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The role of speciation in analytical chemistry
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trends
The role of speciation in analytical chemistry è ik* Agata Kot, Jacek Namiesn
Department of Analytical Chemistry, Technical University of Gdanèsk, 11 / 12 Narutowicza Str., 80-952 Gdanèsk, Poland The growing awareness of the strong dependence of the toxicity of heavy metals upon their chemical forms has led to an increasing interest in the qualitative and quantitative determination of speci¢c metal species. Speciation has therefore become an important topic of present-day analytical research. This article describes the main types and areas of application of speciation analysis, and the use of sequential extraction in speciation studies. Brief characteristics of the basic types of speciation analysis, examples of their applications, and the usage of the various analytical techniques are also included. z2000 Elsevier Science B.V. All rights reserved. Keywords: Speciation; Sequential extraction; Metal speciation analysis
1. Introduction The rapid increase in the levels of environmental pollution over recent decades has resulted in increasing concern for people's well-being and for global ecosystems. The need to determine different species of trace elements in environmental and biological materials is beyond question since the effects or toxicity of an element and its behaviour depend to a great extent on its chemical form and concentration. Originally, most analytical measurements dealt with the total content of a speci¢c element in an analysed sample ( such as lead, mercury, or cadmium, as examples of toxic elements, or cobalt, selenium, or magnesium, as examples of elements necessary for living organisms ). Until recently, analytical methods allowed analysts to determine total contents only, but it was soon realised that this ana*Corresponding author. Tel.: +48 (58) 471010; Fax: +48 (58) 47 2694. E-mail: [email protected]
lytical information was insuf¢cient. Biochemical and toxicological investigation has shown that, for living organisms, the chemical form of a speci¢c element, or the oxidation state in which that element is introduced into the environment, is crucial, as well as the quantities. Therefore, to get information on the activity of speci¢c elements in the environment, more particularly for those in contact with living organisms, it is necessary to determine not only the total content of the element but also to gain an indication of its individual chemical and physical form. Generally, the appearance of multiforms is described by speciation, but the process leading to quantitative estimation of the content of different species is called speciation analysis. According to the of¢cial de¢nition which is currently under discussion at IUPAC [ 1 ] speciation analysis is the process leading to the identi¢cation and determination of the different chemical and physical forms of an element existing in a sample. Although this de¢nition tends to restrict the term speciation to the state of distribution of an element among different chemical species in a sample, in practice the use of this term is much wider, specifying either the transformation and / or the distribution of species, or the analytical activity, to identify chemical species and measure their distribution. For the description of these processes the terms `species transformation' and `species distribution', respectively, are suggested. The analytical activity involved in identifying and measuring species is hence de¢ned as `speciation analysis'.
2. Importance of speciation analysis for health-related measurements It is well known that the toxicity of elements depends upon their physico-chemical forms. It has been well established that some metals ( e.g., Cr, Mn, Fe, Co, Cu, Zn, Mo ) and that metalloids
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( e.g., B, Si, Se ) are essential for living organisms and that they are necessary components of some proteins playing important physiological functions. Their excess, as well as de¢ciency, may have serious consequences for living organisms. Therefore, the determination of the total content of a selected element in the sample is certainly not suf¢cient for the estimation, for example, of toxicity impacts. The case of selenium is a good example of such a situation: this element in small amounts is a prerequisite for humans [ 2 ]. Suf¢cient selenium supplementation can protect against heart disease. Detoxi¢cation effects of Se by interaction with other metals are proved and widely described. The toxicity of Se is modi¢ed by complexation with As, Ag, Cu and Hg, and Se de¢ciency can cause many diseases, such as haemolysis, multiple sclerosis and rheumatic arthritis, and is most critical for the brain and infant growth. However, the transition from the required level ( the recommended dietary level for humans is 50^70 Wg of selenium per day ) to the toxic dose ( approximately 800 Wg of selenium daily ) is quite easy [ 3 ]. In real life the toxicity of selenium towards the environment and human beings depends strongly on the speci¢c form. Selenium can exist naturally in inorganic forms in different oxidation states: 3II ( selenide ), 0 ( elemental selenium ), +IV ( selenite, SeO23 3 ), +VI ( selenate, SeO23 ), as well as in the form of organic com4 pounds and methylated derivatives ^ each form differing widely in its nutritional and toxic impact. In soil and water, selenium exists mainly in the form of inorganic ions, as Se( IV ) and Se(VI ). Total Se levels in environmental samples range from about 0.1^ 400 Wg l31 in natural waters ( the maximum admissible concentration of Se in water is 8^10 Wg l31 , and for drinking water it is 10 Wg l31 ) to 0^80 ng kg31 in soils. As a result of the activity of micro-organisms and plants, the biomethylation process transforms selenium into organic linkages [ 4 ]. Well-known organic species of selenium in our environment are associated with amino acids ( e.g., selenocystamine SeCM, selenocysteine SeCys, and selenomethionine SeM ). The less toxic forms seem to be volatile methylated selenium compounds ( e.g., dimethyl selenide DMSe and dimethyl diselenide DMDSe ), which are metabolised after detoxi¢cation processes. Metals and metalloids are present in all compartments of our environment and the environmental pathways of these elements are of high importance in relation to their toxicity towards £ora and fauna.
Their concentration levels, mobility, and transformation and accumulation processes in the ecosystem depend on parameters such as pH, redox conditions, oxidation states, temperature, presence of organic matter, and microbiological activity. All these factors strongly in£uence the biogeochemical cycles of elements in our environment. Usually organometallic compounds are much more toxic than the ions of the corresponding inorganic compounds. Mercury, lead and tin obey this general rule, whereas arsenic and selenium represent an exception because most organo-arsenicals are less toxic than inorganic arsenic species, and organic forms of Se are ordinarily less toxic than Se(VI ). The toxicity of compounds varies in relation to the compound, e.g., for tin, mono- and dialkylated species are less toxic than trialkylated ones. The toxicity of organometallic species also varies with the organism monitored. For example, trimethyltin is more toxic for insects, triethyltin for mammals, and tributyltin for ¢sh, fungi and bivalves such as shell¢sh [ 5 ].
3. Main types of speciation Generally, speciation analysis plays a unique role in [ 6,7 ]: õ
õ
õ õ
õ õ
õ õ
studies of biogeochemical cycles of chemical compounds; determination of toxicity and ecotoxicity of selected elements; quality control of food products; control of medicines and pharmaceutical products; technological process control; research on the impact of technological installation on the environment; examination of occupational exposure; clinical analysis.
