Ultrasound-assisted dispersive micro solid-phase extraction with nano-TiO2 as adsorbent for the determination of mercury species

Author’s Accepted Manuscript Ultrasound-assisted dispersive micro solid-phase extraction with nano-TiO2 as adsorbent for the determination of mercury species Magdalena Krawczyk, Ewa Stanisz www.elsevier.com/locate/talanta

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S0039-9140(16)30656-7 http://dx.doi.org/10.1016/j.talanta.2016.08.071 TAL16835

To appear in: Talanta Received date: 26 April 2016 Revised date: 18 August 2016 Accepted date: 28 August 2016 Cite this article as: Magdalena Krawczyk and Ewa Stanisz, Ultrasound-assisted dispersive micro solid-phase extraction with nano-TiO2 as adsorbent for the determination of mercury species, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.08.071 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ultrasound-assisted dispersive micro solid-phase extraction with nano-TiO2 as adsorbent for the determination of mercury species Magdalena Krawczyk, Ewa Stanisz* Faculty of Chemical Technology, Poznan University of Technology, Berdychowo 4, 60-965 Poznań, Poland *

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ABSTRACT The combination of ultrasound-assisted dispersive micro solid-phase extraction (USA DMSPE), with the use of TiO2 nanoparticles (NPs) as adsorbent, with cold vapour atomic absorption spectrometry (CV AAS) is proposed for preconcentration and determination of mercury species (Hgtotal, Hg2+ and CH3Hg+) in biological, geological and water samples. The experimental parameters including the amount of TiO2 NPs, pH of sample solution, ultrasonication and centrifugation time, TiO2 slurry solution preparation before injection to CV AAS were investigated. Effective preconcentration of trace mercury with 10 mg of TiO2 was achieved in pH 7.5. After extraction, adsorbent with analytes was mixed with 500 μL of 1 mol L-1 HNO3 to prepare slurry solution. The concentration limit of detection was 0.004 ng mL-1 for Hg2+. The achieved preconcentration factor was 35. The relative standard deviations (RSDs, %) for mercury species in real samples were 4-20%. The accuracy of this method was evaluated by analyses of certified reference materials: DOLT-2 (Dogfish Liver), IAEA-085 (Human hair), SRM 2709 (San Joaquin Soil), SRM 2711 (Montana Soil) and SRM 2704 (Buffalo River Sediment). The measured mercury species contents in reference materials were in satisfactory agreement with the added amounts (according to the t-test for a 95% confidence level). The presented method has been successfully applied for the determination of mercury species in real water samples (lake and river water).

graphical abstract In this study the analytical potential of USA DMSPE with nano-TiO2 as adsorbent combined with cold vapour AAS in the determination of mercury species was evaluated.

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Keywords: TiO2; Dispersive micro solid-phase extraction; Ultrasound; Mercury speciation; CV AAS.

1. Introduction The current trends in analytical chemistry are focused on a search for new approaches that make sample preparation procedures faster, easier, safer, and less expensive, to provide precise results with low limits of detection [1]. Among extraction procedures solid-phase extraction (SPE) has become one of the most frequently studied technique. The technique has been developed to replace traditional liquid-liquid extraction methods for the separation and/or preconcentration of trace metals in aqueous samples. SPE is a highly effective, ecologically-safe method, which has been developed intensively in recent years [1]. Nowadays, in the solid-phase extraction studies a search for new adsorbents is an important issue. In this context, the use of some nanomaterials is a milestone in this evolution [2]. Nanoparticles (NPs) are especially attractive due to the special chemical, electric, optical, thermal and even magnetic properties as well as adsorption capacity. Some of these characteristics have been successfully utilised in sample preparation [3] and separation [4,5] procedures, especially in solid-phase microextraction [6-8]. TiO2 nanoparticles are well-known and widely used for photoelectrochemical and photocatalytic processes [9-11]. Recently, TiO2 has been used as a solid sorbent due to its physical and chemical properties, low cost, non-toxicity, high specific surface area and high adsorption capacity [12]. The material has been reported as an adsorbent for various metals extraction and determination. TiO2 was used in column (or microcolumn) solid-phase extraction for determination of Cr3+ and Cr6+ [13-15]; Au, Ag and Pt [15]; Co, Cd, Cr, Cu, Mn, Ni, V, Ce, Dy, Eu, La and Yb [16]; La, Y, Yb, Eu and Dy [17]; As3+ and As5+ [18]; Au [19]; Cu, Mn, Cr and Ni [20] and Mo [21]. In some cases to facilitate adsorption of the analytes the sorbent is stirred for 10-15 minutes usually with the use of magnetic stirrer or ultrasound bath and used in dynamic conditions as a suspension. The adsorption under dynamic conditions with the use of

