Colorimetric Test for Amides and Nitriles SAUL SOLOWAY
AND
ABRAHAM LIPSCHITZ
Chemistry Department, The City College, New York, X. Y .
c
Reagents. 5% ferric chloride hexahydrate in ethanol 1 M hydroxylammoniuni chloride in methanol
E R T A I S derivatives of carboxylic acids such as esters, anhydrides, acid chlorides, and imides react readily with hydroxylamine to form hydroxamic acids. The hydroxamic acids give violet colors with ferric ion in many solvents and have therefore been the basis of the hydroxamic acid test for some carboxylic acid derivatives (1-3). Amides are also converted to hydroxamic acids with hydroxylamine, but more slowly than the other derivatives mentioned. For test purposes the conversion hai been tried in low boiling solvents such as ethanol, methanol, and water. I t has been the experience of the authors and others ( 1 ) that the usual heating period of a few minutes is insufficient, to detect any convrraion by means of color formation nit'h ferric ion. However, when a higher boiling solvent much as propylene glycol is w e d aa the rraction medium, the amides rract readily with hydro Nitriles react with hydroxylamine to produce amidosinies, \vhich usually give red colors with ferric ion ( 4 ) . Horvever, here again, if a low boiling solvent is used, the test, is negative when the heating period is the usual fen minutes. The substitution of propylene glycol for the lower boiling media causes all the nitriles tried to be rapidly converted to the amidoximes. Some miscellaneous derivatives of carbonic acid and other carboxylic acids, such as ureas, guanidines, hydrazides, and amidines, have been converted to hydroxamic acids with hydroxylamine in propylene glycol, where the reaction previously failed under the conditions of reaction in low boiling solvents. The reaction for the formation of ferric complexes of hydroxamic acids ( 5 )and amidoximee can be formulated as fo1lotr.s:
1 ,If hydroxylammonium chloride in propylene glycol 1 Af potassium hydroxide in methanol 1 ilf potassium hydroxide in propylene glycol All solutions were prepared from C.P. chemicals. Procedure for Blank. Because some compounds give red and violet colors with ferric chloride directly, blanks were run. Thirty milligrams of solid sample or one drop of liquid sample a'as dissolved in 2 ml. of methanol, the solut,ion was heated for 2 minutes at the boiling point and cooled, and 0.5 to 1.0 ml. of 5% ferric chloride solution was added. Phenolic or other compounds usually do not interfere wit.h the hydroxamic acid test because of the instahility of their ferric, complexes in acid solution. The same procedure was followed using propylene glycol instead of methanol as the solvent. PROCEDURE A. Approximately 30 mg. of solid sample or one drop of liquid sample was placed in a test tube, and 1 ml. of hydroxylammonium chloride in methanol and 1ml. of potassium hydroxide in methanol were added. The mixture was heated gentlv a t its boiling point for 2 minutes and cooled, and 0.5 to 1.0 nil. of 5 % ferric chloride solution was added. Enough 2 M methanolic hydrochloric acid was added to dissolve any ferric hydroxide. .4 red or violet color constit,ut'ed a positive test. (kcasionally, off-colors appear in these procedures, due to the forination of colored substances in the test admixed with t h r ferric hydroxamates. A case in point is o-nitroacetanilide, which gives a reddish brown color. T h e same color is obtained if the result of Procedure A on o-nit,roaniline (yellow color) is added to the result of Procedure A on but,-] acetate (violet color). PROCEDURE B. Approsiniat'ely 30 mg. of solid sample or one drop of liquid sample was placed in a test tube, and 2 ml. of 1 -11 hydroxylammonium chloride in propylene glycol were added. The mixture was heated at its hoiling point for 2 minutes and cooled, and 0.5 t o 1.0 nil. of 5% ferric chloride solution was added. A red to violet color constituted a positive test. PROCEDURE C. Approximately 30 mg. of solid sample QI, one drop of liquid sample was placed in a test tube, and 2 ml. of 1 31 hydroxylammonium chloride in propylene glycol and 1 ml. of potassium hydroxide in propylene were added. The mixture was heated a t its boiling point for 2 minutes and cooled, and 0.5 to 1.0 nil. of ferric chloride solution w ~ i sd d e d . A red t o violet rolor constituted a positive test. PROCEDLT~E D. Approximately 30 mg. of a solid sample or one drop of a liquid sample m e p1:tced in a test tube, and 1 nil. of 1 JI hydroxylammoniuni chloride in propylene glycol and 2 to 6 nil. of 1 M potassium hydroxide in propylene glycol were added. The mixture was heated at it's boiling point for 2 minutes and cooled, and 0.5 to 1.0 ml. of ferric chloride solution was added. In many cases ferric hydroxide did not precipitate immediately and t,herefore did not interfere with the ferric hydroxamate complex. If, on the other hand, a typically red t o violet color is not obtained because of ferric hydroxide or because of the instability of ferric hydroxamate in alkaline solution, the mixture should be acidified with 2 M methanolic hydrochloric acid.