Table 1 presents examples of the main application areas of speciation analysis. The harmful effects of the chemical forms of metallic pollutants are observed during the monitoring of different environmental compartments ( air, natural waters, soil, sediments, biota ). Examples such as the determination of organolead in leaded gasoline, organotin emitted from antifouling paints, or the discrimination between ionic mercury and methylmercury in environmental, clinical, and foodstuff samples
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Table 1 Main application areas of speciation analysis Element
Application area of speciation analysis
Aluminium Al
Polymerisation products. Forms of aluminium ( e.g., labile, complexed ) in serum. Forms of aluminium in food products. Antimony Sb Redox forms and organoantimony compounds in the environment and food products. Arsenic As Redox forms and organoarsenic compounds in the environment. Arsenic-bound proteins in serum and haemoglobin. Arsenic in food products. Forms of arsine AsH3 ( arsenious hydride ) in indoor air at the workplace. Cadmium Cd Complex organic cadmium compounds, metallothionine. Chromium Cr Redox forms of chromium, Cr(VI ) in the environment. Chemical forms of chromium coupled with proteins. Iodine I Iodine forms in the environment and biological £uids. Lead Pb Forms of lead compounds in the environment, e.g., trialkylated Pb compounds. Phosphorus P Phosphine ( hydrogen phosphides ) in indoor air at the workplace. Mercury Hg Forms of mercury compounds in the environment and food products ( in particular, methylmercury ). Platinum Pt Inorganic forms in the environment. Metallo-organic forms of cis-platinum in medicine ( therapeutic ). Selenium Se Inorganic and organometallic selenium compounds in the environment and food products. Tin Sn Organometallic forms in the environment and food products ( e.g., shell¢sh ). Actinide series Chemical forms of compounds in the environment and in radioactive waste storage places.
( in view of their different toxicities ) have long been principal areas of interest in speciation. Contamination of food products ( e.g., oysters and mussels by organotin, ¢sh by methylmercury, wine by lead compounds ) is the most prominent area of speciation [ 8 ]: export markets have rigid legal prerequisites for the particular forms of elements or compounds. It is therefore not surprising that recommendations and regulations on the determination of selected species are emerging, such as inorganic arsenic ( Environmental Protection Agency, EPA; Food and Agriculture Organisation,
FAO; World Health Organisation, WHO; European Environment Commission, EEC black list ), n-butyltins ( EEC black list ), organotin compounds ( Deutsches Institut fuër Normung, DIN ), chromium VI ( DIN ) etc. The most studied areas as far as ecotoxicology is concerned are homeostatic control, metabolism, detoxi¢cation of a number of essential ( e.g., Zn, Cu ) and toxic elements ( e.g., Cd, Hg ) by environmental biota, research on metallothioneins and metal-binding proteins, and acid precipitation. Identi¢cation of the volatile chemical species and inhaleable particles in the workplace are central interests in industrial hygiene. Clinical chemistry is apparently a very promising, and still littleexplored, ¢eld for speciation analysis. For example, metals are components of many therapeutic drugs (Tc in labelled antibodies ), and platinum and gold compounds are used in cancer therapy. The release of organotin employed as catalysts in the production process of pharmaceuticals, and as stabilisers of polymers used as packing materials, involves speciation in the analytical control of the ¢nal products. Much attention has been paid to speciation analysis of tin [ 8,9 ]. Organotin compounds exhibit signi¢cant differences in the toxicity depending on the number and nature of organic groups in an RnSnX 43n compound (R denoting an alkyl or aryl group, n being 1^4, thus becoming the four different types of organotin compounds, mono-, di-, tri-, and tetraorganotin species, with X indicating a common anion such as F3, Cl3 , OH3 ). It is estimated that the toxicity of species corresponding to n = 3 in the general formulation, namely tributyland triphenyltin (TBT and TPhT, respectively ), far exceeds that of other tin species of anthropogenic origin, which undergo bioaccumulation processes after they have been introduced into the aquatic environment [ 9 ]. The use of these compounds as antifouling agents in paints (TBT ) and biocides in agriculture (TPhT ) had a considerable impact on the environment in the 1960s. When the reduction in the rate of growth of oysters in coastal Atlantic zones was noticed in 1975 ^ as an effect of tributyltin pollution ^ France was the ¢rst country to ban the application of this compound. Although many member states of the European Community banned the use of TBT antifouling paints around 1990, triorganotins and the products of their degradation are still found in our environment. Attention given to sediment analysis has shown that the sediment can act as the ultimate `sink' for organotin com-
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Table 2 Short characteristics of basic types of speciation analysis Type of spe- Area of application ciation analysis Environmental pollution analyses ( air, water, soil ).
Screening speciation
Group speciation
Environmental pollution analyses. Food pollution analyses. Ecotoxicology. Environmental pollution analyses. Food pollution analyses. Ecotoxicology.
Remarks
Examples
Physical speciation This type of speciation analysis is extremely important from various points of view: chemical process investigations and biochemical processes going on in different elements of environment. Chemical speciation It is the simplest case of speciation analysis, which leads to the detection and determination of one de¢nite analyte.
This case of speciation analysis leads to the determination of the concentration level of the speci¢c group of compounds or elements existing in different compounds and forms and at the speci¢c oxidation level.
Distribution Environmental speciation pollution analyses. Ecotoxicology.
This type of speciation is connected in most cases with the analyses of biological samples.
Individual speciation
The most dif¢cult form of speciation analysis. Fractionation and separation techniques have played a particular role. Unique application of chromatography and coupled techniques in this area of speciation analysis.
Environmental pollution analyses. Food pollution analyses. Ecotoxicology.
pounds released into the aquatic environment, and may therefore create an ecotoxicological risk long after anthropogenic sources are banned from a given area. The monitoring of tin compounds is now required by EC legislation. Different countries have already established the limit values for various
Trace metals analysis ( soluble and suspended fraction ). Trace metals analysis of different forms present in soil and sediment after sequential extraction. Determination of tributyltin in sea water, sediments, tissue. Determination of methylmercury in tissue. Determination of chromium compounds, Cr(VI ). Determination of organic matter in samples by the assignation of summary parameters ( i.e., TOC in water or TH in air ). Determination of level of concentration of different forms of mercury ( elementary, inorganic and organic ). Determination of trace metals in blood serum and blood cells. Determination of heavy metals in plants. Identi¢cation and determination of chemical species de¢ned as to molecular, complex, electronic or nuclear structure.
species. The Dutch government has set the limit value at around 10 ng l31 TBT [ 8 ]. Speciation analysis can be performed in at least ¢ve different types, depending on the aim and scope of the analytical investigation. Brief characteristics of basic types of speciation analysis
Table 3 BCR three-stage sequential extraction scheme [ 17 ] Extraction step
Trace metal fraction
Composition of aquatic extract solution ( concentration in mol l31 )
1 2 3
Metals in soil solution, carbonates, exchangeable metals Iron / manganese oxyhydroxides Organic matter and sul¢des
CH3 COOH ( 0.11 ) NH2 OH.HCl ( 0.1 at pH 2 ) H2 O2 ( 8.8 ), then CH3 COONH4 ( 1.0 ) at pH 2
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and examples of their application are given in Table 2. Depending on their origin, trace elements exist in different mineral forms and chemical compounds, and in different combinations with mineral and organic components of soil and sediments which may vary according to various conditions: for example, pH has an in£uence on the trace-metal forms in which they exist. In acidic soils simple cations and complexes of chlorides and sulfates usually exist, while in neutral and slightly alkaline conditions carbonate complexes dominate.
4. Single and sequential extraction in speciation analysis Since the behaviour of the elements in a soil^ water^plant system depends on their form, the determination of trace metals in soils is often performed by single or sequential extraction. The procedures consist in subjecting a solid sample ( soil or sediment ) to successive attacks with reagents of different chemical properties ( acidity, redox potential, or complexing properties ) with each extract a part of the trace metals associated with the sample [ 10 ]. The best-known sequential extraction scheme
Fig. 1. Basic types of speciation analysis in the area of chemical analysis.