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TiO2 was carried out for extraction and determination of species of As, Se, and Sb [22-24]; As3+ and As5+ [25] as well as Tm, Sm, Ho and Nd [26]. The widespread use of solid-phase microextraction techniques is indisputable but the complete acceptance of the new developments in this area depends on their successful integration with conventional analytical instrumentation, simplification and shortening the procedure duration. Extraction techniques are surface-dependent processes. Usually the potential extraction area in solid-phase procedures is not fully exploited since the adsorbent tends to aggregate [2]. This phenomenon becomes critical when the amount of adsorbent, and consequently the overall extraction capacity, is reduced to the microscale [2]. The use of sorbents under dispersion conditions, that overcomes these shortcomings, was proposed for the first time by Anastassiades et al. [27] in organic analysis. Later, dispersion has been successfully applied in the dispersive solid-phase extraction (DSPE) [2,27-30] and dispersive liquid-liquid microextraction (DLLME) techniques [2,31]. Since then, an increased interest in the field of dispersive-based procedures has been presented [2,30]. In dispersive micro solid-phase extraction (DMSPE) [2,30,32] or unpacked modes [33], the solid sorbent in the mg or µg range is dispersed in the sample solution. This procedure enables more efficient contact between the analytes and sorbent, and shortens the time of sample preparation [2,27-30,32]. In previous studies DMSPE procedures using multiwalled carbon nanotubes (MWCNTs) [34] and silver nanoparticles [35] for the isolation and preconcentartion of Cd and Pb and mercury were proposed. In this paper the combination of ultrasound-assisted DMSPE (USA DMSPE) with TiO2 as adsorbent and cold vapour atomic absorption spectrometry (CV AAS) is presented. To the best of our knowledge, a procedure combining these approaches has not been reported yet. In the method commercially available nanoparticles were used as sorbent material to extract mercury species from sample solutions. Direct addition of adsorbent to the sample solution provided rapid adsorption onto the nanomaterial under short ultrasonication time. After extraction the nanoparticles were separated from the sample solution and injected as slurry solution to CV AAS. Injection of slurry samples containing adsorbent and the analytes allowed to avoid backextraction step that reduced the analysis time and the amount of reagents used. The accuracy of the method was demonstrated by analysis of certified reference materials and determination of total mercury, inorganic mercury and organic mercury (as methylmercury). 3

Finally the developed procedure was applied for extraction and determination of mercury species in water samples.

2. Materials and methods 2.1. Instrumentation The analyses were carried out with a mercury analyser (Model Aula-254, Mercury Instruments, GmbH, Karlsfeld, Germany). A mercury electrodeless low pressure discharge lamp was used as the radiation source. A thermoelectric gas dehumidifier and heating of the optical cell was used for elimination of the moisture and prevention if interferences from water vapour. The operating parameters of the CV AAS instrument during mercury determination after TiO2 USA DMSPE are summarized in Table 1. For SPE procedure TiO2 nanoparticles were weighed using an M2P microanalytical balance (Sartorius, Gottingen, Germany) with a resolution of 1 μg (electronic weighing range up to 2 g). The pH values were measured with a pH-meter (pH 211 Microprocessor, Hanna Instruments, Kehl, Germany) supplied with a glass-combined electrode. A Sonopuls HD 70 ultrasonic cell disruptor/homogenizer (70 W, 20 kHz, Bandelin, Germany) equipped with a 2-mm titanium microtip was used for dispersive extraction processes as well as for slurry preparation before CV AAS detection. A 3-mm titanium microtip was used for ultrasonic solubilization procedure (in TMAH) for mercury species determination. Ultrasonic energy was fixed at any desired level using a power setting in the range of 10-65 W for both titanium microtips. A UniClever focused microwave sample preparation system (Plazmatronika, Wrocław, Poland) operating at 2450 MHz and 300 W maximum output was used for biological certified reference material digestion before total mercury determination [9,36]. The computer-controlled system with continuous temperature, pressure and microwave power monitoring was equipped with high-pressure TFM-PTFE vessel and water cooling system. The vessel capacity was 110 mL and the maximum pressure and maximum temperature were 100 atm and 300 oC, respectively.

2.2. Reagents and solutions

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Compressed argon of UHP 5.5 purity obtained from Air Products (Warsaw, Poland) was employed as a carrier gas without further purification. Standard solutions of inorganic mercury Hg2+ were prepared from 1000 mg L-1 mercury standard solution traceable to SRM from NIST (Certipur, Merck) stabilizing with potassium dichromate (5% m/v, GR, Merck). Standard solutions of methylmercury - CH3Hg+ were prepared from 1000 mg L-1 methylmercury(II) chloride, AA standard solution in H2O (Alfa Aesar, Germany). The stock solutions were stored at 4 oC prior to use. All working standard solutions were prepared daily to prevent any possible species change, the appropriate stock solution were diluted with high-purity water. Titanium dioxide nanopowder TiO2 (≥ 99.5%, P25 Aeroxide, Degussa, Germany) with spherical particles (10-30 nm) was used as adsorbent in dispersive micro solid-phase extraction. Its average particle size was approximately 21 nm and specific surface according to BET 50 ± 15 m2 g-1 with an anatase:rutile ratio of about 80:20. The pH of the sample solutions was adjusted with 65% HNO3 and 30% NaOH (Suprapur, Merck). For microwave-assisted digestion 65% HNO3, 40% HF and 30% H2O2 (Suprapur, Merck) were used. A solution of ca. 25% tetramethylammonium hydroxide (TMAH) (Fluka) in water was used for ultrasonic solubilization. High-purity water: deionized and doubly distilled water (quartz apparatus, Bi18, Heraeus, Germany) was used throughout the experiments. For chemical cold vapour generation (2% m/v) stannous chloride was used as a reducing agent. It was prepared by dissolving the appropriate mass of tin(II) chloride dihydrate (max. 0.000001% Hg, Emsure, Merck) in 2 mol L-1 hydrochloric acid (32% extra pure, Merck) in high-purity water. Additionally, a rinsing solution NH4OCl (0.1% (m/v)) was used.

