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Determination of Nickel in Biological Materials After Mi.crowave Dissolution Using Inductively Coupled Plasma Atomic Emission Spectrometry With Prior Extraction into Butan-1-01
Elisa Vereda Alonso, Amparo Garcia de Torres and Jose M. Can0 Pavon* Department of Analytical Chemistry, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain

A sensitive procedure has been developed for the determination of ultratrace amounts of nickel in biological
materials by inductively coupled plasma atomic emission spectrometry after extraction of the nickel ion into butan-1-01 b y using 1,5-bis(di-2-pyridylmethylene)thiocarbonohydrazide as the extracting reagent. Fast, efficient and complete sample digestion is achieved by an HN03-HCI poly(tetrafluoroethy1ene) bomb dissolution technique using microwave heating. Results obtained for eleven certified reference materials agreed with the certified values.

Keywords: Nickel determination; inductively coupled plasma atomic emission 7,5-bis(di-2-pyridylmethylene)thiocarbonohydrazide; extraction; biological materials

spectrometry;

Liquid-liquid extraction is one of the most frequently used sample pre-treatment techniques in the determination of trace metals by inductively coupled plasma atomic emission spectrometry (ICP- AES) .I,* 1,5-Bis(di-2-pyridylmethylene) thiocarbonohydrazine (DPTH) is a suitable complexing reagent for a number of metal ions. Most of the complexes formed by this reagent are coloured and can be extracted into a variety of organic solvents. Its synthesis and properties have been described in detail,3 as has its use in the extraction of cobalt into chloroform, followed by the determination of this metal by flame atomic absorption spectrometry.4 It has also been used for the spectrophotometric determination of cobaltnickel mixtures.5 In this paper, an ICP-AES method for the determination of ultratrace amounts of nickel after extraction of the nickel ion into butan-1-01 (a solvent commonly used in ICP-AES) containing DPTH as the extracting reagent is described. The complex formed is soluble in the organic phase, which allows the use of aqueous-to-organic phase volume ratios of up to 8. In addition, the extraction step enhances the selectivity. The method thus developed was applied to the determination of nickel in various biological materials. Nine certified reference materials prepared by microwave digestion in sealed poly(tetrafluoroethylene) containers and two certified human urines without mineralization were analysed. The use of closed vessels for the decomposition of biological samples, proposed by Kingston and Jassie6 and Aysola et al. ,' makes it possible to eliminate uncontrolled trace element losses of volatile molecular species that are present in a sample and leads to a decrease in blank values compared with openbeaker work, because contamination from the laboratory environment is much lower and also because closed vessels make it possible to use smaller amounts of reagents.8

with a combined glass+alomel electrode. Separating funnels were shaken on a Gallenkamp flask agitator. For sample digestion, a domestic microwave oven, Panasonic Model NN-8507/8557 with a 700 W magnetron, was used. The oven power (0-100% power in 20% increments) and the time range (0 s to 99.0 min) can be pre-set digitally; up to four sequential steps of different power and duration can be programmed. The oven used has a rotating antenna, so that the power is distributed homogeneously throughout the oven volume. The oven was placed in a laboratory fume-hood.
Reagents

All reagents used were of analytical-reagent grade. Doubly distilled, de-ionized water was used throughout. The ligand for the DPTH solution was synthesized as described elsewhere.3 A stock 0.1% solution in butan-1-01 was prepared by dissolving 0.1 g of DPTH in 9 cm3 of N,N-dimethylformamide and diluting to 100 cm3 with butan- 1-01. A stock solution of Nil*was prepared from the nitrate and standardized gravimetrically with dimethylglyoxime. Standards of working strength were prepared by appropriate dilution as required. Standard solutions were prepared daily. A glycine-HC1 buffer of pH 3.6 was prepared by mixing 25 cm3 of 0.2 mol dm-3 glycine and 2.5 cm3 of 0.2 mol dm-3 HC1 in a 100 cm3 calibrated flask and diluting to the mark with water. A 1 mol dm-3 NaC104 solution was also used.