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is that of Tessier et al. [ 11 ]. This consists of ¢ve steps in which heavy metals are distributed among different phases ^ sorbed metals, carbonates, reducible and oxidisable substrates, extractable organics, sul¢des, and residual minerals. The ability to determine the chemical forms of heavy metals in marine and freshwater sediments is becoming increasingly important. It is also necessary to assess the potential of the sediments as either a sink or a source of heavy metals in the aquatic environment. To date, it has generally been accepted that the most appropriate way to evaluate the different phases in which radionuclides may occur is also based on sequential extraction schemes. As with heavy metals, sequential extraction schemes for radionuclides are based mainly on the procedure proposed by Tessier et al., and modi¢cations of it have been described in the literature subsequently [ 10,12^14 ]. The use of sequential extraction in connection with physical speciation enables one to de¢ne the metal forms available to plants. By de¢ning the speci¢c metals existing in soils which are accessible for plants, and the solubility of their metal forms, an indication of the danger of metal leaching to the groundwater is possible. It is necessary to stress that the results from such analyses can only be compared with the results from analyses carried out in a similar way for soil samples of similar character. The lack of uniformity in the different extraction procedures used does not allow the results to be compared world-wide or the procedures to be validated, which has led to many criticisms in the past. Indeed, the results obtained are highly dependent on the extraction procedures used, and the sample type. This actually requires that such optionally de¢ned procedures should be standardised [ 13 ]. A number of investigators have highlighted pitfalls in the use of sequential extraction, the most serious of which appear to be the poor selectivity of the reagents used and the redistribution of released metals between phases [ 15 ]. The necessity to harmonise the existing methods for speciation analysis of metals in samples in quite complicated matrices is important for the further development of speciation analysis ( with respect to the determination of extractable trace-element contents ). As part of recent attempts to harmonise methodology for leaching / extraction methods through the European Community, the BCR has developed a three-stage sequential extraction pro-
tocol in which metals are divided into acid-soluble / exchangeable, reducible, and oxidisable fractions [ 16 ]. In Table 3, common solutions for the threestage sequential extraction recommended by BCR are presented. The method has proved to be reproducible and gave good recoveries. Reference materials, certi¢ed for metals extractable by the Measurements and Testing Programme ( formerly BCR ) of the European Commission procedure, have been made available [ 16,17 ]. The use of certi¢ed reference materials ( CRMs ) is a widely recognised tool for the veri¢cation of the accuracy of analytical techniques used for speciation analysis. Recently BCR has organised a series of interlaboratory studies to evaluate extraction procedures and to certify reference materials for their extractable element contents [ 18 ].
5. Analytical methodology for speciation analysis The recognition of the fact that the determination of the level of a total metal or of a metalloid is not suf¢cient to evaluate its impact on the environment, its bioavailability and its toxicity has stimulated the development of species-selective analytical methodology during the last decade. For want of better speciation analysis, as with all types of analyses performed daily by analysts around the world, a special meaning attaches to the use of proper sample-preparation techniques. When during the sample-preparation steps a sample undergoes an incorrect ^ from the speciation point of view ^ operation, there is a danger of missing part of the information. For example, instead of the determination of individual ( particular ) chemical species and / or physical forms of an analysed element, it would only be possible to determine the total content of this element. A review of sample-preparation steps, and techniques used for the digestion and extraction carried out for speciation analyses of metals and metalloids, has been published recently [ 19 ]. At present, speciation analysis represents a great challenge for analysts working on the development of new procedures or on the practical application of existing methodologies to trace and microtrace components analyses. In many procedures used so far the recommended strategies permit only the determination of the total content of the selected element in a sample. On the other hand, it should be emphasised that many older procedures, espe-
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Table 4 Application of various analytical methods in metal speciation analysis Technique
Remarks
Application ^ examples
Gas chromatography
Gas chromatography is applied with different detectors, very often non-selective.
High-performance liquid chromatography
The most popular detectors (UV^Vis ) ^ due to their low sensitivity ^ are applied very rarely. Fluorescence or electrochemical detectors are used quite often. Reversed-phase LC.
Determination of alkyl-element compounds of metals and metalloids from the IVB ( Ge, Sn, Pb ), VB (Sb, As ) and VIB (Se ) groups of the periodic system. Separation of metalloporphyrins. The most frequent organometallic analyses: Pb and Hg in air, Pb in atmospheric deposits and urban dust, Sn in surface waters, Pb, Sn in sediments, Hg in biota. Speciation analysis of aluminium in water samples. Determination of Cr and V compounds with the application of spectrophotometric detectors. Determination of Hg( II ), methylmercury and methyl-, ethylmercury in wastewater and sediments. Analysis of selenomethionine in soil extracts and tablets. Metallothionein analysis in liver and kidney tissues. Determination of metal binding to proteins ( Al in blood serum, Se in human serum ) and metal detoxi¢cation in marine organisms ( Cd-, Cu-, Znthioneins in mussels, cyanobacteria ). Analysis of vanadyl and nickel porphyrins in crude oils. Ferritin, haemoglobin, myoglobin, cytochrome c ferritin in pharmaceutical products. Cr species in liver tissue. Cd-thionein analysis. Se( IV ), Se(VI ), TMSe determination in urine. Simultaneous separation of As( III ), As(V ), MMA, DMA and analysis in urine, seafood, natural waters, sediments and soil extracts. Se( IV ), Se(VI ), TMSe, acidic, neutral and basic organoselenium analysis in water samples. Arsenobetaine, arsenocholine, trimethylarsine oxide determination in seafood. Sn( II ), Sn( IV ), TBT in harbour water. Cr species in liver tissue and plasma. Hg( II ), MMHg, MEHg in tap water. Fairly comprehensive speciation analysis of As and Se compounds. Analysis of Hg in mussels. Separation of metallo-, phospho- and selenoproteins, metalloenzymes. Cd and Zn-thioneins in rabbit liver. Metallothionine analysis. Speciation analysis of tin.
Size-exclusion chromatography.
Anion-exchange chromatography.
Cation-exchange chromatography.
Electrophoresis
Technique is based on the separation in an electrical ¢eld and is realised in three basic modes: zone, isotachophoresis and isoelectric. The wider use of electrophoretic techniques is hampered by a lack of an element-speci¢c detector in the on-line mode.
Polarography and cathode and anode inversion voltamperometry
Techniques are used for the differentiation of elements at different oxidation states and in case of the analysis of samples containing humic and fulvic acids. Often used in laboratories in ocean-going scienti¢c ships. Ion-selective electrodes Used for the determination of elements at different Speciation analysis of Cd and Cu in the presence of oxidation states; quite low sensitivity. humic and fulvic acids. Determination of £uorides in the presence of aluminium. Electron auger spectroscopy Very rarely applied. Speciation of iron in physiological £uids in the and EPR presence of ascorbinians and oxygen. Nuclear magnetic resonance Although restricted sensitivity application area of the Speciation analysis of actinides. technique in quantitative analysis becomes more and Determination of Al( III ) compounds, hydroxy and more wide. other couplings of aluminium in geological samples. Complexion of aluminium with nucleosides.
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Table 5 Application of coupled techniques in speciation analysis of metals Technique
Comments on the coupling device
Possible problems
GC^MS
Usually heating of analyte transfer line is required.