0
0
oH \\-here X
=
KH2, XHR'. S R ' I I "
H
3H-C-NOH ll H
+ Fe-*- e(R-C-K0)3Fe II
+ 311-
O
0
?;H
Procedure C was developed specifically for nitriles. I t was found that the ferric anlidoximates were much more sensitive to p H than the ferric hydroxamates. After a nitrile is boiled for 2 minutes with a propylene glycol solution of hydroxylamine and hydroxide ion and cooled, and a drop of ferric chloride in ethanol is added, the solution is usualll- acidified to dissolve the ferric hydroxide. A small excess of 2 .If alcoholic hydrochloric acid practically destroyed the rrd color of the ferric complex. In Procedure C, however. it 15 unnecrssary to acidify the solution because the reaction takes place in a hydroxylammoniuni chloride-hydroxylamine buffer in \\ hich ferric hydroxide does not form on addition of ferric chloiide. In a great many casee the addition of water deepened the color of the ferric complexes in the alcoholic solutions and shifted it toward the violet. This obseIvation indicates that the solvent may share in the complex formed, so that the equations given may be oversimplified. Orange colors, which indicate some reaction, were considered to be douhtful or negative,
It
EXPERlMENTA L
Reaction with hydroxylamine is apparently catalyzed 1 ~ alkali. ~ . Hence, three procedures of increasing alkalinity i n propylene glycol were employed to see whether there was a relationship between structure and reaction rate. 898
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2 Slight warming or standing for some time a t room tempprature causes ferric hydroxide to precipitate &-henimmediate precipitation does not take place. The precipitation of potassium chloride on mixing propylene glycol solutions of hydroxylammoniuni chloride and potassium hydroxide is not immediate as it is in the ,case of methanolic solutions. Pure benzamidoxime and benxhydroxamic acid were prepared. The former gave a red color, while the latter gave a violet-red color with ferric ion. The antimonic., stannous, stannic:, silver, mercuric, lead, cadmium, bismuth, barium, nickel, zinc, :rnd arsenic ions did not form cwlored coniplexes with t,hese (tompounds. A green precipitate WIS given with cupric ion rvith tmth of thePe compounds. EXPERIMENTAL RESULTS
Nitriles. Procedure A did not give red or violet colors with any of the nitriles tested. Procedure B gave positive tests with 2-amino-2-cyanopropane, dicyandiamide, diethyl cyanamide, p-dimethylaminopropionitrile, 6, 6’-iminodipropionitrile, and nicotinonitrile. Procedure C gave positive tests, n-hile Procedure B was negative, with the following nitri1r.s: acetone cyanohydrin, acrylonitrile, paminobenzonitrile, n - h t y l cyanide, n-capronit,rile, 3,4-dimethoxyphenylacetonitril~~,P-chloropropionitrile, cyananiide, 1-cyanoethyl-2-naphtho1, lactonitrile, glycolonitrile, phj-droxypropionitrile, nonyl aldehyde cyanohydrin, 8-isoproposypropionitrile, p-nitrol)rnzonit,rile, P,P’-oxydipropionitrile; octadecane nitrile, potassium dicyanoguanidine, succinonitrile, and 0-,m-, p-tolunitriles. Procedures B and C were h t h positive for 2-cyano-2-aminopropane, 8-dirnethylamirioplopioriit,rile, and nicotinonitrile, while Procedure B was positive and Procedure C was negative for dicyandiamide and diethylcyanamide. Every nitrile tested gave a positive result with either Procedure I3 or C. Amides Other Than Derivatives of Carbonic Acid. Procedure =i gave a positive test with only three of the amides tried: onitroacetanilide, p-nitroacetanilitle, and .\--ethyl p-nit,roacetaniliclca.
