Assessment of Different Fluorimetric Reactions forCyanide Determination in Flow Systems

Esther Miralles, Dolors Prat*, Ramon Compano and Merce GranadosDepartament de Quımica Analıtica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona,Spain

Several fluorimetric reactions for the determination ofcyanide by means of flow injection systems were tested.The first reaction is based on the displacement of8-hydroxyquinoline-5-sulfonic acid (HQS) from thePd(HQS)2 complex by cyanide ion and the subsequentreaction of free HQS with MgII to form a fluorescentspecies. Secondly, the inhibitory effect of cyanide on thereaction between iodine and fluorescein was alsoevaluated as a method to quantify cyanide. The thirdmethod is based on the detection of an isoindole derivativeformed by the reaction of o-phthalaldehyde (OPA) or2,3-naphthalenedialdehyde (NDA) with glycine in thepresence of cyanide. Chemical and hydrodynamicparameters were optimized for each system and theanalytical performance of the methods was established.Detection limits of 5 mg l21 (fluorescein–iodine method),0.40 mg l21 (Pd–HQS–Mg method), 0.25 mg l21 (OPAmethod) and 0.03 mg l21 (NDA method) were obtained.

Keywords: Cyanide; flow injection analysis; fluorimetry

Cyanide is an important environmental contaminant that occursin surface and ground waters as a result of the discharge ofindustrial wastes. Owing to its toxicity, the development orimprovement of methods for its determination in industrialeffluents as well as in natural and drinking waters is a subject ofinterest. In order to detect abnormal cyanide levels rapidly, theuse of on-line monitoring systems is highly desirable. More-over, methods used for cyanide determination should besufficiently sensitive to detect the low concentration levelsallowed by law.

Spectrophotometric methods based on Konig’s reaction arethe most widely used methods for cyanide determination.1–4

Although these methods have shown acceptable sensitivities,the chromogenic reagents are unstable and potentially toxic andthe formation of the detectable product requires about 30 min.The current standard methods for cyanide determination, whichare also based on Konig’s reaction, are complex and very timeconsuming since they require a distillation step prior to thephotometric detection with chloramine-T, pyridine and barbi-turic acid.5,6 Moreover, these methods also suffer seriouslyfrom interferences such as sulfide and thiocyanate. In con-tinuous-flow systems, Konig’s reaction is also commonly used.However, in order to avoid the long reaction periods, some ofthese methods have succeeded in detecting unstable inter-mediate products instead of the final product.2,7 Severalautomated analysers based on flow injection analysis (FIA),either for weak acid dissociable (WAD) cyanide or totalcyanide, with amperometric8 or spectrophotometric detec-tion9,10 have been described in the literature. These methodsinclude a photodissociation stage of stable metal–cyanidecomplexes, and a gas diffusion separation.

Because of its high sensitivity, fluorimetric detection couldbe a suitable alternative to the commonly used spectropho-tometric and amperometric detections. Although several fluori-metric methods have been described for batch determination of

cyanide,11–14 little work has been performed with fluorimetricdetection in on-line systems. The few reported flow injectionmethods are based on the detection of some fluorescentproducts of the modified Konig reaction15 and on a flow-through sensor which suffers from sulfide interference.16 On theother hand, chromatographic methods for cyanide determina-tion using fluorescence detection have also been reported.17–19

These methods are based on the pre- or post-column derivatiza-tion of cyanide with o-phthalaldehyde (OPA) or 2,3-naph-thalenedialdehyde (NDA) and primary amines to afford highlyfluorescent isoindole derivatives.

The aim of this work was to develop sensitive on-linereactions for the fluorimetric monitoring of cyanide in watersamples. For this purpose, three fluorimetric reactions wereexamined. The first depends on the demasking of 8-hydroxy-quinoline-5-sulfonic acid (HQS) by cyanide from the non-fluorescent potassium bis(5-sulfoxino)palladium(ii) chelate[Pd(HQS)2].12 The second is based on the inhibitory effect ofcyanide on the fluorescein–iodine reaction.13 Finally, theisoindole derivative method was also investigated. The influ-ence of chemical, hydrodynamic and instrumental parameterson the reactions, and fluorescent characteristics of the specieswere studied, and the quality parameters for the determinationof cyanide were established.

