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Analytical note

On-line determination of Sb(III) and total Sb using baker's yeast immobilizedon polyurethane foam and hydride generation inductively coupled plasma

optical emission spectrometry

Amauri A. Menegário a,⁎, Ariovaldo José Silva a, Eloísa Pozzi b,Steven F. Durrant c, Cassio H. Abreu Jr. d

a Centro de Estudos Ambientais, Universidade Estadual Paulista, Av. 24-A, 1515, CEP 13506-900, Rio Claro, SP, Brazilb Escola de Engenharia de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense 400, CEP 13566-590, São Carlos, SP, Brazil

c Laboratório de Plasmas Tecnológicos, Campus Experimental de Sorocaba, Universidade Estadual Paulista- UNESP,Avenida Três de Março 511, CEP 18087-180, Sorocaba, SP, Brazil

d Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, Caixa Postal 96, CEP 13400-970, Piracicaba, SP, Brazil

Received 19 January 2006; accepted 15 September 2006

Abstract

The yeast Saccharomyces cerevisiae was immobilized in cubes of polyurethane foam and the ability of this immobilized materialto separate Sb(III) and Sb(V) was investigated. A method based on sequential determination of total Sb (after on-line reduction of Sb(V) toSb(III) with thiourea) and Sb(III) (after on-line solid–liquid phase extraction) by hydride generation inductively coupled plasma opticalemission spectrometry is proposed. A flow system assembled with solenoid valves was used to manage all stages of the process. The effectsof pH, sample loading and elution flow rates on solid–liquid phase extraction of Sb(III) were evaluated. Also, the parameters related to on-line pre-reduction (reaction coil and flow rates) were optimized. Detection limits of 0.8 and 0.15 μg L−1 were obtained for total Sb and Sb(III), respectively. The proposed method was applied to the analysis of river water and effluent samples. The results obtained for thedetermination of total Sb were in agreement with expected values, including the river water Standard Reference Material 1640 certified by theNational Institute of Standards and Technology (NIST). Recoveries of Sb(III) and Sb(V) in spiked samples were between 81±19 and 111±15% when 120 s of sample loading were used.© 2006 Elsevier B.V. All rights reserved.

Keywords: Sb(III); Sb(V); ICP OES; Saccharomyces cerevisiae; Speciation analysis

1. Introduction

Antimony is a potentially toxic element and its compoundsare considered as pollutants of priority interest by environmen-tal agencies. In environmental samples, antimony is usuallypresent mainly as inorganic Sb(III) and Sb(V) [1,2], which havedifferent toxicities: trivalent forms are ten times more toxic thanpentavalent species. Typical concentrations of total dissolvedantimony in non-polluted waters range from ng L−1 to μg L−1

[1]. Thus, methods for determination of antimony species attrace levels are fundamental for environmental studies.

The coupling of hydride generation (HG) with atomicspectrometry techniques provides powerful methods fordetermination of antimony at trace levels. HG can be alsoused for selective generation of antimony species by exploitingthe pH-dependence of stibine generation from Sb(III) and Sb(V)[3–8]. The generation of stibine from Sb(III) and Sb(V)depends on several parameters but, generally, Sb(III) isselectively generated at a pH greater than 2 [9]. Although thisapproach is simple and popular, interferences from transitionmetals in the generation of stibine from Sb(III) can beuncontrollable at low acid concentration [9]. Other approaches

Spectrochimica Acta Part B 61 (2006) 1074–1079www.elsevier.com/locate/sab

⁎ Corresponding author. Tel./fax: +55 1935340122.E-mail address: amenega@rc.unesp.br (A.A. Menegário).

0584-8547/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.sab.2006.09.008

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pyto redox speciation analysis of antimony, including methodsbased on liquid–liquid extraction, liquid–solid extraction,coprecipitation and hyphenated techniques were reviewed bySmichowski et al. [8] and Krachler et al. [10].

Use of immobilized microorganisms as sorbent materials inliquid–solid extraction has been an effective alternative forredox speciation analysis of arsenic [11], chromium [12–15]and iron [16]. In these methods, Saccharomyces cerevisiae,Aspergillus niger, Funaria hygrometrica and Ecklonia maximawere the microorganisms used, while controlled pore glass(CPG), sepiolite and polysilicate were used as supports.