2.3. Standard reference materials and real samples Accuracy of the analytical procedure described in this work was verified using standard reference materials: DOLT-2 (Dogfish Liver) from the National Research Council of Canada (NRCC); IAEA-085 (Human hair) from International Atomic Energy Agency; SRM 2709 (San Joaquin Soil), SRM 2711 (Montana Soil) and SRM 2704 (Buffalo River Sediment) from the National Institute of Standards and Technology (NIST). In DOLT-2 and IAEA-085 the reference values were available for mercury, inorganic mercury and methylmercury. The rest of the SRMs have certified values of the total mercury. The solid samples were analyzed after microwave digestion (total mercury) or after alkaline solubilization (speciation analysis). 5

The lake water samples (the Stęszew Lake, 20 km northeast of Poznań, Poland) and river water samples (from Warta, a river in western-central Poland, flowing through Poznań) were collected in 500 mL Schott Duran glass bottles and immediately after sampling, the 500 mL aliquots were acidified with HNO3 (0.5 mol L-1) and stored in the dark at 4 °C. The samples were filtrated before analysis using Cameo syringe filter with polytetrafluoroethylene membrane and pore size about 0.22 mm (GE Water & Process Technologies, USA). The samples were spiked with inorganic mercury and methylmercury before analysis. The samples were used after spiking directly for speciation analysis, and after microwave digestion, for total mercury determination.

2.4. Analytical procedures

2.4.1. Microwave-assisted digestion for total mercury determination The microwave-assisted digestion for total mercury determination was described in previous work [36] and is therefore briefly summarized here. Approximately 350 mg (tissue, hair sediment, soli) of powdered reference material was placed in the TFM-PTFE vessel of the microwave digestion system and moistened by 1 mL of 30% H2O2. Then, 5 mL of 65% HNO3 (for tissue and hair) was added. In the case of sediment and soil samples 4 mL of 65% HNO3 and 1 or 2 mL of 40% HF (for sediment or soil respectively) were used. The samples were heated for 20 min at 300 W. The water samples (5 mL) were heated for 10 min at 300 W with 1 mL of 30% H2O2 and 1 mL of 65% HNO3. After procedure, the clear digested solution was transferred into 10 mL calibrated flask and diluted to volume with high-purity water. Before further analysis this solution was appropriately diluted depending on the concentration level of the analyte. In all cases, a corresponding blank was also prepared according to the above microwave-assisted digestion procedure. After this procedure it was possible to determine the concentration of total mercury presents in a digested sample.

2.4.2. Ultrasonic solubilization procedure for mercury species determination The ultrasonic solubilization procedure for mercury species determination was described in previous work [36] and is therefore briefly summarized here. Nominal 350 mg of reference material was weighed into 30 mL pre-cleaned polypropylene screw-cupped cups and 5 mL of ca. 6

25% TMAH added. Following the reaction of the sample with TMAH for 5 min, 15 mL of water was added. Suspensions were pretreated by ultrasonication at 50 W ultrasonic power for 5 min. The final concentration of TMAH in sample solution was 6.25%. The ultrasonication was performed in continuous mode with a 3-mm diameter titanium probe immersed into the sample solution. A procedural blank was prepared along with the samples for quality assurance purposes. Before further analysis this solution was appropriately diluted depending on the concentration level of the mercury species. After this procedure it was possible to obtain both forms of mercury (Hg2+ and CH3Hg+) in the sample solution and their subsequent extraction and determination. 2.4.3. Preconcentration and AAS determination procedures Ten milliliters of a sample solution was poured into a centrifuge tube. The pH of the sample was adjusted to 7.50. Then, 10 mg of TiO2 (as 100 μL of 10% m/v suspension) was added. The suspension (4 mL containing 400 mg of TiO2) was prepared before analysis with the use of ultrasound (2-mm titanium microtip, 40 W, 20 kHz, for 40 s). Subsequently, the sample solution with nanomaterial was ultrasonicated for 5 s. Homogenization was achieved, that promoted the interaction between mercury and the nanoparticles. After that the mixture was centrifuged for 5 min at 4500 rpm. The mercury adsorbed on TiO2 settled on a bottom of the tube. A water phase was removed and solid phase was mixed with 500 μL of 1 mol L-1 HNO3 and treated with ultrasound for 5 s (ultrasonic probe; 2-mm titanium microtip, 40 W, 20 kHz) to achieve slurry solution. In order to determine the analyte in the TiO2 slurry solution, 400 μL of the solution was injected for CV AAS determination under the optimized conditions. Calibration was performed by the standard addition technique. Optimized experimental conditions for ultrasound-assisted dispersive micro solid-phase extraction with TiO2 as adsorbent coupled to CV AAS for mercury species determination are presented at Table 1.

