Estimation of Microquantities of Cyanide JOSEPH EPSTEIN, Gassing and Analytical Section, Medical Division, Edgewood Arsenal, M d .
A method is described for the estimation of microquantities of cyanide (or thiocyanate) ion. As little as 0.2 microgram of cyanide can be estimated with an accuracy of 99 * 4% standard deviation. Larger amounts of cyanide may be recovered with less deviation. APPARATUS
A
LTHOUGH many methods have been reported for the microestimation of cyanides, few are both sensitive 'and reliable. The methods of estimation bssed upon the reaction of cyanides with picric acid (8) and the formation of Prussian blue (7) are comparatively insensitive. Good sensitivity has been acquired with the phenolphthalein (4), o-cresolphthalein (6), and Aldridge ( 1 ) methods. The method described herein has the sensitivity of any of these, and in addition has the distinct advantage that it may be carried out in acid, neutral, or slightly dkaline media. Gehauf et aZ. ( 8 ) used a pyridine-pyrazolone mixture for the detection of cyanogen halides. I n this method, cyanogen chloride or bromide reacts with pyridine to form glutaconic aldehyde which forms a . blue colored dye with l-phenyl,3-methyl,5-pyrazolone. By conversion of cyanide to cyanogen chloride with chloramine T solution and by the reaction of this cyanogen chloride with a mixture of pyridine containing 0.1% bis-pyrazolone and 1-phenyl,3-methyl,5-pyrazolone, the author has been able to form a dye which is stable for a t least 0.5 hour at 25" C. and which follows the Beer-Lambert law between the limits of 0.2 and 1.2 micrograms of cyanide ion. The reaction may be expressed by the following equations (6):
+
NaCN
CNCl
A Coleman Universal spectrophotometer No. 11with PC No. 4 filter is suitable. Test tubes fitted with rubber stoppers. Matched square couvettes, 1- to 1.5-cm. cell. REAGENTS
Chloramine T solution, 1%aqueous. Commercial grade of l-phenyl,3-methyl,5-pyrazolone recrystallized twice from 95% ethyl alcohol, melting point 127128" C. Pyndine, C.P. grade. bis-( l-Phenyl-3-methyl-5-pyrazolone) is prepared accordin to the method given by Knorr (3). One-tenth mole (17.4 grams? of recrystallized l-phenyl,3-methyl,5-pyraaoloneis dissolved in 100 ml. of 95% ethyl alcohol, and 0.25 mole (25 grams) of freshly distilled phenylhydrazine is added. The mixture is refluxed for 4 hours. The insoluble portion is the bls-pyrazolone. The mixture is filtered hot and the precipitate washed several times with hot 95% alcohol, melting point >320° C. Pyridine-Pyrazolone Reagent. Five hundred milliliters of a saturated water solution of phenyl methyl pyrazolone are mixed with 100 ml. of pyridine in which 0.1 gram of the bis-pyrazolone has been dissolved previously. The solution Is stored in a dark bottle. On standing, the solution develops a pink color, which does not affect the final result. The reagent should be freshly prepared every 3 days. Standard Cyanide Solutions. A batch of 95y0 sodium cyanide was analyzed by the Liebig method (6)for cyanide, and a solution then made in distilled water containing exactly 10 micrograms of cyanide ion per milliliter of water. This was used as a stock solution. Two-, 4-, 6-, 8-, lo-, and 12-ml. samples were withdrawn from the stock solution and diluted to 100 ml. with distilled water. These solutions contained 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 micrograms per ml. of cyanide ion, respectively. They were used as standard solutions.
+ I
CHa
A
PROCEDURE
Calibration. To 1 ml. of each standard solution in a test tube la added 0.2 ml. of 1%aqueous chloramine T solution. The tube is stoppered immediately and shaken. After one minute,. 6 ml. of the pyradine-pyrazolone reagent are added, the tube is stoppered again, and the reagents are mixed. After 20 minutes the optical density a t 630 mp is compared with that of a reagent blank in a 1- to 1.5-em. cell. The density readings are then plotted against the concentration to make the standard calibration curve. Unknown. One milliliter of the solution containing up to 1.2 micrograms of cyanide ion is treated in the manner described above. The color produced in this tube is read against a reagent blank which is set to zero optical density. The amount of cvanide Dresent in the unknown solution is determineh from the standard curve. The color is stable for a t least 30 minutes (3) after maximum development a t room temperature.
