Microwave-Assisted Sample Preparation for Trace Element Determination, 1st Edition PDF

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Chapter 10 Microwave-Assisted Sample Preparation for Element Speciation Joerg Feldmann1, Abdelkarem Elgazali1,2, Mohamed F. Ezzeldin1, Zuzana Gajdosechova1, Eva Krupp1, Fatai Aborode1, Mohamed M. Lawan1, Andrea Raab1, Asta H. Petursdottir1 and Kenneth Amayo1 1University of Aberdeen, Department of Ch...

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Chapter 10

Microwave-Assisted Sample Preparation for Element Speciation Joerg Feldmann1, Abdelkarem Elgazali1,2, Mohamed F. Ezzeldin1, Zuzana Gajdosechova1, Eva Krupp1, Fatai Aborode1, Mohamed M. Lawan1, Andrea Raab1, Asta H. Petursdottir1 and Kenneth Amayo1 1University

of Aberdeen, Department of Chemistry, TESLA (Trace Element Speciation Laboratory), Aberdeen, Scotland, UK 2University of Benghazi, Faculty of Arts & Science, Department of Chemistry, El-Marj Campus, El-Marj, Libya

10.1. INTRODUCTION 10.1.1. Chemical Speciation? Chemical speciation is defined according to IUPAC [1] as follows: (1) Chemical species: specific form of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure. (2) Speciation analysis: analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample. (3) Speciation of an element: distribution of an element among defined chemical species in a system. In contrast to this, “fractionation analysis” is clearly different from what we understand as speciation and speciation analysis. This way of describing a sample/ analyte is defined by IUPAC [1] as follows. Fractionation is the process of classification of an analyte or a group of analytes from a certain sample according to physical (e.g., size and solubility) or chemical (e.g., bonding and reactivity) properties. These definitions allow to clarifying the differences between the terms “speciation” and “fractionation,” and have been used for understanding the tasks and possibilities of element speciation analysis.

10.1.2. General Analytical Procedure for Speciation Analysis Procedures used for speciation analysis are governed by matrix the species are in, and by analytical techniques that can or will be employed for analysis. For solid matrices (e.g., soil, sediment, food, or plant material), samples can be either Microwave-Assisted Sample Preparation for Trace Element Determination http://dx.doi.org/10.1016/B978-0-444-59420-4.00010-6 Copyright © 2014 Elsevier B.V. All rights reserved.

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directly analyzed for their elemental speciation using direct solid analysis, or, as used more frequently, the element species are separated from matrix prior to analysis. Methods using direct solid analysis allowing element speciation comprise X-ray diffraction techniques which can determine the nature of the mineral structure of an element, but it needs to be crystalline. If the sample is amorphous and/ or the analyte is in trace amounts in sample, X-ray absorption spectroscopy, such as X-ray absorption near edge structure and/or extended X-ray absorption fine structure, can be directly applied to solid or liquid samples. These techniques can reveal in situ the elemental oxidation states, the elements’ coordination number, and their bond length to the next binding ligands. Although these techniques do not need any sample preparation, they cannot give information of minor molecular species of the element and cannot give the entire molecular form. If there is a necessity to determine the molecular structure of element species, there is a need to transfer the elemental species into a solution or a gas phase, which then allows the use of molecular speciation methods for analysis. Hence, the elemental species need to be extracted in the first place.

10.1.3. All Species—Single Species? The ideal goal in speciation analysis is to determine all species of all elements simultaneously, with the same extraction strategy, instrumentation, and analytical method. However, most often the quantitative, precise, and accurate determination of one element species compromises the quality with which another species can be determined. One of the main hindrances in multispecies analysis is the different chemical behavior. Many element species usually present a different behavior, in terms of polarity, solubility, and stability. As an example, arsenic species in fish can occur as “free” molecule in the trivalent or pentavalent form (As(OH)3 or HAsO42−), they can consist of water-soluble species (e.g., arsenobetaine), or they can occur as lipid-soluble (As-fatty acids or As-hydrocarbons). Considering fish samples, different extraction schemes must be used to separate the species free from their biological matrix. Therefore, usually, a targeted extraction method is used for fish tissues, which covers only water- or only lipid-soluble arsenic species. Arsenic speciation is therefore most often focused on a certain form of arsenic. Recently, methods that target the most toxic forms of arsenic, the “inorganic” arsenic, have been in discussion with regards to maximum allowed intake levels for humans, thus regulating maximum levels of inorganic arsenic in food stuff. With regards to organotin determination, the focus of extraction method development has been on both species conservation and quantitative recovery, due to the varying polarity of differently substituted organotin compounds (OTCs). Here, the aim remains to determine all possible OTCs with one analytical method to minimize cost and analysis time. Related to mercury speciation, the main concern lies on methylmercury (abbreviated CH3Hg+, it should be stated that it is most likely not ionic in the