Uptake of antimony species by baker's yeast was evaluated(in batch mode) by Pérez-Corona et al. [17]. Under appropriateconditions, the microorganism selectively retained the Sb(III)species, allowing the separation of Sb(III) and Sb(V) in watersamples. S. cerevisiae immobilized on CPG was successfullyused for redox speciation analysis of arsenic and chromium[11,13]. The use of CPG as a support, however, reduces theselectivity of the microorganism due to interactions of thesupport with anionic species [13].

In the present work S. cerevisiae was immobilized on poly-urethane foam cubes and its use for on-line separation of Sb(III) and Sb(V) was investigated. A flow system incorporat-ing a column containing the immobilized material is pro-posed for determination of Sb(III) and total Sb by hydridegeneration inductively coupled plasma optical emissionspectrometry.

2. Experimental

2.1. Equipment and accessories

AGBCmodel Integra XL inductively coupled plasma opticalemission spectrometer was used. The spectrometer was operatedunder the following conditions: forward power=1.3 kW; plasmagas flow rate=10 Lmin−1; auxiliary gas flow rate=0.5 Lmin−1;argon carrier gas flow rate=0.6 L min−1. Measurements of Sbwere performed at 206.833 nm.

The flow system was assembled with a minipuls 3 peristalticpump (Gilson, Villiers-le-Bel, France), Tygon tubing, 0.8-mminternal diameter polyethylene tubing, three-way solenoidvalves (N-Research, 161T031, Stow, MA, USA) and a gas–liquid phase separator (GBC, Melbourne, Australia). The valveswere controlled by a microcomputer via a program written inQuick BASIC 4.5.

A column made from 47 mm of 4-mm internal diameterpolyethylene tubing was used. The columnwas filled with 5-mmpolyurethane foam cubes and its end was connected to Tygontubing. For cleaning before use, purified water was passedthrough the column (0.8 mL min−1) for a period of about 2 h.

2.2. Reagents and solutions

All solutions were prepared with 18.2 MΩ cm purified waterproduced in a Milli-Q system (Millipore, Bedford, MA, USA).

Fig. 1. Electron micrographs of cubes of polyurethane foam: (a) before immobilization; (b) external surface after immobilization; (c) internal surface after immobilization.

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Single stock solutions of Sb(III) and Sb(V) were preparedfrom SbCl3 (Merck, Darmstadt, Germany ) and SbCl5 (Aldrich,Milwaukee, USA), respectively.Working standard solutions of 0to 1000 μg L−1 of Sb(III) and Sb(V) were prepared in 0.05 molL−1 TRIS buffer [tris(hydroxymethyl) aminomethane, 99.9%](Sigma–Aldrich) at pH 9.0 from single stock standard solutions.

Solutions of 3% (m/v) NaBH4 (Nuclear, São Paulo, Brazil) in0.1 mol L−1 NaOH (Merck) and 1 mol L−1 thiourea (Synth, SãoPaulo, Brazil) in 4 mol L−1 HCl (Merck) were prepared dailyfrom pro-analisi reagents.

S. cerevisiae used as a biosorbent was a commercial drybaker's yeast. The polyurethane foam (bulk density 23 g cm−3)was obtained from a local supplier.

2.3. Samples

Fourwater sampleswere analyzed, three of themcollected fromthe Corumbataí river (1 FHD and 1C FHD) and Atibaia river (RP),and a NIST 1640 Standard Reference Material. The collectionpoint on the Atibaia river is subject to effluent discharges from apetrol refinery. An industrial effluent sample from a Galvanizationplant was also analyzed. Before analysis, samples were filteredthrough a microfiber filter and the pH adjusted to 9.0.

2.4. Immobilization procedure

The procedure used for immobilization of the yeast wasbased on methods described previously for immobilization offungal biomass and Pseudomonas in polyurethane foam[18,19]. These materials have typically been used for removalof metals in bioreactors.

The polyurethane foam was cut to 5 mm3 cubes and washedwith purified water. The cubes (0.20 g) were soaked in a solutionof sucrose (20%, w/v) and placed into a conical flask containing

10 mL of freshly prepared yeast cell suspension (20%, w/v).Subsequently, the cubes were mixed with the suspension and leftundisturbed at 25±2 °C for 2 h. Next, the cubes were removedfrom the beaker, compressed to remove the excess of slurry,washed with water and dried in an oven at 60±5 °C for 12 h.Weighing before and after immobilization revealed that 90mg ofyeast (per gram of foam) was deposited on the support. Fig. 1showsmicrographs obtained by scanning electronmicroscopy atdifferent magnifications of the surface of the supports before (a)and after (b–c) immobilization of the yeast.