Experimental

Apparatus

An LS-50 fluorescence spectrometer (Perkin-Elmer, Beacons-field, Bucks., UK) equipped with a xenon lamp and a Model176.752 flow cell (25 ml inner volume) (Hellma, Mullheim,Germany) were used. Slit-widths were set to 15 and 20 nm in theexcitation and emission monochromators, respectively, unlessstated otherwise.

The flow injection manifold consisted of a Minipuls 3peristaltic pump (Gilson, Villiers le Bel, France) and a Model5041 injection valve (Rheodyne, Cotati, CA, USA) with a 170ml injection volume. Tygon tubes were used for pumping, andall reaction and mixing coils were made from PTFE tubing of0.5 mm id.

A Digilab 517 pH-meter (Crison, Barcelona, Spain) with acombined electrode (Orion, Boston, MA, USA) was used for pHmeasurements.

Reagents

All chemicals used were of analytical-reagent grade unlessstated otherwise.

A stock standard solution of cyanide (1 g l21) was preparedfrom sodium cyanide (Carlo Erba, Milan, Italy) in 0.1 m sodiumhydroxide. Working solutions were prepared daily by dilutionof the stock solution with sodium hydroxide solution. Injectionsamples in all experiments were made to be 1 3 1023 m insodium hydroxide.

The potassium bis(5-sulfoxino)palladium(ii) reagent wassynthesized and purified using the procedure described by

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Hanker et al.12 from HQS (Aldrich, Milwaukee, WI, USA) andpalladium(ii) chloride (Carlo Erba). Solutions of 8 3 1025 mwere prepared daily in borate buffer. MgII solutions wereprepared from Mg(NO3)2 (Merck, Darmstadt, Germany).

An ethanolic iodine (Fluka, Buchs, Switzerland) solution (1g l21) was prepared. Aqueous working solutions of iodine wereprepared daily by appropriate dilution of the stock solution withwater. Fluorescein (Merck) stock solution was prepared in 5 31023 m sodium hydroxide. Working solutions were prepared bydilution with phosphate buffer.

Stock 2 3 1022 m solutions of OPA (Fluka, Biochemica) andNDA (Fluka, Biochemica) were prepared in ethanol and storedin the refrigerator. These were diluted with borate buffer to givethe working solutions. Aqueous 2 3 1022 m glycine (Merck)solutions were prepared and stored in the refrigerator. Dilutionswere prepared daily in the same buffer.

MilliQ-plus Ultrapure water (Millipore, Molsheim, France)of resistivity 18.2 MW cm21 was used throughout.

All glassware used for experiments was previously soaked in10% v/v HNO3 for 24 h and rinsed with ultrapure water.

Pd(HQS)2 Demasking Method. Optimization of the FIAVariables

Cyanide detection is based on the displacement of HQS fromthe non-fluorescent palladium complex by the cyanide ion, andthe subsequent reaction of the liberated ligand with a magne-sium ion to form a highly fluorescent complex (Fig. 1).

It has been reported that HQS forms highly fluorescentcomplexes with several metal ions, and that their fluorescenceintensity can be enhanced in the presence of a cationicsurfactant such as cetyltrimethylammonium chloride(CTAB).20 Therefore, in a preliminary fluorimetric study, theexcitation and emission spectra of MgII, ZnII and CdII–HQScomplexes in water and in CTAB micellar media were recordedin the presence of a large excess of metal ion. The resultsobtained showed that the fluorescence intensity was higher forMgII than for the other ions, and it was found that a micellarmedium of CTAB did not increase the fluorescence intensity.The maximum excitation and emission wavelengths obtainedfor MgII were 355 and 498 nm, respectively. It should be pointedout that neither the excitation maxima nor the effect of CTABagreed with the behaviour described for the MgII–HQScomplex.20 These apparently anomalous results can easily beexplained from differences in the fluorescent species present. Inthe paper that studied the properties of the metal–HQScomplexes,20 a large excess of HQS with respect to MgII waspresent, leading to the formation of an Mg(HQS)2 complex,whereas in the demasking reaction described here the metal ionwas in excess, which resulted in the complex Mg(HQS) beingthe predominant species, and hence, different charges andexcitation maxima.

