Micromethod for Differentiation of Primary and Secondary from Tertiary Aliphatic Amines by Cya noet hy Ia ti o n GEORGE W. STEVENSON and SHERWOOD H. BIERS Deparfment o f Pharmacology and Toxicology, School o f Medicine, University o f California,
b A procedure specific for primary and secondary amino groups detects aquethese aliphatic amines in 1 OW4M ous solution. The pH's of solutions of these amines are decreased 1 pH unit or more b y the addition of acrylonitrile and heating a t 100" C. Acrylonitrile reacts with primary and secondary amines to form the weaker cyanoethylamine bases responsible for the pH decrease. Tertiary amines and some sterically hindered secondary amines d o not react. The method i s rapid, simple, and reliable.
A
it \{as found in this laboratory that cyanoethylamines were much weaker bases than the corresponding ethylamines (8),a nelv method was developed for detection of primary and secondary amines. The method is based upon the lowering of p H of the solution of primary or secondary amine due to reaction of the amine 11-ithacrylonitrile. Because tertiary amines do not react n ith acrylonitrile, they are differentiatd from primary and secondary amines. A number of methods for differentiation of primary, secondary, and tertiary aliphatic ::mines have been described. The method of Hinsberg (6) using benzenesulfonyl chloride has been the classical qualitative procedure. Treatment of amines with nitrous acid a t 0" C. is another commonly used method. Duke has described a sensitive method for differentiation of amines ( 4 ) . .4 number of procedures for the differentiation of amines on a microgram scale have been described b y I'eigl ( 5 ) . FTLR
PROCEDURE
The p H of 10 ml. of lo-* to 10-4M aqueous amine solution is measured with a Beckman Model G p H meter (pHln,t,al). The solution is transferred to a small glass-stoppered tube and 100 p l . of acrylonitrile (hIatheson P.5534) is added. After'being shaken to dissolve the acrylonitrile, the stoppered tube is placed in a boiling water bath for 5 minutes. After the tube is cooled
under tap water, the p H is measured again (PHM). APH pHin,ttaI - ~ H ~ i n a l (1) When the ApH is 0.60 or more, a primary or secondary amine is present. RESULTS
The procedure was applied to ammonia, nine primary amines, 12 secondary amines, and five tertiary amines (the purest grade of Matheson, Eastman, or Brothers). The ApH values obtained using various concentrations are shown in Table I. The ApH n i t h ammonia was no greater than 0.36. The maximum ApH obtained with tertiary amines was 0.29 with triethanolamine. With other tertiary amines sporadic values up to 0.20 were obtained. All primary and secondary amines gave ApH's of a t least 1.6 a t concentrations of 3 ' X or higher except for diisopropylamine, iminodipropionitrile, di-sec-butylamine, and dicyelohexylamine. The maximum ApH of iminodipropionitrile R as 0.80 and that of dicyclohexylamine was 0.52, The relatively low ApH's n-ere due to either partial or complete failure of the cyanoethylation reaction n ith these four amines. On the basis of these data, 0.60 has been considered a sufficiently high ApH value to indicate the presence of a primary or secondary amine, Some secondary amines are only partially cyanoethylated with acrylonitrile and are therefore not differentiated from ammonia or tertiary amines. DISCUSSION
Amine cyanoethylation will be illustrated using a primary amine, RSH2:
+ CHFCHCEN RNHCHZCHZC&i RKHCHzCHgCkN + CHTCHCGN RNHz
--L
RN(CHzCH2C=I$)*
(2) -+
(3)
Whether Reaction 3 takes place depends upon the nature of R. Titration with acid of the acrylonitrile-treated amine solution allows calculation of the pK. of the derivative. Therefore, if Reaction 3 has taken place, the pK, of the
105
Angeles 2 4 , Calif.
