Comparison of digestion methods for the determination of selenium in fish tissue by cathodic stripping voltammetry

Analytica Chimica Acta 408 (2000) 97–102

Comparison of digestion methods for the determination of selenium in fish tissue by cathodic stripping voltammetry David F. Lambert, Nicholas J. Turoczy ∗ School of Ecology and Environment, Deakin University, Warrnambool, Vic. 3280, Australia Received 8 June 1999; received in revised form 24 September 1999; accepted 11 October 1999

Abstract A certified reference material (NIST-RM-50 Albacore Tuna) was analysed for selenium by cathodic stripping voltammetry (CSV), after digestion of the material by a number of methods that avoid the use of perchloric acid. The digestion techniques tested included wet and dry ashing methods, oxygen bomb digestion, ultraviolet (UV) digestion, and methods involving elevated pressure. The only method that reliably produced results that agreed with the certified value for selenium in the reference material was the combination wet/dry ashing method incorporating elevated pressure recommended by the Association of Official Analytical Chemists (AOAC) for determination using hydride generation atomic absorption spectrometry (AAS). Recoveries using the other methods were low and variable, apparently because of incomplete destruction of organic matter and losses caused by volatilisation of selenium. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Selenium; Fish; Digestion; Cathodic stripping voltammetry

1. Introduction Selenium is a metalloid which is an essential micronutrient at low concentrations but becomes toxic at higher concentrations, with the range between being very narrow [1]. Although it has been found difficult to precisely express the nutritional requirement for humans, health authorities around the world generally recommend an intake of about 70 ␮g per day for adults in order to avoid deficiency diseases [2]. However, intakes as low as 350 ␮g per day have been found to produce signs of selenium toxicity [2]. With such a narrow concentration range for dietary suitability, it is essential that reliable and accurate ∗ Corresponding author. Tel.: +61-3556-33-110; fax: +61-3556-33-462. E-mail address: [email protected] (N.J. Turoczy).

methods are available for the assessment of selenium concentrations in food. The present methods of analysis typically involve an acid digestion step followed by instrumental determination. Commonly used instrumental methods include hydride generation coupled with either atomic absorption spectrometry (HG-AAS) [3,4] or with inductively coupled plasma emission spectrometry (HG-ICPES) [5,6], graphite furnace atomic absorption spectrometry (GFAAS) [7,8], differential pulse polarography (DPP) [9,10] and cathodic stripping voltammetry (CSV) [11–19]. Compared to the techniques involving atomic spectrometry, voltammetric methods have certain distinct advantages and disadvantages. Instrumentation is relatively cheap to purchase and operate. The CSV methods for Se are highly sensitive but are highly susceptible to matrix effects and other interferences [13]. Extremely thorough digestion methods are therefore

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essential if CSV is to be used to analyse biological materials. Digestion methods that are suitable for analysis by atomic spectrometry in which the digest is atomised may not be suitable for voltammetric analysis in which selenium is measured in solution. In this study, several methods which have been proposed for the digestion of biological samples for the determination of selenium (by atomic spectrometry or voltammetry) are compared for their suitability to digest fish samples for analysis by CSV. The CSV method of Holak and Specchio [16] was used as the detection method. Methods that have been used for the destruction of organic matter include wet ashing, dry ashing, combustion, and irradiation with ultraviolet (UV) radiation. In the case of analysis of biological tissues for Se, wet acid and wet acid/dry ashing combinations are the predominant methods. Microwave heating [7,9,20] and arrangements involving increased pressure [3,7,9] have been used to increase the speed and completeness of digestion. Digestion methods that avoid the use of perchloric acid are extremely attractive when facilities for using this acid safely are not available. However, although a number of digestion methods have been proposed and tested, it seems that many workers have difficulty in achieving the recoveries reported by others [13,15]. Recoveries are apparently highly sensitive to the precise conditions (e.g. amounts of sample and reagents and heating rates) utilised during digestions. This sensitivity to experimental conditions is clearly undesirable and unsatisfactory. Higham and Tomkins [15] have compared four proposed acid digestion procedures, in both the originally proposed forms and with several variations, for the determination of selenium in tuna using CSV. Relatively poor recoveries were obtained using most of the original procedures, but it was found that significant improvements could be achieved by variations in the quantities of acids and the digestion times used. The best recoveries (approximately 98%) were obtained using a modified version of the procedure described by Holak [11]. This method is an open vessel combination of wet acid/dry ashing in which Mg(NO3 )2 ·6H2 O is used as an ashing aid. Although the procedure was reported to be effective and eliminated the use of perchloric acid, it was very time-consuming as several hours of operator attention are required. Furthermore, recoveries were assessed using a standard additions

