Determination of metal-cyanide complexes by ion-interaction chromatography with fluorimetric detection

Analytica Chimica Acta 403 (2000) 197–204

Determination of metal-cyanide complexes by ion-interaction chromatography with fluorimetric detection E. Miralles, R. Compañó, M. Granados, M.D. Prat ∗ Departament de Qu´ımica Anal´ıtica, Universitat de Barcelona, Mart´ı i Franquès 1, 08028 Barcelona, Spain Received 31 March 1999; received in revised form 2 August 1999; accepted 9 August 1999

Abstract A chromatographic separation/UV-photodissociation/fluorimetric detection system is reported for the determination of cyanide and metal-cyanide complexes. After separation by ion-interaction chromatography, the stable metal complexes are photodissociated with an on-line PTFE reaction coil irradiated by an 8 W standard germicidal lamp. Free cyanide is then detected by reaction with o-phthaldialdehyde and glycine to form a fluorescent isoindole derivative. The method allows the determination of labile cyanide and the cyanide complexes of Fe(II), Fe(III), Ni(II) and Co(II). The detection limits are about 10 ␮g l−1 cyanide, except for Co(CN)6 3− , which is less sensitive. The method has been successfully applied to the analysis of cyanide species in spiked river-water samples. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Cyanide speciation; Liquid chromatography; Photodissociation; Fluorimetry

1. Introduction The widespread use of cyanide compounds in some industrial processes has led to its entering the aquatic environment, either as free ion or as metal complexes. The most important metal-cyanide complexes involved in environmental issues are those of iron, zinc, nickel, copper, cobalt and cadmium, which differ considerably in toxicity and environmental persistence. Thus, weak and labile complexes such as Zn(CN)4 2− , Cd(CN)4 2− and Cu(CN)3 2− , which can easily release cyanide, are more toxic than Ni(CN)4 2− , Fe(CN)6 3− , Fe(CN)6 4− and Co(CN)6 3− . Iron complexes are also known to photodissociate to ∗ Corresponding author. Tel.: +34-3-402-1277; fax: +34-3-404-1233 E-mail address: [email protected] (M.D. Prat)

free cyanide upon exposure to sunlight, which means the toxicity of these species could be higher than expected based on thermodynamic stability. There are different approaches to determine cyanide levels in environmental samples, such as total cyanide, cyanide amenable to chlorination, weak acid dissociable cyanide (WAD) or free cyanide [1]. Although these categories provide some differentiation between cyanide species, a more detailed speciation of cyanide is desirable. Several publications about cyanide analysis have focussed on this subject in recent years. Most of the reported methods are based on liquid chromatographic (LC) separations [2–7], but capillary electrophoresis has also been recently applied to cyanide speciation [8–11]. As metal-cyanide complexes are anionic in nature, anion exchange chromatography or ion interaction chromatography can be used, the latter being the more common approach.

0003-2670/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 6 0 3 - 0

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Fig. 1. Scheme of the LC-UV-photodissociation-fluorimetry set-up and operating conditions. L1 : photodissociation reactor, L2 and L3 : reaction coils, F: fluorimeter, W: waste.

Detection is usually based on direct UV absorbance measurements at 214 nm and the detection limits are in the mg l−1 range. In order to extend these methods to the determination of cyanide species at the ␮g l−1 level, a preconcentration step must be included [2,8]. Fluorimetric detection, in combination with post-column reaction, can be an alternative to overcome sample pre-concentration, as it provides more sensitivity than absorbance measurements. In a previous work a very sensitive fluorimetric method for cyanide determination in flow systems has been reported [12]. The method, which is a modification of one reported by Gamon and Imamichi [13], is based on the formation of a fluorescent isoindole derivative by reaction of free cyanide with o-phthaldialdehyde (OPA) and glycine, and the adaptation to a post-column derivatization of metal-cyanide complexes after their decomposition to free cyanide seems promising. The release of free cyanide from weak metal complexes is easily achieved in acidic media (pH < 4), but dissociation of strong metal complexes, namely those of Fe(II), Fe(III) and Co(III), requires more drastic conditions. Metal-cyanide complexes decompose into free cyanide upon exposition to UV-radiation, and some automated methods of determining total cyanide using on-line UV-photodissociation coupled to gas-diffusion separation systems have been described

[14–17]. With high-intensity mercury lamps and several minutes irradiation time, total dissociation of iron complexes was obtained, whereas low-intensity lamps led to significantly lower recoveries. However, total dissociation is not required when the photodissociation step is applied to chromatographic detection: in these cases the use of low intensity lamps seems more convenient, since no cooling system is required. This paper reports a study on a chromatographic separation coupled to photodissociation and fluorimetric post-column reaction with OPA and glycine for the determination of labile and stable metal-cyanide complexes. The method uses an on-line UV-photoreactor between the LC column and the fluorimetric detection system. The characteristics of the new method have been studied and it has been applied to the analysis of polluted river-water samples.