Analytes should be volatile or after derivatisation before ¢nal determination.
LC^MS
Considerable numbers of interfaces are built up; the most common are particle beam, thermospray, atmospheric pressure chemical ionisation, electrospray. GC^AAS ( quartz GC^FAAS: usually narrow-bore tube furnace and between the two instruments, tube should graphite furnace ) be as short as possible to avoid peak broadening, transfer tube has to be heated to the highest temperature of the oven temperature programme. HPLC^FAAS Simple coupling. The end of capillary is connected with nebuliser. In case of ICP it is necessary to use device for the separation of analytes and mobile phase solvent.
HPLC^EF AAS
FIA^FAAS
Capillary electrophoresis^ FAAS Hydride generation^AAS GC^AES ( with plasmas ICP or MIP excitation source ) GC^ICP-MS
GC^ICP-AES
Examples of application
Determination of lead organic compounds in benzine, metalloporphyrins in petroleum, organotin compounds in environmental matrices. Restrictions concerning £ow rates and the Speciation of organotin composition ( e.g., additives ^ buffers ) of compounds. the mobile phase. Due to the sensitivity insuf¢cient for environmental analysis, a silica tube furnace electrically heated to the atomising temperature is needed ^ a feature that commercial furnaces do not have.
Suitable for organometals ( those of Pb, Sn, As, Se ). Determination of tributyltin in sea water. Determination of trimethyllead. The outlet of the column can contain Determination of organotin molecules which block nebuliser. Organic compounds in water, sedicompounds in the outlet can undergo ments and shell¢sh tissue. incomplete combustion in the burner Speciation analysis of As, Sb, ( blowpipe? ) and then block. Solvents Se, Hg, Pb, Cr. from the mobile phase can cause plasma extinguishing in ICP. The HPLC ef£uent is volatilised to an Limitations in the use of solvents and Determination of tin species. aerosol in a heated silica capillary and buffer solutions in the eluent ( buffers are Analysis of organolead comenters the furnace through a vitreous highly volatile ). Only well-separated spe- pounds. graphite tube. cies can be distinguished. Very simple coupling. The outlet of the column can contain Determination of inorganic molecules which block nebuliser. Organic Se and Cr compounds in compounds in the outlet can undergo water. incomplete combustion in the burner and then block. Solvents from the mobile phase can cause plasma extinguishing in ICP. From the technical point of view the couOnly several applications of pling is quite dif¢cult. The £ow rate of these techniques are mobile phase in CZE is usually not comreported in the literature due patible with the £ow rate of sample in AAS to the connection problems. spectrometry. Coupling relatively simple. This technique does not identify species Determination, with a very that do not give rise to volatile derivatives high sensitivity, of As, Se, Sn, during reduction Bi, Te, Sb, Pb and Hg species. The ef£uent from GC can be introduced Speciation analysis of orgadirectly into the £ame without extinguishnolead in rainwater, snow, ing it. harbour, river, lake and tap water. Speciation of Hg, Se, Sn. As for MIP, GC ef£uent is usually transSpeciation of Sn, Pb, Hg. ported to the plasma via heated line which Determination of Sb, Se, Sn, is either glass-lined or has the analytical Te, Hg, Pb, Bi in natural column passing through the centre. waters, land¢ll and fermentation gas after purge and trap preconcentration. Large plasma gas £ow rate causes dilution Low detection limits because the sensitiv- Speciation of Hg. of the analytes. ity of an atomic emission spectrometer does not overcome the dilution with the plasma gas; large dead volume; poor excitation / ionisation of non-metals.
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Table 5 ( continued ) Technique
Comments on the coupling device
Possible problems
Examples of application
LC^ICP-MS
The necessity to use nebuliser spray chamber ( plasma technique is sensitive to organic solvents present in LC ef£uents ). Different sample introduction techniques are investigated ( glass frit nebulisers, thermospray vaporisers, ultrasonic nebulisers ). As above.
Low ef¢ciency of sample nebulisation, which has a major effect on sensitivity. Instability of the plasma to organic vapours. Deposition of the carbon on the sampling cone and torch.
Speciation of organoarsenic and organochromium. Determination of tributyltin in sea water. Determination of organolead.
As above
Speciation of organoarsenic compounds. Determination of Cr( III ) and Cr(VI ). Determination of metalloprotein. Analysis of organotin compounds in marine mussel samples.
LC^ICP-AES
AAS: atomic absorption spectromety; EF AAS: electrothermal atomic absorption spectrometry ( graphite furnace ); FAAS: £ame atomic absorption spectrometry; MIP: microwave-induced plasma; ICP^AES: inductively coupled plasma atomic emission spectrometry; ICP^MS: inductively coupled plasma mass spectrometry.
cially those used for the determination of organic compounds, were used a long time ago, but without the name `speciation'. In the 1950s, when speciation analysis was connected only with biological cycles of metals in the hydrosphere, two forms of metals were distinguished: metals in soluble forms and metals adsorbed on suspended matter. It was enough to ¢lter the aqueous sample through a ¢lter with pore size 0.45 Wm to obtain the separation of the two phases. Later, owing to the development of electrochemical methods, the differentiation of miscellaneous existing forms of metals in soluble form ^ free metal ion and complex ions ^ became possible. Parallel investigations of the possible equilibrium state between ions and ligands ( organic and inorganic, also ) permitted the conclusion that in a water environment a very wide range of metal forms can appear. Now, speciation analysis includes not only metals, but also other elements in different types of samples [ 1,10 ]. In 1976 a detailed scheme for an analytical procedure for speciation of trace metals in water was proposed [ 20 ]. Seven possible chemical and physical forms presented in water were emphasised: õ õ õ
free metal ions; labile metals coupled with organic complexes; metals permanently coupled with organic complexes;
õ õ
õ õ
labile metals coupled with inorganic complexes; metals permanently coupled with inorganic complexes; metals absorbed in organic matter; metals absorbed in inorganic matter.
Now, according to this scheme, the determinations are performed of trace metal contents in different types of waters, with the application of measurements in the laboratory and in situ. Basic types of speciation analysis in the area of chemical analysis are shown in Fig. 1.