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biological sample), which is the most toxic and most relevant mercury compound due to its biomagnification in food chain. Marine fish is the food commodity with the highest CH3Hg+ concentration. While in fish muscle (e.g., tuna fish) CH3Hg+ is usually around 95% of the total mercury (HgT), in sediments the rate of CH3Hg+:Hg2+ is often at or below 1%. These huge differences can pose analytical problems due to interconversion reactions during extraction and analytical procedures. Therefore, often an extraction step is used to separate CH3Hg+ from an excess of inorganic mercury. In summary, it is extremely difficult to determine all element species of all elements simultaneously. However, if species properties are similar, and extractions as well as analytical methods are the same, multispecies and multielement speciation is possible. This is the case for CH3Hg+ and OTCs in sediments or biota, where analytical methods enable their simultaneous determination. Where a clear target element species is defined, extraction and analytical methods can be tailored to the task, taking into account that all other element species are not included in this analytical method. Therefore, it is important that researcher considers not only the most suited analytical technique but also the most suited sample preparation for the analytical task in question.

10.1.4. Integrity of the Species/Derivatization Prior Analysis The term “species integrity” states that it is possible to identify the original chemical species that was present in a sample. There are a variety of ways in which species integrity can be compromised. Very often losses of organic substituents or functional groups are observed, e.g., tributyltin (TBT) transforms to dibutyltin (DBT) by loss of a butyl group, or loss of a methyl group from CH3Hg+ which leads to Hg2+ formation. Furthermore, the oxidation state of elements, e.g., oxidation states of chromium ((CrIII) or (CrVI)) and arsenic ((AsIII) or (AsV)), can easily be converted. These transformations can occur by natural processes (e.g., through degradation by microorganisms), or during the sample preparation processes due to chemical shift of an equilibrium at higher temperature and different pH. In general, the main goal for speciation analysis is to preserve the original species composition in the sample taking into account the storage conditions like light, temperature, and time, in addition to sample preparation method. Storage of samples is recommended in the cold and dark, with −20 or 4 °C being used for most of samples. However, species conversion has been shown upon defrosting (e.g., arsenic speciation in urine samples). Sediment and biota samples are often dried for species stabilization, but transformation reactions can occur and need to be carefully evaluated. Species integrity is an issue that needs to be investigated with regards to microwave-assisted extraction procedures, with the main parameters being microwave power, temperature, and irradiation time. Closed or open systems must be used in different ways, and the selected solvent is a very important parameter. Examples are given in the following sections.

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For applications aiming for the determination of unstable compounds or complexes, microwave-assisted extraction is not applicable. An example is the determination of metalloenzymes or metal-containing biomolecules. Compounds like arsenic- or mercury-phytochelatins, which act as detoxifying agents in plants against metal pollution, must be extracted using cold solutions to conserve species integrity. Other applications involve the intentional transformation of element species into different compounds. Mostly, these are derivatization reactions that transform an ionic species (e.g., TBT) into a fully alkylated compound which is amenable to gas chromatographic separation and subsequent element detection. These derivatizations are carried out in a way that the original species information is preserved, i.e., even when the final compound differs from the original species, it is possible to still know exactly which compound was originally in the sample. These procedures have to be carefully evaluated to assure that no additional species conversion has taken place, or that artifacts are produced in the process.