2. Experimental 2.1. Apparatus The arrangement of LC separation and detection components used is depicted in Fig. 1. The LC equipment consisted of a double piston pump (Model 420, Gynkotek, Munich, Germany) and an injection valve (Gynkotek MSV 6) fitted with a 25 ␮l injection loop.

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The analytical column used was a 250 mm × 4 mm i.d. C18 reverse phase column packed with 5 ␮m particles (Merck LiChrospher 100 RP-18) and equipped with a C18 precolumn. All chromatographic separations were done at room temperature using a mobile phase flow-rate of 1.0 ml min−1 . The photodissociation reactor consisted of PTFE tubing (2m × 0.5 mm i.d.) coiled around a 8 W standard germicidal mercury lamp (254 nm peak wavelength) Vilver Lourmant T-8C (280 mm × 16 mm). The photoreactor did not need cooling since the 8 W lamp provided low-intensity radiation. The post-column reagents were delivered by means of a Gilson (Villiers le Bel, France) Minipuls 3 peristaltic pump, PTFE tubing (0.5 mm i.d.) for mixing and reaction coils, and Tygon tubes for pumping the reagents. The detection was carried out with an AB2-Series (SLM-Aminco, New York, USA) fluorescence spectrometer operated at 334 nm excitation and 386 nm emission and equipped with a 25 ␮l inner volume flow cell (Hellma, Mülheim, Germany). The flow injection manifold used to optimise the post-column derivatization system was the same as in Fig. 1 without the chromatographic set-up. An injection valve Rheodyne (Cotati, CA, USA) with an injection loop of 70 ␮l was used in these experiments. 2.2. Reagents and solutions All chemicals were of analytical-reagent grade or HPLC-grade unless stated otherwise. They were purchased from Merck (Darmstadt, Germany), Aldrich (Milwaukee, WI, USA), Johnson Mattey (Karlsruhe, Germany) or Fluka (Buchs, Switzerland). Ultrapure water MilliQ-plus (Millipore, Molheim, France) 18.2 Mohm cm−1 was used throughout. Glassware used for experiments was previously soaked in 10% nitric acid for 24 h and rinsed with doubly-deionized water. 2.2.1. Stock solutions (1.000 g l−1 CN− ) of CN− , Fe(CN)6 3− , Fe(CN)6 4− , Ni(CN)4 2− , Co(CN)6 3− , and Ag(CN)2 − were prepared by dissolving the corresponding potassium salts (Merck, Aldrich or Johnson Mattey) in 0.01 M aqueous sodium hydroxide. Solutions of Zn(CN)4 2− ,

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Hg(CN)4 2− and Cd(CN)4 2− were prepared by adding stoichiometric amounts of Zn(CN)2 (Aldrich), Hg(CN)2 (Merck) and Cd(CN)2 (Merck) to a KCN solution in 0.01 M NaOH. The Cu(CN)3 2− species was formed by dissolving CuCN (Johnson Mattey) in a known excess of KCN in 0.01 M NaOH. All these stock solutions were stored at 4◦ C in dark glass bottles. Working solutions were prepared daily by appropriate dilution of the stock solutions in 10−2 M NaOH. 2.2.2. Mobile phase This phase consisted of methanol–tetrahydrofuran– water (10 mM sodium sulphate, 7.2 mM tetrabutylammonium hydroxide (TBAOH), 14.5 mM phosphoric acid, pH adjusted to 7.5 with sodium hydroxide) (30 : 1 : 69 v/v/v). Both the aqueous and the organic phases were separately filtered through 0.22 ␮m Nylon membrane filters and the mixture degassed in an ultrasonic bath prior to use. The final pH measured in the mobile phase was about 8.0. 2.2.3. Post-column reagent (PCR) The fluorimetric reagents consisted of a 2 × 10−3 M solution of glycine at pH 8.7 and a 2 × 10−3 M solution of OPA at pH 8.7.They were prepared by diluting with a 0.1 M borate buffer a 0.02 M aqueous stock solution of glycine or a 0.04 M solution of OPA in ethanol. Working solutions were prepared daily. 2.3. Procedure For the chromatographic analysis, standards and samples were filtered through a 0.45 ␮m membrane filter and then injected into the LC system. The column effluent (1 ml min−1 ) merged with a 0.05 M HCl solution (0.3 ml min−1 ) and was pumped through the 2 m UV-photodissociation coil for the decomposition of inert complexes. Then the released cyanide reacted with the post-column reagents (0.55 ml min−1 glycine solution and 0.55 ml min−1 OPA solution). The total effluent flowed through the 3 m reaction coil to the detector. Fluorescence intensity was measured at 386 nm using an excitation wavelength of 334 nm (excitation and emission slit widths of 8 nm). Calibration was done by injecting standard solutions of KCN, and Fe(II), Fe(III), Ni(II) and Co(III) complexes.

