THE DETECTION OF NITROGEN IN ORGANIC COMPOUNDS' KENNETH N. CAMPBELL, SR., M. ST. CHARLES, S.N.D.,z and BARBARA K. CAMPBELL3 University of Notre Dame, Notre Dame, Indiana
Tms paper embodies the results of a study on the fusion of organic compounds and on the detection of nitrogen as cyanide. It is part of a project designed to improve the procedures used in student courses in qualitative organic analysis. Two methods have been more widely used than others for the decomposition of organic compounds into inorganic ions. The Lassaigne sodium fusion is the best known and in general it is satisfactory, although it is reported to give poor results with azo and diazo compounds (I), with nitro compounds (8), and with pyrroles. There is a great deal of variation in the procedures given in qualitative organic analysis textbooks for the sodium fusion. The other method involves fusion with magnesiumpotassium carbonate mixture, and has been investigated in detail by Baker and Barkenbus (S), who obtained very satisfactory results with it. Because nitrogen may be taken up from the air (4) Baker and Barkenbus carried out their fusions in an atmosphere of ether. Tucker (4a), whose conclusions are similar to the ones described here, gives an excellent discussion of the methods of fusion. Cyanide ion is usually detected by Prussian blue formation. In this test an alkaline solution of cyanide ion is treated with ferric and ferrous ions and the mixture is then acidified, whereupon a blue precipitate or a blue coloration is formed. Many modifications have been proposed. Several workers (5-9) have found that it is not necessary to add ferric ion, as aerial oxidation of the ferrous ion provides enough ferric ion for the test, and an excess of ferric ion seems to be detrimental. Other workers recommend the use of as smaU volumes as possible to avoid dilution of cyanide ion (lo), and the addition of potassium fluoride has been thought to increase the sensitivity of the test by forming a com~ l e xwhich removes most of the ferric ions (11). The acidification step has been carried out in thk hot (193) and in the cold (6), using sulfuric acid (If), hydrochloric acid, or nitric acid. In spite of aU the work that has beeu donr on the P ~ s s i a nhlue test, n romparison of theproerdures givm in st:mdard textl~ooks(?, 15-15,9) Presented hefore the Analytical Division of the American Chemioal Society a t the 113th meeting at Chicago, Illinois, April, 1948. Present address: St. Mary'sHigh School, Warren, Ohio. a Present address: Indiana University, South Bend, Indiana.
indicates that there is no general agreement as to the optimum conditions to use. In recent years considerable work has been done on the detection of cyanide ion with organic reagents that are oxidized to colored compounds in the presence of cyanide and cupric ions. Among the reagents that have been proposed for this purpose are phenolphthalin (26, I?), benzidine (IS), guaiac (19), and fluorescin (20). In general these reagents have been used to detect cyanide in water supplies, and for this purpose are much more sensitive than the Prussian blue test. They have not been used extensively to detect cyanide in fusions of organic compounds, however. In the latest edition of Shriner and Fuson's text (8) the benzidine test is given. In the present work wecompared the various procedures given in representative texts for carrying out the sodium fusion, and we also investigated the Castellana magnesium-potassium carbonate fusion. We then studied each step in the formation of Prussian blue, to evaluate the suggestions of various authors as to suitable modifications. Finally we tried out several of the organic reagents recommended for cyanide ion detection. As a result of this work, it was concluded that for student use the best method is sodium fusion,' followed by the Prussian blue test, or alternatively, by the benzidine test, and a procedure was developed which has been highly successful for the past several years in the course in quaIitative organic anaIysis. No difficulty has ever been experienced with nitro or azo compounds, nor has the thiocyanate complication ever been encountered. In the course of the present work several thousand fusions were carried out; only representative results are recorded here. FUSION METHODS
Sodium Fusion. The procedures given by Mulliken (1.9. Kamm (LO. and Shriner and Fuson (16) were comiarkd. The pi&dure of Mulliken was f&d to be the most satisfactory, and modifications of it were then tried. As a result of numerous tests it was found that best resulrs were obtnind when the amount of codium wag redured from 0.25 g. ro 0.1 g. or l r s ~(a cube about 4 mm. on an edge) and when the organic compound (5 a
'As a result of his work on micro qualitative analysis, H. K. Alber prefers sodium fusion for decomposing an organic compound. See Mierochemie ver. Mikrochim. A d a , 29, 294-328; C. A., 37,3365.