The manifold used is illustrated in Fig. 2(a). Other configura-tions were tested but better responses were obtained with theproposed manifold. The sample was injected into an aqueouscarrier stream, which then merged with the Pd(HQS)2-bufferedsolution in the reaction coil L1. The resulting solution wassubsequently mixed with an MgII solution in the reaction coil L2to produce the fluorescent species. In order to start the FIAstudy, the conditions were as follows: Pd(HQS)2, 1.6 3 1024 min borate buffer of pH 9.0, MgII, 0.1 m.

Effect of coil length flow rate

It was observed that an increase in the length of L2 as well as adecrease in the global flow rate produced an increase in thefluorescence response. The total flow rate was studied between1.3 and 3 ml min21, and for each value tested, the length of L2was varied between 2.5 and 11 m. It was observed that a coil L2of 7 m and a flow rate of 0.5 ml min21 in each channel producedthe optimum signal on the basis of peak intensity anddispersion. Moreover, the length of L1 was found not to becritical, which showed that the displacement reaction was fast.A coil L1 of 2 m was selected for further studies.

Effect of chemical parameters

The effect of pH was studied in the range 7–10.5. The responseincreased with increasing pH up to 9 and the optimum responsewas achieved in the pH range 9–10; between these values,variations in fluorescence intensity were not significant. Forfurther studies, Pd(HQS)2 was prepared in boric acid–NaOHbuffer of pH 9.8, which fell to pH 9.6 in the waste solutionowing to the acidity of the Mg(NO3)2 solution.

The effect of MgII concentration was investigated over therange from 2 3 1023 to 0.8 m. The results showed that theresponse increased up to a concentration of 0.2 m MgII and thenremained constant. Hence, a value of 0.3 m was adopted insubsequent experiments.

The concentration of Pd(HQS)2 was varied in the range from3 3 1025 to 3 3 1024 m. Between 6 3 1025 m and 3 3 1024

m, no significant difference was observed in the response. An 83 1025 m solution was used in further studies, which guarantees

Fig. 1 Reactions for cyanide determination with Pd(HQS)2 and MgII.

Fig. 2 Diagrams of the flow injection systems. L1, L2, Reaction coils; V,injection valve; W, waste. Manifold (a) Pd(HQS)2 demasking method:reagent A: Pd(HQS)2-buffered solution, reagent B: MgII solution; fluor-escein–iodine method: reagent A: iodine solution, reagent B: fluorescein-buffered solution; isoindole derivative method: reagent A: OPA-bufferedsolution, reagent B: glycine-buffered solution. Manifold (b) isoindolederivative method: reagent A: OPA- or NDA-buffered solution, reagent B:glycine-buffered solution.

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an excess of palladium complex with respect to cyanide in theworking interval of concentrations.

Effect of temperature

In order to study the effect of temperature, the reaction coil L2was placed in a thermostated bath. The bath temperature wasvaried over the range from room temperature to 55 °C. Workingwith the selected reaction coil (L2 7 m), the effect of temperatureon the reaction was not significant; however when a shorterreaction coil was used (1.5 m) the response increased withincreasing temperature. Since similar responses were obtainedusing a long coil at room temperature and a short coil in athermostated bath at 50 °C, the former conditions were used forconvenience. The working conditions selected are summarizedin Table 1.

Fluorescein–Iodine Method. Optimization of the FIAVariables

Iodine reacts with fluorescein to produce a non-fluorescentreddish species. The products of the electrophilic aromaticsubstitution have not been characterized. If cyanide is present,iodine reacts with cyanide (I2 + CN2 ? ICN + I2), and anincrease in the fluorescence intensity is observed because of theinhibition of the reaction of iodine with fluorescein.

