-
Table 11. Phosphorus Compounds Detected by Sodium PerborateDianisidine Molybdate Method
Detection Limit,
PO, EqUlv.,
Compoiind 1 Sarin 0.5 0 3 Phenylphosphonic acid 1 O B Tetraethyl pyrophos1 phate 0 7 Octylphenyl acid phosphaten 5 1 4 Phenylphosphonot hionic dichloride 1 0 5 4 1 3 Parathionb O,O,S-triethyl -12 0 phosphorodithioate 80 Tributyl phosphate 200 72 0 a Mixture of mono- and diary1 acid phosphates, approximately equimolar. 0,O-Diethyl-0-p-nitrophenyl phosphorothionate. The liquid mixture should be boiled for 45 seconds before solvents ale driven off t o ensure complete hydrolysis.
appears (30 seconds). The tube is aircooled by being waved vigorously (20 seconds), and then the bottom half is water-cooled (10 seconds). Two drops of dianisidine molybdate reagent are added. 4 reddish-brown precipitate indicates the presence of phosphorus compounds. At microgram concentrations it can be viewed as a light cloudiness against a black background. The test appears within 30 seconds. SENSITIVITY.The compounds studied by this method are listed in Table 11. I n the detection of sarin, a nerve gas which is more volatile than most of the conipounds studied, this method was far superior to Methods A and B. With
other members of this subgroup, the sensitivity equaled t h a t of Method A. With difficultly hydrolyzed compounds such as esters, it tvas ineffective. Requiring only two reagents, i t is the most convenient method where applicable. IKTERFERENCES. The method does not permit the presence of a large excess of organic matter n-hich is likely to be carbonized and obscure the test. Most of the interferences observed by West and Robinson in detecting inorganic phosphate are observed here. The test tolerated as much as 230 y of sodium metasilicate; more than this gave a positive reaction. The use of dianisidiiie molybdate reagent a t full strength caused cloudiness in the blanks. Apparently this was due to a reaction with sodium borate. DISCUSSION
The best test for organic phosphorus compounds t h a t has been previously described appears t o be t h a t of Feigl (W), in which the u n k n o m is ignited with calcium oxide a t red heat, and the resulting calcium phosphate is detected by the benzidine molybdate method. The procedure requires five reagents, a considerable amount of time, and is said to apply to nonvolatile compounds only. Where it is applicable, its sensitivity approximately equals t h a t of the tests which utilize o-dianisidine molybdate. Calcium oxide as a degradative reagent is relatively ineffective because it is not an oxidant or even a strong base. The Feigl test relies on conversion of the phosphorus compound to a salt before the unknown escapes through
volatilization. The salt can then be ignited strongly and air-oxidized to calcium phosphate. The types of compounds that will give salts in this way are about the same as those to nhicli Method C is applicable: acids, acyl halides, and anhydrides. It is unlikely t h a t trialkyl phosphates, dialkyl alkylphosphonates, or many of their thio analogs would respond. Method A appears to be the niobt general one now available for organic compounds of phosphorus, and is also the most selective with respect to other hetero-elements (4). Method B is most sensitive, while Method C is the most convenient and selective for acids and easily hydrolyzed acyl derivatives. All three methods are much more nearly independent of the volatility of the unknon-n than previously described procedures. ACKNOWLEDGMENT
This work has been sponsored by t h r United States Army Chemical Corps, whose technical advice and permission for publication are appreciated by the authors. LITERATURE CITED
(1) Boltz, D. F., hIellon, RI. G I . ~ N A L CHEM 19,873 (1947). (2) Feigl, F., “Spot Tests,” 4th ed , 5’01. 2 , p. 77, Elsevier, Sew Tork,
1954.
G. A1 , “Organophosphorus Compounds,” p. 203, Kiley, Yew York, 1950. (4) Robinson, J. IT., West, P. W , in press. (3) Kosolapoff,
RECEIVEDfor review December 7, 1956. Accepted February 18, 1957.
Detection of Nerve Gases by Chemiluminescence JEROME GOLDENSON Chemical Corps Chemical Warfare laboratories, Army Chemical Center,
b Nerve gases can b e detected by the chemiluminescence produced in the presence of a solution containing 5amino 2,3 dihydro 1,4 phthalazinedione (luminol) and sodium perborate; 0.5 y of nerve gas can be readily detected. A plot reasonably close to a straight line was obtained when the amounts of nerve gas were plotted against the maximum luminosity values, indicating that the reaction may also b e used for quantitative purposes. The most promising potential use indicated for the reaction is the applica-
-
-
-
-
Md.
tion to continuous automatic sampling of the atmosphere.