Experimental
Apparatus

The ICP-AES measurements were made on a Perkin-Elmer 40 sequential emission spectrometer controlled by an IBM XT-286 computer. A Meinhard nebulizer was used each time butan-1-01 was employed. The r.f. generator of the nebulizer is internally mounted with a 'free-running' oscillator-type with a nominal central frequency of 40 MHz and a nominal operating power level of 900 W. The pH measurements were made with the aid of a Crison Digit-501 pH meter furnished

* To whom correspondence should be addressed.

Procedures Liquid-liquid extraction of nickel In 100 cm3 separating funnels were placed between 0.3 and 20 pg of Ni" plus 1 cm3 of 1 mol dm-3 NaC104, 5 cm3 of the glycine-HC1 buffer of pH 3.6 and distilled water up to a final volume of 20 cm3. Then, 10 cm3 of the 0.1% butan-1-01 solution of DPTH were added and the funnel was shaken vigorously on the mechanical agitator for 10 min, after which the phases were allowed to separate and the organic phase was collected. The organic phase was pumped into the plasma bulk by means of a peristaltic pump for the determination of nickel. Emission readings for nickel were taken on the 341.476 nm line (this wavelength was chosen as it resulted in the largest differences between the samples and the noise signals). A calibration graph was prepared from nickel standards treated in the same way.

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Table 1 Working conditions of the microwave oven used with the different samples studied

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Sample* BCR CRM 062 Olive Europoea (Olive Leaves) BCR CRM 060 Lagarosiphon Y a j o r (Aquatic Plant) BCR CRM 061 Platihypnidium Ripariodides (Aquatic Plant) NRCC CRM DORM-1 Dogfish Muscle NRCC CRIPISOLT-1 Dogfish Liver NRCC CRM TORT-1 Lobster Hepatopancreas NIST SRM 1572Citrus Leaves BCR CRM 186Pig Kidney BCR CRM 184Bovine Muscle

Power and time 2minat480 W + 4min at 180 W 2.5 rnin at 480 W 4.5 rnin at 360 W 2min at 700 W + 4minat 360 W 4minat360W+ 10minat180W 4minat360W + 10minat 180W 4minat360W+ 10minat180W 4minat360W+ 10minat180W 4 min at 360 W + 10 min at 180W 4minat360W+ 10minat180W

No. o treatments f

* BCR = Community Bureau of Reference; NRCC = National Research Council Canada; NIST = National Institute o Standards and f Technology; CRM = certified reference material; and SRM = standard reference material.
100
100

s 75 . ;
g
.c
w
25
C

5
c

75

0 .w
c

5 50
25
I I I I I
I I I I
I I I I I I

50

8 10

0.5 1.0 1.5 2.0 2.5 3.0

[NaC1041/10-2 rnol dm-3

2 4

Shaking time/min

[DPTH]/10-3 mot dm-3

PH

Fig. 1 Extraction ofnickel into butan-1-01versus pH using DPTH as f extracting reagent. Concentration o nickel in the aqueous phase: 2 pg cm-3; volume of aqueous phase: 20 cm3; volume of organic phase: 10cm3

Fig. 2 Influence of experimental variables on the extraction of the NP-DPTH com lex into butan-1-01. ( a ) Influence of perchlorate influence of shaking time; and (c) influence of concentration; reagent concentration (organic phase). Nickel concentration, 2 pg ~ m (aqueous phase); initial volume of aqueous phase, 20 cm3; - ~ initial volume of organic phase, 10 cm3 (total in each instance)