Kinetic aspects

As the only literature reference about this application13 did notcontain information about kinetic aspects of the reaction andpreliminary studies indicated that the kinetics of the reactionsinvolved in this system could have a strong influence on thefluorimetric response, some parameters were examined using abatch system. The experimental conditions were taken from theliterature and were as follows: fluorescein, 4 3 1026 m, iodine,5.5 3 1026 m; phosphate buffer of pH 6.5; borate buffer of pH8.5. These pH values were selected as the first was the optimumgiven in the literature and the second was the optimum found forthe fluorescence intensity of fluorescein.

As can be seen in Fig. 3, the extinction of fluorescence wasfast and a stable signal was reached in several minutes whennearly equimolar concentrations of fluorescein and iodine at pH6.5 were used. Visually, the solution rapidly evolved to areddish colour. On the other hand, higher pH values presentedslow kinetics. Different iodine : fluorescein molar ratios werealso tested at pH 6.5. It was observed that when a 1 : 1 molarratio was used the fluorescence intensity decreased five timeswith respect to a solution containing only fluorescein whereas a

2 : 1 iodine : fluorescein molar ratio caused the completeextinction of the fluorescence. Although an excess of iodineleads to a lower baseline, it is not advisable to work with a highiodine : fluorescein ratio since cyanide could react with iodinewithout causing an increase in the fluorescence intensity.

The order of addition was also varied. It was observed that theintensity was maximum when fluorescein was added to thereaction product between cyanide and iodine. A minor responsewas obtained when iodine was added to a mixture of fluoresceinand cyanide, and no signal was obtained when cyanide wasadded to the reaction product between fluorescein and iodine.

Effect of coil length and flow rate

The study was conducted with the flow-injection manifolddepicted in Fig. 2(a). As shown by the study described above,other mixing orders are not possible: the sample containingcyanide reacts with iodine and then the fluorescein-bufferedsolution is added to obtain the signal. Initially, 1.2 3 1025 mfluorescein in phosphate buffer of pH 6.5 and 1.2 3 1025 maqueous iodine solution were used.

The length of the first reaction coil, L1, was varied between20 cm and 1.5 m. An increase in length only caused dispersioneffects; hence, an L1 of 50 cm was selected for furtherexperiments. As expected, owing to the kinetics of the reactionbetween iodine and fluorescein, an increase in the length of L2produced a lower baseline because the amount of iodo-fluorescein formed was higher. However, no significantdifferences were obtained in the peak intensity between L2 = 50cm and L2 = 4 m. A coil L2 of 2.5 m was selected for furtherexperiments. The flow rate, which was investigated in theinterval 0.5–1.5 ml min21 in each channel, did not have asignificant effect on the signals either. A flow rate of 1 ml min21

in each channel was adopted.

Effect of chemical parameters

Because of the complexity of a flow system in which kineticsplays an important role, a factorial design experiment with four

Table 1 Operational conditions selected for cyanide determination

Method Conditions selected

Pd(HQS)2–MgII Borate buffer of pH 9.8 83 1025 m Pd(HQS)2,0.3 m MgII 1.5 ml min21, L1 2 m, L2 7 mlex = 355 nm, lem = 498 nm

Fluorescein–iodine Phosphate buffer of pH 6.5 1.23 1025 mfluorescein, 1.23 1025 m iodine 3 ml min21,L1 50 cm, L2 2.5 m lex = 488 nm,lem = 513 nm

OPA–glycine Borate buffer of pH 8.2 23 1023 m glycine,23 1023 m OPA (10% ethanol) 1.5ml min21, L1 2 m, L2 5 m lex = 331 nm,lem = 379 nm

NDA–glycine Borate buffer of pH 9 1 mm glycine,43 1024 m NDA (4% ethanol)1.5 ml min21, L1 2 m, L2 15 mlex = 418 nm, lem = 480 nm

Fig. 3 Decrease in the fluorescence intensity as a function of time of asolution containing 4 3 1025 m fluorescein and 5.5 3 1025 m iodine, in (a)phosphate buffer of pH 6.5 and (b) borate buffer of pH 8.5.