method, which does not test for complete release of bound metal. In this study, the method proposed by Higham and Tomkins was evaluated using a certified reference material (NIST-RM-50 Albacore Tuna). The method proposed by Higham and Tomkins is similar to that recommended by the Association of Analytical Chemists (AOAC) for the dissolution of fish samples for subsequent analysis for selenium by HG-AAS [3]. However, the AOAC method involves different quantities of reagents and a closed vessel step. Furthermore, the AOAC method is for the determination of Se by HG-AAS. The AOAC method was therefore evaluated in this study using CSV as the detection method. A variation of the AOAC method in which an open vessel was substituted for a closed vessel was also evaluated. As the methods described above are quite time-consuming and labour intensive, other methods of digestion were also investigated in this study. Combustion in an oxygen bomb has been described for the determination of mercury in fish [21]. This method of sample destruction is very fast if a small number of samples need to be processed, and produces a digest which is suitable for voltammetry. The method was therefore investigated for suitability for the determination of Se in fish. UV irradiation has previously been used to destroy small quantities of organic matter in water samples in preparation for CSV analysis [17–19]. In this study, the use of UV irradiation to destroy organic matter in partially digested fish samples was investigated. Pressure-aided digestion techniques involving nitric acid and nitric acid/H2 O2 mixtures were also evaluated. A pressure cooker was used to heat the samples inside polytetrafluoroethylene (PTFE)-lined steel bombs. A similar digestion technique (using a microwave oven rather than a pressure cooker) has been reported to be satisfactory for the decomposition of marine biological tissues for subsequent analysis by GFAAS [8].

2. Experimental Deionised water (resistivity >18 M cm) was produced by passing singly distilled water through a Milli-Q water purification system (Millipore). Concentrated hydrochloric acid, concentrated nitric acid,

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magnesium nitrate hexahydrate and magnesium oxide (AnalaR grade) were obtained from BDH chemicals. Thirty percent hydrogen peroxide (AnalaR) and copper standard solution (1.000 mg l−1 , spectrosol grade) were obtained from Ajax Chemicals. A stock selenite solution (1.00 g Se l−1 ) was prepared by dissolving anhydrous sodium selenite (AnalaR, Aldrich) in deionised water. Working standards were prepared on the day of use by appropriate dilution of the stock solution. Glass and plastic vessels were cleaned by soaking for at least 24 h in 1 M HNO3 followed by rinsing with deionised water. Metal utensils were soaked in 5% (v/v) Extran 300 and rinsed with deionised water. The electrodes for the voltammetric determination were cleaned between determinations by soaking in 0.01 M HNO3 and then rinsing with deionised water. Differential pulse cathodic stripping voltammetry (DPCSV) was performed using a PAR Model 364 Voltammetric Analyser in combination with a PAR 303A SMDE electrode stand. The hanging mercury drop electrode (HMDE) mode (medium drop size) of the electrode stand was selected. An Ag/AgCl 3 M KCl electrode was used as the reference electrode and a platinum wire was used as the auxiliary electrode. Voltammograms were recorded on a Hewlett–Packard 7045A XY recorder. Essentially, the voltammetric procedure described by Holak and Specchio [16] was used to analyse the digests. A similar method had previously been described by van den Berg and Khan [17] for the determination of selenium in seawater. A suitable aliquot of the digest was pipetted into a voltammetric cell, and sufficient quantities of concentrated HCl and 0.10 g l−1 Cu2+ solution were added so that after dilution to 10.0 ml, the analyte solution contained 0.10 M HCl and 0.10 mg l−1 Cu2+ . After purging the solution with high purity nitrogen (BOC Gases) for 8 min, an accumulation potential of –0.4 V versus SCE was applied for a period of 60 s, during which time the solution was stirred at 240 rpm using a magnetic stirrer and a PTFE-coated stirrer bar. The stirrer was then stopped, and the solution was allowed to settle for a period of 15 s. The accumulated Hg–Cu–Se product was stripped from the HMDE using a negative-going differential pulse scan (pulse height = 50 mV) at a scan rate of 5 mV s−1 . Under these conditions, a peak for Se appeared at a potential of −0.7 V versus SCE. The