6. Application of different analytical techniques to speciation studies The successful approach for speciation analysis depends on two factors: selectivity ( to be sure of determining the proper species ) and sensitivity ( to match the analyte's level in the sample ). The breakthrough in terms of these two key issues was achieved using on-line coupling of chromatographic or electrophoresis technique with an element-selective detector ( atomic absorption, emission, £uorescence, or mass spectrometry, inductively coupled plasma, or microwaveinduced plasma ). The development of speciation techniques, especially the chromatographic ones, was the
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most important factor in the sudden evolution of speciation studies. Speciation analysis now uses the following separation techniques: gas chromatography ( GC ), supercritical £uid chromatography (SFC ), liquid chromatography ( partition, ionexchange, gel, af¢nity ), capillary zone electrophoresis ( CZE ), and ¢eld-£ow fractionation ( FFF ) [ 10,21,22 ]. For volatile, thermally stable species of various elements in a large variety of biological and environmental matrices GC-related analysis is the universally accepted approach. To change polar species into volatile ones which may be separated by GC, a derivatisation step is required. The widespread derivatisation procedures involve formation of volatile hydrides by borohydride reduction ( e.g., As, Sb, Se, Ge, Sn ). Another possibility is ethylation by sodium tetraethyl borate ( e.g., Sn ) or derivatisation by Grignard reagents ( e.g., Sn, Hg, Pb ) [ 10,21 ]. For non-volatile analytes various modes of liquid chromatography can be used, e.g., normal and reversed-phase liquid chromatography, ionexchange chromatography, size-exclusion chromatography. There are hardly limits to the application of other techniques such as SFC or electrophoresis, with special emphasis on CZE and FFF. There is no space to present here all the possible applications of the techniques applied to speciation analysis, so in Table 4 only selected examples of the application of the various analytical methods used in metal speciation analysis are shown. Excellent comprehensive reviews of the state of the art of analytical techniques employed for elemental speciation studies have been presented in the literature by several authors [ 10,21,23^25 ]. Coupled techniques play a particular role in speciation analysis. Chromatographic separation methods are hyphenated with extremely sensitive and selective detectors. The absolute detection limits of, e.g., LC^ICP^MS are in the sub-nanogram to picogram range. Absolute detection limits in picograms, even to the sub-pg range, are gained when GC, SFC and CE are coupled to ICP^MS [ 26 ]. Thus, coupled techniques have gained an important place in different areas of speciation analysis [ 26^ 30 ]. Speciation studies are contributing signi¢cantly to the progress of the life sciences, as is well re£ected by the continuously growing number of papers that has been produced in recent years. Only selected applications of coupled techniques applied to speciation analysis of metals described in the literature are presented in Table 5.
7. Conclusions There is no doubt that speciation analysis now offers a great challenge for analysts. The proper approach for the sequential extraction and application of appropriate analytical techniques and instruments can encourage wider use of speciation analysis in the laboratory. Elemental speciation information is crucial today because the toxicity and biological activity of many elements depend not only on their quantities, but also on their oxidation states and / or chemical forms. Thus speciation analysis can increase the information capacity of collected results.
References [ 1 ] D.M. Templeton, F. Ariese, R. Cornelis, L.-G. Danielsson, H. Huntan, H.P. van Leeuwen, Pure Appl. Chem. ( submitted ). [ 2 ] B.A.N. Popp, F.J. Sansone, T.M. Rust, D.A. Merritt, Anal. Chem. 67 ( 1995 ) 405. [ 3 ] M. Potin-Gautier, Analusis 25 ( 1997 ) M22. [ 4 ] G.A. Pedersen, E.M. Larsen, Fresenius' J. Anal. Chem. 358 ( 1997 ) 591. [ 5 ] I. Havezov, Fresenius' J. Anal. Chem. 355 ( 1996 ) 452. [ 6 ] L. Ebdon, S.J. Hill, C. Rivas, Trends Anal. Chem. 17 ( 1998 ) 277. [ 7 ] R. Jobinèski, Appl. Spectrosc. 51 ( 1997 ) 260A. [ 8 ] Ph. Quevauviller, O.F.X. Donard, in S. Caroli ( Editor ), Element Speciation in Bioinorganic Chemistry, Wiley, New York, 1996, 331. [ 9 ] S.J. de Mora, ( Editor ) Tributyltin: Case Study of an Environmental Contaminant, Cambridge University Press, Cambridge, 1996. [ 10 ] A. Ure, Ph. Quevauviller, H. Muntau, B. Griepink, Int. J. Environ. Anal. Chem. 51 ( 1993 ) 129. [ 11 ] A. Tessier, P.G.C. Campbell, N. Bisson, Anal. Chem. 51 ( 1979 ) 844. [ 12 ] U. Forstner, Int. J. Environ. Anal. Chem. 51 ( 1993 ) 5. [ 13 ] Ph. Quevauviller, Trends Anal. Chem. 17 ( 1998 ) 289. [ 14 ] M. Vidal, G. Rauret, Int. J. Environ. Anal. Chem. 51 ( 1993 ) 85. [ 15 ] C.M. Davidson, A.L. Duncan, D. Littlejohn, A.M. Ure, L.M. Garden, Anal. Chim. Acta 363 ( 1998 ) 45. [ 16 ] Ph. Quevauviller, G. Rauret, J.F. Lopez-Sanchez, R. Rubio, A. Ure, H. Muntan, Sci. Total Environ. 205 ( 1997 ) 223. [ 17 ] Ph. Quevauviller, Spectrochim. Acta Part B 53 ( 1998 ) 1261. [ 18 ] Ph. Quevauviller, Method Performance Studies for Speciation Analysis, Royal Society of Chemistry, Cambridge, 1998. [ 19 ] R. Jobinèski, Z. Marczenko, Spectrochemical Trace Analysis for Metals and Metalloids, Elsevier, Amsterdam, 1996. [ 20 ] G.E. Batley, T.M. Florence, Anal. Lett. 9 ( 1976 ) 379.
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[ 21 ] S. Caroli, in S. Caroli ( Editor ), Element Speciation in Bioinorganic Chemistry, Wiley, New York, 1996, p. 1. [ 22 ] R. Jobinèski, F.C. Adams, Spectrochim. Acta Part B. 52 ( 1997 ) 1865. [ 23 ] J.A.C. Broekaert, Mikrochim. Acta 120 ( 1995 ) 21. [ 24 ] R. Jobinèski, F.C. Adams, Trends Anal. Chem. 12 ( 1993 ) 41. [ 25 ] Y.K. Chau, Analyst ( London ) 117 ( 1992 ) 571.
[ 26 ] G.K. Zoorob, J.W. McKiernan, J.A. Caruso, Mikrochim. Acta 128 ( 1998 ) 145. [ 27 ] K. Pyrzynèska, Mikrochim. Acta 122 ( 1996 ) 279. [ 28 ] L. Moens, T. Smaele, R. Dams, P. Van Den Broeck, P. Sandra, Anal. Chem. 69 ( 1997 ) 1604. [ 29 ] T. Guerin, M. Astruc, A. Batel, M. Borsier, Talanta 44 ( 1997 ) 2201. [ 30 ] I. Rodriguez-Pereiro, A. Wasik, R. Lobinski, J. Chromatogr. A 795 ( 1998 ) 359.