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factors at two levels was used to investigate the reaction.21 ThepH factor was studied at 6.5 and 7.5. Among the differentprotonated species of fluorescein, only the totally deprotonatedspecies exhibits fluorescence (pKa = 2.2, 4.4, 6.7) as wasdeduced from the sigmoidal curve obtained from the variationof the fluorescence intensity of fluorescein as a function of pH.The iodine : fluorescein molar ratio was expected to have animportant effect. Hence, 1 : 1 and 2 : 1 molar ratios were tested.Fluorescein concentration was also studied at two levels: 1.2 31025 and 6 3 1026 m; in previous studies it was observed thathigher concentrations of fluorescein caused poorer signalsowing to the increase in baseline noise. The fourth factor wasthe length of coil L2 which was studied at 2.5 and 4 m. Acyanide concentration of 50 mg l21 was used in this study.Owing to the high emission intensity of fluorescein, excitationand emission slits were kept at 5 nm. Data of the factorial designexperiment are given in Table 2.

After the 24 experiments, the Statgraphics Plus 2.0 program(Statistical Graphics 1994–96) was used to calculate the effectsand their interactions. The estimated effects and the results of ananalysis of variance (ANOVA) test are shown in Table 3. Sixeffects had P-values less than 0.05, indicating that they weresignificantly different from zero at the 95% confidence level.pH and iodine : fluorescein ratio were the most important factors

and there was a strong interaction between them. Although thefluorescein concentration was found to be significant, no furthervariations in this factor were made because this experiment aswell as previous studies provided sufficient information. Thesmall interaction between reagents ratio and L2 was probablybecause longer reaction coils caused a slightly lower baseline.The reagents ratio and fluorescein interaction had a P-valueclose to 0.05, indicating that this interaction is less importantthan the others. Consequently, pH and iodine : fluorescein ratiowere studied in more detail by carrying out a two factor fourlevel variation. The pH was varied over the range 5.5–7.5, andthe iodine : fluorescein molar ratio between 2 : 1 and 0.5 : 1 ateach of the pH values studied. Slightly higher signals wereobtained at pH 7 and a 0.5 : 1 ratio; however, pH 6.5 and a 1 : 1ratio were selected on the basis of signal-to-noise ratio. It shouldbe pointed out that one of the reasons for the high backgroundlevel is the fact that, at the working pH, fluorescein is present inthe solution partially as a fluorescent species. The workingconditions selected are summarized in Table 1.

Isoindole Derivative Method. Optimization of the FIAVariables

In the proposed mechanism of the reaction,22 the aromaticdialdehyde first reacts with the primary amine and then anucleophile group forms the 2-substituted benz[f]isoindolerapidly and in good yield (Fig. 4). According to this mechanism,a manifold in which cyanide was injected over the reactionproduct between OPA and glycine was tested [Fig. 2(b)], andcompared with the manifold proposed in the literature in whichcyanide is injected into the OPA channel18,19 [Fig. 2(a)].

The excitation and emission spectra of the isoindole deriva-tives of OPA–glycine and NDA–glycine were obtained underthe conditions of pH and reagent concentrations given in theliterature. The maximum excitation and emission wavelengthsobtained were 331 and 379 nm, respectively, for the OPAderivative and 418 and 480 nm, respectively, for the NDAderivative. These wavelengths agreed with published val-ues.22

The optimization was performed with OPA and glycine.Later, the results were compared with those obtained with NDAbecause of the high cost of the NDA reagent. A flow rate of 0.5ml min21 in each channel was selected since faster rates causeda decrease in the signal.

When manifold (a) was used, an increase in the length of L1caused a decrease in the peak height, probably owing to thedispersion of the sample since no reaction occurs betweencyanide and OPA. An increase in the length of L2 up to 10 mwas found to increase the peak height. Since raising the

Table 2 Results of the 24 factorial experiment*

Run pH [F]† [I2] : [F]‡ L2 Responsenumber A B C D (fluorescence)

1 2 2 2 2 4.462 + 2 2 2 6.993 2 + 2 2 4.974 + + 2 2 8.455 2 2 + 2 2.756 + 2 + 2 4.577 2 + + 2 3.248 + + + 2 4.789 2 2 2 + 4.44

10 + 2 2 + 8.0811 2 + 2 + 5.5612 + + 2 + 8.7113 2 2 + + 2.8014 + 2 + + 4.0915 2 + + + 2.6516 + + + + 3.99

* Levels used for the experiment: pH (2) 6.5, pH (+) 7.5; [F] (2) 6 31026 m, [F] (+) 1.2 3 1025 m; [I2] : [F] (2) 1, [I2] : [F] (+) 2; L2 (2) 2.5 m,L2 (+) 4 m. † Fluorescein concentration. ‡ Molar ratio.