(8,

Determination of nickel in biological samples The samples were placed in Parr 478 microwave acid digestion bombs. The bombs were cleaned before use with 2 mol dm-3 nitric acid for 1 d followed by repeated rinsing with water. All glassware used was treated in the same way. Samples (about 200 mg) were weighed directly into the digestion vessels on a digital electronic balance and 4 cm3 of concentrated nitric acid were added. The mixture was allowed to stand for 30 min after which 2 cm3 of concentrated hydrochloric acid (12 mol dm-3) were added. The bomb was sealed and placed in the microwave oven with a beaker filled with 10 cm3 of water. This procedure minimizes the risk of damage to the magnetron in that small sample loads can cause magnetron failure, because most of the microwave power is reflected back to the waveguide and magnetron. The working conditions of the microwave oven are listed in Table 1 . After digestion, the solutionswere evaporated by heating to a small volume (about 0.5 cm3) to eliminate the nitric acid. The pH was adjusted to 3.6 with 1mol dm-3 NaOH solution and 10 cm3 of buffer plus 2 cm3 of 1mol dm-3 NaC104solution were added. The solutions were diluted to 250 cm3 with de-ionized water in a calibrated flask. Then, in a 250 cm3 separating funnel were placed 125 cm3 of sample solution and 15 cm3 of a 0.05% butan-1-01 solution of DPTH. Thefunnel was shaken vigorously on the mechanical agitator for 5 min, after which the phases were allowed to separate and the organic phase was collected in order to measure the Ni concentration by ICP-AES at 341.476 nm, as described above. Human urine can be analysed without mineralization. The urine was acidified with concentrated nitric acid until 1% v/v and stored frozen until required for the determination of nickel. In 250 cm3 separating funnels were placed 100-150 cm3 of acidified urine, which was analysed as described above. In all instances it is convenient to analyse the reagent and buffer solutions previously in order to test for the presence of nickel.

Results and Discussion

Effect of pH on the Extraction

The influence of pH on the extraction of the NP-DPTH complex into butan-1-01 was studied over the range 1.0-12.5. The results (Fig. 1) showed that the extraction is quantitative between pH 3.0 and 7.0, and that it takes place to a much smaller extent outside this range.
Selection of the Buffer Solution

Three buffer solutions were tested: one of pH 3.6 consisting of glycine-HC1 and two of pH 3.8 consisting of potassium hydrogen phthalate-HC1 and acetic acid-soldium acetate. All yielded very similar results. Nevertheless, the glycine-HC1 buffer was chosen on account of its greater complexing ability, which could be of use in overcoming interferences.
Effect of Perchlorate Concentration, Shaking Time and Reagent Concentration

The extraction of the nickel-DPTH complex into an acidic medium was investigated by varying the NaC104 concentration up to 6.5 x 10-2 mol dm-3. The extracted fraction increased on increasing the perchlorate concentration to 3 X 10-2 mol dm-3 [Fig. 2(a)];however, further increases in the concentration, which were obtained by adding 1cm3 of 1mol dm-3 NaC104 to an aqueous volume of 20 cm3, had no appreciable effect for subsequent experiments. A study of the influence of the shaking time [Fig. 2(b)] revealed that the extent of extraction levelled off after 3 min. A shaking time of 5 rnin was selected for subsequent experiments. Next, the reagent concentration in the organic phase was varied while keeping the final volume at 10 cm3. The-results obtained [Fig. 2(c)] showed that the extracted fraction remained constant for DPTH concentrations equal to or

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greater than 6.83 x 10-4 mol dm-3 (0.03%). A concentration of 0.05% was used in practice, in order to prevent depletion of the reagent by other extractable ions that might be present in the aqueous medium.
Effect of Phase-volume Ratio

