Detection and Estimation of Nerve Gases by Fluorescence Reaction BERNARD GEHAUF' and JEROME GOLDENSON Chemical Corps Chemical Warfare laborafories, Army Chemical Center, Md.
,To meet the many problems imposed by the high toxicity and rapid physiological action of nerve gases such as sarin, a fluorometric method has been developed having a sensitivity a t least 50 times greater than that shown by the best available colorimetric method. The method is based on the formation of a highly fluorescent solution of indoxyl by the reaction of the nerve gas with indole and sodium perborate. Other factors favoring the method are simplicity, rapidity, and freedom from blanks. The problem of interference from extraneous substances was not investigated thoroughly, but indications are that no serious difficulties will be encountered from this source.
A
H
I
H Indole I
OH I
H Indoxyl (highly fluorescent) I1
foinis a blue color indicating further oxidation to indigo. Re-examination in ultraviolet radiation a t this stage reveals a noticeable loss of fluorescence intensity, which proceeds to practically zero after long standing. That the brilliant fluorescent effect thus observed is due to the presence of indoxyl is demonstrated by adding isatin to the reaction niixtuie a t the stage OH
OH I
I
H
H Indigo white (highly fliiorescent) 111
3PECIFIC COLORIMETRIC REACTION
suitable for the detection and analysis of neive gases such as sarin Jvas first described by Schonemann (4). This reaction was based on the oxidation of o-tolidine to a colored substance by alkaline peroxide solution in the presence of the nerve gas. A quantitative method based on this reaction and use of o-dianisidine vias developed by Gehauf and coworkers (1). It had the desired specificity and a sensitivity comparable to the best colorinietric methods, but it was realized that a new order of sensitivity would have to be attained if the many problems imposed b y the high toxicity and rapid physiological action of the nerve gases ( 2 , 3 ) were to be met. Because fluorometric methods are many times more sensitive than colorimetric methods, the possibility of finding a suitable fluorescent reaction was explored. Investigation of a large number of possible reagents in this work revealed that indole could be converted to indigo by reaction with alkaline peroxide and nerve gases such as sarin, soinan, and tabun. These compounds have structures either of the type R(R'O)(PO)F or (R,S)(R'O)(PO)CS (2> 3 ) . Th'is mas of particular theoretical interest, because the synthesis of indigo directly from indole had previously been accomDeceased. 276
plished only by means of ozone. HOKever, froni the standpoint of colorinietric analysis, the reaction is of little value because of the great insolubility of indigo in ordinary solvents, as \\-ell as other factors. More careful investigation of the reaction revealed that several steps were involved in the formation of indigo by this method, as shoum by the folloning reactions:
ANALYTICAL CHEMISTRY
I
H
Compounds I1 and I11 are slightly colored (yellow), but are characterized by an intense blue-green fluorescence when irradiated by ultraviolet radiation of 3000 to 4000A. I n passing from I to 11, the reaction requires high oxidation activity for its accomplishment. The next oxidation steps (I11 and IV) do not require such activity and probably obtain the necessary oxygen from the excess of peroxide present in the reaction mixture. I n practice, it was convenient to use a reagent solution made up of indole and sodium perborate in equal parts of acetone and water. When this is treated ivith a small amount of a nerve gas, a brilliant green fluorescence appears immediately when examined in the radiation of a n ultraviolet lamp; the fluorescence increases to full intensity in less than 1 minute. I n ordinary light the solution appears pale yellow a t this stage, and after a few minutes
I
H Indigo (nonfluorescent) IV
of optimum fluorescence. Instead of indigo, indirubin is formed, which shows marked differences in color and solubility. The reaction involved is as follows:
Indoxyl (keto form)
H Isatin 0
Indirubin V
0
The preparation of coinpound V from these intermediates has been reported in the literature ( 5 ) . REAGENTS A N D APPARATUS
Sodiiini perborate, reagent grade. Indole (rxtra pure), obtained 11.0111 Givaudaii-I~ela\~anria, Inc., 330 W. 42nd Yt., Sew York, S . T. Acetone, c.P., further purified by treatment with activated charcoal and distillation. Quinine, USP grade. Sarin, CHz(CaH7O)(PO)F,purity 98% or Iietter based on phosphorus, fluorine, and isopropoxy group analysis,' prepared for this work bv Oreanic Branch. Chemical and Radiologicd Laboratories, Army Chemical Center, Md. Electronic photofluorometer, Model 12, Coleman Electric Co., Maywood, Ill., with D-type cuvettes and filters B1 (passing 365-mp band) and PCl ( a blue-green filter opaque to 365-mp band). EXPERIMENTAL
Sensitivity of Fluorescence Reaction. Qualitative tests were made t o determine t h e relative sensitivity of t h e fluorescence reaction as compared with t h e colorimetric method ( I ) . T w o test tubes half filled n i t h t h e ieagents Jvere exposed t o vapors of sarin b y siTspending a rod wet ivith t h e reagent for a few seconds in t h e space above t h e liquid sarin. T h e rod n a s then placed in t h e test tube, and t h e tubes were stoppered and shaken. Jf-ith the indole-perborate reagent a stiong brilliant green fluorescence was obtained, \Thereas with the o-dianisidine-peiborate reagent no orange color, or a t the most only a faint yellow, n a s observed. A large number of such tests indicated that the fluorescence method was many times more sensitive than the colorimetric method. Variation i n Reagent Concsntration. Solutions containing large a n i o m t s of perborate developed a maximum fluorescence in a f e n seconds, which then faded rapidly. T h a t this mas a direct effect of p H was shown by t h e even more rapid deTelopment and fading in solutions t o which sodium hydroxide h a d been added. Conversely, lowering t h e alkalinity b y using smaller amounts of sodium perborate slowed up the development of fluorescence to 30 seconds and also prolonged the period of maximum luminosity. The optiniuni pH appears to be about 9.5. Variations in the amount of indole had little effect on the rate of reaction. Acetone, in amounts up to 50%, favored the development of fluorescence but retarded the indigo formation. Glyceiine and alcohol had a like effect. These tests of variations in composition of reagents indicated the possibility of controlling conditions so that the reaction could either be stopped a t the
Table I.
