Postcolumn liquid chromatographic method for the determination of cyanide with fluorimetric detection

255

Analytica Chimica Acta, 251 (1991) 255-259 Elsevier Science Publishers B.V., Amsterdam

Postcolumn liquid chromatographic method for the determination of cyanide with fluorimetric detection Keiji Gamoh * Faculiy

of Education, Kochi University, Akebono-cho,

Kochi 780 (Japan)

Senya Imamichi Analytical Applications

Department,

Shimadzu

Corporation, (Received

Kuwabara-rho-l,

14th February

Nishinokyo,

Nakagyo-ku,

Kyoto 604 (Japan)

1991)

Abstract A postcolumn liquid chromatographic procedure for the determination of cyanide with fluorimetric detection was developed. The method is based on both the separation of cyanide ion in the ion-exclusion mode and the postcolumn fluorescence derivatization of cyanide ion with o-phthalaldehyde or naphthalenedicarboxaldehyde in the presence of amino acid. Various parameters such as the kind of amino acid, pH of the reagent buffer and concentration of the sodium salt of EDTA were optimized to provide quantitative conversion of cyanide ion to the fluorescent derivative. Keywords:

Fluorimetry;

Liquid

chromatography;

Cyanide

Many detection schemes for the determination of cyanide ion have been used, based on cyanideselective electrodes [1,2], coulometry [3,4], gas chromatography [5], liquid chromatography (LC) [6], etc. Ion chromatography with amperometric detection has also been used for the cyanide and/or metal cyanide determination [7,8]. Wang et al. [9] reported the potentiometric determination of cyanide and sulphide using an anion-selective electrode. Silinger reported [lo] a method that uses sodium hypochlorite to oxidize free cyanide ion to cyanate ion before chromatographic separation. However, the optimum temperature, the stability of cyanate ion formed and the interferences were not elucidated. Sano et al. [ll] reported a fluorimetric method for the determination of cyanide as the isoindole derivative, formed by reaction with naphthalenedicarboxaldehyde (NDA) in the presence of taurine. However, it is necessary to remove 0003-2670/91/$03.50

Q 1991 - Elsevier

Science Publishers

the contaminants or to block the thiols with Nethylmaleimide prior to derivatization in the fluorimetric determination because NDA reacts readily with other nucleophiles in the sample matrix. Recently a convenient precolumn LC procedure was reported in which cyanide ion is converted into the 1-cyanobenz[ f lisoindole derivative by NDA in the presence of glycine followed by fluorimetric detection [12]. The detection of the cyanide ion in its derivatized form is more sensitive and selective than electrochemical detection of the cyanide ion itself. However, in the precolumn LC procedure it is necessary to clean up the analytical samples before or after derivatization, in case the sample matrix is complex. In this paper, a postcolumn LC procedure for cyanide ion determination with fluorimetric detection is described. This method is based on both the separation of cyanide ion in the ion-exclusion mode and the postcolumn fluorescence derivatizaB.V. All rights reserved

256

tion of cyanide ion with o-phthalaldehyde (OPA) or NDA in the presence of an amino acid. A postcolumn LC method would be advantageous because the selectivity for the separation of the cyanide ion is high and it is not necessary to clean up the sample before injection. Various parameters such as the kind of amino acid, the pH of the reagent buffer and the concentration of the sodium salt of EDTA (Na,EDTA) were optimized to provide quantitative conversion of cyanide ion to the fluorescent derivative.

EXPERIMENTAL

Chemicals All reagents and chemicals were of analyticalreagent grade or better and were used as received. The water used in the chromatographic mobile phase was doubly distilled. OPA and amino acicids were obtained from Wako (Tokyo) and NDA from Tokyo Kasei Kogyo (Tokyo). Standard cyanide ion solutions were made by dissolution in 100 mM sodium hydroxide solution. A cyanide solution of concentration lop6 M or higher (in 100 mM sodium hydroxide) was stable for at least 24 h, and was prepared daily. The mobile phase and postcolumn reactor solutions were filtered through 0.45pm membrane filters and vacuum deaerated. A carbonate-borate buffer was prepared by mixing 100 ml of 0.1 M sodium carbonate solution and 100 ml of 0.1 M sodium borate solution and adjusting the pH with sodium hydroxide.

