 12.4 12.3
0.054
0.060
0,050
REAGESTS
Boric Acid. Prepare 1 liter of saturated solution. Let the solution stand overnight a t a temperature of 23' C. and theii filter through a rapid pa er at 23" C. The boric acid may bc standardized with 0.2 $sodium hydroxide solution. Measure out 10 ml., add mannitol, and titrate with 0.2 N sodium hydroxide to a phenolphthalein end poiit. A titration of 42.65 nil. should be obtained. Potassium-Sodium Hydroxide Mixture. Five liters of solution contain 280.0 grams of potassium hydroxide and 395.0 grams of sodium hydroxide. Pipet 10 ml. into a 250-ml. volumetric flask, make up to the mark with distilled water, and mix well. Pipet 25 ml. into a titrating flask. Add 20 ml. (buret) of 0.2 N sulfuric acid and boil t o remove traces of carbon dioxide. Cool, add phenolphthalein, and titrate. with 0.2 N sodium hydroxide. Adjust the strength of the initial caustic solution until the 25-ml. aliquot is equivalent to 13.86 ml. of 0.2 N sulfuric acid. Sulfuric-Tartaric Acid Mixture. Dissolve 800 grams of tartaric acid in 1500 ml. of water, add 935 ml. of 2 N sulfuric acid, and dilute to 2500 ml. Pipet 10 ml. into a 250-ml. volu-
metric flask. Make up to the mark. Mix well and pipet a 25ml. aliquot into a titrating flask. Heat to boiling, add phenolphthalein, and titrate with 0.2 N sodium hydroxide. The 25ml. aliquot (1 ml. of acid mixture) is equivalent to 24.55 of 0.2 N sodium hydroxide. The above reagents appear to be relatively stable. S o appreciable alteration has been noted in solutions used over a 6-month period. PROCEDURE FOR DETERiMINATlON O F COPPER
In Steel. Weigh a 0.500-gram sample of steel into a 400-ml. beaker and add 15 ml. (buret) of boric acid solution and 7 nil. of concentrated nitric acid. Cover with a borosilicate glass cover glass and evaporate to dryness. Remove the cover glass and invert it upon the hot plate (270" (3.1. Bake the sample and cover for 3 minutes. Samples baked for 7 minutes yield equally
V O L U M E 23, NO. 7, J U L Y 1 9 5 1 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.
1035 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,