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have been given of specific colorimetric ( 2 ) and fluorescence (3)reactions suitable for the detection and determination of the nerve gases which are organic pentavalent phosphorus compounds having structures either of the type R(R’O)(PO)F or (R2N)(R’O)(PO)CS (4, 8) with very reactive P-F or P-CN linkages. These reactions are outlined in Table I. -4 paper indicator test described ESCRIPTIONS
in a German report (10) has come to be known as the Schonemann reaction. It was based on the oxidation of otolidine to a colored substance by alkaline peroxide in the presence of the nerve gas. A greatly improved reaction based on the use of o-dianisidine was devrloped by Gehauf and others ( 2 ) . It had the desired specificity and a sensitivity comparable t o the best colorimetric methods, but it was realized t h a t a new order of sensitivity would be needed to meet some of the problems imposed by the high toxicity and rapid VOL. 2 9 , NO. 6, JUNE 1957
877
Table I.
Reactions for Assay of Nerve Gases
Approx.
Reaction Schonemann (101
Gehnuf and others ( 2 ) Gehauf and Goldenson ( 3 ) This paprr
Minimum Sensitivity, Reactants o-Tolidine, alkaline peroxide, nerve gas o-Dianisidine, alkaline peroxide, nerve gas Indole, alkaline peroxide, nerve gas Luminol, alkaline peroxide, nerve gas
Assay Method Colorimetric (yellow) Colorimetric (orange-red) Fluorescent (blue-green) Chemiluminescent (blue-green)
Y
... 0.5 0 03 0.5
II. Nerve Gas Concentration Related to Luminescence Microphotometer Reading, pa., at Indicated Time in Concn. of Nerve Gas in Reagent Minutes5 Mixture, y/ML 0.25 0.5 0.75 1 1.5 2 Table
0 31 16 6.2 3.1 0.62 6.2
a
0.01 0.01 0.73 0.35 0.15
0.01 0.60 0.36 0.17 0.06 0.02 0.18
0.06 0.02 0.15 FolloKing addition of nerve gas.
physiological action of the nerve gases. This led to the development of the more sensitive fluorescent reaction mentioned in the table. Later the colorimetric and fluorescence reactions were used in devices for continuous monitoring of the atmosphere ( I , 11). For this purpose, the colorimetric and fluorescence reactions have the disadvantage that sources of visible or ultraviolet light are required to illuminate the sample solution. When the chemiluminescence reaction described here was discovered, its use for detection purposes was studied further because i t did not require such sources of light. I n the commonly used luminol reaction (6), chemiluminescence is produced by treatment of a dilute aqueous alkaline solution of luminol with hydrogen peroxide and potassium ferricyanide. The light produced is normally a greenish-blue color, but may be intensified and/or changed in color by addition of accelerators and fluorescent dyes (5-7). Nerve gases such as sarin give the chemiluminescence in place of the potassium ferricyanide. It is possible that the Fe-CN linkage acts in this reaction as postulated previously (2) for the P-CN and P-F linkages in the nerve gases. The addition of fluorescein (0.002 gram per 100 ml.) to the reagents does not appreciably increase the luminosity upon addition of the nerve gas. EXPERIMENTAL
Roberts (9) investigated the sensitivity of the luminol reaction in order t o 878
ANALYTICAL CHEMISTRY
0.01 0.40 0.24
0.10 0.055 0.015 0.12
0.01 00.25 25 0 . li 0 . 08 08 0.05 0.01 0.08
0.01 0.15
0 10 0.10 0.06 0 06 0.04 0.01 0.01 0.06
0.01 0 . 1100 0.0 077 0.04 0 04 0.04
determine the possibility of using it in an automatic alarm. He found an approximate minimum sensitivity of about 0.5 y of nerve gas. For this sensitivity test, B luminol-perborate reagent was prepared having the following composition: 0.05 gram of luminol (Eastman White Label); 0.01 gram of sodium perborate (c.P.); 0.1 gram of trisodium phosphate (12 HzO) in 100 ml. of distilled chlorine-free water. As the luminol is insoluble in water and only slightly soluble in alcohol, ether, and benzene, it is necessary to dissolve it in an alkaline, aqueous medium. It is soluble in a perborate solution, but the addition of trisodium phosphate makes it easily soluble. The luminol is somewhat soluble