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trends
The role of speciation in analytical chemistry è ik* Agata Kot, Jacek Namiesn
Department of Analytical Chemistry, Technical University of Gdanèsk, 11 / 12 Narutowicza Str., 80-952 Gdanèsk, Poland The growing awareness of the strong dependence of the toxicity of heavy metals upon their chemical forms has led to an increasing interest in the qualitative and quantitative determination of speci¢c metal species. Speciation has therefore become an important topic of present-day analytical research. This article describes the main types and areas of application of speciation analysis, and the use of sequential extraction in speciation studies. Brief characteristics of the basic types of speciation analysis, examples of their applications, and the usage of the various analytical techniques are also included. z2000 Elsevier Science B.V. All rights reserved. Keywords: Speciation; Sequential extraction; Metal speciation analysis
1. Introduction The rapid increase in the levels of environmental pollution over recent decades has resulted in increasing concern for people's well-being and for global ecosystems. The need to determine different species of trace elements in environmental and biological materials is beyond question since the effects or toxicity of an element and its behaviour depend to a great extent on its chemical form and concentration. Originally, most analytical measurements dealt with the total content of a speci¢c element in an analysed sample ( such as lead, mercury, or cadmium, as examples of toxic elements, or cobalt, selenium, or magnesium, as examples of elements necessary for living organisms ). Until recently, analytical methods allowed analysts to determine total contents only, but it was soon realised that this ana*Corresponding author. Tel.: +48 (58) 471010; Fax: +48 (58) 47 2694. E-mail: [email protected]
lytical information was insuf¢cient. Biochemical and toxicological investigation has shown that, for living organisms, the chemical form of a speci¢c element, or the oxidation state in which that element is introduced into the environment, is crucial, as well as the quantities. Therefore, to get information on the activity of speci¢c elements in the environment, more particularly for those in contact with living organisms, it is necessary to determine not only the total content of the element but also to gain an indication of its individual chemical and physical form. Generally, the appearance of multiforms is described by speciation, but the process leading to quantitative estimation of the content of different species is called speciation analysis. According to the of¢cial de¢nition which is currently under discussion at IUPAC [ 1 ] speciation analysis is the process leading to the identi¢cation and determination of the different chemical and physical forms of an element existing in a sample. Although this de¢nition tends to restrict the term speciation to the state of distribution of an element among different chemical species in a sample, in practice the use of this term is much wider, specifying either the transformation and / or the distribution of species, or the analytical activity, to identify chemical species and measure their distribution. For the description of these processes the terms `species transformation' and `species distribution', respectively, are suggested. The analytical activity involved in identifying and measuring species is hence de¢ned as `speciation analysis'.
2. Importance of speciation analysis for health-related measurements It is well known that the toxicity of elements depends upon their physico-chemical forms. It has been well established that some metals ( e.g., Cr, Mn, Fe, Co, Cu, Zn, Mo ) and that metalloids
0165-9936/00/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 9 ) 0 0 1 9 5 - 8
ß 2000 Elsevier Science B.V. All rights reserved.
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( e.g., B, Si, Se ) are essential for living organisms and that they are necessary components of some proteins playing important physiological functions. Their excess, as well as de¢ciency, may have serious consequences for living organisms. Therefore, the determination of the total content of a selected element in the sample is certainly not suf¢cient for the estimation, for example, of toxicity impacts. The case of selenium is a good example of such a situation: this element in small amounts is a prerequisite for humans [ 2 ]. Suf¢cient selenium supplementation can protect against heart disease. Detoxi¢cation effects of Se by interaction with other metals are proved and widely described. The toxicity of Se is modi¢ed by complexation with As, Ag, Cu and Hg, and Se de¢ciency can cause many diseases, such as haemolysis, multiple sclerosis and rheumatic arthritis, and is most critical for the brain and infant growth. However, the transition from the required level ( the recommended dietary level for humans is 50^70 Wg of selenium per day ) to the toxic dose ( approximately 800 Wg of selenium daily ) is quite easy [ 3 ]. In real life the toxicity of selenium towards the environment and human beings depends strongly on the speci¢c form. Selenium can exist naturally in inorganic forms in different oxidation states: 3II ( selenide ), 0 ( elemental selenium ), +IV ( selenite, SeO23 3 ), +VI ( selenate, SeO23 ), as well as in the form of organic com4 pounds and methylated derivatives ^ each form differing widely in its nutritional and toxic impact. In soil and water, selenium exists mainly in the form of inorganic ions, as Se( IV ) and Se(VI ). Total Se levels in environmental samples range from about 0.1^ 400 Wg l31 in natural waters ( the maximum admissible concentration of Se in water is 8^10 Wg l31 , and for drinking water it is 10 Wg l31 ) to 0^80 ng kg31 in soils. As a result of the activity of micro-organisms and plants, the biomethylation process transforms selenium into organic linkages [ 4 ]. Well-known organic species of selenium in our environment are associated with amino acids ( e.g., selenocystamine SeCM, selenocysteine SeCys, and selenomethionine SeM ). The less toxic forms seem to be volatile methylated selenium compounds ( e.g., dimethyl selenide DMSe and dimethyl diselenide DMDSe ), which are metabolised after detoxi¢cation processes. Metals and metalloids are present in all compartments of our environment and the environmental pathways of these elements are of high importance in relation to their toxicity towards £ora and fauna.
Their concentration levels, mobility, and transformation and accumulation processes in the ecosystem depend on parameters such as pH, redox conditions, oxidation states, temperature, presence of organic matter, and microbiological activity. All these factors strongly in£uence the biogeochemical cycles of elements in our environment. Usually organometallic compounds are much more toxic than the ions of the corresponding inorganic compounds. Mercury, lead and tin obey this general rule, whereas arsenic and selenium represent an exception because most organo-arsenicals are less toxic than inorganic arsenic species, and organic forms of Se are ordinarily less toxic than Se(VI ). The toxicity of compounds varies in relation to the compound, e.g., for tin, mono- and dialkylated species are less toxic than trialkylated ones. The toxicity of organometallic species also varies with the organism monitored. For example, trimethyltin is more toxic for insects, triethyltin for mammals, and tributyltin for ¢sh, fungi and bivalves such as shell¢sh [ 5 ].
3. Main types of speciation Generally, speciation analysis plays a unique role in [ 6,7 ]: õ
õ
õ õ
õ õ
õ õ
studies of biogeochemical cycles of chemical compounds; determination of toxicity and ecotoxicity of selected elements; quality control of food products; control of medicines and pharmaceutical products; technological process control; research on the impact of technological installation on the environment; examination of occupational exposure; clinical analysis.
Table 1 presents examples of the main application areas of speciation analysis. The harmful effects of the chemical forms of metallic pollutants are observed during the monitoring of different environmental compartments ( air, natural waters, soil, sediments, biota ). Examples such as the determination of organolead in leaded gasoline, organotin emitted from antifouling paints, or the discrimination between ionic mercury and methylmercury in environmental, clinical, and foodstuff samples
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Table 1 Main application areas of speciation analysis Element
Application area of speciation analysis
Aluminium Al
Polymerisation products. Forms of aluminium ( e.g., labile, complexed ) in serum. Forms of aluminium in food products. Antimony Sb Redox forms and organoantimony compounds in the environment and food products. Arsenic As Redox forms and organoarsenic compounds in the environment. Arsenic-bound proteins in serum and haemoglobin. Arsenic in food products. Forms of arsine AsH3 ( arsenious hydride ) in indoor air at the workplace. Cadmium Cd Complex organic cadmium compounds, metallothionine. Chromium Cr Redox forms of chromium, Cr(VI ) in the environment. Chemical forms of chromium coupled with proteins. Iodine I Iodine forms in the environment and biological £uids. Lead Pb Forms of lead compounds in the environment, e.g., trialkylated Pb compounds. Phosphorus P Phosphine ( hydrogen phosphides ) in indoor air at the workplace. Mercury Hg Forms of mercury compounds in the environment and food products ( in particular, methylmercury ). Platinum Pt Inorganic forms in the environment. Metallo-organic forms of cis-platinum in medicine ( therapeutic ). Selenium Se Inorganic and organometallic selenium compounds in the environment and food products. Tin Sn Organometallic forms in the environment and food products ( e.g., shell¢sh ). Actinide series Chemical forms of compounds in the environment and in radioactive waste storage places.