\N' C 'N
H
A
I
I+l
+ HzO +O=C-CH=C-b-C=O
C1-
HI
\"
?!ILL
I
CN 0
0
cCsN(? I1
C-N-
-C=j=O
Hz/C
I
I
EXPERIMENTAL
CHa
0
11
H H H H
N=C
I
CHa
Stability of Dye. h small quantity of the blue colored dye formed in the reaction was isolated and purified. Dilute solutions of this dyestuff were found to be stable between p H 7 and 9. Below pH 7 , the color changed progressively from a blue to a red color; above pH 9, the color faded slowly to an orange. The stability of the dyestuff in solutions of p H between 7 and 9 WM unaffected by excesses of pyridine or pyrazolone. Excess chloramine T, however, rapidly bleached the dye. Pyrazolone reduced chloramine T, and a mixture of pyrazolone and chloramine T
0
H
I1
C-NC=N
I
CH3
With a slight modification of the procedure, thiocyanates may also be determined by this method. 272
V O L U M E 19, NO. 4, A P R I L 1 9 4 7
273 hydrogen-ion concentrations. The color waa developed according to the procedure described without neutralization. Results in Table I1 indicate that solutions between a pH of 2.8 and 9.0 containing cyanide can be analyzed by this method. Ratio of Pyridine tb grazolone. The time for developing a maximum stable color was found to be a minimum when the mixture contained a ratio of pyridine t o pyrazolone of 1 to 5 (Table
Table I. Effect of Delay in Addition of PyridinePyrazolone Solution on Final Color Time of Addition Min. 1 3 6
CN - Added Microgram 0.87 0.87 0.87
Found Microgram 0.87 0.86 0.85
Recovery
% 100 98.9 97.8
111). (in which pyrazolone was in excess) did not affect the stability of the dyestuff. Production of Color. To produce color, it was necessary first to reduce the excess chloramine T. This could be accomplished by the addition of pyrazolone. However, although there is a preferential reaction of pyrazolone with chloramine T, pyrazolone also reduced cyanogen chloride. Cyanogen chloride fortunately preferentially reacted with pyridine; hence, the addition of a mixture of pyrazolone and pyridine enabled the pyrazolone to destroy the excess chloramine T solution while the pyridine reacted with the cyanogen chloride to form the intermediate aldehyde. The aldehyde then combined with the excess pyrazolone to form the blue colored dyestuff, Stabilization of Color. It was found that only a commercial form of pyrazolone obtained from D o n Chemical Co. formed a atable dye. Eastman Kodak pyrazolone or Dow pyrazolone which had been recrystallized from alcohol, water, or chloroform formed a fleeting blue color. Apparently, the commercial pyrazolone contained an impurity which had been stabilizing the dye. This was proved when the addition of a small amount of the alcohol filtrate of recrystallization of Dow pyrazolone to the pyridine-pyrazolone mixture produced a stable color. Since I-phenyl,3-methyl,5-pyrazolone is made from phenylhydrazine and acetoacetic ester, it was thought that phenylhydrazine might be the stabilizer. Small quantities of phenylhydrazine (20 to 200 micrograms) were added to the pyridine-pyrazolone mixture and tested. Although phenylhydrazine stabilized the color considerably, nevertheless the appearance of the final color was altered to a green, due to the combination of the yellow oxidized phenylhydrazine and the blue dye. When a small quantity of pyrazolone dissolved in ethyl alcohol was allowed to react with phenylhydrazine at room temperature for 3 days, a yellow compound resulted. This compound was insoluble in hot alcohol and was separated from the excess of pyrazolone and phenylhydrazine by washing with several portions of hot alcohol. -4crystal of this material was added to the pyridine-pyrazolone mixture and tested. The blue color which developed was stable. An analysis of the compound (by C. A. Rush, Technical Command, Edgemood Arsenal, Md.) showed it to have the composition of bis-(l-phenyl,3-methyl,5-pyrazolone). Found Calculated