in acetone. However, the addition of 401, acetone by volume to the reagent quenches the luminescence obtained from addition of nerve gas to the reagent. The reagent blank luminosity is somewhat larger when the solution is fresh. After standing 1 to 2 hours, it drops to a value which remains constant for at least 1 day. The reagent used in this work was allowed to age for 3 hours prior to use. The chlorine in tap water interferred with the reaction by giving a transient blue flash of light of much greater intensity than large amounts of nerve gas. The addition of ordinary distilled water yields some luminescence measurable with a photomultiplier tube. However, the addition of a small amount of thiosulfate to the water eliminated this interference. The sensitivity of the reagent to chlorine is of the order of 0.001 y per ml. An Aminco microphotometer was set up as follows to measure the chemiluminescence of the reaction. The cover was removed from the 931-A photo-
multiplier tube to expose the entire area of the light-sensitive surface. A cell (19 X 4 X 65 mm.) was constructed to fit the filter holder nearest to the phototube. The calibration was set at 100 and the phototube voltage was set a t the maximum. Portions of 0.5 ml. of known nerve gas solutions in isopropyl alcohol were added to 2.5 ml. of the luminol-perborate reagent in the specially constructed cell. A transient blue-green luminescence was formed which reached peak intensity in 15 seconds and persisted for about 2 minutes. The photometer readings obtained are given in Table 11. The maximum readings were plotted against nerve gas concentration. The points were reasonably close to a straight line, indicating that the reaction may also be used for quantitative purposes. DISCUSSION
I n regard to the practicai application of the chemiluminescence test, the preliminary work indicates that its use for a visual test would be limited because i t must be observed in the dark. However, Huntress has carried out the reaction on a moist towel, and there is a possibility that the reaction might be adapted to a paper detector for special uses. The most promising potential use indicated for the reaction is the application to continuous automatic sampling of the atmosphere. The colorimetric and fluorescence reactions have the disadvantage that sources of visible or ultraviolet light are required to illuminate the s.-tmple solution. By use of the chemihminescence reaction, such sources of light would be unnecessary. Also, by use of a barrier-type photocell and a springoperated pump for obtaining the air sample, a device not requiring a power supply could probably be designed. Some difficulty might be experienced in application of this reaction to quantitative assessment, because the rate of generation and complete conversion of the available “fuel” to chemiluminescence would require careful control. For some applications, a device to integrate the total chemiluminescent energy produced may be satisfactory. LITERATURE CITED
Foley, G. M., Eanes, R. D., Smith, H. R., Cherry, R. H., ANAL. Cmhi., submitted for publication. (2) Gehauf, B., Epstein, J., Wilson, G. B.. Witten. B.. Sass. S.. Bauer. V.’E., Rueggeberg, W.’H. C., Ibid.;
(1)
29, 278 (1957). (3) Gehauf, B., Goldenson, J., Ibid., 29, 276 (1957). (4) . . Holmstead, B., Chem. Eng. News 31, 4676 (1953): (5) Huntress, R. H., Stanley, L. N., Parker, A. S., J. Chem. Educ. 11, 142 (1934). (6) Lacy, H. L., Millison, H. E., Heiss,
F. H., U. S. Patent 2,420,286 (May 8 , 1947). ( 7 ) Plotnikov. I.. Iiubal. J.. Phot. Korr. 74, 97 (1938). (8) Riser, A., Protar 16, 11/12 (1950). (9) Roberts, W. B., unpublished work at Army Chemical Center, Md. I
,
(10) Sclionemann, R. B. R., “New Reaction for Detection of Metalloid-
Nonmetal Labile Halogen Linkage,” tr. by Wheeler, CYL., Office of Publication Board, U. S. Dept. of Commerce, P B 119887 (August 1944).
(11) Young, J. C., Parsons, J. R., Reehcr, H., “Abstracts of Papers,” 130th meeting. ACS. Atlantic Citv. a , -N. .I Septemuder 1 9 k , p. 20B. 7
RECEIVED for review October 23, 1950. Accepted February 12, 1957.