( in view of their different toxicities ) have long been principal areas of interest in speciation. Contamination of food products ( e.g., oysters and mussels by organotin, ¢sh by methylmercury, wine by lead compounds ) is the most prominent area of speciation [ 8 ]: export markets have rigid legal prerequisites for the particular forms of elements or compounds. It is therefore not surprising that recommendations and regulations on the determination of selected species are emerging, such as inorganic arsenic ( Environmental Protection Agency, EPA; Food and Agriculture Organisation,
FAO; World Health Organisation, WHO; European Environment Commission, EEC black list ), n-butyltins ( EEC black list ), organotin compounds ( Deutsches Institut fuër Normung, DIN ), chromium VI ( DIN ) etc. The most studied areas as far as ecotoxicology is concerned are homeostatic control, metabolism, detoxi¢cation of a number of essential ( e.g., Zn, Cu ) and toxic elements ( e.g., Cd, Hg ) by environmental biota, research on metallothioneins and metal-binding proteins, and acid precipitation. Identi¢cation of the volatile chemical species and inhaleable particles in the workplace are central interests in industrial hygiene. Clinical chemistry is apparently a very promising, and still littleexplored, ¢eld for speciation analysis. For example, metals are components of many therapeutic drugs (Tc in labelled antibodies ), and platinum and gold compounds are used in cancer therapy. The release of organotin employed as catalysts in the production process of pharmaceuticals, and as stabilisers of polymers used as packing materials, involves speciation in the analytical control of the ¢nal products. Much attention has been paid to speciation analysis of tin [ 8,9 ]. Organotin compounds exhibit signi¢cant differences in the toxicity depending on the number and nature of organic groups in an RnSnX 43n compound (R denoting an alkyl or aryl group, n being 1^4, thus becoming the four different types of organotin compounds, mono-, di-, tri-, and tetraorganotin species, with X indicating a common anion such as F3, Cl3 , OH3 ). It is estimated that the toxicity of species corresponding to n = 3 in the general formulation, namely tributyland triphenyltin (TBT and TPhT, respectively ), far exceeds that of other tin species of anthropogenic origin, which undergo bioaccumulation processes after they have been introduced into the aquatic environment [ 9 ]. The use of these compounds as antifouling agents in paints (TBT ) and biocides in agriculture (TPhT ) had a considerable impact on the environment in the 1960s. When the reduction in the rate of growth of oysters in coastal Atlantic zones was noticed in 1975 ^ as an effect of tributyltin pollution ^ France was the ¢rst country to ban the application of this compound. Although many member states of the European Community banned the use of TBT antifouling paints around 1990, triorganotins and the products of their degradation are still found in our environment. Attention given to sediment analysis has shown that the sediment can act as the ultimate `sink' for organotin com-
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Table 2 Short characteristics of basic types of speciation analysis Type of spe- Area of application ciation analysis Environmental pollution analyses ( air, water, soil ).
Screening speciation
Group speciation
Environmental pollution analyses. Food pollution analyses. Ecotoxicology. Environmental pollution analyses. Food pollution analyses. Ecotoxicology.
Remarks
Examples
Physical speciation This type of speciation analysis is extremely important from various points of view: chemical process investigations and biochemical processes going on in different elements of environment. Chemical speciation It is the simplest case of speciation analysis, which leads to the detection and determination of one de¢nite analyte.
This case of speciation analysis leads to the determination of the concentration level of the speci¢c group of compounds or elements existing in different compounds and forms and at the speci¢c oxidation level.
Distribution Environmental speciation pollution analyses. Ecotoxicology.
This type of speciation is connected in most cases with the analyses of biological samples.
Individual speciation
The most dif¢cult form of speciation analysis. Fractionation and separation techniques have played a particular role. Unique application of chromatography and coupled techniques in this area of speciation analysis.
Environmental pollution analyses. Food pollution analyses. Ecotoxicology.
pounds released into the aquatic environment, and may therefore create an ecotoxicological risk long after anthropogenic sources are banned from a given area. The monitoring of tin compounds is now required by EC legislation. Different countries have already established the limit values for various
Trace metals analysis ( soluble and suspended fraction ). Trace metals analysis of different forms present in soil and sediment after sequential extraction. Determination of tributyltin in sea water, sediments, tissue. Determination of methylmercury in tissue. Determination of chromium compounds, Cr(VI ). Determination of organic matter in samples by the assignation of summary parameters ( i.e., TOC in water or TH in air ). Determination of level of concentration of different forms of mercury ( elementary, inorganic and organic ). Determination of trace metals in blood serum and blood cells. Determination of heavy metals in plants. Identi¢cation and determination of chemical species de¢ned as to molecular, complex, electronic or nuclear structure.
species. The Dutch government has set the limit value at around 10 ng l31 TBT [ 8 ]. Speciation analysis can be performed in at least ¢ve different types, depending on the aim and scope of the analytical investigation. Brief characteristics of basic types of speciation analysis
Table 3 BCR three-stage sequential extraction scheme [ 17 ] Extraction step
Trace metal fraction
Composition of aquatic extract solution ( concentration in mol l31 )
1 2 3
Metals in soil solution, carbonates, exchangeable metals Iron / manganese oxyhydroxides Organic matter and sul¢des
CH3 COOH ( 0.11 ) NH2 OH.HCl ( 0.1 at pH 2 ) H2 O2 ( 8.8 ), then CH3 COONH4 ( 1.0 ) at pH 2
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and examples of their application are given in Table 2. Depending on their origin, trace elements exist in different mineral forms and chemical compounds, and in different combinations with mineral and organic components of soil and sediments which may vary according to various conditions: for example, pH has an in£uence on the trace-metal forms in which they exist. In acidic soils simple cations and complexes of chlorides and sulfates usually exist, while in neutral and slightly alkaline conditions carbonate complexes dominate.
4. Single and sequential extraction in speciation analysis Since the behaviour of the elements in a soil^ water^plant system depends on their form, the determination of trace metals in soils is often performed by single or sequential extraction. The procedures consist in subjecting a solid sample ( soil or sediment ) to successive attacks with reagents of different chemical properties ( acidity, redox potential, or complexing properties ) with each extract a part of the trace metals associated with the sample [ 10 ]. The best-known sequential extraction scheme
Fig. 1. Basic types of speciation analysis in the area of chemical analysis.