Table II. pH of Amine Solutions as a Function of Their Per Cent Cyanoethylation
Cyanoethylatcd
%
Dimethylamine pH
0 25 50 75 100
11.19 11.03 10.94 10.74 8.87
Diethylamine pH 11.22 11.08 10.07 10.62 9.61
dicyanoethylated primary amine (pK. 4 to 5 ) distinguishes it from the monocyanoethylated secondary amine (pK. 6.5 to 8). I n practice, this is feasible only if solutions are a t least 10-2N in base. Some hindered primary nminrs are not dicyanoethylatcd and therefore not distinguished from secondary amines. Of the primary tunines in t>hisseries, isopropylamine. sec-liutylamine, cyclohesylaniine, benzylamine, and 2-aminoethanol were not dicyanrrethylated. Many functional groups other than the primary or secondary alipliatic amino group react with acrylonitrile, but a catalyst, usually an alkaline catalyst, is required ( 2 ) . Reactions 2 and 3 require no catalyst. Ahis far a s is known, the uncatalyzed reaction of acrylonitrile to give a reduction of p H of an aqueous system occurs only with primary and secondary aliphatic amino groups. The reduction of p H is novel in that it is brought allout by weakening t'he base already present rather than by the addition of acid or removal of the 1 m e . The decrease in pK, on introduction of the @-cyanoethyl group has been found to be in excess of 3 p K units (8). The relation between the pK,'s and structures of the various cyanoamines have h e n studied (7, 8). The relation between the degree of cyanoethylat'ion of the amine in solution and the p H is indicated in Table 11. Aqueous solutions of dimethylamine were mixed with dimethylaminopropionitrile, and solutions of diethylamine -cere mixed with diethylaniinopropionitrile in the stated proportions t o VOL. 31, NO. 12, DECEMBER 1959
2095
Table 1.
lo-* Primary amines Methylamine Ethylamine Propylamine Isopropylamine Butylamine .set-Butylamine
2-Aminoethanol Cyclohexylamine Benzylamine Pecoiidary amines Dimethy lamine Diethylamine Dipropylamine Dibutylamine Diisopropylamine Di-sec-bu tylamine Diisobu tylamine 2-Ethylaminoethanol Piperidine hforpholine Iminodipropionitrile Dicyclohexylamine Tertiary amines Trimethylamine Triethylamine Triethanolamine
Table 111.
..imine Diniethylamine 2-Ethylaminoethanol Piperidine
4.14 2.88 3.16 1.81 2.54 1.86 2.28 L70 2.30
3.83 2.87 2.92 1.80 2.52 1.76 2.28 1.66
2.29
3.51 2.90 2.71 1.71 2.59 1.78 2.26 1.72 2.23
3.13 1.83 2.44
3.15 1.79 2.43 2.79 0.28
3.02 1.66 2.39 2.81 0.23
2.88 1.48 2.20 2 58 0.26
2 53 1.41 1.91 2.33 0.30
...
...
...
3.35 2.48 2.65
0.28
Use of pH Paper in the Cyanoethylation Procedure
10-2
x 10-3 5 x 10-3 10-2 5 x 10-3 5
ANALYTICAL CHEMISTRY
pHm>tmi Meter Paper
PHr)aai Meter Paper
11.12 10.88 10 82 10.59 11 48 11.27
7.99 7.73 8.58 8.58 8.79 8.51
10.9 10.3 10.8 10.5 11.2 11.0
...
0.47 0.33
...
0.02 0.01 0.24 0.17 0.15
0.02 0.08 0.18 0.08 0.09
C'oncn, Ai.
2.14 1.48 1.63
0.17 0.09 0.16 0.12 0.15
0.05 0.00 0.17
0.20
1.59 2.17 1.69 2.17
0.08 0.04 0.27 0.12 0.10
0.09 0.02 0.29 0.05 0.07
0.14
2.50
...
2.90
...
3.05 2.67 2.21 1.63
2.24 2.45 2.37 0.03 0.52
2.23 2.71 3.00 0.51
...
3 X lo-'
2.25 2.33 2.67 2.73 0.17 0.20
2.22 2..76 3.14 0.69
*.
...
0.02
2.24 2.69 3.27 0.80
give total amine concentrations of 10-*51. The pH's of these solutions were measured. With these two amines 75% cyanoethylation or more had to be achieved to attain a ApH of 0.60. Experience in this laboratory indicates that the reaction takes place with the free base, but not n i t h salts of the base. Amine salts, including amino acids, have been detected using the procedure after addition of a n equivalent amount of sodium hydroxide to their solutions, thus adjusting the p H of the sample to the approximate p H of a solution of the pure base. Strong acids or bases, if first neutralized, do not interfere with this modified procedure. The extent of interference from organic acids or bases in using the procedure Rill be determined by the extent of buffering by the acid or base in the region of the ApH of the test base. The extent of interference of ammonia or tertiary amines with the procedure when there is a rnixture of these with a test base of comparable base strength can be roughly determined from the data in Table 11. 2096
6 X lo-'
3 X
4.27 2.76 2.78 1.82 2.55 1.85 2.29 1.77 2.33
...
