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pyridine and benzidine to form a highly colored polymethine dye is still in use for the spectrophotometric determination of low concentrations of cyanide.5.
ANALYTICAL SCIENCES OCTOBER 2004, VOL. 20 2004 © The Japan Society for Analytical Chemistry

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Cyanide Reaction with Ninhydrin: Elucidation of Reaction and Interference Mechanisms Gabi DROCHIOIU,*† Ionel MANGALAGIU,* Ecaterina AVRAM,** Karin POPA,* Alin Constantin DIRTU,*** and Ioan DRUTA* *Organic Chemistry and Biochemistry Department, Al. I. Cuza University of Iasi, 11 Carol I, Iasi-700506, Romania **Petru Poni Institute of Macromolecular Chemistry, 41a Ghica Voda, Iasi-700487, Romania ***Analytical Chemistry Department, Al. I. Cuza University of Iasi, 11 Carol I, Iasi-700506, Romania

A new sensitive spectrophotometric method has recently been developed for the trace determination of cyanide with ninhydrin. Cyanide ion was supposed to act as a specific base catalyst. Nevertheless, this paper demonstrates that the reported assay is based on a novel reaction of cyanide with 2,2-dihydroxy-1,3-indanedione, which affords purple or blue colored salts of 2-cyano-1,2,3-trihydroxy-2H indene. Hydrindantin is merely an intermediary of the reaction. The formation of a stable and isolable ninhydrin-cyanide compound has been confirmed by its preparation in crystalline form. Also, it is thoroughly characterized by elemental as well as MS, IR, UV/VIS and 1H NMR analyses. The Ruhemann’s sequence of reactions of cyanide with ninhydrin has been reconsidered and an adequate mechanism of the reaction is proposed. As a consequence, the interference of oxidizers as well as copper, silver and mercury ions with the cyanide determination has been elucidated. (Received March 2, 2004; Accepted July 26, 2004)

Introduction Although cyanides and hydrocyanic acid are most toxic, they are nevertheless extensively used in many industries as well as in agriculture.1 Hydrocyanic acid was reported to be present in cigarette smoke as well.2 The origin of blood cyanide concentrations may also be from hydrocyanic acid inhalation from the combustion fumes of plastics.3,4 Due to its acute toxicity, it is very important to monitor the cyanide concentration using specific and sensitive analytical methods. At present, the well-known Aldridge’s method based on the formation of cyanogen bromide and its subsequent reaction with pyridine and benzidine to form a highly colored polymethine dye is still in use for the spectrophotometric determination of low concentrations of cyanide.5 Its variants involve the use of reagents, such as pyrazalone6 or barbituric acid,7 instead of benzidine. Cyanide may be qualitatively8,9 and/or quantitatively2,10,11 determined by measuring the absorbance of chromophores formed by the interaction of cyanide ion with suitable reagents. A specific method for cyanide is based on cyanide reactions with p-nitrobenzaldehyde and oVarious indirect methods have been dinitrobenzene.10 developed based on the discharge of the color of metal complexes by cyanide as a ligand-exchange reaction.1 A direct and sensitive method for the determination of cyanide is reported based on the conversion to ammonia, followed by UV absorption spectrometry.12 Another spectrophotometric method is based on the conversion of cyanide to cyanate using sodium † To whom correspondence should be addressed. E-mail: [email protected]

hypochlorite, followed by acid hydrolysis to form ammonium sulfate. The formed ammonium sulfate is determined based on the Berthelot reaction using salicylic acid, sodium hypochlorite, and sodium nitroprusside to form indophenol.1 Also, a sensitive gas-chromatographic method for the determination of cyanide in biological specimens, based on its conversion to cyanogen chloride,13 as well as a fluorometric procedure14 were described. One of the most recent spectrophotometric methods reported for the trace determination of cyanide is the reaction of cyanide with ninhydrin in an alkaline medium.15 This method is very sensitive, highly specific, and relatively free from interference by various species, and does not require heating or extraction. Nevertheless, the authors have claimed that cyanide ion can act only as a specific base catalyst. Consequently, the analytical behaviors of the reaction, especially the interference and the opportunity to be used in biological media, cannot be explained. Previously,16 we also developed a highly sensitive and selective analytical procedure for cyanide based on its reaction with ninhydrin, and showed that a novel reaction may occur,17 not a catalytic one.18 Being very simple, accurate, fast, selective and sensitive, this method is also useful in the determination of as little as 0.025 µg ml–1 CN– in biological samples.19 Nevertheless, the process whereby the purple or blue compounds are formed from ninhydrin remained obscure. In the present work, the formation of a stable and isolable ninhydrin-cyanide compound was confirmed by its preparation in crystalline form. Also, most of the reaction products were isolated and characterized both spectroscopically and by elemental analyses. Further on, the reaction sequence was compared with that in the literature.20 Thus, we established the mechanism of reaction, which is precious for a cyanide assay, and explained scientifically the interference. In addition, the

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novel cyanide assay in environmental and biological samples based on its reaction with 2,2-dihydroxy-1,3-indanedione has been greatly improved.