C 69.2 69.4
Ha 5.3 5.2
The time adopted for the reading of the sample was 20 minutee after the pyridine-pyrazolone mixture was added. . Interference of Foreign Substances. Substances which may be converted to cyanogen chloride will interfere with the test. Reducing agents, if present in not too large a quantity, will be oxidized by the excess chloramine T and will not interfere. Phosphates, carbonates, chlorides, ammonium salts, borates, sodium salts, cyanates, oxalates, ferricyanides, ferrocyanides, and sulfates have been tested and found noninterfering. Iron salts hasten the reaction of the chloramine T with cyanide, but do not interfere with the final determination. Estimation of Thiocyanate. Thiocyanates may also be determined by this method since, on reaction with chloramine T
Table 11. Recovery of Cyanide from Solutions of Different Hydrogen-Ion Concentration PH 2.8 3.4 5.1 6.1 7.4 9.0
CN (Recovered) Microgram 0.73 0.71 0.72 0.74 0.73 0.73
Recovery
% 100 97.4 98.6 101.2 100 100
Table 111. Effect of Ratio of Pyridine-Pyrazolone Mixture on Time of Development of Maximum Color Time of Development of Maximum Color Min. 40 30 25 20 15 15 15
Ratio of Pyridine to Pyraeolone 1: 1 1:2 1:3 1:4 1:b 1:6 1:7
Table IV. C N - Added Microgram 0.2
Nz 16.1 16.2
A small quantity of bis-pyrazolone was therefore added to the pyridine-pyrazolone mixture. Rate of Reaction between Chloramine T and Cyanide Ion. The reaction between 0.2 ml. of 1% chloramine T solution and the cyanide ion was found to reach a maximum in 1 minute a t temperatures as low as 15"C. Because of the volatility of cyanogen chloride, it is desirable to add the pyridine-pyrazolone solution as soon as possible after the reaction with chloramine T has reached a maximum. However, a t 2.5' C. the loss in color calculated as cyanide is only 27, when the addition of pyridinepyrazolone solution is delayed 4 minutes (Table I). The time adopted for reaction with chloramine T was taken as 1 minute, although not too serious an error is encountered if the time is between 1 and 5 minutes. Effect of pH of Cyanide Solution on Depth of Final Color. Similar quantit,ies of cyanide sdution were made up a t various
C N - Added Miorogram 0.73 0.73 0.73 0.73 0.73 0.73
Length of Stability of Color Min. 30 30 30 30
30 30 30
Recovery of Known Amounts of Cyanide C N - Found Microgram 0.205 0,200 0.195 0.195 0.200 0.205 0.200
C N - Added Microgram 0.8
0.200
0.200 0.200 0.4
0.395 0.390 0.385 0.390 0.410 0.395 0.395 0.395
1.0
0.6
0.615 0,615 0.580 0.600 0,605 0.600 0,580 0.595 0.610 0.610
1.2
C N - Found Microgram 0,805 0.805 0.805 0.810 0.780 0.780 0.780 0.785 0,820 0.820 0.820 0,790 1,000 1,050 1.030 1.000 1,000 0,960 0.960 0.960 0.950 0.950 0.976 0,975 1,025 1.195 1.230 1.195 1.195 1.160 1.160 1.165 1,190 1,190 1.190
0
ANALYTICAL CHEMISTRY
274
thiocyanate will also yield cyanogen chloride. The reaction between thiocyanate and chloramine T, however, is slow and a maximum color developed from the cyanogen chloride formed is not reached until the chlorinating agent and the thiocyanate have been in contact for 30 minutes. Addition of 20 micrograms of the catalyst, ferric chloride, to the thiocyanate solution prior to or simult'aneous with t'he addition of 0.2 ml. of 1% chloramine T decreased t'he time for maximum reaction between thiocyanate ion and chloramine T a t 25" C. to 3 minutes. Amounts of ferric chloride up to 50 micrograms did not further decrease the reaction time.