10.2. MERCURY SPECIATION 10.2.1. Mercury Speciation for Monitoring Mercury Exposure Mercury (Hg) is potentially one of the most toxic elements to organisms as it has strong ability to bioaccumulate and the enrichment of highly toxic Hg compounds in aquatic food chain poses a serious environmental problem [2]. The most common chemical forms of Hg in the environment are elemental mercury (Hg0), inorganic mercury (Hg2+), monomethyl mercury (CH3Hg+), and dimethylmercury ((CH3)2Hg). Hg is naturally present in the Earth’s crust and, during its biogeochemical cycle, the Hg vapor is released to the atmosphere where it is oxidized to Hg2+. Water-soluble Hg2+ is removed from the atmosphere mainly through wet deposition on the terrestrial and aquatic surfaces. The majority of Hg2+ deposited on the aquatic surface is reduced to Hg0 and revolatilized, while the rest is subjected to sulfate- and iron-reducing bacterial methylation, mainly in wetlands and sediments [3]. Additionally to natural sources of Hg, the anthropogenic emissions and releases are extensively increasing the concentration of Hg in the environment. Hg exposures can be estimated by measuring the pollutant levels in the biological tissues and body fluids which accumulate Hg such as hair, urine, blood, nails, and human milk. The concentration could determine the internal dose, which can be used to evaluate the adverse effects caused by Hg. In addition, these tissues are very useful for monitoring Hg exposure in individuals or in general population [4]. Due to its simple, integrative, and noninvasive sampling for determination of longterm exposure to Hg, hair analysis became increasingly popular in the recent years.

10.2.1.1. Key Challenges and Analytical Procedure Reliable analysis of data based on proper monitoring methods is required in order to correctly evaluate and elucidate the degree of Hg contamination in

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biological and environmental samples. The methods used are as follows: (1) proper choice of sample, (2) appropriate sample collection, (3) storage and transport, (4) sample preparation methods, (5) analytical techniques, (6) experienced/trained staff, and (7) clean laboratory and tools with glassware and containers free from contaminations [5]. Prior to sample analysis, the accuracy of the method should be evaluated using suitable certified reference materials (CRM), for example, NIES 13 and IAEA 085 are used during the determination of Hg in hair. Increased accuracy using gas chromatography coupled to inductively coupled plasma mass spectrometry (GC-ICP-MS) can be achieved by so called species-specific isotope labeling utilising simultaneous monitoring of different mercury isotopes [6]. The quality of results is generally associated with sample pretreatment. For the analysis of biological and environmental samples, a digestion/leaching (alkaline or acid) step is required to extract Hg species from sample matrix prior to detection. However, for ionic Hg species (not fully alkylated Hg species), a derivatization reaction is required in order to generate volatile Hg species. There are, however, potential sources of error related to nonquantitative recoveries of Hg species, artifact formation, and CH3Hg+ transformation during sample preparation and derivatization steps [7]. To overcome the potential problems related to accuracy of Hg speciation data, especially for the CH3Hg+ content, the use of isotope dilution mass spectrometry (IDMS) allows minimizing the uncertainties since quantitative recoveries are not required and rearrangement reactions are easily detected [7]. However, in order to prevent preferential extraction of labeled species, sufficient time must be allowed for the endogenous species and spike to equilibrate. 10.2.1.1.1. Derivatization (Ethylation and Propylation) In earlier studies, CH3Hg+ has been the most investigated organomercury compound. For its determination in environmental samples, sodium tetraethylborate (NaBEt4 or NaB(C2H5)4) has been used as derivatization reagent [8]. Thus, when ethylation is performed with NaBEt4, Hg2+ is transformed to Hg(C2H5)2, while CH3Hg+ generates CH3Hg(C2H5), according to Eqns (10.1) and (10.2). 2+

+

Hg + 2NaB(C2H5)4 → Hg(C2H5)2 + 2Na + 2B(C2H5)3 +

+

CH3Hg + NaB(C2H5)4 → CH3HgC2H5 + Na + B(C2H5)3

(10.1) (10.2)

The main drawback of ethylation reaction is that it does not distinguish between Hg2+ and C2H5Hg+. Both species may coexist in the environment [9]. It was reported that derivatization using NaBEt4 generates the formation of CH3Hg+ from Hg2+, especially if Hg2+ is found at high concentrations (probably due to impurities in NaBEt4) [10]. In addition, the