3. Results and Discussion

3.1 Optimization of TiO2 USA DMSPE conditions The parameters that affect the extraction efficiency, including the sample volume, the amount of TiO2 (solid sorbent), the sample pH, homogenization (ultrasonication) and centrifugation time as well as slurry preparation step before injection to CV AAS, were 7

investigated. Initial conditions with an analytes concentration of 2 ng Hg2+ mL-1 and 1 ng CH3Hg+ mL-1 were used. Regarding the amount of applied TiO2, 4 mL of a suspension containing 400 mg was prepared. 100 μL of the suspension added to the sample solution corresponded to 10 mg of TiO2 per sample. All the experiments were performed in triplicate (n=3).

3.1.1. Sample volume The first parameter that can affect the extraction efficiency is volume of the aqueous sample. The effect of sample volume on the sorption of analyte on the TiO2 nanoparticles was studied using three different volumes (2.5 mL, 5 mL and 10 mL) of an aqueous standard solution containing 2 ng of Hg2+. The recoveries obtained for three different volumes of sample did not differ more than 4%. Due to the construction of the centrifuge and vials it was not possible to apply higher sample volume. Considering the convenience of operation as well as an obtained enrichment factor a sample volume of 10 mL was used in the course of the study.

3.1.2. Particles amount To achieve an optimum amount of TiO2 necessary for quantitative enrichment of mercury, the introduced amount of nanoparticles was varied from 2.5 to 20 mg per sample (10 mL). It was found that effective adsorption of Hg2+ (the highest analytical signal) was achieved with amounts of nanomaterial from 10 mg (Fig. 1.). For further application, the amount of TiO2 equal to10 mg was used (that corresponding to 100 µL of 10% TiO2 slurry solution added to 10 mL of the sample). TiO2 nanoparticles with 80% of anatase were chosen due to the possible mechanism of interaction with analyte and possibility of effective adsorption connected with high specific surface area. The adsorbent is known for its physical and chemical stability in acidic and alkaline solutions, low cost, nontoxicity and fast rate of adsorption and desorption [12,37]. Additionally anatase crystalline form of TiO2 is used as material with a high photocatalytic activity due to its strong oxidizing power and favorable band gap energy [38-40]. In literature two types of hydroxyl groups are described to exist on the surface of the adsorbent [16,41,42]. Terminal hydroxyl groups are assumed to be bound to one Ti4+ site and the bridged OH are bound to two Ti4+ sites. Since the bridged (OH) should be strongly polarized by the cations, it is expected to be acidic in character, whereas the terminal (OH) could be 8

predominantly basic. Both of them may be involved in the adsorptive process of metals analytes from aqueous samples [16,41,42].

3.1.3. Sample pH The pH value in the aqueous solutions plays an important role in the adsorption of mercury on nano-TiO2.The effect of the sample pH was investigated in the range of 2-12. The modification of pH was carried out by adding the appropriate amounts of NaOH or HNO3. Fig. 2 shows the pH dependence on Hg2+ adsorption with the use of nano-TiO2. The adsorption of analyte (absorbance values) on adsorbent increased gradually in the pH range of 2.0-7.5. For pH between 7.5 and 12 absorbance is not statistically different. Since the analytical signals reached maximum at pH 7.5, this value was chosen for further experiments. TiO2 used in the course of the study consists of 80% of anatase. The binding mechanisms of mercury to the nanoparticles is mainly depended on the type of active sites on the surface of adsorbent and the concentrations of the produced sorbate species at measured pH value [38]. The point of zero charge (PZC) for the anatase nanoparticles is reported in the range of 5.9 -6.9 [12]. When pH is below this value the anatase nanoparticles are composed of the mixture of positively charged TiOH2+ and neutral species of TiOH0. Above PZC (basic pH) the main species of the nanoparticles include the neutral species of TiOH0 and the negatively charged TiO- [25,43]. At pH 7.5 the surface of adsorbent is mostly covered with the neutral TiOH0 and negatively charged TiO- [38]. At pH below 4 main form of mercury is Hg 2+; in the pH range of 2-6 exist Hg2+, HgOH+ and Hg(OH)20. At high pH levels (e.g., 6-12), mainly the hydrolysis of Hg2+ ions in the form of Hg(OH)20 takes place [44,45]. Thus, the effect of sample pH on mercury adsorption can be explained by considering that the compound adsorbed is a non-charged species such as Hg(OH2) 0 and its adsorption is not altered by the surface charge [46].

3.1.4. Ultrasonication and centrifugation time In the USA DMSPE procedure mass-transfer takes place during the ultrasound-assisted dispersing step. Even a short ultrasonication time enables disaggregation the large agglomerates,

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and reduction of the particles size. Therefore an increase in the area-to-volume ratio can be achieved [2]. For mercury species determination, ultrasonication time should be chosen to make the solid phase well dispersed into the aqueous solution. However, too long time may cause increase temperature of the solution and loss of the analyte [35]. During the ultrasonication step an ultrasonic probe (2-mm diameter) was immersed into the sample solution (10 mL) containing mercury and TiO2 nanoparticles (as adsorbent). The ultrasonication was performed at 40 W ultrasonic power in continuous mode. The solution became milky due to the dispersion of nanomaterial into the aqueous phase. The effect of ultrasonication time was evaluated in the range of 3-30 s and the results showed that the absorbance increased in the first 5 seconds and then after exceeding this value analytical signals remained approximately constant (Fig. 3). Hence, the time of ultrasonication step equal to 5 s was chosen for further study. During the study the absorbance was also investigated as a function of centrifugation time (at the rate of 4500 rpm) (Fig. 3.). The experiments were performed in a range of 1-10 min. With increasing time the absorbance increased rapidly (volume of sedimented phase increased). Over 5 min the absorbance was constant, indicating settling of solid phase to the bottom of the centrifuge tube. Thus, 5 min was selected as the final centrifugation time (at the rate of 4500 rpm).