Experimental Instrument An UVIKON 933-KONTRON double-beam UV/VIS spectrophotometer with 1-cm matched cells of quartz was used for spectral measurements. In addition, some reaction steps were performed on a CINTRA 10e UV-visible spectrometer in order to establish the most adequate reaction mechanism. The FT-IR spectra were taken on a “Jasco 660-Plus” Fouriertransform infrared spectrometer using KBr-diluted samples against a KBr standard (1 – 2% of analyzing sample). The measured wave number range was 350 – 4000 cm–1 with a resolution of 4 cm–1 and a scanning speed of 2 mm/s. 1H-NMR spectra were recorded with a LUCY (DPX300) 300 MHz spectrometer using D2O, acetone-d6, and DMSO-d6, respectively, as solvents. Mass spectra were carried out with a VESTEC-201, 2000 amu, with an ionization source, EI-CI. The pH values were measured with a CG 837-Schott pH meter. Elemental analyses were obtained from Organic Chemistry Laboratory of Al. I. Cuza University of Iasi. Column chromatography was performed with silica-gel grade 230 – 400 mesh. Melting points were obtained in open capillary tubes, and were uncorrected. Reagents All chemicals were of analytical reagent grade (Merck), and all solutions were prepared with milliQ grade water with R = 18.2 Ω. A standard solution of cyanide was prepared according to Nagaraja et al.15 A 0.5% aqueous solution of ninhydrin in 2% sodium carbonate was prepared. Also, a 2.5 mol dm–3 sodium hydroxide solution was used. Nitrogen was bubbled into each solution to release the interfering oxygen. Synthesis of a cyanide-ninhydrin adduct Potassium cyanide and ninhydrin were mixed in a 2:1 molar ratio. Thus, 1.78 g (10 mM) of ninhydrin, 1, was added to 1.3 g of KCN (20 mM) solved in a 2% solution of sodium carbonate. The white powder of ninhydrin dissolved immediately to form a deep red-colored solution. Upon adding a hydrochloric acid solution, white-pink crystals separated (compound 6). The reaction, which was carried out under nitrogen, proved to be quantitative. The precipitate was washed out several times with milliQ grade water on the filter paper and dried at room temperature. All of the operations were also carried out under nitrogen. The thus-obtained crystals melted at 124 – 126˚C (uncorrected). The structure of the product 6 as well as that of the other compounds in the reaction sequence was established by elemental and spectral (MS, IR, 1H NMR) analyses. When dissolved in solutions of sodium carbonate and sodium hydroxide, respectively, compound 6 changed its color to red (5, λmax 485 nm) and blue (4, λmax 590 nm). Upon evaporating the solvent under a vacuum, the colored compounds were obtained in crystalline form. Hydrindantin formation Small amounts of cyanide are supposed to catalyze the formation of hydrindantin.15,22 In order to prove the correct mechanism of hydrindantin formation, we additionally treated 1 mM ninhydrin (178 mg) with 65 mg of KCN, an equivalent amount of cyanide, in the presence of sodium carbonate.

Fig. 1 Effect of the cyanide concentration on the shape of the absorption curve.

Separately, the amount of cyanide was doubled. Both redcolored solutions were diluted accordingly, and their absorbances were read over the wavelength range from 400 nm to 600 nm. In addition, the reaction of ascorbic acid with ninhydrin to afford hydrindantin was also performed. The three spectra were compared with each other. It was assumed that cyanide reacted stoichiometrically with ninhydrin to form hydrindantin as a major product. Recommended procedure Samples of 10 – 100 µL of solutions containing less than 0.02 µg cyanide were pipetted into an Eppendorf vial of 1.5 mL, and 0.5 – 1.0 mL of ninhydrin reagent was added. To increase the sensitivity, 0.5 – 1.0 mL of sodium hydroxide was sometimes added. The resulting solution was bubbled with nitrogen, capped and let to develop color. The deep-red or deep-blue color was measured at 485 nm and 590 nm, respectively. A reagent blank with no cyanide and a standard with 0.01 µg mL–1 cyanide were used.

