A Specific Fluorometric Method For the Detection of Cyanide SIR: There are numerous published procedures for the estimation of cyanide. These methods may be classified as noncolorimetric methods (titrimetric involving a visual or instrumental end point detection, polarographic, and gas chromatographic methods) ; and colorimetric methods (based on the formation of metal compleses, the Konig reaction, or other miscellaneous procedures), S o n e of these methods is truly specific for cyanide ion ( 1 ) . The two tnethods based on the Konig synthesis (the reaction of cyanogen bromide or chloride with pyridine and an aromatic amine to form a dye) permit the determination of cyanide directly on the original sample, and generally are considered the best for small amounts of cyanide. 130th involve, however, the use of multistage (more than three) processes and/or involve the use of carcinogens. Recently Hanker, Gamson, and Klapper ( 2 ) described a fluorometric method for the estimation of cyanide based on conversion to cyanogen chloride using chloramine-T, followed by reaction with nicotinamide; and lleditsch described a method based upon reaction with chloranilic acid (3). Seither procedure is specific for cyanide. There developed a need in these laboratories for a simple, direct, one-step method for specifically detecting submicrogram quantities of cyanide. Preliminary esperinients indicated that quinone monosiine benzene sulfonate ester (I) satisfied these requirements. .Addition of as little as 0.5 p g . of cyanide to this reagent yielded a highly fluorescent product ([I) which is easily detected. Only cyanide of over 30 anions tested reacts with I to give a fluorescent product. Hence, the method is truly specific for cyanide ion. EXPERIMENTAL
Reagents. All esperiments were performed with reagent grade chemicnls and triply distilled water. All anions were added in the form of the C.P. Podium or potassium salt. Quinone m o 11o sim e benzene C. u 1f o n a t e ester (I) was prepared by reacting equal niolar quantities of the sodium salt of nitrosophenol and benzene sulfonyl chloride in tetrahydrofuran for 1 9 minutes. Five milliliters of pyridine was added. and after 15 minutes the product was isolated in ice 918
ANALYTICAL CHEMISTRY
water, dried, and recrystallized with CC14. The melting point was 130-1” C., and the elemental analysis was: Calcd. for CI2H9O4SS: C, 54.7; H, 3.42. Found: C, 54.0; H, 3.5. Procedure. T o 3 ml. of a 3.4 X 10-4Jf solution of I in dimethyl sulfoside is added 0.1 ml. of the unknown solution. If a green fluorescence develops, cyanide is present. This green fluorescence is read in an spectrophotoAminco - Bowman fluorometer at escitation and etnission wavelengths of 440 and 500 mp, respectively. From calibration plots of fluorescence readings L I S . cyanide concentration, the amount of cyanide present in the unknown may be calculated. DISCUSSION
Only cyanide of over 30 anions tested reacted 11-ithI to give a green fluorescent product. The other ions tested either gave no reaction (Type I : iodide, chloride, bromide. hypochlorite, chlorate, perchlorate, iodate, phthalate, silicate, sulfate, bisulfate, thiocyanate, tartrate, nitrate, fluoride, phosphate, phosphite, thiosulfate, citrate, bromate, sulfide), or a yellow color with no fluorescence [Type 11: periodate, arsenite, sulfite, ferricyanide, cyanate. carbonate, bicarbonate, borate, perborate, bismuthate, tellurite, dichromate, ferrocyanide (rose color), sulfide (yellowish brown)]. I t is interesting to note that sulfide, thiocyanate, and ferrocyanide, which are conimon interferences in most tests for cyanide, give either no reaction, or a yellowish-brown or rose color with I. ;\I1 materials were tested in concentrations up to 10-2J1. Thus, this method is truly specific for cyanide. As little as 0.5 pg. of cyanide per ml. of total solution may be detected by this procedure. A 3.4 x 10-4Alfconcentration of I in diniethylsulfoside was optimum for niasimum sensitivity. A number of other solvents were tested, and the order of fluorescence intensity increased in the general order of solvents used: ethyl formate
