Polarographic Determination of 2-Cyanopyridine JOHN M. S. JARVIE, ROBERT A. OSTERYOUNG,
and
GEORGE J. JANZ
W a l k e r Laboratory, Department o f Chemistry, Rensselaer Polytechnic Institute, Troy,
N. Y.
of this method to the determination of 2-cyanopyridine requires a knowledge of the p H of the solution. The present work indicates that the optimum pH range for the purpose is between 5 and 6. At the higher ~ € 1 the , 2-cyanopyridine wave coalesces with the buffer wave, making an accurate determination of the vave height more difficult. Comparison of these results with the polarography of pyridine and related compounds is of interest. From the data for pj-ridine and nicotinamide reported by Knobloch ( 5 ) , an evtremely high value for the ratio of wave height to concentration is obtained (270 and 50, respectively). The value for picolinic acid, estimated from the work of Tompkins and Schmidt ( 6 ) is much lower (14). Reference to Table I shows that the ratio for 2cyanopyridine is more nearly in accord with that for picolinic acid than for pyridine and nicotinamide. Interpretation of the exact electrode mechanism must await further investigation.
A precise and accurate method is described for the polarographic determination of 2-cyanopyridine. The optimum pH range for the determination is between pH 5 and 6 .
T
HE formation of 2-cyanopyridine by the addition of cymo-
gen to butadiene a t moderately high temperatures has been reported by Janz and coworkers (1-3). Owing to the nonbasic nature of 2-cyanopyridineJ the conventional aqueous and nonaqueous potentiometric titration methods were not applicable for determination of the product yields. The present note describes a study of the polarography of 2-cmnopyridine as a method for the quantitative determination of this compound. EXPERIMENTAL
The 2-cyanopyridine (Aldrich Chemical Co., Inc. j was purified I y fractionation in a Podbielniak semimicro distillation column. The fraction collected a t !04" to 105" C. (10 mm.) was used for the polarography; (melting point, 27.0-27.50"; literature (1 ) 29O, 26" C.; ny 1.5221).
Table I.
Results of Polarography of 2-Cyanopyridine
Concentration of 2-Cyanopyridine,
.l4 x 10'
PH
12.4 9.29 6.2 2.32 12.4 9.29 4.65 2.32 9.29 4.65 2.32 9.29 4.65 2.32
5.65 5.65 5.65 5.65 6.2 6.2 fi.2 6.2 7.1 7.1 7.1 7.4 7.4 7.4
Ei/z, Volts us.
S.C.E.
Wave Height, pa.
Wave Height, mM
-1.35 -1.35 -1.35 -1.35 -1.36 -1.36 -1.36 -1.36 -1.39 -1.39 - 1 40 -1.41 -1.41 -1.42
17.60 12.84 8.20 3.28 16.00 11.52 4.72 2.76 10.48 5.44 2.32 10.48 4.60 1.68
14.21 13.82 13.23 14.14 12 90 12.40 10 15 11.90 11 28 11.70 10 00 11 28 9.89 7 24
L L '
9I
APPLIED -1 2 emf
Figure 1.
The preparation of the polarographic solutions was carried out by making an approximately 10-2 solution of 2-cyanopyridine in ethyl alcohol. This solution was used as a bulk solution for the preparation of the solutions used in the polarography. Aliquots of this bulk solution were added to 50 ml. of a prepared aqueous buffer solution (0.21M potassium dihydrogen phosphate) and made up to 100 ml. with ethyl alcohol. Small amounts of concentrated sodium hydroxide solution were added to attain the desired pH. The polarograph used was a Sargent pen recording instrument, Model XXI. The polarographic solution was thermostated at 25' i~ 0.1" C. and the solution w-as deoxygenated by passing a stream of nitrogen through for 15 minutes. The characteristics of the capillary used were a drop time of 2.4 seconds and a mass of mercury of 3.06 mg. flowing per second into distilled water on open circuit a t a mercury height of 56.2 em. The applied potential is referred to the saturated calomel electrode as zero volts.
5 C -1.4 . E (vot?r)
u
-I 6
Typical Polarograms
An illustration of the precision and accuracy of the polarographic method for 2-cyanopyridine is given by the results summarized in Table 11. In each case a weighed sample (0.2413
Table 11.
Polarographic Determination of 2-Cyanopyridine Given, gram
2-Cyanopyridine Found, gram
Purity,
pH
Wave Height, pa.
5.65 7.40
16 40 12 95
0.2413 0 2413
0.2408 0.2423
99.8 100.4
%
RESULTS A S D DISCUSSION
gram) of purified 2-cyanopyridine was analyzed as an unknown. The results leave little to be desired in this method for the quantitative determination of 2-cyanopyridine. This is of especial interest, since, owing to the nonbasic properties of 2-cyanopyridine, the conventional volumetric methods are not applicable. I n the application of this niethod of 2-cyanopyridine determina-
The results for the polarography of 2-cyanopyridine obtained at various concentrations and a t different p H valu.es are summarized in Table I, and the three typical polarograms to illustrate the type of wave are shown in Figure 1. Inspection of these data shows that both the half-wave potential and the wave height are dependent on the p H of the solutions. .4pplication 264
V O L U M E 28, NO. 2, F E B R U A R Y 1 9 5 6 tion of an unknown mixture, it is advisable to separate the 2-cyanopyridine fraction by distillation as described elsewhere
(4).
265 LITERATURE CITED
(1) Hawkins, P.
J.,and Janz. G. J.,J . Am.