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Quantification was performed by means of calibration graphs obtained in the peak area mode.

3. Results and discussion 3.1. Detection system The optimal conditions for the on-line fluorimetric reaction between free cyanide and the post-column reagents were taken from an earlier study [12]. The present study of the detection system focused on the photodissociation step and on the compatibility of the mobile phase of the LC system with the post-column reaction. A flow injection system was used in the experiments. Standard solutions of metal-cyanide complexes were directly injected into a carrier stream, which simulated the chromatographic mobile phase. The carrier solutions assayed consisted of mixtures of sodium phosphate buffer aqueous solutions containing TBAOH and methanol or acetonitrile, which are commonly used as organic modifiers in the mobile phases for the separation of metal-cyanide species. It was observed that acetonitrile in the effluent led to a high base-line fluorescence signal, which impaired the detection of cyanide at low concentration levels. On the contrary, methanol had no important effect in the base line, although a 25% decrease in the peak height was observed when methanol was increased from 0% to 30%, with no important loss of sensitivity. On the basis of these results, methanol/water mobile phases were used in this study. The effect of pH on the derivatization reaction in the methanol–water medium was also tested. At a 25% percentage of methanol, the maximum signal was obtained in the range 8–9 (pH measured at the system outlet). Further studies were performed using a boric buffer solution pH 8.7, which drifted to pH 8.5 after mixing in the coil L3 . Labile metal-cyanide complexes, such as those of Zn(II), Cd(II) and Cu(II), are easily dissociated in flow systems without irradiation (12). Therefore, the the effect of UV-irradiation on the dissociation of metal-cyanide complexes was studied with the moderately strong Ni(II) complex and the strong cyanide complexes of Fe(II), Fe(III) and Co(III). The para-

Fig. 2. Effect of pH and coil length on cyanide recovery (a) and fluorescence intensity (b).

meters examined in optimising the conditions were pH in the UV-photodissociation step and reaction coil length. The photodissociation was first carried out in a water–methanol (about 20% methanol) medium at two pH values (3 and 8). These conditions were obtained by merging the carrier stream (Fig. 1) with either 0.05 M HCl or pure water. The results of cyanide recoveries are shown in Fig. 2a. Recovery percentages from metal complexes were calculated by comparison of their peaks with peaks obtained from an equivalent KCN standard solution (50 ␮g l−1 CN− ). The results indicated that, with a 2 m reaction coil, about 80% of iron complexes dissociate in both acidic and neutral media. The dissociation of the Ni(CN)6 4− complex was also about 80% in acidic solution but drastically

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decreased to 15% at pH 8. For the Co(III) complex recoveries were low, but in acidic medium higher values were obtained. Additional studies in a higher acidic medium (HCl 1 M) and in a 0.6 M H3 PO4 –0.4 M H4 P2 O7 medium, which has been reported to enhance cyanide recovery from iron complexes [15], indicated that there were no significant differences between these media and 0.05 M HCl. The influence of the photoreactor coil length on cyanide recovery and on peak height is shown in Fig. 2. The selection of this parameter involves a compromise between obtaining a high dissociation of metal complexes (Fig. 2a) and avoiding excessive dispersion in the reactor with a resulting loss of sensitivity and of chromatographic resolution. In fact the parameter to optimise for sensitivity is peak height, i.e. the measured fluorescence intensity (Fig. 2b). The shortest photoreactor coil assayed (1 m) provided low peak heights because of poor cyanide recoveries. Although the 4 m photoreactor led to the best recoveries, the measured peak heights were lower, as a result of the band-broadening effect. The best results were achieved using a 2 m photoreactor, which led to higher fluorescence signals, although dissociation was not as effective as that observed with a 4 m one. A 2 m reactor was used in subsequent experiments. In the selected conditions, similar responses were obtained from Fe(III), Fe(II) and Ni(II) complexes (Fig. 2b). The lower values obtained from the Co(III) complex are due to the fact that this complex is more kinetically inert and thermodynamically stable than iron and nickel complexes.