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height of this peak varied linearly with concentration of selenium over the concentration range 0–4 ␮g l−1 . In order to quantify the selenium concentration, appropriate standard additions of a 1.00 mg Se l−1 selenite solution were added to the analyte solution, the solution being repurged for 2 min before remeasurement. Digestion methods were evaluated using NIST-RM50 Albacore Tuna certified reference material. The following basic methods were evaluated. 2.1. Higham and Tomkins method A 250 mg sample of material was weighed into a 150 ml tall form beaker. 10.0 ml of HNO3 , 4.0 g of Mg(NO3 )2 ·6H2 O and a few antibumping granules were added, the beaker was covered with a watchglass, and the sample was allowed to predigest at room temperature for 18 h. Using a hotplate, the digest was then slowly evaporated to dryness over a period of 5 h. The hotplate was then turned up to maximum until no more fumes evolved. The beaker was placed in a muffle furnace at 500◦ C for 30 min, removed, and allowed to cool. 5 ml of 6 M HCl was added to the beaker, which was then placed on a steam bath until the white residue had dissolved. This treatment using HCl reduces Se(VI) to Se(IV) which is the form necessary for the voltammetric analysis. Such a step is employed with each of the digestion methods tested, although the details vary from case to case. After the reduction step, the solution was transferred to a 25 ml volumetric flask and diluted to the mark with deionised water. 2.2. AOAC method 300 mg of sample was weighed into a 70 ml PTFE-lined steel closed digestion vessel, and 5 ml of concentrated HNO3 and a few anti-bumping granules were added. The digestion vessel was sealed, and the sample was allowed to predigest for 18 h at room temperature. The digestion vessel was transferred to a muffle furnace, heated at 150◦ C for 2 h, removed and allowed to cool, and the digest was transferred to a 50 ml beaker. 1 ml of 75 mg ml−1 magnesium nitrate solution (prepared by mixing 3.75 g of magnesium oxide with 30 ml of H2 O, slowly adding 10 ml of HNO3 and diluting to 50 ml) was added, and the di-

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gest was heated for 4–5 h on a hotplate at 60◦ C until dry. The temperature was increased and maintained until the brown NO2 fumes were no longer evolved. The sample was allowed to cool to room temperature, transferred to a muffle furnace and heated to 500◦ C for 30 min. After cooling, 2 ml of 8 M HCl was added to dissolve the residue. The solution was heated on a steam bath for 10 min, allowed to cool, transferred to a 50 ml volumetric flask, and diluted to the mark with water. 2.3. AOAC method (open version) The AOAC method described above was also performed using 50 ml beakers instead of closed digestion vessels for the 18 h predigestion, and heating for 2 h at 150◦ C. 2.4. Oxygen bomb method The method used by Carter and Rajendram [21] to decompose samples of fish for mercury analysis was adapted. Samples were digested using a 300 ml stainless steel calorimeter bomb. Preliminary experiments indicated that 500 mg samples combusted completely (i.e. without visible charring). Pellets of about 500 mg (density approximately 1.6 g cm−3 ) were formed using a Beckman K-series evacuable die of 10 mm diameter in a Beckman Model 00-25 press at 1500 kg ‘pressure’. The pellets were accurately weighed, wrapped in one quarter of a Whatman No. 42 filter paper, and placed in the crucible of the bomb, in contact with a stainless steel element. Deionised H2 O (10 ml) was added to the bottom of the bomb to act as an absorbent. The bomb was sealed, and charged with 20 atm of oxygen. The current through the element was increased until an increase in the temperature of the bomb was detected, indicating ignition of the sample. The element was turned off, and the bomb allowed to cool for a few minutes. The bomb was rotated end over end for 5 min at 12 rpm using a mechanical rotator. Excess gas was then slowly released from the bomb. The bomb was opened, and the solution was transferred to a 100 ml beaker. A few antibumping granules and 10 ml of 6 M HCl were added, and the beaker was gently heated to approximately 70◦ C for 15 min. The digest was allowed to cool, and was transferred to a 50 ml vol-