K. GAMOH

S. IMAMICHI

Apparatus Figure 1 gives a schematic diagram of the LC system. It consists of a Shimadzu (Kyoto) LC-6A solvent-delivery system with a Model 7125 solvent-injection valve (Rheodyne, Cotati, CA) equipped with a 20-/J loop. The analytical column was a Shim-pack SCR-102H ion-exclusion column (Shimadzu), used at 40 o C in a Shimadzu CTO-6A column oven. The mobile phase was 10 mM perchloric acid at a flow-rate of 1.0 ml n-tin-‘. Shimadzu Model LC-6A reagent-delivery pumps were used. The first postcolumn reagent was 2 mM OPA or NDA and 0.5 mM Na,EDTA in 20% ethanol in carbonate-borate buffer, added at 0.3 ml mm’. The second postcolumn reagent was 2 mM amino acid and 0.5 mM Na,EDTA in carbonate-borate buffer, added at 0.3 ml/mm. These reagents were stored under refrigeration for no more than a few days. Reaction coils were employed to combine the postcolumn reagent with the mobile phase and were 0.5 mm i.d. tubing about 100 cm long (the length of the reaction coil was chosen as the minimum length yielding a maximum peak height for the cyanide ion signal). All tubing was made of stainless steel. The reaction system was maintained at 40°C in the reaction box. A Shimadzu Model RF-535 fluorimetric detector was used for the detection of the fluorescent derivative of cyanide ion. The cyanide ion-OPA derivative was determined with a detector monitoring the fluorescence intensity at 400 nm, when excited at 330 nm. The cyanide ion-NDA derivative was monitored at 490 nm, when excited at 420 nm. The optimum reaction conditions were elucidated by injecting 5 ~1 of a standard solution of cyanide ion into the column.

RESULTS

Fig. 1. Schematic diagram of the postcolumn LC system. I= LC delivery pump; 2 = sample injector; 3 = analytical column; 4 = column oven; 5 = pumps; 6 = OPA (or NDA) solution; 7 = amino acid solution; 8 = reaction coils; 9 = reaction box; 10 = flu&metric detector.

AND

AND

DISCUSSION

Effect of amino acids in the reagent solution on fluorescence intensity As described in previous papers, cyanide ion reacted with OPA [13] or NDA [14] in the presence of amino acids to afford the 1-cyanoisoindole derivative or 1-cyanobenz[ f lisoindole derivative,

DETERMINATION

TABLE

OF CYANIDE

BY POSTCOLUMN

1

Effect of amino acids in the second intensity of cyanide ion Amino Glycine Taurine Valine Aspartic

acid

acid

solution

25-l

LC

on fluorescence

NDAa

OPA a

loo 41 10 10

33 14 5 5

a Either OPA or NDA was used as the first reaction reagent. Each value represents the relative fluorescence intensity, which was calculated from each peak area of the cyanide ion.

respectively. Table 1 shows the effect of various amino acids in the second reagent solution of the fluorescence intensity of cyanide ions when OPA or NDA was used as the first reaction solution in this system. As glycine gave the highest sensitivity among the four, it was selected as the amino acid source.

Effect of pH of reagent buffer and reaction temperature on fluorescence intensity De Montigny et al. [15] have already extensively investigated the effect of pH on the NDACN- derivatization of alanine with a precolumn method. In this work, the effect of the pH of the reagent buffer on the fluorescence intensity in the postcolumn method was examined. Figure 2 shows the peak response in the pH range 8.0-11.0 when NDA-glycine was used as the second reagent. Although it has been reported that the pH-rate profile exhibited a bell-shaped curve with the maximum rate occurring at pH 9.5 [15], the present results indicated that at pH > 9.5 the fluorescence intensity decreased. The optimum pH was 9.0. Based on the value of cyanide ion (9.2), it was expected that a higher sensitivity would be observed at pH > 9.2. The pH-fluorescence intensity profile obtained with the present postcolumn reaction did not always coincide with the previous data for the precolumn reaction. Although this phenomenon cannot be explained, the fluorogenic reaction of cyanide ion with OPA (or NDA)-amino acid may be effected in the hydrogen cyanide form.

The postcolumn reaction temperature had a slight effect on the peak response in the range from room temperature to 80” C. The reaction was subsequently applied at 50 o C, based on both the maximum peak response and the minimum background response. Effect and concentration of Na,EDTA in the reagent buffer on fluorescence intensity As the flow line in the system was made of stainless steel, metal ions may be eluted in the acidic mobile phase, so that it may be possible to form metal-cyanide complexes. The effect of Na,EDTA at concentrations of O-l mM on the fluorescene intensity of cyanide ions was examined. The peak height increased at concentrations up to 0.5 mM and showed a plateau between 0.5 and 1 mM. Therefore, the optimum concentration of Na,EDTA in the reagent solution was chosen as 0.5 mM. It was assumed that the increased response indicates that after the cyanide ion eluted from the analytical column has formed a metal-cyanide complex, the cyanide ion will be released from the complex in the presence of Na ,EDTA.