3.2. Chromatographic separation Most of the methods for analysing cyanide species involve ion-interaction chromatography on a C18 column using mobile phases consisting of acetonitrile–water or methanol–water mixtures with TBA+ as ion-interaction reagent. As indicated above, mobile phases containing methanol were selected to perform the separation. Initial conditions were selected from literature data (2) and consisted of methanol–tetrahydrofuran–water (6.8 mM TBAOH, 13.5 mM sodium sulphate, 13.5 mM phosphoric acid, adjusted to pH 7.5 with sodium hydroxide) (25 : 1 : 74). Although separation of the complexes

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Fig. 3. Effect of methanol (a) and TBAOH (b) on cyanide species retention times.

was achieved, this mobile phase was not entirely satisfactory because of long retention times. Therefore, in order to optimise separation of the related compounds, a series of experiments was performed to adjust the composition of the mobile phase. Fig. 3a shows the effect of methanol content on the retention of the metal complexes in the concentration range 25–35%, while THF was kept constant at 1%, as well as the concentrations of TBAOH, sodium sulphate and phosphoric acid. Retention times and separation factors decrease as the % methanol increase, this effect being strongest between 25% and 30%. Fig. 3b shows the effect of TBAOH concentration in methanol/tetrahydrofuran/water (30 : 1 : 69) in the concentration range 0.725–7.25 mM in the aqueous phase. As expected, retention times and separation factors decreased as the TBAOH increased. 30% methanol and 7.25 mM TBAOH, which provided good separation at reasonable retention times, were

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Table 1 Effect of sodium sulphate concentration on retention times of cyanide speciesa Retention time (min)

CN−

WAD Fe(CN)6 4− Co(CN)6 3− Fe(CN)6 3− Ni(CN)4 2− a

Na2 SO4 0.010 M

Na2 SO4 0.015 M

Na2 SO4 0.020 M

3.4 5.4 9.8 11.3 15.5

3.4 5.0 9.1 10.7 15.3

3.4 4.8 8.2 9.3 13.4

Mobile phase composition: MeOH : THF : water (7.25 × 10−3 M TBAOH, 0.015 M H3 PO4 , Na2 SO4 , pH = 7.5) (30 : 1 : 69 v/v/v).

the selected conditions. The pH of the mobile phase was also investigated in the range 7.0–8.0 and no significant variations in retention times were observed. All the assayed mobile phases contained 1% THF, in order to improve peak shapes. Since retention times are affected by the ionic strength of the mobile phase, this factor was also studied. The concentration of the buffer system was kept constant and the Na2 SO4 concentration was varied. The effect of ionic strength on the retention times of the cyanide species is presented in Table 1. The results proved that the resolution between CN− and Fe(CN)6 4− peaks, as well as between Co(CN)6 3− and Fe(CN)6 3− , slightly improved by decreasing the Na2 SO4 concentration from 20 to 10 mM, whereas no significant increase in the analysis time was observed. Therefore, 10 mM Na2 SO4 was selected. The elution order did not vary under any of the assayed conditions and was as follows: CN− = Cd(CN)4 2− = Zn(CN)4 2− = Cu(CN)3 2− < Fe(CN)6 3− < Co(CN)6 3− < Fe(CN)6 4− < Ni(CN)4 2− . The weak and labile cyanides, Cd(CN)4 2− and Zn(CN)4 2− , completely dissociate under the chromatographic conditions used, and thus cyanide from these complexes is indistinguishable from free cyanide. The uncomplexed cyanide peak also includes the cyanide from the Cu(I) complex. It has been reported that Cu(CN)3 2− could be partially dissociated along the chromatographic separation [18], but our results do not prove whether the copper complex dissociates in the column or coelutes with free cyanide and is totally dissociated in the post-column reactor. Thus, complexes of cadmium, zinc and copper will be determined in the free cyanide fraction, which provides information about the most toxic cyanide species, i.e. the so-called WAD cyanide.