l’rocc~dures B, C, anti I ) g:ive po’itive tests with acet,aniitle, benzaniide, acetyl-p-aniPitiinr, ac~c,t\.l-o-:riiiinobenzoicacid, iicetJ.1vi-aininophenol, pbroniol)c,~izaniicle, chloroacetamide, cinnanianiide, propionamide, f‘ormamide,nialonamide, nicot,inamide, 0-, vi-, and p-nitrobenzaiuides, octadecanamide, salicylamide, sebacic amide, methylene diacet:iniitlr. .V..\--dimethylforni:tniicle, and furylacrylamide. l’i,oc*edure B gave negative twtr, {vhile Procedures C antl D gave positive tests with t h t t follo\vi~igcompounds: L\T-acetyl-paniinoacet,ophenone, o-arrtylaiiiiriodil,hrn31, S-acetyldiphenj-lamine, iV-acetyl-l-naphthyliiiiii~ie,.V-acetyl-2-napht,hylaniine, o-acetyltoluide, p-bromoac~etariilicIr, acetanilide, o-diaretylbianiridine, m- and p-chloroacrt:tnilidca, S-c.yclohexylbenzaniide, .\--c.yclohexylacetamidr, S,.\“-tliacct~-l-p-phenylenediamine, os:ilylclimilide, pethox~-aI:citiriiiliilri, .~-phenyl-p-phenYll)ropioiiamide, 2,4,6-tril-~romoac:et:i11iliclc~, :ind o-iodot)enzamide. I’rowdures B and C were ncsgative, while Procedure D gave positiw tests with the remaining compounds test,ed: AV,LV’diucet,ylbenzidine, benzanilidr, ~~-~iiethoxyhenzanilide, AV-m-tolylbenzaniide, and N-o-toly1t~e11z:i1i~icle. I,:very amide tested giivc L: i)ositivc result with one of the procedures. Proteins. With the excc,lition of the egg albumin, a thricerecrystallized sample, the proteins tested were commercial gr:ttlrs of doubtful purity. Procedure A gave negative t w t p with all the proteins tried. Procedures B, C, and D gave positive tests with casein and t r?psin. I’vocdure R was negative, hut C and D gave positive tests with rciiiiiii :md bovine serum alburiiin.
899 Procedures B a n d C were negative,, hut I)was positive with the remaining proteins-namely, egg albumin and pepsin. Guanidines. Procedures A, R , :ind C \vert’ negative with all guanidine derivatives tested. Procedure D was positive i n all rases with the exception of g~anidine carbonate. The guuunidines tried were: guanidine carbonat’e, S,?\i’-diphenylguanidiIle, pheiiylbiguanide carbonate, phenylguanidine hydrochloride, o-tolylt)iguanitle hJ~drochloride, and phenylbiguanidine hydrochloride Guanidine carbonate pave a pnsitive t e a t i n Procedure D if, instead of boiling for 2 minutes, the reaction mixture was simply brought to a boil quickly, and inimediately cooled under the titp. Ureas. Procedure A failed with these dt~rivativt~s. Procedures B, C, and L) \yere positive for ure:t only. Procedurrs C and D were positive For thioui.e:L, S,.V‘-diphenyI thiourea, and phenylurea. Phenyl thiourea failed t o givcb :L positive test’ with all procedures given. However, when P I Y J ( W ~ UB,~ P C,S and D were modified its in the case of guanitline carbonate, a positive test resulted. Miscellaneous. m-~itrot)en~hvdrazidegave :t positive test with Procedure B. .kcetamidine hydrochloride gave a positive test i n Procedure D. The few purines and pyrimidines tested gave negative results n-ith all the procedures noted. The compounds tried were uric acid, 1):rrbit.uricacid, xanthine, and caffeine. DISCC‘SSIOS
The more drastic procedures descrilwd herein have estended the scope of the hydroxamic acid test to include the amide, nitrile, arid other functional groups. However, it is first necessary to apply Procedure A (reaction with hydro. to an unknown compound or mixture under the conditions of which esters, acid chlorides, anhydrides, and imides are readily convertrtl t o hydroxamic acids. If these latter groups are present t h e - \vi11 also give positive reactions under the more drastic conditions, and hence will cmnstitute interferences in the detection of amides, nitriles, etc. Some approximate generalizations on the relative rates of reaction of substituted amides and nitriles with hydroxylamine can be drawn from the variation i n prowdure. The results indicate that t,he presence of basic groups in nitriles causes considerable amidoxime formation when the compound is boiled for 2 minutes wit,h hydroxylamnionium c*hloridein propylene glycol (Procedure B), whereas the unsuhstituted nitriles do not yield enough aniidoxime to be detected visually as the ferric complex. This diff cwnce is probably due to the increased concentration of “free” hydroxylamine in solution I J ~proton transfer fium hy~lros~~lammonium ion to the bmic group.