2.5. Flow system

The flow system for determination of total Sb and selectivedetermination of Sb(III) by hydride generation inductively coupled

Table 1Analytical sequence for determination of Sbtotal and Sb(III)

Valve (V) switch position Step Signalacquisition

Time(s)

V1 V2 V3 V4 V5 Pre-reduction Separation

45 Off Off Off On Off Mixing sampleand thiourea

– –

05 Off Off Off On Off Mixing sampleand thiourea

– Measurement

120 Off On Off Off Off – Sampleloading

–

90 On Off Off Off Off – Cleaning thecolumn

–

85 Off Off On Off On – Elution ofSb(III)

–

05 Off Off On Off On – Elution ofSb(III)

Measurement

90 On Off Off Off Off – Conditioningthe column

–

Fig. 2. Flow system for determination of total Sb and Sb(III). The circles represent the valves (V1 to V5) and the rectangle the phase separator. The arrows indicate thepumping action and X the confluence between NaBH4 and the acidified sample. Lp=reaction coil=129 cm and Lm=mixing coil=30 cm. HCl+Thiourea=0.5 mLmin−1; sample (V5)=1.0 mL min−1; sample (V2)=0.8 mL min−1; water=0.8 mL min−1; eluent=2.1 mL min−1; NaBH4 3.0% (m/v)=0.8 mL min−1.

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pyplasma optical emission spectrometry is shown in Fig. 2. The flowsystem governs two processes: (a) the on-line reduction of Sb(V)for determination of total Sb and (b) the solid–liquid phase extrac-tion using the yeast cells immobilized for selective determinationof Sb(III). Initially, for determination of total Sb, an aliquot of thesample solution (valve V5) was continuously pumped through thesystem, mixing with thiourea solution in the Lp reactor and thenreceiving the 3% (w/v) NaBH4 solution at the confluence point X.Volatile species produced from the sample were separated in thephase separator and carried by argon to the ICP. In a second step,for selective determination of Sb(III), another aliquot of the samplesolution (valveV2) was continuously pumped through the columnand, subsequently, the column was cleaned with water. Insequence, the analyte was eluted with 2 mol L−1 HCl, and theSbH3 was generated from Sb(III) atX. All the steps were managedby switching on or off the valves V1 to V5, as shown in Table 1.Unless otherwise specified, pre-reduction solution (4 mol L−1

HCl +1 mol L−1 Thiourea), sample in valve V5, sample in valveV2, water, eluent (2 mol L−1 HCl) and NaBH4 solution werepumped at flow rates of 0.5, 1.0, 0.8, 0.8, 2.1 and 0.8 mL min−1,respectively.

3. Results and discussion

To evaluate the capability of the material to retain Sb(III) or Sb(V) species or both, a preliminary test using a column(4 mm×47 mm) containing only the support (polyurethanefoam) and another containing the immobilized yeast was carriedout. In this test, 1000 μg L−1 single standard solutions of Sb(III)or Sb(V) were loaded onto the columns at 0.8 mL min−1 and theretained species were eluted by pumping 0.9 mL of 2 mol L−1

HCl at 0.8 mL min−1 directly to the ICP (without hydridegeneration). For the column filled with immobilized baker'syeast, the recoveries of Sb(III) and Sb(V) were 41±14 and 6±1,respectively. The recovery of Sb(III) was lower than 5%when the

column containing only the support was used. These resultsillustrate that Sb(III) was selectively retained by the yeast,similarly to the results of batch experiments reported previously[17]. Thus, aiming to separate Sb(III) and Sb(V), the influence ofthe pH and flow parameters on the retention of the analytes wasinvestigated using part of the flow system shown in Fig. 1 (valvesV4 andV5 remaining switched off). The experiments were carriedout by loading 1.8 mL of 100 μg L−1 single standard solution ofSb(III) or Sb(V).