Fluorometric Method for Estimation of Cyanide Application to Estimation of Free Hydrogen Cyanide in Ethy I Dimethylphospho ra m idocya nidate Va por JACOB S. HANKER, ROBERT M. GAMSON, and HAROLD KLAPPER Chemical Warfare laboratories, Army Chemical Center, Md.
b Appreciable quantities of free hydrogen cyanide were found as an impurity in vapor streams of tabun (ethyl dimethylphosphoramidocyanidate). As existing procedures for estimating microquantities of cyanide were unsatisfactory, nicotinamide as a reagent for cyanide in the von Braun reaction was investigated. The free hydrogen cyanide i s separated from the tabun vapor and determined b y the nicotinamide-chloramine-T procedure. This involves conversion of cyanide to cyanogen chloride by chloramine-T. The cyanogen chloride cleaves the pyridine ring of nicotinamide, giving a product which has a strong blue fluorescence in alkaline medium. A plot of fluorescence intensity vs. cyanide concentration approximately follows the Bouguer-Beer law. Tabun concentration i s determined b y the Schb’nemann reaction. The analytical procedure i s confirmed by a total cyanide determination-i.e., free hydrogen cyanide plus cyanide obtained upon hydrolysis of tabun. The method i s more rapid and sensitive than procedures previously described.
D
cyanide by conversion to cyanogen chloride and the subsequent action of the cyanogen chloride on pyridine compounds [von Braun reaction ( 9 ) ] yielding yellow glutaconic aldehydes] which may condense with aromatic amines [Konig reaction ( I d , IS)] or compounds having active methylene groups to form intensely colored products, have been reported by Aldridge ( I ) and Gehauf, Falkof, and Witten (6, 7). The latter suggested the possibility of using a quantitative colorimetric cyanide method with 3-methyl-1-phenyl-5-pyrazolone to condense with the glutaconic aldehyde, yielding an intense blue color. ProETERMINATION Of
cedures for estimation of microgram quantities of cyanide and tabun (GA) by this pyridine-pyrazolone method Fere developed by Epstein (4, 6, IO). I n attempts to use the pyridinepyrazolone procedure for the measurement of tabun-hydrogen cyanide ratios in tabun vapor, several difficulties were noted; lack of reproducibility, length of time required for color development, variation of color intensity with active chlorine concentration, and instability of the reagents. Although Gehauf, Falkof, and Witten (7) tested nicotinamide as a chromogenic reagent in this reaction, they did not mention the appearance of a fluorescence. As nicotinamide has been estimated fluorometrically using cyanogen bromide (3, 14) as a reagent, it was studied as a reagent for the fluorometric estimation of cyanide and tabun by the von Braun reaction. EXPERIMENTAL
Effect of Cyanogen Chloride on Nicotinamide. Initially, a yellow color was produced by the action of cyanogen chloride on nicotinamide in buffered solutions a t p H 7, and a plot of intensity us. cyanogen chloride concentration approximately followed Beer’s law. When viewed under ultraviolet radiation] the yellow solutions had a weak greenish fluorescence. Upon addition of alkali they became colorless under visible light but intensely fluorescent with a bluish hue when viewed under ultraviolet radiation. The intensity of fluorescence was proportional to the intensity of the yellow coIor present before the addition of alkali. Determination of cyanide in strongly alkaline medium is desirable for two reasons: Alkaline solutions are more efficient for the collection of hydrogen
cyanide and they promote rapid hydrolysis of the tabun:
0 I’
(CH,)2S-P-O-
I
+ HCN
CZHr-0 However, neither the yellow color nor the fluorescence was produced when the reagents were added directly to alkaline cyanide solutions. After collection of the tabun and hydrogen cyanide in strong alkali, the p H was reduced and the solution simultaneously buffered by addition of potassium bicarbonate prior t o addition of the chloramine-?’ and nicotinamide. Effect of pH on Fluorescence. Addition of 6117 alkali to the sample solution, after addition of the nicotinamide and chloramine-?‘ reagents, was necessary to destroy the yellow color
20 0 180
160 140 120 100
80 I
2
3
4 5 6 7 8 9 N OF KOH ADDED
1
0
Figure 1. Variation of fluorescence intensity with normality of alkali added after addition of nicotinamide-chloramine-T reagents to solution containing 5 y of cyanide VOL. 29, NO. 6, JUNE 1957
879