2-dimethy laminoethanol
.4mine Concentration, Mole/Liter 5 X
4.32 3.02 2.83 2.01 2.71 1.90 2.31 1.54 2.41
0.23
2-Diethylaminoethanol
ApH's of Amine Solutions Treated with Acrylonitrile
7.5 i.2 8 0 i .1 8.3 7.3
All the primary aliphatic aniiiies tested have undergone cyanoethylation. Of the secondary amines tested, those compounds which failed to cj-anoeth! 1ate completely were all a-carbon substituted except for iininodipropionitrile. The magnitude of the ApH values obtained indicated that some cyanoethylation may have occurred. Kitrilotripropionitrile, the product which should be obtained from iniinodipropionitrile by cyanoethylation, has been obtained only in low yields by cyanoethylation of ammonia (8, 9). A 12y0 yield of diisopropylaniinopropionitrile was obtained by Burckhalter et al. ( 3 ) . The low reactivity of these hindered secondary amines makes their differentiation from tertiary amines difficult with methods which depend on derivative formation. The conditions chosen for the cyanoethylation procedure are somewhat arbitrary. Carrying out cyanoethylation also a t room temperature al1on.s differentiation of amines of differing reactivity, but heating to 100' C. was found necessary for the general
2 X lo-' 1.40 1.26 1.31 1.02 2.00 0.07 1.13 1.77 1.52 0.75 1.20 1.12 1.98 0.01 0.25 2.18 1.19 1.94 1.81 0.07 0.29 0.04 0.07 0.19 0.01
...
8X 0.65 0.92 0.71 0.35 0.13 0.30 0.32 0.94
.. 0.25 0.82 0.31
...
0.16 0.24 0.47 1.63 1.25 0.02 0.39 0.19 0.12 0.13
...
...
scheme because of the slow reaction of some compounds. Heating as long as 30 minutes did not change the results. Much smaller volumes than 10 ml. of solution could be used and it is possible that the sensitivity of the method might be further increased by exclusion of carbon dioxide from solutions. Because the acrylonitrile contains essentially no acidic or basic material, its maximum concentration is limited only by its water solubility of 7.35% or 1.4714 at 25' C. (1). The 100 pl. used in the procedure gives a n 0.80% or 0.15M solution. For 10-2M amine the acrylonitrileamine mole ratio is 15. This ratio has been found sufficient in all cases. For a higher concentration of amine, up to nine times this concentration of acrylonitrile could be used. Mole ratios of 1.5 or more nith amine concentrations of 10-2M give ApH values of 1 or more, but mole ratios of 15 have been used to assure complete reaction. A p H meter need not be used as the method of p H determination. Indicators or p H paper can be used. For example, short-range p H paper has been used successfully with amine concentrations of and 5 X 10-3Af. Results using both p H paper and a p H meter with the same solutions are compared in Table 111. It is apparent that cyanoethylation is the most consistently sensitive procedure available for the detection of primary and secondary amines and their differentiation from ammonia and tertiary amines. The simplicity
of the cyanoethylation procedure and the reliability of the measurement used (the measurement of pH) are also very advantageous. Observational uncertainties are inherent in other methods in which precipitation, separation of oils, gas evolution, etc., are utilized. The major limitations of the cyanoethylation method are its failure to distinguish primary amines from secondary amines, to detect some secondary amines and thereby distinguish them from ammonia or tertiary amines, and to detect primary or secondary amines, when in mixtures with a too high
proportion of ammonia or tertiary amine. LITERATURE CITED
(1) American Cyanamid Co., Xew York, N. Y., “Chemistry of Acrylonitrile,” 1951. ( 2 ) Bruson, H. A., Org. Reactions 5 , 79 (1949).
, - - - - I
( 3 ) Burckhalter, J. H., Jones, E. M., Holcomb, W. F., Sweet, L. A.; J. Am. Chem. SOC.65, 2014 (1943). (4) Duke, F.R., IND. ENG.CHEbf., ANAL. ED. 17,196 (1945). (5) Feigl, F., “Qualitative Analysis by
Spot Tests,” Elsevier, New York, 1947.
( 6 ) Hinsherg, O., Kessler, J., Ber. 38, 906 (1905). (7) Soloway, S.,Lipschitz, A., J . Org. Chem. 23,613 (1058). (8) St,evenson, G. R’., \\-illismson, D., J . Am. Chem. SOC.80, 5943 (1958;.
:9) Riedeman, 0. F., Montgomery, W. H., Zbid., 67, 1994 (1945). RECEIVEDfor review August 31, 1959. Accept,ed October 6, 1959. From portions of a thesis submitted hy Sherwood H. Biers to the University of California, Los Angeles, in partial fulfillment of t,he requirements for the degree of master of science. Investigation supported by research grant B-1106 from the Sational Institute of Neurological Diseases and Blindne~sof the h’ational Iristjtutes of Heal.th, U.S.Puhlic Health Errvice.