3.1. Separation of Sb(III) and Sb(V)

The effect of pH on retention of Sb(III) and Sb(V) byimmobilized yeast was evaluated by using flow rates of 0.9 and2.1 mL min−1 for sample loading and elution, respectively.Signals from the elution step showed that the retention of Sb(III)considerably increased (from 9 kCPS to 27 kCPS) when the pHof the solutions was increased from 3.0 to 10.5. Minimalretention of Sb(V) was achieved for single standard solutionsat pH 9.0. The relative elution signals (%) found for the singlestandard solutions of Sb(V) at different pHs (considering asa reference the signal from Sb(III) solutions at the same pH)are shown in Table 2. These results indicate that the bestcondition for separation of Sb(III) and Sb(V) was attained atpH 9.0.

The retention of Sb(III) systematically increased when loadingflow rates from 1.2 to 0.4 mLmin−1 were used. For a flow rate of0.4 mL min−1, however, the retention of Sb(V) also increased,resulting in a poor separation of the analytes. Based on theseresults, a pH of 9.0 and a sample flow rate of 0.8 mL min−1 wereselected as the most favorable for selective retention of Sb(III).

Significant reductions of the peak height were observed forelution flow rates lower than 2.1 mL min−1. On the other hand,the risk of leaks increased considerably for flow rates higher than2.1 mL min−1.

3.2. On-line pre-reduction of Sb(V)

Determination of Sb in real samples requires a pre-reductionstep of the analyte, because the efficiency to produce SbH3

depends on state of oxidation of the element. Thiourea has been

Table 2Effect of pH on separation of Sb(III) and Sb(V)

pH Relative signal Sb(V)/Sb(III), %a

3.0 35±17.0 36±49.0 3.7±0.710.5 6.5±0.3

a. Mean±SD, n=3 from 100 μg L−1 single standard solutions of Sb(III) or Sb(V).

Table 4Determination of Sb(III) and Sb(V) (mean±SD) in samples of river water andeffluent using 120 and 360 s of sample loading

Found, μg L−1 Reference value,μg L−1

Sample Sb(III) Sb(V) Sbtotal Sbtotal

River water-SRM-NIST-1640

1.4±0.2 12.3±0.3 13.7±0.2 13.79±0.42a

River water-RP b0.5 2.4±0.3 2.4±0.3 2.5±0.1b

River water-RP b0.2 2.4±0.3 2.4±0.3 2.5±0.1b

Effluent b0.5 2.9±0.2 2.9±0.2 2.7±0.1b

Effluent 0.3±0.1 2.6±0.2 2.9±0.2 2.7±0.1b

a. Certified value, expressed as μg kg−1.b. Determination by Inductively Coupled Plasma Mass Spectrometry (mean±SD,n=3) [24].

Table 3Effect of sample flow rate and the size of the reaction coil on pre-reduction of Sb(V)

Reaction coil, cm

Relative signala Flow rate, mL min−1 29 79 129

Sb(V)/Sb(III), % 2.1 49±1 60.3±0.3 68.2±0.4Sb(V)/Sb(III), % 1.6 – – 87.3±0.7Sb(V)/Sb(III), % 1.0 – – 97±1

a. Mean±SD, n=3 from 100 μg L−1 single standard solutions of Sb(III) or Sb(V). Thiourea 1 mol L−1 and 100 μg L−1 single standard solution of Sb(III) andSb(V).

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proposed as an effective pre-reducer of Sb(V) and as a maskingagent to minimize interference from transition metals [20–22].Thus, aiming to assess optimal conditions to reduce Sb(V) to Sb(III) with thiourea (in 4 mol L−1 HCl medium), different sampleflow rates and sizes of reaction coil were tested. The test wascarried out using part of the flow system shown in Fig. 1 (valvesV1, V2 and V3 remaining off).

Table 3 shows the relative signals of Sb(V) when differentsample flow rates and sizes of reaction coil (Lp, Fig. 1) were used.These results show that quantitative reduction of Sb(V) wasattained when a sample flow rate of 1.0 ml min−1 and a reactioncoil of 129 cm were used.

3.3. Analytical figures

Pre-concentration factors for Sb(III) of 2.5, 5.3 and 8.7 wereobtained for loading times of 120, 360 and 720 s, respectively. Thepre-concentration factor was calculated by ratioing the slope of theanalytical curve obtainedwith pre-concentrationwith that obtainedby direct hydride generation (without pre-concentration).

Detection limits (3σ) of 0.5 and 0.8 μg L−1 were obtained forSb(III) and total Sb, respectively, using the analytical sequenceshown in Table 1. The process (including determination of totalSb and selective determination of Sb(III)) was characterized bya sample throughput of 8 to 9 samples per hour. The detectionlimit of Sb(III) was improved to 0.2 and 0.15 by increasing thesample loading time to 360 and 720 s, respectively, but thiscondition resulted in a lower sample throughput.