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mg.) was added all a t once to the molten sodium, rather than in portions. Use of a larger amount of organic compound was apt to lead to incomplete fusion, and a larger quantity of sodium made the resulting stock solution too alkaline. Castellana Fusions. These were carried out as described by Baker and Barkenbus (5), using an atmosphere of ether. In our hands this fusion method was not as satisfactory as sodium fusion. The cyanide tests were much weaker and with compounds difficult to fuse, such as sulfanilic acid, poor results were obtained frequently. When the color tests with organic reagents were used, false positive tests from nonnitrogenous com~oundswere much more common with the ~astellanafusion than with sodium fusion, and no improvement was noted when care was taken to exclude all air during the fusion. The technique of the Castelfor ~ to lana fusion was much more difficult -.~t , ,. ~ d ~ ~-~ master, and in carryins out the pmssian blue test on . sulfur-containing compounds it was always necessary to remove the sulfide ion; this was not the case with sodium fusions. Also, the reagent tends to cake on storage.
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PRUSSIAN BLUE FORMATION
The following points were investigated: (a) the optimum pH of the original stock solution, (b) the optimum dilution of the original stock solution, (c) the effect of adding ferric chloride, (d) the effect of adding potassium fluoride, (e) the best acid to use in the acidification step, and whether acidification should be done in the hot or the cold. Alkalinity of the Stock Solution. I t was found that best results were obtained when the pH of the sodium fusion stock solution was 13. Since the pH of the solutions varied from about 9 t o higher than 14, due to variations in the amount of sodium used, the pH was adjusted to 13 (as shown on Hydrion E paper) before addition of ferrous ammonium sulfate. Kolthoff (21) and Neubauer (5) found excess alkali detrimental to the sensitivity but did not determine the optimum pH. The Optimum Dilution of the Stock Solution. The stock solution was routinely made up t o 20 ml., and 1ml. portions of this were used. When this was diluted with an equal volume of water the Prussian blne test was frequently negative. On the other hand, when more than 1 ml. of stock solution mas used the specified quantities of reagents were too small and the test was frequently negative. For these reasons, we used graduated droppers to measure the amounts taken, and used all the reagents in rather concentrated form to avoid excessive dilution of the cyanide solution. Effect of Adding Fem'c Chloride. Addition of ferric chloride was found to be definitely detrimental to a good Prussian blue test; this is in agreement with the results found by Neubauer (5), Mulliken and Gabriel (6), Vorlander (7), and Davidson ( 8 ) ; and in disagreement with the procedures given by Cheronis (22), Shriner and Fuson (IS), and Kolthoff (21). Even when freshly prepared ferrous ammonium sulfate solution is
used, as in the present work, enough ferric ions are present t o give Prussian blue, and an excess of ferric ions makes the test less sensitive. Effect of Adding Potassium Fluoride. Viehoever and Johns (11) first reported that potassium fluoride increases t,he seusitivit,yof the Prussian blue test. This is probably due to the fact that the fluoride ion forms a very stable complex witb ferric ion. When most of the ferric ions are thus removed the so-called soluble Prussian blue is formed rather than ferric ferrocyanide (23): Removal of most of the ferric ions also minimizes oxidation of ferrocyanide ion to ferricyanide ion (8): . . we+++L A "
8
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We confirm the fact that the addition of ~otassium fluoride ~~ t , ~~ improves ~ . the ~ Prussian ~ blue ~ test. False negative tests were eliminated and ~olutiouswhich were very weak in cyanide ion still gave a good blue color. The optimum amount of potassium fluoride for a 1-ml. sample of stock solution is 2 drops of a 30 per cent solution. Less than this is not as effective, and more is detrimental, presumably because too many ferric ions are removed. Acidification. The mode of acidification was found to be extremely important in securing good results. The best results were obtained when the test mixture was acidified hot, witb sulfuric acid. Use of cold solntions gave much poorer results, and the mixture had to stand for some time before the Prussian blue color d o veloped. Sulfuric acid was much more satisfactory than either hydrochloric or nitric acids; the latter reagent frequently gave greenish rather than blue solutions. The concentration of the sulfuric acid was important. The use of 1 N acid, as recommended by some (241, gave many false negative tests, 6 N acid was still not very satisfactory, and the best results were obtained with 30 per cent acid. The acid must be added dropwise, so that an excess is avoided; careless addition of acid leads to weak tests. This point is not sufficiently emphasized in most texts. ORGANIC REAGENTS FOR CYANIDE ION