3.1.5. TiO2 slurry solution preparation In the course of the study the injection of slurry samples for CV AAS analysis was performed. Therefore, it was necessary to prepare the TiO2 slurry (containing adsorbed analyte) after extraction step prior to determination. For this purpose, in order to stabilize the analyte during the ultrasonic slurry preparation, HNO3 at a concentration of 1 mol L-1 was chosen. During the study a volume range of 250-1000 μL was checked. Slurry volume of less than 250 μL was too small to obtain satisfactory precision (expressed as RSD, below 10%). On the other hand, unnecessary dilution of the samples causing decrease of analytical signals was observed for volumes above 500 μL. Therefore, in this study 500 μL of 1 mol L-1 HNO3 was chosen as solution for slurry preparation before CV AAS analysis. After centrifugation and removing water phase, the solid phase was mixed with 500 μL of 1 mol L-1 HNO3 and treated with ultrasound for 5 s (2-mm titanium microtip, 40 W, 20 kHz) to

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achieve stable slurry solution. In order to determine the analyte in the TiO2 slurry solution, 400 μL of the solution was injected to CV AAS detector.

3.1.6. Methylmercury extraction conditions As it was in the case of Hg2+, for methylmercury (initial concentration of analyte: 1 ng CH3Hg+ mL-1) the same parameters affected the extraction efficiency have been studied. The analytical signals were no obtained at pH 7.5 suggesting that CH3Hg+ cannot be extracted using TiO2. Also increase the concentration of the analyte above the commonly found in environmental samples, has not resulted in increase of analytical signals of methylmercury. In accordance with the results obtained, it was possible to conduct indirect determination of organic mercury using the proposed procedure: USA DMSPE (section 3.1.7.).

3.1.7. Indirect determination of methylmercury The methylmercury concentration (CH3Hg+) was calculated as the difference between total (Hg) and inorganic mercury (Hg2+) from the equation [36]: Hgtotal – Hginorganic = Hgorganic (Hg – Hg2+ = CH3Hg+ ) where Hgtotal and Hginorganic are the quantities of mercury. The total mercury determination was conducted after microwave-assisted digestion of the samples (section 2.4.1.). After ultrasonic solubilization procedure (section 2.4.2.) both forms of mercury (Hg2+ and CH3Hg+) were obtain in the sample solution.

3.2. CV AAS conditions To achieve slurry solution that was injected to CV AAS, the solid phase with adsorbed analytes was mixed with 500 μL of 1 mol L-1 HNO3 and treated with ultrasound. In order to determine the mercury in the TiO2 slurry solution, 400 μL of the solution was injected for the determination (sections 2.4.3. and 3.1.5.). In the course of the study the CV AAS analysis was conducted according to the manufacturer's recommendations. Mercury determination was performed by the reduction of mercury (Hg2+) to Hg0 with 2% (w/v) SnCl2 as a reducing agent and detected by AAS. After extraction, TiO2 slurry solution (400 μL) was placed in the reaction flask for cold vapour generation. Mercury vapour was generated in 2 mol L-1 HCl medium. The Hg0 was transferred to the quartz cell by an argon stream (70 mL min−1). A rinsing solution NH4OCl (0.1% (m/v)) was 11

used. The absorbance signals were integrated and peak height absorbance was recorded and used for calculations. Analytical blank was also carried through the whole procedure to correct possible contamination from the reagents used for the sample preparation. Not significant increase of background signals were observed due to matrix effects for the slurry solution. Quantification of mercury was made with the use of standard addition technique.

3.3. Analytical figures of merit The analytical characteristics of the proposed USA DMSPE CV AAS method were investigated under the optimized experimental conditions (Table 1). The relative standard deviation (RSD) for seven replicate measurements of 2 ng mL−1 in standard solution was 6%. The detection limit (LOD), calculated as the concentration of the analyte yielding a signal equivalent to three times the standard deviation of the blank value (n=5) for 10 mL of real sample solution was 0.004 ng mL−1 for Hg2+. The limit of quantification (LOQ) calculated as the concentration of the analyte yielding a signal equivalent to ten times the standard deviation of the blank value (n=5) for 10 mL of real sample solution was 0.013 ng mL−1 for Hg2+. The calibration was performed under the optimized conditions using standard addition technique for samples after USA DMSPE extraction. An acceptable correlation coefficient was found 0.9986. Preconcentration factor was calculated using the ratio of the analyte concentration in the dissolved solid phase (C1) to the initial concentration of analyte (C0) within the sample solution: PF = C1/C0 [47] and was obtained as high as 35.