Table V. No. of Determinations 10 8 10
12 13 8
.
Added Microorams 0.2 0.4 0.6 0.8 1.0 1.2
0.7
Average Recovery
Standard Deviation
5%
%
99 99 100 100 98 99
4 1.3
2.5 1.9
2.8 2.0
DISCUSSION
0.6
Ninety-eight to 1 0 0 ~ oof cyanide in quantities ranging from 0.2 to 1.2 microgram may be recovered by this method with a standard deviation of no greater than 470,as shown in Table V. This method has not been tried for determining cyanide in distillates from animal organs.
0.5 0.L
9
3
Recovery of Cyanide
0.3 0.2 0.1
0
ACKNOWLEDGMENT 0.2
0.4
0.6
0.8
1.0
1.2
1.4
YI~CGRA!S X'AhTDDE ION
Figu e 1. Calibration Curve
The author wishes to express his gratitude to S. D. Silver, chief, Gassing and Analytical Section, Medical Division, and B. Gehauf, Technical Command, for valuable suggestions. Grateful acknowlednment is also due Mrs. Marv " Rumert Van Hollen and Mrs. Aurora Bransford for technical assistance. I
The procedure for thiocyanates is the following:
.
To 1 ml. of a solution containing up to 2.5 micrograms of thiocyanate ion are added 0.2 ml. of 0.1% ferric chloride solution and 0.2 ml. of 1% chloramine T solution. The tube is stoppered and shaken. After 3 minutes' contact time, 6 ml. of the pyridinepyrazolone mixture are added. The tube is stoppered again and the reagents are mixed. After 20 minutes, readings are taken in a spectrophotometer set a t wave length 630 mp with the optical density of the reagent blank set zero. The color is stable for a t least 30 minutes a t room temperature. RESULTS
Figure 1 gives a typical calibration curve. Table 1V shows the recoveries of known amounts of cyanide.
..
LITERATURE CITED
(1) Aldridge, W. N., Analyst, 69, 262 (1944). (2) Gehauf, B., Witten, B., and Falkof, M.,personal communication, June 1944. (3) Knorr, L., Be?., 17, 2044 (1884). (4) Kolthoff, I. M., 2. anal. Chem., 57, 11 (1918). ( 5 ) Kolthoff, I. M., and Sandell, E. B., "Textbook of Quantitative
Inorganic Analysis", revised edition, New York, Macmillan
Go., 1943. (6) Nicholson, R. I., Analyst, 66, 189 (1941) (7) Viehoever, A., and Johns, C. O., J . Am. Chem. SOC.,37, 601 (1916). (8) Waller, A. D., Ibid., 35,406 (1910).
Volumetric Semimicrodetermination of Sulfur in Organic Compounds By Use of the Oxygen Bomb E. C. WAGNER
T
AND
SARAH H. MILES, Department of Chemistry and Chemical Engineering, University of Pennsylcania, Philadelphia, Pa.
HE procedure described depends upon combustion of the sample in the Parr oxygen bomb and determination of the resulting sulfuric acid by precipitation as benzidine sulfate and titration with standard alkali. Similar macro- and microprocedures are already available (3,6, 14); in the development of the method presented, the conditions appropriate to the analysis of samples of semimicro size were determined. The semimicro scale of operation appears to be more favorable to satisfactory execution of this volumetric procedure than either the macro or micro scale. In the macroprocedure the size of the precipitate, combined with its tendency to become matted and compacted upon the filter, results in clogging, difficulty in filtration, and often high results caused by occluded benzidine hydro-
chloride not satisfactorily removable by washing. In the microprocedure accuracy is decreased because the end point of the titration is only moderately sharp and the titration is small. Neither disadvantage is apparent in the semimicroprocedure; filtration is rapid, washing is easy and apparently effective, and the titration yields results which are not affected by any noticeable irregularity. A preliminary study of the volumetric beyidine method on a semimicro scale, with respect to several points on which the literature records disagreements, led to conclusions which were applied in elaboration of the procedure, and which for brevity are stated without supporting data;