8

Fig. 2. Effect of pH of reagent of cyanide ions.

9

buffer

10

on fluorescence

11

intensity

258

K. GAMOH

AND

S. IMAMICHI

1

I

I

I

0

5

10

Retention

I

15 / min

time

Fig. 4. Chromatogram of a river water sample spiked with cyanide ion (0.5 pg 1-l). Peak 1= cyanide ion.

I 0

I 5 Retention

I

I

10

15 time

/

tin

Fig. 3. Chromatogram of a standard solution of cyanide ion (60 pg I-‘). Peak 1= cyanide ion.

Chromatographic separation and quantitative response Both NDA and OPA can be used as the first reaction reagent for the post-column derivatization of cyanide ion. From the point of view of sensitivity, NDA should be used if the determination of relatively trace amounts of cyanide ion in analytical samples is required. Figure 3 shows a chromatogram obtained from a standard solution of cyanide ions (60 pg 1-l) using the system described above when NDA was used as the first reagent. The retention time was 13.5 mm. According to the calibration graph obtained by measuring peak areas, the relative standard deviations (R.S.D.; n = 10) were 1.4% at the 10 pg 1-l level for the cyanide ion solution and 3.1% at the 0.5 pg 1-l level. The calibration graph for cyanide ion was linear in the range 0.5 pg l-l-2 mg 1-l. The detection limit was found to be about 0.1 pg l- ’ with a signal-to-noise ratio of 3. Application of the proposed method One of the major advantages of the proposed method is its high sensitivity and high selectivity

for the separation of ionic compounds compared with other methods for the determination of cyanide. The present LC technique was employed for the determination of cyanide in some real samples. Figure 4 shows a chromatogram from a river water sample to which cyanide ion was added (0.5 pg 1-l). The recoveries were 93.5-96.4% (n = 10; R.S.D. = 2.4%). Figure 5 shows a chromatogram from an extract of Japanese apricot diluted tenfold with 100 mM sodium hydroxide solution. From the sample, 2.1 ng of cyanide ion in 10 ~1 was determined. By use of the ion-exclusion mode described above, analytical samples could be in-

1

I

I

0

5

I 10

I

15

Retention time / min Fig. 5. Chromatogram of an extract of Japanese apricot sample; sample diluted 1: 100 with 100 mM NaOH. Peak 1 = cyanide ion.

DETERMINATION

OF CYANIDE

BY POSTCOLUMN

259

LC

jetted directly without further clean-up steps prior to injection. In conclusion, the proposed method is the first example of the use of NDA reagent in the postcolumn LC determination of cyanide. It is based on both the ion-exclusion separation mode and fluorimetric detection, which are satisfactory with respect to high selectivity and sensitivity. The method is useful for the determination of cyanide in various types of samples.

REFERENCES 1 Testing Methods for Industrial Wastewater, JIS K0102, Japanese Standard Association, Tokyo, 1986, p. 116. 2 American Public Health Association, American Water Works Association and Water Pollution Control Federation, Standard Methods for the Examination of Water and

3 4 5 6 7 8 9 10 11 12 13 14 15

Wastewater, American Public Health Association, Washington, DC, 16th edn., 1985, p. 327. M. Nonomura, Ind. Water Jpn., (1984) 37. T.J. Rohm and R. Davidson, Anal. Lett., 11 (1978) 1023. J.C. Valentour, V. Aggarwal an 1. Sunshine, Anal. Chem., 46 (1974) 924. Y. Suzuki and T. Inoue, Bunseki Kagaku, 33 (1984) 425. A.M. Bond, I.D. Heritage, G.G. Wallace and M.J. McCormick, Anal. Chem., 54 (1982) 582. R.D. Rocklin and E.L. Johnson, Anal. Chem., 55 (1983) 4. W. Wang, Y. Chen and M. Wu, Analyst, 109 (1984) 281. P. Silinger, Plat. Surf. Finish., 72 (1985) 82. A. Sano, M. Takezawa and S. Takitani, Talanta, 34 (1987) 743. K. Gamoh and H. Sawamoto, Anal. Sci., 4 (1988) 665. J.J. D’Amico, B.R. Stuls, P.G. Ruminski and K.V. Wood, J. Heterocycl. Chem., 20 (1983) 1283. R.G. Carlson, K. Srinivasachar, R.S. Givens and B.K. Matuszewski, J. Org. Chem., 51 (1986) 3978. P. de Montigny, J.F. Stobaugh, R.S. Givens, R.G. Carlson, K. Srinivasachar, L.A. Stemson and T. Higuchi, Anal. Chem., 59 (1987) 1096.