SH,OH
+
+ base eH2SOH .+ base H
+
P-.Iiiiiiiol,enzonitrile is an exception to this generalization. This cwmpound, a vinl-log of cyanamide, is a very weak base hecauw of’ the resonance interaction of the functional groups, This resonance interaction also decreases the positivit,y of the atom, whivh i n turii should decrease the rate of nitrile c:i~~Iion int(,r:ivtioii of this group with hyclrosylaniine. The oiily compounds tested which gave a positive result in niethnriol (Procedure .I)were o- antl p-nitroacetanilides and p nitro-.\--c.t hylacetanilide, which are vinylogs of S-acetylnitramide. Like t.he latter arid the imides, these compounds have two elc~ctronegat~ivrgroups attached to the replaceable nitrogen. rlssuniing that the mechanism of these reactions is an attack by the hydi,osylaniine on the positive cwbonyl carbon atom, this structure feature should increase the rate of reaction with hydrosylaniine. The results indicate that there is a considerable difference in the rate of reaction of unsubstituted nitriles and unsuhstituted
ANALYTICAL CHEMISTRY
900 amides. The latter gave positive tests in Procedure B. whereas the former did not. The single exception found is o-iodobenzamide, where again the slower rate may be due to the large iodo group in close proximity to the amide carbon, thereby decreasing its positivity. It seems anomalous that dicyandianiide and diethyl cyanamide gave positive tests in Procedure B, but m-ere negative in Procedure C. This phenomenon is probably due to the greater instability of the resulting aniidoximes as the solution becomes progressively alkaline. The approximate order of the rates of hydroxylaminolysis can be summarized in the following series: A 1. Acylated aniline with electronegative groups in 0- and ppositions, greater than R 1. Nitriles with,basic groups 2. Unsubstituted amides 3. Ai-alkyl substituted amides greatey than
c,
1. Unsubstituted nitriles 2. N-aryl alkyl amides
greater than L)
1. lY-aryl aryl amides
The variation in the rate of this reaction with proteins may or may not be meaningful because of the purity of the samples used. If crystalline or highly purified samples were to give the same results, this reaction might be useful in pointing up structural differences in certain amide linkages of proteins. Finally, these results show that a n appreciation of such factors as temperature, reaction time, stability of reaction product, and p H may make the difference betn-een a positive and a negative qualitative analytical result. ACKVOWLEDG.MENT
Most of the compounds tested mere from commercial sources. The authors acknowledge with thanks gifts of experimental samples from the American Cyanamid Co., Armour Chemical Corp , hlonsanto Chemical Co., and Rohm & Haas Co. The egg albumin sample was kindly furnished by Harry D‘agreich, Chemistry Department, City College. LITERATURE CITED
(1) Buckles, R. E., and Thelen, J. C., ANAL.CHEM., 22,676 (1950). (2) Davidson, D., J. Chem. Edzccation, 17,81 (1940). 13) Feigl, Fritz, “Laboratory Manual of Spot Tests.” p. 186, Xem
York, Academic Press, 1943.
A41thoughonly a few of the less common derivatives of carboxylic acids were tried, the indications are that the test may be used to detect amidines, hydrazides, and possibly other derivatives. It appears to be general for ureas and guanidines.