Cheiii. Soc. 7 4 , 1790
(1962). ACKNOWLEDG\IENT
Support of this work by a grant-in-aid from the Xational Science Foundation is gratefully acknowledged. The purchase of the Sargent pen recording polarograph was made possible through nn apparatus grant to one of the authors (R.-i.O.) by Trustees Research Committee, Rensselaer Polytechnic Institute The authors wish to thank S. C. Waite. J r . , for assistance in sonip of the measurements.
(2) Janz, G. J., and Hawkins, P. J.,.Vatwe 162, 28 (1948). (3) Jans, G. J.,Alscah,K. G., and Keenan, A. G.. Can. J . Research B25,272 (1947). (4) Jarvie, J. A I . S., Fitsgerald. W. E , and Janz, G . J..,J, d r r i . C ' h e r r ~ . Soc., in
press.
(5) Knobloch, E., Collection Czechosloc. C h e m . Corrirrutris. 12, 407
(e)
(1947).
Tompkins, C., and Schmidt, C. L. A , . r n i o . Calif. ( B e r k e l e y ) Publs. Phusiol. 8, 229 (1944).
R E C E I V E Dfor r e v i m J u n e 1, 1925.
Accepted Soveinher 10. 19%
Colorimetric Ninhydrin Reaction for Measlrrement of Alpha-Amino Nitrogen HAROLD KALANT Department o f Pathological Chemistry, University o f Toronto, Toronto, Canada
The photometric ninhydrin reaction of Cocking and Yemm can be applied safely to the measurement of free amino nitrogen in tungstic acid filtrates of plasma. Excess of ninhydrin or of cyanide reagent exerts a deleterious effect on color development, as does the presence of peroxides. hletal salts were not found to interfere when a citrate buffer was employed. Other protein piecipitants tested were not found satisfactory. A r k rial plasma from normal fasted dogs gave an average valiie of 4.91 mg. per 100 ml.
C
OLORIMETRIC measurement of a-amino nitrogen by means of the purple color formed on reaction with ninhydrin was originally proposed by Harding and 3lacLean ( 2 ) , arid first placed on a sound quantitative basis by Moore and Stein ( 5 ) . This procedure has undergone n ~ ~ m e r o uimprovements s in recent years. The use of reducing agents such as stannous chloride ( 5 ) , ascorbic acid ( I O ) , potassium cyanide (1, 9, If), or reduced ninhydrin ( 6 ) , and addition of various organic solvents such as pyridine (9, 10)or methyl Cellosolve ( I , 5 ) t o the reaction mixtiire result in uniform xield of the same colored product for equiniolecular amounts of almost all of the a-amino acids. An adequate buffer, generally a citrate buffer a t pH 5 (1, 4,5 ) , permits i!se of the method with biological fluids or protein hydrolyrates. The method of Cocking and Teinm ( 1 , 11), which incorporates all these features, has been employed in this laboratory t)ecnuse of its simplicity and accuracy. INFLUENCE O F PEROXIDES
Temm and Cocking ( 1 1 ) specify that the methyl Cellosolve employed should give a faint or negative peroxide test with 10% potassium iodide. The importance of this warning is shown in the following observations. After a long period of successful use of the method as described by the authors, it was found that the reference standard solutions of pure amino acids had begun t o j-ield progressively less color. This decrease became increasingly rapid, until after a month the absorbance was only 15% of its usual value. At first, increasing the amount of ninhydrin reagent above the recommended quantity of 0.2 ml. restored the full color yield, but later, amounts of 1.5 or even 2.0 nil. failed to do PO.
The meth)-l C~elloeolveused in preparation of the ninhydrin :1nd cyanide reagent E had been originally redistilled and stored in a dark bottle. It was n o x found, however, t o have developed a high content of peroxide as indicated by liberation of free iodine from acidified potassiiirn iodide solution, decolorization of I h r k oside of nickel, Prussian blue test, and blackening of inetnllic~ mercury. Tests for reactive carbonyl groups Tere ncgntive. After distillation from a zinc-copper couple the Cellosolve was fi,ee of peroxide, and reagent solutions made with it p1r.e the. iisual fiill color yield in the ninhydrin reaction. Hydroquinone added to the stock reagents in concentrations of 0.001 t o 0.01% does not impair the color reaction, and may serve to prevent peroxide formation. Storage of the Cellosolve under nitrogen, or in small filled dark bottles. is proh:tbl>. also advisable. INFLUENCE OF CY 4YIDE CONCENTR 4TIO\
In view of the harmful effect of peroxides, the influence of variation in the amount of cyanide reagent was determined t - q ing replicate samples of a standard amino acid solution, the reaction was carried out Kith 0.5, 1.0, 1.5, and 2.0 ml. of 2 x 10-4.1P cyanide-Cellosolve reagent. All reaction nii.ctiirrq were
Table I.
Effect of Added Substances on Colorimetric Ninhydrin Reaction
Added hfaterial Zl-one Cobalt nitrate Copper sulfate Ferrous sulfate Versene (satd. rollition in 0 2.11 citrate buffer, p H 5 )
Absorbance 0 208 0 201 0 248 0 ?Oli 0 217
Each reaction mixture contained 5.4 y of a-amino nitrowen. .-\brorhance of each was determined by comparison with a reagent bTank containin. the same added material.
made t o the same volume with meth:.l Cellosolve before heating. \Then peroxide-free reagents were employed, mavimal color yield was obtained with all but the smallest amount of cyanide reagent. When the reagents were made with Cellosolve contaminated with peroxides, use of increasing amounts of cyanide