Test YO,
1 2 3 4
5 6 7 8 9 10
Effect of Varying Amount of Indole, Perborate, and Acetone on Development of Fluorescence IsoMax. Indole Perborate Sarin propyl, Max. FliioromSolution, Solution, Water, Acetone, Solution, Alcohol, Reading, eter 111. MI. 111. 111. M1. 111. See. Reading 1
1
m
1 1 1 1 1 0.5 2 1 1
0.5
i 5
1
-
2
6
i
3 8
5 ...
2 2
6.5
2 2
.. .. .. ..
5
..
..
3 6
3
fluorescent stage or, a t least. stabilized long enough for measurements. Use of Silica G e l for Sampling. T h e possibility of using silica gel t o adsorb nerve gas vapors from t h e atmosphere prior t o applying t h e indole test !vas explored. Small amounts of nerve gas vapors were drawn by means of a p u m p through small glass tubes packed n-ith about 0.5 gram of fine-mesh silica gel. A4ddition of indole-perborate solution to the exposed tubes gave a visible fluorescence which was confined to the forward pait of the tube, showing that adsorption was complete. Evposed tubes were also extracted eitlici by passing acetone through the tubcs or b y transferring the contents into 1 nil. of acetone. -4ddition of the indole-perborate reagent gave a positive test from all exposed tubes and a negative one on unevposed tubes. The intensity of fluorescence observed visually was apparently proportionate to the amount of nerve gas vapors sampled. Quantitative Measurements. Some preliminary work was carried out t o determine if t h e fluorescenee reaction could be made t h e basis of a quantitative method. T h e following solutions were prepared: Sarin in anhydrous isopropyl alcohol 10 i y per ml. Sodium perborate in water 2 5 mg. per nil. Indole in acetone 10 mg. per ml. Quinine in 0.1N sulfuric acid 0.001 mg. per ml. Measured amounts of perborate and indole solutions were mixed in a small flask and added to a measured aniount of the sarin solution contained in another flask. The solutions were mixed, immediately transferred to a cuvette, and the maximum fluorescence developed was read b y means of a photofluorometer equipped with a B1 primary filter and a PC1 secondary filter. The sensitivity of the fluorometer was set to read 30 with the quinine solution. The maximum readings were attained in
1 ..
..
1 1 1 1 1 1 1 1 1
3
66
25
20
..
..
20
32 35
38 -19 -17 47 38 36
56
49
53
22
25
20
.. ..
..
Table 11. Data Relating Sarin Concentration with Phototluorometer Readings LTaximuin FliioromSarin Isoeter ReadingSolripropyl Corr. tion, .4lcohol, Not con. for 111. 111. for blank blank 0 0 10 24iblank) 0
0 7
0 9 1.0
0 3
0 1
0 0
43
67 i0 85
55 61
from 30 to SO seconds, after which tiicy decreased. The results obtained (Tahle I) SILOW that tests 4 and 9 gave the maxiiiium readings. However, in test 9 the niaxiniuni reading took about 56 seconds to develop, coinpared to about, 25 swoiitis for test 4. As more time to mako the reading is desired, the conditions of tcst 9 were adopted as optimum for a pi,eliniinary survey to determine if this fluorescence reaction could he used for quant'itative assays. Some t1,ials nit'li different filters inilicated that optiniuni sensitivity foi, tliis fluorescmt subst,ance \vas obtaiiialde irith a primary fiker, B1, passing the 365-1np niercui'y band, interposed het w e n the exciting source and the w n ple cuvette, and PC1, a blue-green filter which does not pass the 365-mp band, interposed between the sample cuvette and phototube. From cxperiments with several filters, it appeared that the emitted fluorescent radiation ranged from about 460 to 490 mp. The data summarized in Table I1 were obtained n-ith the B l and PC1 filters. The solutions described bc,fore were used, with 4 nil. of the indole eolution and 5 nil. of the perborate solution being mixed first, then added to a measured amount of the sarin solution, followed b y ti,ansfer to a photofluoromctcr VOL. 29, NO. 2, FEBRUARY 1957
e
277
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.
278
ANALYTICAL CHEMISTRY
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