Microdetermination of Phosphate in the Range of 1 to 10 Micrograms ARTHUR A. HIRATA’ and DAVID APPLEMAN College o f Agriculture, University of California, tos Angeles, Calif.
b A reliable method was needed for determining phosphorus in biological materials containing less than 10 y of phosphorus per available sample. The method of Bernhart and Wreath lacked the sensitivity required for determining such small amocnts. A detailed study of factors affecting the phosphomolybdate chromogen formation led to changes in concentrations of reagents and measuring at a different wave length. The modified method had greatly increased sensitivity; it is possible to determine phosphorus in biological and other materials in the range of 1 to 10 y with high accuracy and precision.
color development. Therefore, a detailed study was mad? of the various factors that influence the formation of the phosphomolybdate complex. The absorbance of the complex was measured at different wave lengths. As a result, changes were introduced in the concentra.tions of the reagents used to form the phosphoinolybdate complex, and in the wave length at which the absorbance of the complex is measured. These modifications increase the sensitivity of the Bernhart and Wreath method many fold, extending its usefulness into the micro region. It is now possible to determine phosphorus in the range of 1 to 10 y with a high degree of accuracy and precision.
and Wreath (2) developed a method for the determination of phosphate, taking advantage of the fact that acetone enhances the absorbance of the phosphomolybdate complex. The method has simplicity of operational procedure, stability of the color developed, excellent reproducibility, and stability of the reagent over several months. These points have been confirmed in this laboratory. The method, however, lacks the sensitivity necessary to determine with a high degree of :muracy less than 10 y of phosphorus. The original report (2) gives little information on the conditions that affect
REAGENTS
ERSHART
1 Present address, Division of Chemistry and Chemical Engineering, California
Institute of Technology, Pasadena, Calif.
MOLYBDATE REAGEST. Dissolve 1.545 grams of ammonium molybdate, (NH4)6M07024 4H20, in 400 ml. of distilled water with the aid of 0.8 mi. of 70% HCIO, and dilute to 1 liter with water. ACETONE,double distilled, stored in deep freeze. PERCHLORIC ACID, 7OY0 (w./v.) , 11.7N1reagent grade. AMMONIUM HYDROXIDE, concentrated, reagent grade (approximately 14N). PHOSPHATE STAKDARD. Dissolve 219.7 mg. of reagent grade potassium dihydrogen phosphate (dried a t 110” C.) in water and dilute to 1 liter (50 y of phosphorus per ml.). Dilute this stock solution with water to obtain standard phosphate solutions 1.0 ‘to 10 y of phosphorus per ml.
ANALYTICAL PROCEDURE
PROCEDURE A. Pipet a maximum of 3.3 ml. of inorganic phosphate solution (ivater for blank) into a 10-ml. volumetric flask. Add 0.3 to 0.6 ml. of 707, perchloric acid. Add water to bring the total volume to approximately 4 ml. and mix, Add 2.0 ml. of molybdate reagent and mix thorouglily. ildd 4.0 ml. of ice-cold acetone, inserting tlie tip of the pipet beneath the surface of the solution. Add water to the 10-nil. mark. Stopper and mix thoroughly. A piece of Saran Wrap (The Dow Chemical Co., hlidland, Xich.) may he used as a stopper for the flask. LIeasure the absorbance a t 320 mu in a Ueckinan I)U spectrophotometer against the blank. The absorbance of the blank against r a t e r should read 0.570 with 1-cm. light path. The ahsorhance of the phosphomolybdate-acetone complex against the blank is 0.324 per 5 y of phosphorus, and remains stable for 8 hours when the phosphorus content in 10 ml. is less than 4 y . With more than 4 y of phoqphorus per 10 ml. the absorbance increases 3531, in 8 hours. PROCEDTJRE 13. Three instead of 4.0 ml. of acetone may be used. I n this case, however, the perchloric acid should. not exceed 0.4 ml. per 10-ml. total volume. The absorbance of the blank should read 0.410. The absorbance of the phosphomolybdate-acetone complex remains the same as in Procedure A. PHOSPHORUS DETERMINATION IN ORGANIC COMPOUNDS
Only organic phosphorus compounds that were hydrolyzed by treating with 707, perchloric acid for 40 minutes a t VOL. 31, NO. 12, DECEMBER 1959
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