Results and Discussion Novel reaction of cyanide with ninhydrin Hydrindantin, 3, seemed to be the major product of the reaction when ninhydrin was treated 1:1 with potassium cyanide. Both the solution of hydrindantin produced in the reaction with ascorbic acid and that of cyanide-ninhydrin 1:1 adduct absorbed at 485 nm. Nevertheless, the former was unstable and its color vanished in the long run. Under these circumstances, we confirmed hydrindantin formation, as previously shown by Nagaraja et al.15 Nevertheless, once formed, hydrindantin possibly reacted with another molecule of cyanide to afford another dye with a characteristic peak at 460 nm, which was superposed with the other at 485 nm. Its size was 94% of the intensity of the 485 nm peak. This peak was previously not reported. Upon doubling the amount of cyanide, the absorbance at 485 nm increased 2.12 times, and that at 460 nm 2.98 times (Fig. 1). Therefore, hydrindantin was the main, but not the sole, product of the reaction, and it reacted with the excess of cyanide to

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Scheme 2 Degradation of 2-cyano-1,2,3-trihydroxy-2H indene under aerobic condition and at a high pH value.

Scheme 1 Reaction sequence for the formation of 2-cyano-1,2,3trihydroxy-2H indene, 6.

complete the formation of the novel compound (Scheme 1). Even if this final product of the reaction proved to be quite stable, it could be decomposed to hydrocyanic acid or cyanide ions and hydrindantin. Thus, upon heating a solution of this adduct in 2% sodium carbonate with a reagent for cyanide, different from the ninhydrin reagent described above, the reaction for cyanide ion was positive. Analyses Elemental (C, H, and N) analyses were compatible with the proposed structure for 6. Having shown that the red and blue colors of 6 were obtained simply by a change in the pH, we considered that the colors were due to the anions of 2-cyano1,2,3-trihydroxy-2H indene. A similar discussion was made previously for the indene structure of hydrindantin.21 The transformation of ninhydrin, 1, into hydrindantin, 3, which is a 2H indene form, a more stable structure, would allow for two ionizable groups. Therefore, the red color is attributable to the monovalent anion, and the blue color to the divalent anion. The ease of oxidation upon exposure to air and oxidizers is consistent with the indene structure of 3 – 6. The fragmentation schema was also in best agreement with the proposed structure for 6. The molecular weight of 6 of 189 Da was determined by mass spectrometry. The main fragments were found to be at 104, 188, 76, 133, 134, and 106 units, respectively. The 1H-NMR spectrum also confirmed the structure of 2cyano-1,2,3-trihydroxy-2H indene. The high peak at 7.90 ppm was assigned to the four aromatic protons. Its shape suggested that they are almost equivalent due to the specific structure of 6, which contains the two enolic groups. Contrary, a structure with two ketone groups would have generated different signals in the 1H-NMR spectrum for the aromatic protons. The three OH protons generated a large band, which was situated from 4.50 ppm to 5.33 ppm in the spectrum, in accordance with the proposed structure. The high peak at 700 cm–1 in the IR spectrum was assigned to 1,2-disubstituted ethylene. The cyan group gave a characteristic signal at 2220 – 2250 cm–1, while the hydroxyl group absorbed at 1200 cm–1. The OH groups associated by H-bonds presented an intense band at 3200 – 3500 cm–1. Moreover, all of the other signals in the IR spectrum were in good agreement with the proposed structure of 6. Also, all of the properties of 6 supported its structure, which was confirmed spectroscopically. Thus, the red color of 5, which is an anion form of 6, vanished upon shaking air into its solution, particularly upon heating. Compound 6 proved to be a powerful reducer due to its HO groups in positions 1 and 3,

