Sensitive Test for Detection and Determination of Malononitrile and its Derivatives Muwaffak Haddadin, Usama Khalidi, Nabila Turjuman, and Raymond Ghougassian Department of Biochemistry, American University of Beirut, Medical School, Beirut, Lebanon
0-Chlorobenzalmalononitrile (CS) is used sometimes as an irritant agent by the military and law enforcement agencies in various parts of the world. Thin layer ( I , 2) and gas chromatographic ( 3 ) procedures have been published as well as ultraviolet and infrared spectra ( 4 ) . A color test with 4(4'-nitrobenzy1)pyridine giving a yellow to orange color has been described ( I ) ; however, this test is neither very specific nor is it very sensitive. We describe here a reaction of malononitrile and its derivatives including CS with benzofurazan oxide in alkalide medium to given an intensive violet color. This test is suitable for quantitative determinations and can be used for field paper strip tests as well as for the staining of the thin layer and paper chromatograms.
EXPERIMENTAL The chemicals used were malononitrile (Merck-Darmstadt), ochlorobenzalmalononitrile (CS) and ethyl cyanoacetate (K & K Laboratories), ethylacetoacetate (L-Light and Co. Ltd.), acetoin (Fluka AG), aspartic acid (Sigma Chemical Co.), malonic acid (Eastman Organic Chemicals), and diethyl malonate (BDH Chemicals, Ltd.). Benzalmalononitrile (BMN) was synthesized by dissolving equimolar quantities of benzaldehyde and malononitrile in n- propanol. The condensation was catalyzed by the addition of 1 drop of IN NaOH ( 5 , 6 ) . The product crystallized into fine needles and was filtered and washed with propanol. I t had the correct melting point (84') and needed no further crystallization. The benzofurazan oxide was synthesized by dissolving 0.64 mole KOH in 500 ml ethyl alcohol with warming. Then 0.58 mole of onitroaniline was dissolved in the warm solution which was cool6cf to zero in an ice bath. Commercial NaOCl (Clorox) 1200 ml were added slowly to the stirred solution keeping the temperature below 10".The flocculent yellow precipitate was collected, washed with water, and air dried. I t was recrystallized from 80% ethanol. Melting point was 72-73' ( 7 ) . Reagents. The following reagents were prepared: 1?/0(w/v) benzofurazan oxide in absolute ethanol; 1 N NaOH; CS standard stock solution (13 itmoles per ml in absolute ethanol); and a CS working standard (15 dilution of the stock solution, 0.5-1 ml used per tube). Procedure. The sample to be measured, dissolved in absolute ethanol. is placed in a dry test tube to which is added 0.1 ml of 1% benzofurazan oxide in absolute ethanol, followed by 0.5 ml of 1N NaOH; H20 is added to a total volume of 10 ml. The violet color developed is read a t 580 nm, in the range between 20 and 30 min after the addition of the NaOH. .4 qualitative test can be run in test tubes or by spotting on paper or thin layer chromatography plates.
RESULTS AND DISCUSSION Benzofurazan oxide reacts in alkaline medium with active hydrogens. The following compounds were tested and shown to give negative results: potassium thiocyanate, maionic acid, ethyl acetoacetate, acetoin, aspartic acid, and chloroacetophenone (CN). Diethyl malonate produced a (1) W. D. Ludemann, M. H. Stutz, and S. Sass, Anal. Cbem., 41, 679 (1969). (2) H. G. Euienhofer,J. Cbromatogr., 36, 198 (1968). (3) S. Sass, T. L. Fisher, M. J. Jascot, and J . Herban, Anal. Cbem., 43, 462 (1971). (4) V. R . Sreenivasan and R . Boese, J. Forensic Sci., 15, 433 (1970). ( 5 ) B. B. Corson and R . W . Stoughton, J. Amer. Chem. Soc., 5 0 , 2825 (1928). (6) S. Patai and E. Rapoport. J. Chem. Soc..383 (1962). ( 7 ) F. 5. Maliory. Org. Syn., 37, 1 (1957).
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yellow color and a precipitate. Ethyl cyanoacetate produces a faint red color. BMN, CS, and malononitrile gave a distinct violet color to the same extent. BMN and CS are known to hydrolyze rapidly in alkaline medium to produce malononitrile. The reaction presumably involves a splitting of the heterocyclic ring of the benzofurazan oxide with the formation of an addition product with the active hydrogen. The presence of the two cyano groups together with the active hydrogens in the malononitrile is probably responsible for the high absorption of the addition product with the benzofurazan oxide with a broad maximum a t 580 nm. Under our assay conditions, the absorbance was proportional to the amount of the malononitrile group up to 0.4 pmole/ml. The color developed reached a maximum in 20 to 30 minutes after which it slowly started to fade and ended by producing a red color. A similar red color was produced when the reaction was run in weaker alkaline medium. It is therefore important to read the reaction in 20 to 30 minutes. When the reaction was run in 9 replicates using 2.6 pmoles of malononitrile and made up to 10 mi, the absorbance a t 580 nm was 0.50 f 0.22, a relative standard deviation less than 5%. The reagent is also useful for the detection of malononitrile, BMN, CS, and related compounds on TLC and paper chromatography where it is possible to detect 5 X mole/cm' by spot test. This is more sensitive than the detection of CS on fluorescent TLC by ultraviolet light. Paper strips prepared by soaking in 1% benzofurazan oxide in alcohol and dried can be used for field testing. In this case, the area suspected of contamination can be wiped with the dry strip followed by dropping 1N NaOH on the paper. An intensive violet color develops on the paper specially on the edges of the drop if malononitrile or one of its derivatives is present. The paper strips are stable to storage a t room temperature.
ACKNOWLEDGMENT The authors thank Makhlouf Haddadin from the Chemistry Department in A.U.B. for a sample and help in the synthesis of benzofurazan oxide.
RECEIVEDfor review March 28, 1974. Accepted June 26, 1974. This research was supported by a grant from the Lebanese Council for Scientific Research. M.H. is Visiting Professor, University of Jordan, supported by the Harkness Commonwealth Fund.
CORRECTION
Signal-to-Noise Ratio Theory of Fixed-Time Spectrophotometric Reaction Rate Measurements
In this article by J. D. Ingle and S. R. Crouch, Anal. Chem., 45, 333 (1973), the exponent in Equation 11 should and the exponent in Equation 16 be lo9 instead of should be lo9 instead of
NOVEMBER 1974