Table 3 Estimated effects and analysis of variance of the 24 factorial experiment

Estimated Sum of Degrees ofSource effects squares freedom Mean square F-ratio P-value

Average 5.03 ± 0.08A : pH 2.35 ± 0.15 22.07 1 22.07 230.64 0B : [F] 0.52 ± 0.15 1.09 1 1.09 11.36 0.0199C : [I2] : [F] 22.85 ± 0.15 32.46 1 32.46 339.28 0D : L2 0.01 ± 0.15 7.6 3 1024 1 7.6 3 1024 0.01 0.9326AB 0.03 ± 0.15 3.3 3 1023 1 3.3 3 1023 0.03 0.8598AC 20.85 ± 0.15 2.90 1 2.90 30.29 0.0027AD 0.01 ± 0.15 1.6 3 1024 1 1.6 3 1024 0 0.9693BC 20.41 ± 0.15 0.67 1 0.67 6.99 0.0458BD 20.15 ± 0.15 8.5 3 1022 1 8.5 3 1022 0.89 0.3878CD 20.47 ± 0.15 0.87 1 0.87 9.09 0.0296Total error 0.48 5 9.6 3 1022

Total 60.62 15

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temperature had been proposed in the literature to increase thesignal, its effect was studied. A shorter reaction coil L2 could beused if it was placed in a thermostated bath at 50 °C. This studywas conducted at pH 9 and 8.4, and the same variations wereobtained at each working pH.

When the manifold (b) was tested, L1 was varied in the range1–3.5 m. A coil L1 of 2 m was selected for further work but noimportant differences were observed, which showed that thereaction between OPA and glycine was relatively fast. Thevariation of L2 showed that 5 m were sufficient to afford themaximum intensity. The reaction product between OPA andglycine suffers degradation;22 therefore, the possibility of usinga solution containing both reagents was eliminated. This factmight also limit the length of L1. The effect of temperature onthe reaction was also investigated. For this purpose, L1 and L2were placed both individually and together inside a thermo-stated bath. Although the temperature did not have a significanteffect on the signal, it was found that an increase in thetemperature in L1 caused a slight decrease in the finalresponse.

Manifold (b) was finally selected since the responsesobtained were slightly higher than in manifold (a) and it alsoallowed operations to be carried out at room temperature andprovided a greater sample throughput.

Effect of chemical parameters

In the following investigation the pH of the buffer wasoptimized. A 2 3 1023 m OPA solution (10% ethanol) wasprepared in the buffer solution. A 2 3 1023 m glycine solutionwas also prepared in the same buffer. Phosphate buffer was usedover the pH range 6.5–8 and borate buffer for the pH range8–10. It was found that the pH profile increased rapidly up to pH8.0, the maximum being constant up to pH 8.3 and then theresponse decreased slowly. This behaviour differs slightly fromthat reported by Gamoh and Imamichi.18 Borate buffer of pH8.2 was used in further experiments.

It has been reported that glycine gave the best sensitivityamong various amino acids.18 In this work, ethylamine andammonium ion were also examined as the second reagent when

OPA was used. Among them, glycine gave the best response. Ifethylamine was used the response decreased slightly, butvirtually no response was obtained with the ammonium ion.Glycine was used for further experiments.

OPA and glycine concentrations were both varied at threelevels (5 3 1024–2 3 1023–8 3 1023 m). The amount ofmethanol in the OPA solution was kept constant at 10%. Thelower concentration tested for glycine did not produce goodresults, neither did an 8 3 1023 m equimolar ratio, but similarresponses were obtained with other combinations. Concentra-tions of 2 3 1023 m for each reagent were selected.