3.4. Accuracy verification and mercury species determination in real samples The accuracy of the proposed method was evaluated by analyzing four standard reference materials with different matrices: DOLT-2 (Dogfish Liver), IAEA-085 (Human hair), SRM 2709 (San Joaquin Soil), SRM 2711 (Montana Soil) and SRM 2704 (Buffalo River Sediment). In DOLT-2 and IAEA-085 the reference values were available for total mercury and methylmercury. The rest of the SRMs have certified values of the total mercury. In the case of speciation analysis the mercury species recoveries of certificate values were 119% and 106% for CH3Hg+, 94% and 109% for Hg2+, 103% and 106% for total Hg for DOLT-2 and IAEA-085, respectively. Recoveries for the rest of the SRMs were in the range of 101-102% for total mercury. Obtained results are considered satisfactory for the low concentration levels of 12

mercury species (Table 2). The precision expressed as relative standard deviation (RSD, %) was the highest for methylmercury determination (6-20%) that may be connected with the specificity of indirect analysis for this form of the analyte. Obtained RSDs for total Hg and Hg2+ were in the range of 4-11%. The certified reference materials used in the research are characterized by a complex matrix containing high concentrations of other elements (e.g. Al, Ca, Cd, Cr, Cu, Fe, Mg, Mn, Na, Ni, Pb, Zn) in the range of mg kg-1, or even % (for soil and sediment matrices). The obtained recoveries of mercury species, in the presence of the elements, proved that the interferences from foreign ions can be ignored. Due to the satisfactory results, the additional study in the terms of matrix influence was not carried out. Obtained results show that the proposed method can be applied for the preconcentration and determination of mercury species in biological and environmental samples with complex matrices.

3.5. Mercury species determination in real samples

The applicability of the developed method was assessed by preconcentration and determination of mercury species in two real samples: lake and river water. The real samples were acidified with HNO3 (0.5 mol L-1) and stored in the dark at 4 oC. The

samples

were

filtrated

before

analysis

using

Cameo

syringe

filter

with

polytetrafluoroethylene membrane and pore size about 0.22 mm (GE Water & Process Technologies, USA). Trace mercury in samples was preconcentrated and determinate by using procedures described in Section 2.4. However, the mercury species were not detected according to the optimized procedure, indicating that their concentrations were too low in the real water samples. This may be due to the fact that in uncontaminated water mercury naturally occurs at very low levels. Mercury concentrations in contaminated waters can be in the μg L-1 range depending on season and suspended solids content [48]. According to Polish legislation acceptable mercury concentration in the drinking water is 0.001 mg L-1. United States Environmental Protection Agency gives the value of this parameter as 2 µg L-1. Both values refer to the total concentration of mercury. In order to evaluate applicability of proposed method, water samples were spiked with 2 ng mL-1 of Hg2+ or 1 ng mL-1 of CH3Hg+ (for species determination) or with the sum of two forms (for total mercury determination) and were analyzed using the same procedures (Section 13

2.4.). The analytical data are reported in Table 3. It can be seen that the recoveries for the mercury species in these samples are in the range of 97-118%. Therefore, the matrix effect from real water samples and differences between added and detected values were not significant.

3.6. Comparison with other methods A comparison of the present work with some of the alternative methods for extraction and determination of mercury is summarized in Table 4. It is obvious that direct comparison of detection limits is often misleading owing to the use of different analytical techniques, chemical vapour generation systems or sorbents. However, it is known that detection limits that can be achieved using CV AAS after preconcentration on nano-TiO2 are better than those obtained for conventional CV AAS. An on-line procedure for the determination of total mercury in environmental and biological samples was described by Ferrúa et al. [49]. This methodology combines cold vapour generation associated to atomic absorption spectrometry (CV AAS) with preconcentration of the analyte on a minicolumn packed with activated carbon. After preconcentration procedure the analyte was quantitatively eluted from the minicolumn with nitric acid. Solid-phase preconcentration using (tri-octylmethylammonium chloride)-activated carbon as adsorbent and determination of Hg2+ in drinking water by X-ray fluorescence spectrometry was proposed by Aranda et al. [50]. El-Sheikh et al. [51] reports for the first time the use of oxidized and non-oxidized multiwalled carbon nanotubes of different geometrical dimensions for Hg2+ adsorption and preconcentration in water samples. Mercury was determined using cold vapour atomic absorption spectrometry.

Preconcentration of Hg2+ from natural water and milk sample by histidine

functionalized multiwalled carbon nanotubes and its determination using cold vapour atomic absorption spectrometry was described by Moghimi et al. [52]. Selective extraction and preconcentration of ultra-trace level of mercury ions in water and fish samples using Fe3O4-magnetite-nanoparticles functionalized with triazene groups prior to its determination by inductively coupled plasma optical emission spectrometry was described by Rofouei et al. [53]. The integration of cell sample introduction and magnetic nanoparticle solid

phase microextraction (MSPME) on a microfluidic chip, combined with electrothermal vaporization-inductively coupled plasma mass spectrometry (ETV ICP MS) detection of Cd, Pb, 14