The linearity of the proposed system for selective determi-nation of Sb(III) was tested by using standard solutions at 0, 30,50, 100, 200, 400 and 1000 μg L−1. The analytical signals werecharacterized by a linear response (y=176+60.2 x, R2 =0.9996)up to 400 μgL−1. For the 1000 μg L−1 solution, the sensitivity(angular coefficient of the curve) decreased to 42 CPS per μgL− 1, possibly due to losses of Sb(III) in the retention step orsignal suppression in generation of the hydride or both.

Throughout this study two columns filled with materialimmediately after the immobilization procedure were used.Whennot in use the columns were stored in a refrigerator (4 °C). Thefirst column maintained its properties for 160 pre-concentrationcycles (5 months) and after 7 months there was a deterioration inits performance, probably due to alteration of its biomass

characteristics (shown by the appearance of black specks on thematerial). Therefore, a second column was made with a freshimmobilized mass, having an efficiency identical to the first.

3.4. Speciation analysis of Sb(III) and Sb(V)

Table 4 presents results of the analyses of the water andeffluent samples. Analytical curves plotted with single standardsolutions of Sb(III), at pH=9.0, were used for determination oftotal Sb and Sb(III) in the samples. The concentration of Sb(V)was calculated by subtracting the concentration of Sb(III) fromthe total Sb concentration. Thus, in this approach, Sb(III) can bedetected in the samples at a concentration of 0.2 μg L−1 when asample loading time of 360 s is used, while the detection limit ofSb(V) depends on the detection limits of total Sb (0.8 μg L−1).The total Sb concentrations obtained showed excellentagreement with the reference values. For Sb(V) in the NISTSRM 1640 the measured value (12.3±0.3 μg L−1) wascomparable to the value previously reported by Miravet et al.(13.1±0.3 μg L−1, n=3) [23], demonstrating the accuracy ofthe determination of this species.

Recovery data for Sb(III), Sb(V) and total Sb in syntheticsamples (mixed standard solution, pH=9) and spiked samplesare presented in Table 5. Recoveries of total Sb, Sb(V) and Sb(III) were found to be higher than 80%, which confirms thereliability of the proposed approach for redox speciationanalysis of Sb. Miravet et al. [23] also undertook recoverytests for Sb(V) and Sb(III) for NIST-SRM-1640 to evaluate aseparation method for Sb species based on ion chromatography-hydride generation atomic fluorescence spectrometry andreported similar values (88 and 95% for spikes of 5 μg L−1)to those of our proposed method (94 to 107%).

4. Conclusion

The results obtained in this work demonstrate that baker'syeast immobilized on polyurethane foam can be used as aselective sorbent material to retain Sb(III) in the presence of Sb(V). Both the support and the immobilized microorganism arecheap and easy to obtain. The proposed immobilizationprocedure avoids chemical hazard and is extremely simplecompared to other processes. In the proposed method forspeciation analysis of Sb(III) and Sb(V) the pre-reduction stepsand selective solid–liquid phase extraction of Sb are performedon-line and almost simultaneously, minimizing error due tosample preparation and manipulation. Owing to the pre-concentration step of Sb(III), this analyte may be determinedin very low concentration compared to a direct determinationusing hydride generation. The generation of stibine isperformed at a high concentration of HCl and in the presenceof thiourea, providing suitable conditions to control interfer-ences from transition metals.

Acknowledgement

The authors thank the Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP) for financial support.

Table 5Recovery of Sb(III) and Sb(V) in mixed standard solution and spiked realsamples

Spike, μg L−1 Recoverya, %

Sample Sb(III) Sb(V) Sbtotal Sb(III) Sb(V) Sbtotal

Mixed solution-1 30 30 60 99±9 97±11 98±3Mixed solution-2 20 40 60 113±9 94±4 100±1Mixed solution-3 40 20 60 100±11 97±25 99±7River water-1 FHD 30 30 60 90±6 99±15 97±4River water-1C FHD 30 30 60 97±7 101±16 96±4River water-SRM-NIST-1640

10 10 20 94±6 107±7 101±7

Effluent 2 2 4 81±19 111±15 97±8

a. Mean±SD, n=3.

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