cuvette for measurement of the maximum fluorescence. A reference solution containing 0.00025 mg. of quinine per nil. of 0.lATsulfuric acid was used with the instrument set to read 7 5 . The b k n k reading of 24 remained constant throughout the entire reading period of 1 hour. This blank was due to irrelevant fluorescence inherent in the components of the reagents (indole, acetone, and isopropyl alcohol), and not to any spontaneous reaction. A straight line plot vias obtained when the concentration of sarin was plotted against fluorometer readings. Under the conditions of this trial, as little as 0.05 y of sarin in 10 ml. of solution may be determined. The range of concentrations shown in Table I1 does not represent the range of the method, but only of the fluorometer, which in this case was set a t moderate sensitivity. DISCUSSION
The results obtained indicate that this fluorometric method can be used for detection and estimation of small amounts of the nerve gases. The chief virtue of the fluorometric method lies in its inherent sensitivity, which may be hundreds of times greater than that of any colorimetric method. The quantitative data do not indicate the extreme limit of sensitivity that can be achieved, but do show that measurements can be
made of very small amounts. Refinements in procedure and instruments may possibly result in a fluorometric method that will be capable of giving accurate tests on as little as 0.001 y. This method has one defect-the short life of the indoxyr or fluorescent stagewhich requires the immediate reading of the solution when all the reagents have been added. This can be done satisfactorily. but it would be much more convenient in some cases if the fluorescent stage in the reaction could be prolonged to permit dilution of strong tests. Some promising results n-ere obtained in stabilizing this fluorescent stage, particularly by the addition of substances like glycerine, which indicate that the fluorescent compound is subject to stabilization. A complete study of interferences has not been made. Because the basic chemical reaction involved is the same as in the dianisidine method ( I ) , it would be expected that many of the same interferences might be encountered. However, qualitative tests indicate that the indole method is subject to less interference. There is little or no interference from spontaneous color formation; also, certain organic compounds that might be expected to suppress the indoxyl reaction appear to have little or no effect. These include such oxidizable substances as dextrose, glycerine, and alcohol. Carbonates, n-hich
interfered with the colorimetric method, have no effect on the fluorescent method, although large amounts of carbonate cause a change in fluorescent color. The stability of reagents has not been fully studied, but no decomposition of the mixed reagents was noted within a period of several hours. I n quantitative work the indole and perborate solutions can be kept separate until the time of the test. Indole in pure acetone did not decompose in 24 hours, and the water solution of perborate is stable for 8 hours. LITERATURE CITED
(1) Gehauf, B., Epstein, J., Wilson, G. B.,
Witten, B., Sass, S., Bauer, V. E., Rueggeberg, W. H. C., ANAL. CHEY.29, 278 (1957). 12) Holmstead, B., Chem. Eng. News 31, 4676 (1953 ). (3) Riser, A.,Proiar 16, 11/12 (1950). (4)Schonemann, R. B. R., “New Reaction for Detection of Metalloid-Nonmetal Labile Halogen Linkage,” tr. by Wheeler, C. L., Office of Publication Board, U. S. Dept. of Commerce, PB 119887, Sugust 1944. (5) Thorpe, J. F., Linstead, R. P., “The Synthetic Dyestuffs,” 7th ed., Griffin, London, 1933. RECEIVEDfor review July 21, 1956. Accepted November 1, 1956. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., February 1956.
Reaction for Colorimetric Estimation of Some Phosphorus Compounds BERNARD GEHAUF’, JOSEPH EPSTEIN, G. B. WILSON, BENJAMIN WITTEN, SAMUEL SASS, V. E. BAUER, and W. H. C. RUEGGEBERG* Chemical Warfare laboratories, Army Chemical Center,
,The acceleration of the rate of oxidation of amine bases such as benzidine by various organophosphorus compounds has been made the basis of a sensitive method for quantitative estimation of the phosphorus compound. The reaction has been applied to a number of phosphono and phosphoro halides, phosphoroanhydrides, and phosphorophenolates. Selected carbon anhydrides and carbonyl halides also respond to this test with only slight modification of the procedure. The mechanism of the test reaction and the relevant chemistry to enable the reaction to be applied to other compounds are discussed.
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T
Md.
for sensitive and specific detection tests for the German nerve gases, ethyl phosphorodimethylamidocyanidate (tabun) and isopropyl methylphosphonofluoridate (sarin) has brought to light a comparatively little-known or -used reaction which has been found to have analytical (detection and estimation) application t o many phosphorus-containing compounds. A spot test reaction, which produces a yellow color when a n aqueous alkaline peroxide solution is added to the nerve gas in the presence of a n oxidizable amine base such as benzidine, was first described by Schonemann and transmitted through an inHE URGENT NEED
telligence report (4). A summary of the efforts of the authors to understand and extend the original observation made by Schonemann is given here. Included is a typical procedure using these reactions for construction of a calibration curve for sarin, tabun, tetraethyl pyrophosphate (TEPP), or the insecticidal preparation hexaethyl tetraphosphate (HETP). The reaction has been applied in these laboratories to the estimation of microgram quantities of a number of Deceased. Present address, Atlas Powder CO., Wilmington, Del. 1
2
anh) diides, both of phosphorus and carbon, anti acid chlorides of carbon, phosphorus, and sulfur, as well as to phosphono- and phosphorofluoridates. Of special significance is the fact that the compound must contain a particular structure to participate effectively in this reaction. For the phosphorus compounds listed herein which have insecticidal properties by virtue of their ability to inactivate the enzyme cholinesterase. estimation of concentrations of the iiisecticides via this reaction parallels those obtained by measurement of the anticholinesterase properties (and touicity) of the compound. REACTION MECHANISM
Schonemann, t o explain the oxidative pon-er given to peroxide by the nerve gases, considered them to be dipoles with the fluorine or cyanide positive in nature. He postulated that the reaction between the nerve gas and peroxide was such as to liberate nascent oxygen (which could then oxidize the amine base) much in the same manner as bromine or chlorine would oxidize peroxides to oxygen. It is more likely that the reaction proceeds through the formation of a peracid which is responsible for the oxidation of the amine:
If,then, benzidine or a related base is added t o the reaction mixture, the oxidation of the amine takes place either in preference to the hydrogen peroxide and a colored material is formed (as shown in Equation a),or by the oxygen formed as a result of the interaction between the intermediate and excess hydrogen peroxide (as shown in Equation 4). I n either case, the net result is an appreciable acceleration of the rate of oxidation by the mixture over that observed when hydrogen peroxide is used alone. It is perhaps worthy of mention, too, that alkaline solutions of hydrogen peroxide and isopropyl methylphosphonofluoridate oxidize thiosulfate ion to sulfate ion. Feigl attributes such oxidation to peracids ( 3 ) . The exact nature of the colored material formed in the reaction has not been established. FVhen benzidine was used as the amine base, the colored ma-
I n support of this mechanism, studies on the reaction of isopropyl niethylphosphonofluoridate with hydrogen peroxide in the absence of an oxidizable amine have shown that:
terial formed in the reaction had spectrophotometric characteristics in the visible region very similar, but not identical, to that of 4,4’-diaminoazobenzene.
Two moles of hydrogen peroxide are consumed and 1 mole of oxygen is evolved per mole of reacted phosphonofluoridate. The reaction does not occur to a measurable extent in acidic solutions of hydrogen peroxide-Le., p H