The volume of the aqueous phase was varied from 10 to 150 cm3 (0.05% m/v DPTH); phase volume ratios between 1 and 15 were tested. Nickel could be quantitatively extracted up to a phase ratio of 8, above which phase separation was unsatisfactory and the procedure was, therefore, inapplicable. On the other hand, when the volume of the aqueous phase was increased, the volume of the organic phase decreased, because butan-1-01 is not totally miscible with water; however, the quantitative extraction of nickel could be verified from the calibration graphs. When a phase ratio of 8 was used (80 cm3 of aqueous phase and 10 cm3 of organic phase), only approximately 2.5 ml of organic phase were obtained after extraction; hence the phase ratio is actually 32. This phase ratio allows the sensitivity of the direct non-extractive method to be increased by a factor of approximately 32.
Determination of Nickel by ICP-AES A wavelength of 341.476 nm was selected as it resulted in the largest differences between the sample and the noise signals (minimum background equivalent concentration). The nebulizer was then optimized: the optimum signal-to-noise ratio was obtained at a value of 1.9 on an arbitrary scale from 0 to 6; the plasma gas flow rate was 12 dm3 min-1; and the auxiliary gas flow rate was 0.5 dm3 min-l. Other experimental variables were optimized, for which the following values were selected: torch height, first position on an arbitrary scale from 1to 4 (the fourth position is the ignition position); photomultiplier voltage, 750 V; element time, 360 ms; integration time, 100 ms; read delay, 15 s; and spectral range, 1-00nm.

The limits of detection and determination of the method were established according to American Chemical Society Committee of Environmental Improvement definitions.9 The detection limit thus achieved for an aqueous-to-volume phase ratio of 8 was 0.2 ng cm-3 (aqueous phase) and the determination limit was 1.7 ng cm-3 (aqueous phase). Relative standard deviations were 3.4% for 2.5 ng cm-3 and 1.8% for 25 ng cm-3 (all concentrations are referred to the aqueous phase).
Study of Interferences

The effect of various ions on the determination of nickel by the proposed method was examined under the optimum working conditions. For this study, different amounts of the ionic species tested were added to a 10 ng cm-3 solution of Ni in the aqueous phase, the volume of which was 160 cm3, whereas that of the organic phase was 20 cm3. The starting point was an m/m interferent-to-Ni ratio of 4000; if any interference occurred, the ratio was gradually lowered until the interference disappeared. The tolerance limits found (Table 2) show that nickel can be determined in the presence of a variety of ions including most of those that commonly occur with nickel in natural and synthetic samples. Cobalt interferes with the determination of nickel up to at least a ratio of 40; hence, another wavelength, 352.454 nm, was chosen to eliminate this interference. With this wavelength, the tolerated ratio of cobalt can be increased to 1000.

Determination of Nickel at 352.454 n m

Table 2 Tolerated levels of foreign species in the determination of NiII*

Species Alkali metal ions, alkaline earth metal ions, A Cr1I1, P , bromide, chloride, fluoride, nitrate, sulfate, phosphate, thiourea, thiosulfate Mo"', Vv, Selv,ascorbic acid, oxalate A1 1 pbll MnII Bill1 s1 Hg",Agl,Sblll,Cull Fell, Fell1 Zn" * Nil1 = 10 ng ~ r n in ~the aqueous phase. ? ,
9

Tolerated ratio (dm)

The different instrumental variables for this wavelength were optimized. The following values were selected: nebulizer flow rate, 2.0 on an arbitrary scale from 0 to 6; plasma gas flow rate, 12 dm3 min-1; auxiliary gas flow rate, 0.5 dm3 min-1; torch height, first position on an arbitrary scale from 1 to 4; photomultiplier voltage, 900 V; element time, 360 ms; integration time, 40 ms; read delay, 15 s; and spectral range, 1.00 nm. The detection limit thus achieved for an aqueous-to-volume phase ratio of 8 at this wavelength was 3.2 ng cm-3 (aqueous phase) and the determination limit was 8.1 ng cm-3 (aqueous phase). Relative standard deviations were 3.9% for 10 ng cm-3 and 3.2% for 25 ng cm-3 (all concentrations are referred to the aqueous phase).