umetric flask and made to the mark with deionised water. 2.5. UV irradiation method Digestion using UV irradiation with nitric acid and hydrogen peroxide was evaluated. The effects of irradiation time and the amounts of hydrogen peroxide were investigated. Irradiation was performed inside a chamber containing a 1000 W Oliphant UV lamp. Samples were introduced into the chamber inside 130 ml silica tubes, which were arranged symmetrically around the lamp. 100 mg samples were weighed into the silica tubes, and 10 ml of HNO3 was added to each. Each tube was covered with a 50 ml beaker, and the samples were allowed to predigest for 18 h. An aliquot of H2 O2 (0, 0.2, 0.5 or 1.0 ml) was added, and the digestion tubes were placed in the digestion chamber. After a period of irradiation (15, 30, or 60 min), the digests were allowed to cool and additional H2 O2 was added, followed by further irradiation. This cycle was repeated, giving a total of three additions of H2 O2 . This procedure aims at ensuring that oxidising conditions are maintained throughout the digestion. The digests were transferred to beakers, and 2 ml of 4 M HCl added. The solutions were heated under conditions of mild reflux for 15 min, cooled, and diluted to 50 ml in a volumetric flask. Table 1 summarises the properties of the digests obtained under the different conditions tested. Clear, colourless digests were obtained for a range of experimental conditions, although H2 O2 was always required. If a longer irradiation time was employed, less H2 O2 was necessary in order to achieve a clear, colourless digest. However, all the digests were analysed voltammetrically, irrespective of colour. Table 1 Properties of digests after UV irradiationa Aliquots of H2 O2 (ml)

0 0.2 0.5 1.0 a

Irradiation time (min) 15

30

60

A A A B

A A B B

A B B B

A: clear, coloured digest produced, B: clear colourless digest produced.

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2.6. Pressure-aided HNO3 and HNO3 /H2 O2 digestion

Table 3 Results of analysis of NIST-RM-50 Albacore Tuna for selenium using several digestion methods, and comparison with the certified value

Pressure-aided wet digestion using nitric acid or nitric acid/hydrogen peroxide was investigated. The nitric acid decomposition involved the addition of 10 ml of concentrated HNO3 to 100 mg of sample. The nitric acid/hydrogen peroxide decomposition was identical except that 3 ml of 30% hydrogen peroxide solution was also added. The sample was weighed into a 70 ml PTFE-lined steel digestion vessel, the reagents added, and the bomb sealed tightly. The digestion vessel was placed in a pressure cooker, and heated at 122◦ C for 45 min. After cooling, the digestion vessel was opened and the contents were transferred to a 50 ml beaker. 2 ml of 4 M HCl and a few antibumping granules were added, a watchglass placed on top, and the beaker was heated on a hotplate under conditions of mild reflux for 15 min. The digest was transferred to a 50 ml volumetric flask and diluted to the mark with deionised water.

Digestion method

[Se] (␮g g−1 )a

Higham and Tomkins AOAC open AOAC closed Bomb Certified value

2.7 ± 0.4 0.9 ± 1.0 3.1 ± 0.2 1.7 ± 1.4 3.6 ± 0.4

3. Results and discussion Table 2 summarises some of the properties of the digests that were obtained using each of the digestion techniques evaluated. Each of the basic digestion techniques produced a clear digest using at least one of the variations tested. However, not all of these digests were suitable for voltammetric analysis. For example, the HNO3 /H2 O2 pressure-aided digestion technique produced a clear solution that when analysed yielded a large interfering peak over the potential range from −0.5 to −0.8 V versus SCE. This interfering peak was not observed in reagent blanks and is therefore

a

Mean ±95% confidence interval, n = 4.

attributed to the incomplete destruction of organic matter or a product of decomposition. Similarly, selenium was unable to be determined in any of the samples digested using UV irradiation: no peak current was observed in any sample digest, even when selenite was added to the digest. However, reproducible peaks and linear standard addition plots were produced when selenite was added to the digested reagent blank. It is therefore concluded that this digestion technique had also failed because of incomplete destruction of organic matter or because of a product of decomposition. It is therefore evident that the production of a clear and colourless digest is not necessarily sufficient to produce a suitable digest for CSV. The Higham and Tomkins method, the AOAC ‘open’ and ‘closed’ methods, and the bomb digestion method each produced digests in which selenium was detected using DPCSV. Table 3 compares the results of the determinations obtained using these methods. Only the AOAC ‘closed’ method produced results compatible with the certified value and with relatively low variance. The other methods produced lower estimations with greater variability. This appears to be due to variable losses of selenium during the digestion process, as with each of the methods, the upper

Table 2 Properties of digests after various methods of digestiona Technique

Clear digest produced?