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presence of dissolved organic compounds from sample matrix may present a strong interference in ethylation reaction [10]. An alternative is the use of propylation reaction for Hg species derivatization using sodium tetrapropylborate (NaBPr4) which is more tolerant to some interferences [7,10]. The advantages of NaBPr4 are the easy handling for in situ derivatization and extraction, combined with the possibility to distinguish between C2H5Hg+ and Hg2+. Solutions of NaBPr4 are also more stable than NaBEt4, making this reagent a suitable alternative to NaBEt4 for Hg species derivatization. 10.2.1.1.2. Species-Specific Isotope Dilution Mass Spectrometry IDMS has been considered the best analytical method for element speciation. It is based on the measurement of isotope ratio in the spiked samples where its isotopic composition has been altered by the addition of a known amount of an isotopically enriched element. Employing IDMS, the concentration of chemical species can be measured very precisely even if the derivatization is not quantitative [10].

10.2.1.2. Application Using Microwave-Assisted Extraction Microwave-based procedures have been used in recent years as extraction methods for determination of Hg species in human hair. Microwave leaching procedures have been found useful when combined with the subsequent determination of CH3Hg+ in hair samples, by means of IDMS analysis (Figure 10.1). In this method, about 20 mg of washed and dried hair sample are weighed in 20 ml glass vials. Then, hair is spiked with a calculated amount of enriched CH3Hg+ (CH3201Hg+) standard solution. An aliquot of 5 ml of 25% tetramethylammonium hydroxide (TMAH) solution in water is added into the glass vials. The vial is closed and microwave radiation is applied according to the following program: 55 °C for 20 min and 60 °C for 20 min. Then, 1 ml of extract is transferred to clean glass vials and 5 ml of acetate buffer (0.1 mol/l, pH ≈ 3.9) is added. Thereafter, 1 ml of isooctane is added and the Hg species are subjected to the propylation reaction using 1 ml of 1% NaBPr4. The solution is vigorously shaken for 5 min and the mixture is centrifuged for 10 min. The isooctane layer containing the derivatized Hg species is transferred into amber grass vials and analyzed by GC-ICP-MS. In order to determine the accuracy and precision of CH3Hg+ determination, hair CRM (e.g., NIES-13) is used, which is subjected to the same preparation

Hair sample

Washing and drying

Spiking with enriched CH Hg

5 ml 25 TMAH in water

Microwave leaching

Aqueous derivatization by NaBPr

Centrifugation and organic layer extraction

Speciation analysis by GC-ICP-MS

FIGURE 10.1 Sample preparation method for CH3Hg+ determination in hair using microwave-assisted extraction and species-specific IDMS.

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method and analysis as the samples. A typical chromatogram obtained for the spiked NIES-13 is shown in Figure 10.2.

10.2.2. Mercury Speciation in Marine Biota 10.2.2.1. What are the Key Species and Why? CH3Hg+ is formed by natural process of biomethylation of Hg2+ found in water and sediment and it possess the highest human toxicity from all Hg species. In general, more than 95% of HgT present in the fish and sea mammals is in the form of CH3Hg+ which undergoes biomagnification along the food chain [11]. The advisory levels of Hg are shown in Table 10.1 with a reference to the different countries. While the consumption of two meals containing fish per week is highly recommended in order to gain cardiovascular protection, researches showed that Hg in contaminated fish inhibits essential functions of omega-3 [18]. In order to establish which species should be avoided, Groth [19] conducted an analysis on available data of Hg levels in 51 fish and shellfish samples. Table 10.2 summarizes fish varieties with very high Hg levels exceeding the safe recommendations by regulatory bodies mentioned in Table 10.1. In addition, various studies have shown 40–90% differences in Hg levels between the same fish species inhabiting different geographic region [13]. CH3HgC3H7

250 CH3HgC3H7 250000

Intensity (103cps)

200

200000 150000

150

Hg(C3H7)2 100000 199

Hg

100

50000

200

Hg

201

Hg

0

202

140

50

160

180

200

220

Hg

240

0 141

144

147

150 Time (s)

153

156

FIGURE 10.2 Chromatogram for mercury speciation analysis using GC-ICP-MS for CRM NIES-13 spiked with enriched CH3201Hg+. (For color version of this figure, the reader is referred to the online version of this book.)

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TABLE 10.1 Advisory Mercury Levels in North America, European Union, and Japan Regulatory Body

Mercury Advisory Level

References

US Environmental ...