>4OOo

3000 2000 1500 1000 200

Analysis of Biological Materials

In order to ascertain the accuracy of the method, the optimized procedure was applied to the following certified biological materials: Community Bureau of Reference

Table 3 Application of the proposed method to the determination of nickel in biological samples

Sample BCR CRM 062 Olive Europoea (Olive Leaves) BCR CRM 060 Lagarosiphon Major (Aquatic Plant) BCR CRM 061 Platihypnidium Ripariodides (Aquatic Plant) NRCC CRM DORM-1 Dogfish Muscle NRCC CRM DOLT-1 Dogfish Liver NRCC CRM TORT-1 Lobster Hepatopancreas NIST SRM 1572 Citrus Leaves BCR CRM 186Pig Kidney BCR CRM 184Bovine Muscle NIST SRM 2670 Freeze-Dried Urine (Toxic Metals at Normal Levels) NIST SRM 2670 Freeze-Dried Urine (Toxic Metals at Elevated Levels) * Mean of three separate determinations. t Values in pg crn-3.

Ni certifiedpg g-1 8.0 40.0 420.0 1.20 0.26 2.3 0.6 0.42 0.27 0.07t 0.30t

Ni found*/pgg-1 7.9 zk 0.4 38.8 f 1.7 430.2 k 30.0 1.40 k 0.15 0.31 f 0.11 2.6 f 0.1 0.6 _+ 0.1 0.39 0.03 0.30 f 0.01 0.09 0.02-l 0.28 f 0.Olt

* *

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(BCR), Certified Reference Materials (CRMs) 062 Olive Europoea (Olive Leaves); 060 Lagarosiphon Major (Aquatic Plant); 061 Platihypnidium Ripariodides (Aquatic Plant); 186 Pig Kidney; 184 Bovine Muscle; National Research Council Canada (NRCC), CRMs DORM-1 Dogfish Muscle; DOLT-1 Dogfish Liver; TORT-1 Lobster Hepatopancreas; National Institute of Standards and Technology (NIST), Standard Reference Materials (SRMs) 1572 Citrus Leaves; and 2670 Freeze-Dried Urine (Toxic Metals at Normal and Elevated Levels). The results obtained are given in Table 3, and agree well with the certified values. The limits of detection found for the different biological samples dissolved in the aqueous phase are (in ng (3111-3): CRM 062 Olive Leaves, 0.4; CRM 060 Aquatic Plant, 0.2; CRM 061 Aquatic Plant, 0.2; DORM-1 Dogfish Muscle, 1.2; DOLT-1 Dogfish Liver, 1.7; TORT-1 Lobster Hepatopancreas, 1.1; SRM 1572 Citrus Leaves, 0.5; CRM 186 Pig Kidney, 1.1; CRM 184 Bovine Muscle, 0.9; and SRM 2670 Freeze-Dried Urine, 0.5. We thank the Direccion General de Investigacidn Cientifica y Tkcnica (DGICYT) for supporting this study (Projects PB870711 and PB90-0809), and also the Junta de Andalucia.

References
Miyazaki, A., Kimura, A., Banzho, K., and Umezaki, Y., Anal. Chim. Acta, 1982, 114,213. Whiteley, R. V., and Merril, R. M., Fresenius Z . Anal. Chem., 1982, 311, 7. Bonilla Abascal, J. R., Garcia de Torres, A., and Can0 Pavon, J. M., Microchem. J., 1981,26,55. Bustos, A., Sanchez-Rojas, F., Bosch Ojeda, C., Garcia de Torres, A., and Can0 Pavon, J. M., J. Anal. At. Specrrom., 1987, 2,253. Can0 Pavon, J. M., Garcia de Torres, A., and Bosch Ojeda, C., Analyst, 1985, 110, 1137. Kingston, H. M., and Jassie, L. B., Anal. Chem., 1986,58,261. Aysola, P., Anderson, P., and Langford, C.H., Anal. Chem., 1987,59, 1582. Introduction to Microwave Sample Preparation, eds. Kingston, H. M., and Jassie, L. B., American Chemical Society, Washington, DC, 1988. ACS Committee of Environmental Improvement, Anal. Chem., 1980,52,2242.

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Paper I f06438J Received December 23, I991 Accepted February 26, 1992