(4) Nordmann, E., Ber., 17,2746 (1884). (5) Sidgwick, N;V., “Organic Chemistry of Nitrogen,” p. 198. S e r r Tork. Oxford University Press, 1937. KECEIVED for review June 17, 1951.
lccepted November 8, 1953
Microdetermination of Azide by a Kjeldahl Procedure LEONARD P. PEPKOWITZ’ University of California, Los Alamos Scientijic Laboratory, Los Alarnos, iV. iV. ELATIVELY few methods for the determination of the azide
€3 group have been reported and to the writer’s knoa ledge no
micromethod has been proposed. Methods based on the gravimetric determination of silver azide ( 6 ) , titration with iodine ( 2 ) , titration with silver nitrate ( 3 , 6), titration of the excess silver nitrate ( 9 ) , titration with nitrous acid (8), titration with permanganate (IO), and liberation of free nitrogen by the decomposition of azide with ceric ammonium nitrate ( I ) have been published. The described method is based on Kjeldahl-type procedure and has the advantage of being a micromethod so that only small quantities of explosive materials need be handled. The method is simple and accurate and should be applicable to the semimicro and macro scale as well. The method is based on the observation ( I f ) that hydrazoic acid in the presence of a reducing agent liberates one third of the azide nitrogen as ammonia and the remaining two thirds as free nitrogen. The reaction is quantitative and in spite of the poor factor offers a satisfactory microprocedure because of the accuracy with which ammonia can be determined by the Kjeldahl technique. R E4GERTS
Concentrated sulfuric acid, nitrogen-free. Selenium oxychloride, 12 grams per liter of concentrated sulfuric acid. Sodium thiosulfate, :337,. Perchloric acid, 3594, dillited from t h e 80 to 72% reagent grade with distilled water. Boric acid, 2y0. Mixed indicator, 5 parts of 0.1 Jf bromocresol green and 1 part of 0.1 Mmethyl red (4). Standard hvdrochloric acid, 0.05 1 Present address, Knolls Atoiiiic Power Laboratory, General Electric Co., Scheneotady, N. Y .
Sodium hydroxide, 30%. Ceric sulfate, sat.urated aqueous solution. PROCEDURE
Keigh 5 to 10 mg. of sample on a 3.5 X 4.5mm. piece of cigarette paper and introduce t h e sample together with the paper into a clean dry 18 X 150 mm. rimless test tube. Add 3 drops of 33% sodium thiosulfate and mix thoroughly t o wet t h e sample. Then add 1 ml. of concentrated sulfuric acid and 0.5 ml. of the selenium oxychloride solution. Mix well by swirling the solution and proceed with t h e digestion as described by Pepkowitz and Shive ( 7 ) . DIGESTION.Heat t h e solution moderately with a microburner for a minute or two to determine whether the sample will froth excessively. Following the subsidence of the frothing, if it occurs, boil t h e sulfuric acid solution vigorously for 10 to 15 minutes. This period depends on the length of time necessary for the material to go int.0 solution, clear, and pass from t h e black stage through the muddy brown stage and finally t o a clear ruby wine color. I n this final st,age a colorless liquid condenses on the walls of t h e test t.ube and t,he evolut,ion of white fumes is materially decreased. Cool the digest t o room temperature and add 2 drops of 35% perchloric acid directly to the digest in order to minimize the loss of perchloric acid by volatilization from the sides of the test tube during the subsequent heating. Gently heat below the boiling point until the digest clears and becomes colorless. Cool t,he solution and dilute with a few milliliters of distilled water. Determine t h e ammonia in the digest by t h e usual microdistillation procedure using 2% boric acid as the absorbing solution. Titrate the ammonium borate with 0.05 S hydrochloric acid using the bromocresol green-methyl red indicator (4). I n some lead azide preparations a proteinaceous material, usually gelatin, is added t o limit the size of the crystals in order t o prevent spontaneous detonation. This protein nitrogen will be included wit,h the azide nitrogen by the described procedure. To correct for the nonazide nitrogen, weigh 10 mg. of t h e sample in a microplatinum boat, as cigarette paper is an effective reducing agent,, and carefully slide the boat to t h e bottom of the digestion test tube. Add 1 nil. of R saturated aqueous solution of