which remind us of the similar behavior of the two HO enolic groups in ascorbic acid. Moreover, compound 6 was quite stable in the solid state or at a low pH value. The solution changed color from red to blue with increasing pH. The color again turned to white if the pH was decreased. Bromine, chlorine and other oxidizers destroyed the colored compound to form HCl and HBr. The presence of some reducers, such as ascorbic acid, enhanced the color stability. Under anaerobic reaction conditions, the red color was stable indefinitely, even at 100˚C. Also, under more alkaline conditions, the reaction gave a blue solution, which was stable for weeks in the absence of air. Therefore, cyanide reduced ninhydrin, 1, to hydrindantin, 3, which reacted with another cyanide molecule to form a stabilized 2H indene, 6. This one is stable under anaerobic conditions, but can be easily oxidized to Ruhemann’s compound, 7, which affords 8 and 9 with increasing pH (Scheme 2). Spectral and elemental characteristics 2-Cyano-1,2,3-trihydroxy-2H indene (6). White-pink crystals, m.p. 124 – 126˚C. IR (KBr): ν– = 1200 cm–1 (OH group); 3250 cm–1 (OH group with H bond); 2240 cm–1 (CN group); 700 cm–1 (1,2-disubstituted ethylene). MS 104 [the highest peak, which was assigned to C7H4O·+ resulted from M–(H+HCN+CO+·CHO)]; 188 (M–1); 76 (C6H4+); 133 (C6H4C+(OH)CO or 134 [M–(HCN+CO)]; 106 C6H4(CHO)C≡O+); [M–(HCN+2C=O)]; 78 (C6H6+); 189 (the molecular peak); 161 (M–H–HCN); 51 (C4H3+-aromatic). 1H-NMR (DMSO-d6, 50˚C, δ): 7.90 ppm (aromatic 4H); 4.50 ppm to 5.33 ppm (3HOH). C10H7NO3: calcd. C 63.49; H 3.73; N 7.40; found C 63.10; H 3.55; N 7.55. 2-(2-Cyano-2-hydroxy-acetyl)-benzoic acid (8). Pale-brown crystals, m.p. 158˚C. IR (KBr): ν– = 1770 cm–1 (carboxylic C=O group); 1620 and 1640 cm–1 (C=O group); 2700 – 2850 cm–1 (carboxylic OH group); 1075 cm–1 (OH group); 1300 cm–1 (carboxylic OH group); 3400 – 3450 cm–1 (OH group with H bond); 2210 cm–1 (CN group); 745 cm–1 (1,2-disubstituted benzene); 1540 and 1600 cm–1, respectively (characteristic peaks for the aromatic rings); 3000 – 3100 cm–1 (aromatic); 1360, 1390 and 2860 cm–1 (C–H bond). MS 106 (the main signal), which was assigned to M–(CO2+OHC–CN); 135 was assigned to M–(CO2+CN); 77 (C6H5+); 105 (C6H5–C≡O+); 134 (C6H5C(OH)CO+); and 51 (C4H3+-aromatic, a low peak). 1HNMR (DMSO-d6, 50˚C, δ): 5.67 ppm (alcoholic OH); 2.15 ppm (aliphatic proton); 7.50, 7.62, 7.70, and 7.95 ppm, respectively (aromatic protons); 8.45 ppm (carboxylic proton). C10H7NO4: calcd. C 58.54; H 3.44; N 6.83; found C 58.30; H 3.50; N 6.80. The reaction mechanism The experimental data demonstrated that cyanide ion attacks nucleophylically the C=O group at position 1 of ninhydrin, 1. Then, the negative charged oxygen at position 2 (due to alkaline medium) attacks nucleophylically the carbon atom of the cyan group to release cyanate ion –OCN, and to afford an isomer of

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Fig. 3

Ninhydrin reaction with small amounts of cyanide.

Fig. 2 Spectra of cyanide-ninhydrin complex in 2% Na 2CO3 30 min (1) and 24 h (2) after ninhydrin was added.