Fig. 4. Chromatogram of a mixture of cyanide complexes. Mobile phase: Methanol:tetrahydrofuran:water (7.25 × 10−3 M TBAOH, 0.015 M H3 PO4 , 0.010 M Na2 SO4 , pH = 7.5) (30 : 1:69 v/v/v). Solute concentration as CN− : (1) CN− 200 ␮g l−1 , (2) Fe(CN)6 4− 280 ␮g l−1 , (3) Co(CN)6 3− 950 ␮g l−1 , (4) Fe(CN)6 3− 560 ␮g l−1 , (5) Ni(CN)4 3− 220 ␮g l−1 .

Observed elution behaviour was similar to that reported by Haddad and Kalabaheti [2] using a Waters Nova-Pak C18 column with a similar mobile phase. The most significant difference was for Cu(I) and Fe(II) complexes; Fe(II) was eluted earlier in the Waters column and was retained longer in the LiChrosphere column. This elution order is quite different from that obtained using water–acetonitrile mobile phases at a similar percentage of the organic modifier [3].

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Table 2 Figures of merit of the chromatographic method

tR (min) tR RSD (%) n = 6 Peak area RSD (%) LOD (␮g l−1 CN− )a LOQ (␮g l−1 CN− )b a b

WAD CN−

Fe(CN)6 4−

Co(CN)6 3−

Fe(CN)6 3−

Ni(CN)4 2−

3.4 1.0 6.3 10 20

5.4 1.0 1.6 8 20

9.8 0.4 6.7 53 132

11.3 0.5 3.1 14 28

15.5 0.9 5.1 9 18

Determined as three times the standard deviation of the base line noise. Vinj = 25 ␮l. Determined as ten times the standard deviation of the base line noise. Vinj = 25 ␮l.

Table 3 Cyanide recovery from spiked river-water samples [WAD CN− ]a

[Fe(CN)6 4− ]a

[Co(CN)6 3− ]a

[Fe(CN)6 3− ]a

[Ni(CN)2 2− ]a

Added

Foundb

Added

Foundb

Added

Foundb

Added

Foundb

Added

Foundb

50 100

47 ± 4 95 ± 2

112 224

106 ± 5 212 ± 6

398 795

390 ± 10 821 ± 40

140 280

148 ± 4 288 ± 7

71 172

78 ± 3 145 ± 5

a b

Concentration values expressed as ␮g l−1 of cyanide. Mean value ± standard deviation (n = 3).

Fig. 4 shows a chromatogram obtained from a mixture of several cyanide species under the selected conditions (see procedure). As can be seen, well defined peaks and good resolution were obtained. Under these conditions a chromatographic run takes less than 20 min. Retention time and peak area reproducibility data for cyanide species from six replicate measurements are summarised in Table 2. Calibration graphs are linear in the concentration ranges assayed, from 20 to 500 ␮g l−1 . In the case of Co(III), concentration was varied between 150 and 1000 ␮g l−1 . The detection and quantification limits, determined at signal-to-noise ratio of 3 and 10, respectively, are also given. All concentrations refer to cyanide. The mass detection limits for a 25 ␮l injection volume are in the range 0.20–0.35 ng cyanide, with the exception of Co(III) complex which shows at 1.32 ng, owing to its low photodissociation efficiency. Sulphide ion, which also reacts with glycine and OPA to form an isoindole derivative, and thiocyanate ion, which decomposes to cyanide and sulphide under irradiation at λ < 300 nm, were investigated because they are potential interfering species. The results showed that they do not interfere with the determination of cyanide species. Thiocyanate elutes between Fe(II) and Co(III) complexes and the three peaks were

completely resolved. However, no sulphide peaks were detected through the analysis time after injection of a 3 mg l−1 sulphide solution. 3.3. Analysis of cyanide species in water samples The proposed method was applied to the analysis of water samples from the Llobregat river, one of the drinking water sources of Barcelona. It flows into the NW Mediterranean Sea (Catalonia, Spain). The Llobregat basin involves agriculture and industrial areas. Samples were collected in 1–l polyethylene bottles, preserved by addition of NaOH to pH 12, filtered through 0.45 ␮m membrane filters and stored at 4◦ C. Analyses were carried out as soon as possible. Since direct analysis of these natural samples proved that they do not contain measurable cyanide species they were spiked with different amounts of the cyanide species. The analysis of spiked samples (Table 3) shows that good recoveries were obtained.

Acknowledgements The authors thank Agbar Company for supplying samples. They also thank CICYT (project

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E. Miralles et al. / Analytica Chimica Acta 403 (2000) 197–204

AMB98-0327) and CIRIT (project SGR97-394) for supporting this study.

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