and Hg was proposed by Chen et al. [54]. Ma et al. [55] developed method of magnetic solid phase extraction combined with inductively coupled plasma mass spectrometry for the speciation of mercury in environmental water and human hair samples. The authors successfully prepared and used magnetic nanoparticles Fe3O4@SiO2 modified with γ-mercaptopropyltrimethoxysilane. The sorption performance of the prepared Fe3O4@SiO2@γ-MPTS nanoparticles towards CH3Hg+ and Hg2+ was investigated. It was found that CH3Hg+ and Hg2+ could be simultaneously retained on the Fe3O4@SiO2@γ-MPTS MNPs, and the quantitative elution of CH3Hg+ and total mercury was achieved by using 1.5 molL-1 HCl containing 0.01% and 3% thiourea, respectively. The levels of Hg2+ were obtained by subtracting CH3Hg+ from total mercury. Hg2+ and CH3Hg+ at the sub-ppb level were adsorbed quantitatively from aqueous solution on a column packed with dithizone immobilized on sodium dodecyl sulfate coated alumina [56]. The trapped mercury was then back-extracted into the aqueous phase with 1 mol L-1 hydrobromic acid and determined by cold vapour atomic absorption spectrometry. The proposed analytical procedure was applied for determination of Hg2+ and CH3Hg+ in river and tap water. Fe3O4 magnetic nanoparticles were used in mercury speciation analysis by high performance liquid chromatography-cold vapour generation atomic fluorescence spectrometry [57]. Inorganic mercury (Hg2+), methylmercury, ethylmercury and phenylmercury were selected as model compounds to validate the methodology. Separation of these mercury species was accomplished on a RP-C18 column with a mixture of acetonitrile and water (10:90) at pH 6.8 containing 0.12% (m/v) L-cysteine as the mobile phase. Acknowledgements This work was supported by the 03/31/DSPB/0316 grant from Polish Ministry of Science and Higher Education.

15

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[56] J.L. Manzoori, M.H. Sorouraddin, A.M. Haji Shabani, Determination of mercury by cold vapour atomic absorption spectrometry after preconcentration with dithizone immobilized on surfactant-coated alumina, J. Anal. At. Spectrom. 13 (1998) 305-308. [57] X. Ai, Y. Wang, X. Hou, L. Yang, C. Zheng, L. Wu, Advanced oxidation using Fe3O4 magnetic nanoparticles and its application in mercury speciation analysis by high performance liquid chromatography-cold vapor generation atomic fluorescence spectrometry, Analyst 138 (2013) 3494-3501.

Fig. 1. Effect of amount of TiO2 on the determination of Hg2+ with the use of USA DMSPE procedure. Conditions: pH=8.0, sample volume 10 mL, ultrasonication time 10 s, centrifugation 4 min. The error bar is the standard deviation (SD, n= 3). Fig. 2. Effect of sample pH on the determination of Hg2+ with the use of USA DMSPE procedure. Conditions: sample volume 10 mL, 10 mg of TiO2, ultrasonication time 10 s, centrifugation 4 min. The error bar is the standard deviation (SD, n= 3). Fig. 3. Effect of ultrasonication and centrifugation time on the determination of Hg2+ with the use of USA DMSPE procedure. Conditions: pH=7.0, sample volume 10 mL, 10 mg of TiO2, centrifugation 3 min or ultrasonication 5 s. The error bar is the standard deviation (SD, n= 3). Table 1 Optimized experimental conditions for ultrasound-assisted dispersive micro solid-phase extraction (USA DMSPE) with TiO2 as adsorbent coupled to CV AAS for mercury species determination (parameters for preparation of real samples and reference materials are also presented). Sample preparation Microwave-assisted digestion for total mercury determination (hair, tissue, soila, sedimentb)

350 mg sample, 1 mL 30% H2O2, 5 mL 65% HNO3, 20 min, 300 W.

Microwave-assisted digestion for total mercury determination (water samples after spiking)

5 mL sample, 1 mL 30% H2O2, 1 mL 65% HNO3, 10 min, 300 W.

Ultrasonic solubilization for mercury species determination (tissue, hair)

350 mg sample, 5 mL ca. 25% TMAH, 5 min, 50 W, 15 mL H2O. 21

Direct analysis (for mercury species water samples determination after spiking) USA DMSPE with TiO2 Sample volume (mL) 10 Amount of TiO2 (mg) 10 pH of sample solution 7.5 Ultrasonication time (s) 5 Centrifugation time (min) / rpm 5 / 4500 -1 Solution for TiO2 slurry / concentration (mol L ) HNO3 / 1 / 500 / final vol. (μL) Cold vapour generation SnCl2 concentration 2% HCl concentration 2 mol L-1 Sample volume (slurry solution) 400 μL Detection Aula-254 Mercury Instruments; mercury EDL, 10 mA; wavelength, 253.7 nm; AAS spectral bandpass, 0.4 nm; quartz cell temperature, 50 oC; measurement mode, peak height. a,b

1 mL 30% H2O2 + 4 mL 65% HNO3 + 1 or 2 mL 40% HF (for sediment or soil respectively).