Colourless digest produced?

Selenium detected in digested samples?

Higham and Tomkins AOAC open AOAC closed Bomb UV irradiation Pressure-aided HNO3 /H2 O2 Pressure-aided HNO3

Y Y Y Y Y Y Y

Y Y Y Y Y/N (see Table 1) Y N

Y Y Y Y N N N

a

Y: yes, N: no.

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range of the estimate is similar to the certified value. The success or failure of these methods seems to be extremely dependent on precise experimental conditions, which probably explains the disagreements reported in the literature. In the case of the method proposed by Higham and Tomkins, an additional problem is suspected. After ashing at 500◦ C, a residue of MgO is formed, which is subsequently dissolved in HCl. It was found that the residue was extremely difficult to dissolve using the 5 ml of 6 M HCl suggested. Examination of the stoichiometry of this reaction together with the quantities of reagents recommended indicates that insufficient HCl is added, which leads to incomplete dissolution of MgO and probably unsuccessful conversion of Se(VI) to Se(IV). Holak [11] recommended the use of 10 ml of 6 M HCl, which is almost twice the stoichiometric quantity required for dissolution of the MgO.

4. Conclusion Analysis of fish tissue for selenium by DPCSV requires a digestion method that is extremely thorough in decomposing organic matter, and yet does not result in conditions under which selenium is lost. Of the methods tested, only the AOAC recommended digestion method produced acceptable results for NIST-RM-50 Albacore Tuna. The other digestion methods tested were found to produce digests that were unsuitable

for DPCSV analysis or resulted in variable losses of selenium. References [1] D.W. Bryce, A. Izquierdo, M.D. Luque de Castro, Fresenius’ J. Anal. Chem. 351 (1995) 433. [2] C. Reilly, Selenium in Food and Health, Blackie, London, 1996, Chapters 4 and 8. [3] Association of Official Analytical Chemists, Official Methods of Analysis, Vol. 1, Arlington, 1990, p. 237. [4] H. Narasaki, M. Ikeda, Anal. Chem. 56 (1984) 2059. [5] P. Schramel, L. Xu, Fresenius’ J. Anal. Chem. 340 (1991) 41. [6] I. Al-Saleh, I. Al-Doush, J. Trace Elem. Electrolytes Health Dis. 14 (1997) 6. [7] P. Hocquellet, M. Candillier, Analyst 116 (1991) 505. [8] M. Deaker, W. Maher, Anal. Chim. Acta 350 (1997) 287. [9] H. Aydin, G.H. Tan, Analyst 116 (1991) 941. [10] W.G. Lan, M.K. Wong, Y.M. Sin, Talanta 41 (1994) 53. [11] W. Holak, J. Assoc. Off. Anal. Chem. 59 (1976) 650. [12] S.B. Adeloju, A.M. Bond, M.H. Briggs, H.C. Hughes, Anal. Chem. 55 (1983) 2076. [13] S.B. Adeloju, A.M. Bond, M.H. Briggs, Anal. Chem. 56 (1984) 2397. [14] N. Peerzadu, L. Pessina, Anal. Lett. 23 (1990) 2027. [15] A.M. Higham, R.P.T. Tomkins, Food Chem. 48 (1993) 85. [16] W. Holak, J.J. Specchio, Analyst 119 (1994) 2179. [17] C.M.G. van den Berg, S.H. Kahn, Anal. Chim. Acta 231 (1990) 221. [18] G. Mattsson, L. Nyholm, A. Olin, U. Ornemark, Talanta 42 (1995) 817. [19] C. Elleouet, F. Quentel, C. Madec, Water Res. 30 (1996) 909. [20] V. Ducros, D. Ruffieux, N. Belin, A. Favier, Analyst 119 (1994) 1715. [21] R.J. Carter, V.S. Rajendram, Sci. Total Environ. 125 (1992) 33.