hydrindantin (1,2-dihydroxy-3-keto-3H indene). This one turns into the 2H indene isomer, which is more stable. The process whereby ninhydrin is rapid dissolved in a solution of KCN with the formation of a purple solution was thus explained by the 2H indene structure with the two ionizable HO groups at positions 1 and 3. Then, cyanide ion attacks hydrindantin at the C=O group at position 2 to form 2-cyano-1,2,3-trihydroxy-2H indene or its anions according to the pH value. The motive force of the reaction seems to be the high stability of the indene structure over the other structures involved in this study. When we put ninhydrin in a sodium carbonate solution for 48 h, hydrindantin have also been formed. It was red in color and, upon bubbling air, its color vanished and ninhydrin resulted. The colorless solution was able to react again with cyanide. The cyan group also stabilizes the indene structure. Upon blocking with urea or alcohol of one of the HO groups at position 2 of ninhydrin, 1, the reaction between cyanide and ninhydrin was hindered. In addition, Ruhemann’s compound 7 cannot form 2-cyano-1,2,3trihydroxy-2H indene, even if it reacts with cyanide, because one HO group at position 2 was changed by a cyan group. The spectra of colored solutions of the cyanide-ninhydrin adduct showed a gradual disappearance of the yellow color, which is characteristic of ninhydrin in the presence of sodium carbonate (Fig. 2). In addition, the absorbance at 485 nm, slowly decreased, and was practically absent after 2 weeks from the beginning of the experiments. Contrary, the absorbance at 460 nm was constant over this interval. Thus, Fig. 2 shows the good stability of the 2H indene derivative, as compared to hydrindantin. To prove the fact that the cyanate ion is really released as a by-product, we identified it in the solution as ammonia after hydrolysis with sulfuric acid and distillation with 32% sodium hydroxide, as recommended by Deepa and coworkers.1 Thus, the possibility of other reaction mechanisms has been excluded. Stability of the colored product Constant absorbance values were obtained 15 – 30 min after adding a ninhydrin reagent with sodium carbonate as a function of the cyanide concentration in the samples. When sodium hydroxide was added to a deep-red colored solution, the solution became instantaneously blue in color. Due to nitrogen addition, the colors remained stable indefinitely. An increase in

temperature of up to 100˚C resulted in no fading of the color. Upon bubbling air in each solution, the colors gradually faded within a few minutes. Nevertheless, when no nitrogen was used, the stability of the colored solutions was low, especially in the presence of small amounts of cyanide (Fig. 3). The experimental data suggested the tendency of hydrindantin formation, which is less stable. Nevertheless, each time the 2H indene derivative was present. Its formation as well as the presence of hydrindantin clarified the properties of the ninhydrin reagent and possible interference. Interference studies The interfering effects of common anions and cations, which may co-exist with cyanide, were studied. Also, the effect of amino acids, ascorbic acid, and oxidizers, which may be present in biological fluids, was investigated. In the free cyanide determination, varying concentrations of interfering species were introduced into 0.2 µg of cyanide, and the recovery of cyanide was established following the procedure described under the determination of free cyanide. Copper formed a water-soluble, greenish 1:2 molar complex with ninhydrin in a 2% sodium carbonate solution, which was unable to react with cyanide (Scheme 3). Nevertheless, when the ninhydrin was present in a large excess, a reaction with cyanide was again possible. An excess of Cu2+ resulted in the formation of a bluish precipitate of copper carbonate. Upon adding sodium hydroxide, pH increased dramatically and a 1:1 molar complex of copper with ninhydrin occurred. Nevertheless, the deep-green solution of the copper-ninhydrin complex turned immediately into a dark-green precipitate. Once formed, the red or blue color of cyanide-ninhydrin adduct was not disturbed by copper ions. Therefore, it was established that copper ions interfere with cyanide determination due to its reaction with ninhydrin, not with 2-cyano-1,2,3-trihydroxy-2H indene. Silver and mercuric ions also interfere with cyanide determination, making precipitates with the ninhydrin reagent. Nevertheless, their interference was abolished by adding hydroxylamine. The presence of some reducers, such as ascorbic acid, enhanced the color stability. Nevertheless, large amounts of ascorbic acid reacted with ninhydrin to form hydrindantin, which masked the formation of red-colored salts of 6. In the present method, ascorbic acid could be tolerated up

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References Scheme 3 solutions.

Reaction of ninhydrin with copper ions in alkaline

to 50 mg mL–1. Application of the recommended method Previously, samples of industrial and river water as well as blood and urine were analyzed by the recommended assay. The results of the determinations were in good agreement with those made with a well-known spectrophotometric method.11,16,17 Hydroxylamine was added in the ninhydrin reagent only in the case of samples of water being analyzed. Small amounts of copper in blood and serum did not interfere with the determination of cyanide.

Conclusions The reaction of ninhydrin with cyanide has been reconsidered, and evidence supporting an indene structure of a novel compounds, 2-cyano-1,2,3-trihydroxy-2H indene has been obtained. The main reaction product has been isolated in crystalline form and thoroughly characterized. Therefore, an adequate reaction mechanism was advanced, in which hydrindantin is merely an intermediary of the reaction. The interference of oxidizers as well as copper, silver and mercury ions with the cyanide determination has been elucidated. These data could be important to clarify the analytical properties of the novel compounds involved and to improve the procedure for cyanide determination in the environment and in body fluids.

Acknowledgements We gratefully acknowledge the financial support of the CNCSIS Bucharest (Grant 97/2003). The authors also express appreciation to Professor Michael Przybylski for technical

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