Significanceg

Value of t-test

Table 2 Accuracy verification of the method for mercury species determination in selected standard reference materials by USA DMSPE with TiO2 coupled to CV AAS using the optimized parameters. Recovery and relative standard deviation (RSD) values also are shown. Obtained values: average value ± standard deviation (n=3). Determined Certified Reference material

Hg form

Hg (μg g-1)

DOLT-2

Hgtotala

2.05 ± 0.12

103

6

1.99 ± 0.10

0.866 NS

Hginorg.b

1.224 ± 0.109

94

9

1.297d ± 0.113

1.160 NS

CH3Hg+c 0.826 ± 0.162

119

20

0.693 ± 0.053

IAEA-085

Hgtotal

a

24.6 ± 1.5

Recovery RSD (%) (%)

106

6

Hg (μg g-1)

e

1.422 NS

22.4 - 24.0 / 23.2

f

1.617 NS 22

Hginorg.b

0.37 ± 0.04

109

11

0.3d

24.2 ± 1.5

106

6

21.9 - 23.9 / 22.9

1.501 NS

a

1.3 ± 0.07

93

5

1.4 ± 0.08

2.474 NS

CH3Hg

e

3.031 NS

+c

f

SRM 2709

Hgtotal

SRM 2711

Hgtotala

6.18 ± 0.25

99

4

6.25 ± 0.19

0.485 NS

SRM 2704

Hgtotala

1.54 ± 0.11

105

7

1.47 ± 0.07

1.102 NS

a

Determined after microwave-assisted digestion. Determined after ultrasonic solubilization. c Indirect analysis, Hgtotal – Hginorg. = CH3Hg+. d Calculated according certificate as difference between total Hg and organic Hg values. e 95% confidence interval, mg kg-1. f Recommended value, mg kg-1. g Significance of t-test (n=3) at 95% confidence level, tcritical=4.303; NS: not significant.

Real sample

Lake water

Hg form

Hg (ng mL-1)

Hgtotala

3.08 ± 0.15

103

5

3.00d

0.924 NS

2.10 ± 0.19

105

9

2.00

0.912 NS

CH3Hg+c

0.98 ± 0.13

98

13

1.00

0.266 NS

Hgtotala

3.11 ± 0.21

104

7

3.00d

0.907 NS

Hginorg.b

1.93 ± 0.09

97

5

2.00

1.347 NS

1.18 ± 0.23

118

19

1.00

1.355 NS

Hginorg. River water

b

+c

CH3Hg

Recovery RSD (%) (%)

Hg (ng mL-1)

Value of t-test

Table 3 Mercury species determination in selected water samples (after spiking) by USA DMSPE coupled to CV AAS using the optimized parameters. Recovery and relative standard deviation (RSD) values also are shown. Obtained values: average value ± standard deviation (n=3). Determined Added

Significancee

b

a

Determined after microwave-assisted digestion. Determined after ultrasonic solubilization. c Indirect analysis, Hgtotal – Hginorg. = CH3Hg+. d Calculated according added values as the sum of Hg inorganic and Hg organic. e Significance of t-test (n=3) at 95% confidence level, tcritical=4.303; NS: not significant. b

23

Table 4 Comparison of the proposed method with other reported in the literature.

Detection technique

LOD (ng L-1)

Ref.

CV AAS

10

49

XRF spectrometry

1

50

Solid-phase extraction with multiwalled carbon nanotubes

CV AAS

12.3a

51

Batch preconcentration procedure with histidine functionalized multiwalled carbon nanotubes

CV AAS

40000

52

Dispersible solid phase extraction with Fe3O4-magnetite-nanoparticles functionalized with triazene groups

ICP OES

40

53

Magnetic nanoparticle solid phase microextraction on a microfluidic chip

ETV ICP MS

0.86

54

Solid-phase extraction with magnetic nanoparticles Fe3O4@SiO2 modified with γ-mercaptopropyltrimethoxysilane

ICP MS

1.9

55

Preconcentration on a column packed with dithizone immobilized on sodium dodecyl sulfate coated alumina

CV AAS

44 (in river water) 28.1 (in tap water)

56

Preconcentration on a column packed with Fe3O4 magnetic nanoparticles

HPLC CV AFS

700

57

Ultrasound-assisted dispersive micro solid-phase extraction with nano-TiO2

Slurry sampling HR-CS ET AAS

4 13a

This work

Preconcentration technique Solid-phase extraction with activated carbon Solid-phase extraction with (trioctylmethylammonium chloride)activated carbon

a

Limit of quantification.

24

25

0.140

0.120

Absorbance

0.100

0.080

0.060

0.040

0.020

0.000 0

5

10 15 Amount of TiO2 (mg)

20

25

Fig. 1.

26

0.140

0.120

Absorbance

0.100

0.080

0.060

0.040

0.020

0.000 0

2

4

6 8 Sample pH

10

12

14

Fig. 2.

27

Ultrasonication time (s) 0

5

10

15

20

25

30

35

0.100

Absorbance

0.080

0.060

0.040

centrifugation time ultrasonication time

0.020

0.000 0

1

2

3

4

5

6

7

8

9

10

11

12

Centrifugation time (min)

Fig. 3.

Highlights 

A new solid-phase extraction procedure for Hg species determination was proposed.



Ultrasound-assisted dispersion of nano-TiO2 was used.



The effect of ultrasound on the extraction was studied.



No back-extraction was needed before CV AAS determination of Hg species.



An application to Hg species determination in complex matrices was presented.

28

29