NDA as reagent

It has been reported that excellent sensitivity is achieved withNDA.18 Therefore, some parameters were examined usingNDA in manifold (b). Owing to the low solubility of NDA inwater, a 4 3 1024 m NDA solution was used in borate buffer. Itwas observed that the peak height increased with increasingreaction coil length L2 up to 14 m working at room temperature.The effect of pH was also examined. The optimum pH was 9.0,which coincides with the value reported by Gamoh andImamichi.18 The working conditions selected either for OPA orNDA are summarized in Table 1.

Characteristics of the Methods

Calibration graphs, detection limits and precisions were ob-tained for cyanide under the conditions given in Table 1.Calibration graphs were obtained from peak heights of triplicateinjections of 170 ml of standard cyanide solutions. The graphswere linear up to the maximum concentration of cyanideinvestigated (nearly 200 mg l21). Detection and quantificationlimits for the fluorescein–iodine method were calculated as 3sand 10s, respectively, above the blank value (where s is thestandard deviation for a blank solution with n = 10). In othermethods, no blank signal was observed and the detection andquantification limits were calculated as the concentration ofcyanide that produced an analytical signal three times thestandard deviation of the background signal. The relativestandard deviation (n = 10) was calculated at a cyanideconcentration of ten times the quantification limit. Table 4indicates the results obtained.

When no chromatographic separation is performed, themonitoring of cyanide requires the removal of interferences.Most of the on-line monitoring systems include a first step inwhich cyanide is separated as HCN in a gas diffusion unit.2,7–9

The gas diffusion method separates cyanide from most ionicspecies in the sample solution. However, it does not separatecyanide from sulfide since H2S also diffuses across themembrane. Hence, the sulfide interference was evaluated. Forthis purpose, different mixtures of cyanide : sulfide, with aconstant cyanide concentration of 40 mg l21, were injected intoFig. 4 Fluorogenic reaction of cyanide with OPA and primary amines.

Table 4 Quality parameters of the different methods for cyanide determination in flow systems

Linearity/mg l21

LOD†/ LOQ‡/ Precision§ (%) Throughput/Method Range Calibration graph mg l21 mg l21 (n = 10) samples h21

Fluorescein–iodine 17–200* If = 0.392 + 0.055 cCN2 5 17 3.6 30r = 0.9932

Pd(HQS)2–MgII 2.3–160* If = 20.012 + 0.064 cCN2 0.40 2.25 1.8 10r = 0.9999

OPA–glycine 1–200* If = 20.032 + 0.184 cCN2 0.25 1.05 1.1 12r = 0.9998

NDA–glycine 0.15–50* If = 0.140 + 1.178 cCN2 0.03 0.15 1.5 6r = 0.9999

* Maximum concentrations tested. † LOD = Limit of detection. ‡ LOQ = Limit of quantification. § Calculated at 10 3 LOQ.

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the flow system. The OPA–glycine and the NDA–glycinemethods tolerated a 25- and 5-fold excess amount of sulfide,respectively, but in the Pd(HQS)2–MgII and fluorescein-iodinesystems even a 1 : 1 ratio caused a > 50% increase in theanalytical signal. Therefore, in terms of selectivity, the OPA–glycine system is preferable.

Conclusions

Any of the studied methods offers the required sensitivity toquantify cyanide ion under the maximum value allowed indrinking waters (50 mg l21, Directive 80/778/CEE). Althoughfrom the point of view of sensitivity NDA should be the reagentof choice, the cost of this reagent limits its application to routineanalysis. On the other hand, owing to the fluorescence of thebaseline, the iodine–fluorescein method provides the lowestsignal-to-noise ratio. The OPA–glycine and Pd(HQS)2–Mgmethods show similar analytical performances and are the mostpromising for cyanide determination in water samples. How-ever, the OPA method seems to be less sensitive to sulfideinterference.

In order to apply these detection systems to determine freeand labile bound cyanide in natural waters, coupling to a gasdiffusion separation step is required and further work is beingcarried out along these lines.

The authors thank CICYT for supporting this study and E.M.also thanks CIRIT for an FPI grant.

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Paper 6/08422BReceived December 16, 1996

Accepted March 5, 1997

558 Analyst, June 1997, Vol. 122

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