Four of these reagents for the detection of cyanide ion were investigated, using the stock solutions from sodium fusions and from Castellana fusions. The phenolphthalin reagent of Childs and Ball (16) when correctly prepared and stored, gave good results, and no false positive tests were encountered. It is more sensitive than Prussian blue, but as it must be stored and handled a t temperatures below 15'C., and as sulfide ion must he removed before this reagent is used, it is not recommended for student use. Theroux's modified phenolphthalin reagent (17) was totally unsatisfactory, as false positive tests were repeatedly obtained on such compounds as cyclohexanol, decane, ethyl bromide, etc. Guaiac-copper sulfate reagent (19) similarly gave false positive tests. Stamm (20) has recommended a fluorescin reagent as
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263
very sensitive for the detection of cyanide. I n our hands it gave many false positive tests with nonnitrogenous compounds especially when used in conjunction with the Castellana fusion. I t was the most sensitive reagent tried, and like the other organic reagents, required removal of sulfide ion. Benzidine-copper Acetate Test. This test showed itself to be more sensitive than the Prussian blue test, and free from some of the drawbacks of the other organic reagents tried; it has promise as a qualitative organic analysis reagent. Two solutions (18) are required for this test. Solution I was prepared by dissolving 286 mg. of copper acetate in 100 ml. of water; solution I1 was made by dissolving 150 mg. of henaidine in 100 ml. of hot water and adding 3 4 drops of glacial acetic acid. The two solutions were kept separate until used; the final reagent was prepared by mixing equal volumes of the two solutions. When the stock solution was added dropwise to the reagent a blue color developed when cyanide ion was present. Addition of the reagent to the stock solution was less satisfactory (19) and substitution of sulfuric acid for acetic acid made the test less sensitive. The test in general gave excellent results, especially with sodium fusion filtrates; the results with Castellana fusion filtrates were less positive. No false positive tests were obtained with this reagent, in contrast to the other organic reagents tried. It has one disadvantage over the Prnssian blue test; it is necessary to remove sulfide ion. This is best done with lead acetate, but an excess of lead acetate should be avoided. Results with the henaidine test are snmmarized in Table 1. a
TABLE 1 Comparison of Benzidine and Prussian Blue Teats o n Filtrates from Sodium Fusions and Castellana Fusions Compound
Diphenylamine Naphthylamine Dimethvlmiline 3,SDinitrabeneoio acid Aaobensene Acetoxime Phthalimide Diphenylthiaures. Sulfanilic acid Benzoin Cvclohexanol
for thirty seconds longer. Alcohol (1 ml., 95%) is added t o the cold tube to decompose excess sodium, followed by 5 ml. of water, and the mixture is heated to boiling and filtered hot. The filtrate is diluted to 20 ml. with distilled water t o give the stock solution which is used for all subsequent tests. Technique of Prussian Blue Test. One milliliter of stock solution is placed in a small pyrex test tube, and the pH is adjusted to 13, as shown on Hydrion E paper. Two drops of freshly prepared saturated ferrous ammonium sulfate solution are added, and two drops of 30 per cent potassium fluorjde solution. The mixture is heated to boiling, and acidified while hot with 30 per cent sulfuric acid, added dropwise, to give the blue solution characteristic of Prnssian blue. With the test as developed it is not necessary to remove sulfide ion. Benzidine-Copper Acetate Test. To 0.5 ml. of the benaidine-copper acetate reagent is added 1 drop of 20 per cent acetic acid and the stock solution to he tested is added dropwise; if cyanide ion is present the blue color develops when 1 to 15 drops of stock solution have been added. If sulfur is present it must be removed before running the test. This is best done by adding 1 drop of saturated lead acetate solution to 1 ml. of stock solution, filtering off the lead sulfide. REFERENCES (1) GRAEBE, C., Ber., 17,1178 (1884). R. L., AND R. C. FUSON,"Systematic Identifica(2) SHRINER, tion of Organic Compounds," 3rd ed., John Wiley and Sons, New York, 1948. Ind. Eng. Chem., Anal. (3) BAKER,R. H., AND C. BARKENBUS, Ed., 9, 135 (1937). D . G.. AND F. SCHNEIDER. ibid.. 10. 104 (1938). (4) FOULKE. (4i) TUCKER,S. H., J. CHEM.EDUC.,i 2 , 2 1 i (1'945). (5) NEUBAUER, C., Z. anal. Chem., 9, 429 (1870); 11, 361
.
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(6) MULLIKEN, S. P., AND C. L. GABRIEL, Chem.-Ztg., 36, 1186 114101
VORLANDER, D., Ber., 46, 187 (1913). DAVIDSON, D., J. CHEM.EDUC.,14, 238 (1937). MCELVAIN, S. M., "The Chartracterization oi Organic Compounds," The MmMillan Co., New York, 1945. BERL,E., AND M. DELPY,Ber., 43, 1430 (1910). VIEHOEVER, A., AND C. 0. JOHNS,J. Am. Chem. Soc., 37,
-Sodium Fua'on4 a s l e l l u n a FusimPrussian Bemidine Prussian BmLidine
++
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++ ++ +++
++ ++ +++ + ++
Neg. Nea.
Neg. Keg.
++ ++ ++
++
Doubtful
++ ++
+ Neg.
Doubtful Neg. Neg,
Doubtful Doubtful
+ +
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++
+++ +
Neg. Neg.
FINAL PROCEDURE
Fuaion Technique. The preferred method of fusion is as follows: A clean dry pyrex ignition tube (16 X 125 mm.) is clamped in a vertical position so that the bottom of the tube reaches the hottest point of a microhurner flame. A cube of sodium about 4 mm. on an edge (0.1 g. or less) is placed in the tube and the flame lighted. When the sodium vapor reaches about 1 cm. up the tube 5 mg. of a solid (1-2 drops of a liquid) is added directly t o the molten sodium and heating is continued
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(12) LINK,A,, AND R. MONKEL, Z. anal. Chem., 17,455 (1878). (13) MULLIKEN, S. P., "Identification of Pure Organio Compounds," John Wiley and Sons, New York, 1916, Vol. 11. (14) KAMM,O., "Qualitative Organic Analysis," 2nd ed., John Wiley and Sons, Xew York. (15) SHRINER, R. L., AND R. C. FUSON,"Identification of Organic Compounds," 2nd ed., John Wiley and Sons, Xera (16) CHILDS,A. E., AND W. C. BALL,Analpst, 60, 296 (1935). F. R., E. F. ELDRIDGE, AND W. L. MALLMANN, (17) THEROIJX, "Analysis of Water and Sewage," 2nd ed., McGrm-Hill Co., New York, 1936, pp. 79, 106. Z . angew. Chem., 34, 3 (18) SIEVERTS,A., AND A. HERMSDORF, 1 1001 \ ,.Y*L,.
ANDERSON, G., Z. anal. Chem., 55, 459 (1916). STAMM, J., J . p h a m . chim., 30, 203 (1924). KOLTHOFP, J. M., Z. anal. C h m . , 5 7 , l (1918). CHERONIS, N., "Semimicro Qualitative Organic Analysis," The Crowell Publishing Co., New York, 1947. L., Z. anal. Chem., 75, 167 (1928). (23) SZEBELLEDY, (24) FISHER,H., "Laboratory Manual of Organic Chemistry," 4th ed., John Wiley and Sons, New York, 1938. (19) (20) (21) (22)