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is that of Tessier et al. [ 11 ]. This consists of ¢ve steps in which heavy metals are distributed among different phases ^ sorbed metals, carbonates, reducible and oxidisable substrates, extractable organics, sul¢des, and residual minerals. The ability to determine the chemical forms of heavy metals in marine and freshwater sediments is becoming increasingly important. It is also necessary to assess the potential of the sediments as either a sink or a source of heavy metals in the aquatic environment. To date, it has generally been accepted that the most appropriate way to evaluate the different phases in which radionuclides may occur is also based on sequential extraction schemes. As with heavy metals, sequential extraction schemes for radionuclides are based mainly on the procedure proposed by Tessier et al., and modi¢cations of it have been described in the literature subsequently [ 10,12^14 ]. The use of sequential extraction in connection with physical speciation enables one to de¢ne the metal forms available to plants. By de¢ning the speci¢c metals existing in soils which are accessible for plants, and the solubility of their metal forms, an indication of the danger of metal leaching to the groundwater is possible. It is necessary to stress that the results from such analyses can only be compared with the results from analyses carried out in a similar way for soil samples of similar character. The lack of uniformity in the different extraction procedures used does not allow the results to be compared world-wide or the procedures to be validated, which has led to many criticisms in the past. Indeed, the results obtained are highly dependent on the extraction procedures used, and the sample type. This actually requires that such optionally de¢ned procedures should be standardised [ 13 ]. A number of investigators have highlighted pitfalls in the use of sequential extraction, the most serious of which appear to be the poor selectivity of the reagents used and the redistribution of released metals between phases [ 15 ]. The necessity to harmonise the existing methods for speciation analysis of metals in samples in quite complicated matrices is important for the further development of speciation analysis ( with respect to the determination of extractable trace-element contents ). As part of recent attempts to harmonise methodology for leaching / extraction methods through the European Community, the BCR has developed a three-stage sequential extraction pro-
tocol in which metals are divided into acid-soluble / exchangeable, reducible, and oxidisable fractions [ 16 ]. In Table 3, common solutions for the threestage sequential extraction recommended by BCR are presented. The method has proved to be reproducible and gave good recoveries. Reference materials, certi¢ed for metals extractable by the Measurements and Testing Programme ( formerly BCR ) of the European Commission procedure, have been made available [ 16,17 ]. The use of certi¢ed reference materials ( CRMs ) is a widely recognised tool for the veri¢cation of the accuracy of analytical techniques used for speciation analysis. Recently BCR has organised a series of interlaboratory studies to evaluate extraction procedures and to certify reference materials for their extractable element contents [ 18 ].
5. Analytical methodology for speciation analysis The recognition of the fact that the determination of the level of a total metal or of a metalloid is not suf¢cient to evaluate its impact on the environment, its bioavailability and its toxicity has stimulated the development of species-selective analytical methodology during the last decade. For want of better speciation analysis, as with all types of analyses performed daily by analysts around the world, a special meaning attaches to the use of proper sample-preparation techniques. When during the sample-preparation steps a sample undergoes an incorrect ^ from the speciation point of view ^ operation, there is a danger of missing part of the information. For example, instead of the determination of individual ( particular ) chemical species and / or physical forms of an analysed element, it would only be possible to determine the total content of this element. A review of sample-preparation steps, and techniques used for the digestion and extraction carried out for speciation analyses of metals and metalloids, has been published recently [ 19 ]. At present, speciation analysis represents a great challenge for analysts working on the development of new procedures or on the practical application of existing methodologies to trace and microtrace components analyses. In many procedures used so far the recommended strategies permit only the determination of the total content of the selected element in a sample. On the other hand, it should be emphasised that many older procedures, espe-
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Table 4 Application of various analytical methods in metal speciation analysis Technique
Remarks
Application ^ examples
Gas chromatography
Gas chromatography is applied with different detectors, very often non-selective.
High-performance liquid chromatography
The most popular detectors (UV^Vis ) ^ due to their low sensitivity ^ are applied very rarely. Fluorescence or electrochemical detectors are used quite often. Reversed-phase LC.
Determination of alkyl-element compounds of metals and metalloids from the IVB ( Ge, Sn, Pb ), VB (Sb, As ) and VIB (Se ) groups of the periodic system. Separation of metalloporphyrins. The most frequent organometallic analyses: Pb and Hg in air, Pb in atmospheric deposits and urban dust, Sn in surface waters, Pb, Sn in sediments, Hg in biota. Speciation analysis of aluminium in water samples. Determination of Cr and V compounds with the application of spectrophotometric detectors. Determination of Hg( II ), methylmercury and methyl-, ethylmercury in wastewater and sediments. Analysis of selenomethionine in soil extracts and tablets. Metallothionein analysis in liver and kidney tissues. Determination of metal binding to proteins ( Al in blood serum, Se in human serum ) and metal detoxi¢cation in marine organisms ( Cd-, Cu-, Znthioneins in mussels, cyanobacteria ). Analysis of vanadyl and nickel porphyrins in crude oils. Ferritin, haemoglobin, myoglobin, cytochrome c ferritin in pharmaceutical products. Cr species in liver tissue. Cd-thionein analysis. Se( IV ), Se(VI ), TMSe determination in urine. Simultaneous separation of As( III ), As(V ), MMA, DMA and analysis in urine, seafood, natural waters, sediments and soil extracts. Se( IV ), Se(VI ), TMSe, acidic, neutral and basic organoselenium analysis in water samples. Arsenobetaine, arsenocholine, trimethylarsine oxide determination in seafood. Sn( II ), Sn( IV ), TBT in harbour water. Cr species in liver tissue and plasma. Hg( II ), MMHg, MEHg in tap water. Fairly comprehensive speciation analysis of As and Se compounds. Analysis of Hg in mussels. Separation of metallo-, phospho- and selenoproteins, metalloenzymes. Cd and Zn-thioneins in rabbit liver. Metallothionine analysis. Speciation analysis of tin.
Size-exclusion chromatography.
Anion-exchange chromatography.
Cation-exchange chromatography.
Electrophoresis
Technique is based on the separation in an electrical ¢eld and is realised in three basic modes: zone, isotachophoresis and isoelectric. The wider use of electrophoretic techniques is hampered by a lack of an element-speci¢c detector in the on-line mode.
Polarography and cathode and anode inversion voltamperometry
Techniques are used for the differentiation of elements at different oxidation states and in case of the analysis of samples containing humic and fulvic acids. Often used in laboratories in ocean-going scienti¢c ships. Ion-selective electrodes Used for the determination of elements at different Speciation analysis of Cd and Cu in the presence of oxidation states; quite low sensitivity. humic and fulvic acids. Determination of £uorides in the presence of aluminium. Electron auger spectroscopy Very rarely applied. Speciation of iron in physiological £uids in the and EPR presence of ascorbinians and oxygen. Nuclear magnetic resonance Although restricted sensitivity application area of the Speciation analysis of actinides. technique in quantitative analysis becomes more and Determination of Al( III ) compounds, hydroxy and more wide. other couplings of aluminium in geological samples. Complexion of aluminium with nucleosides.
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Table 5 Application of coupled techniques in speciation analysis of metals Technique
Comments on the coupling device
Possible problems
GC^MS
Usually heating of analyte transfer line is required.
Analytes should be volatile or after derivatisation before ¢nal determination.
LC^MS
Considerable numbers of interfaces are built up; the most common are particle beam, thermospray, atmospheric pressure chemical ionisation, electrospray. GC^AAS ( quartz GC^FAAS: usually narrow-bore tube furnace and between the two instruments, tube should graphite furnace ) be as short as possible to avoid peak broadening, transfer tube has to be heated to the highest temperature of the oven temperature programme. HPLC^FAAS Simple coupling. The end of capillary is connected with nebuliser. In case of ICP it is necessary to use device for the separation of analytes and mobile phase solvent.
HPLC^EF AAS
FIA^FAAS
Capillary electrophoresis^ FAAS Hydride generation^AAS GC^AES ( with plasmas ICP or MIP excitation source ) GC^ICP-MS
GC^ICP-AES
Examples of application
Determination of lead organic compounds in benzine, metalloporphyrins in petroleum, organotin compounds in environmental matrices. Restrictions concerning £ow rates and the Speciation of organotin composition ( e.g., additives ^ buffers ) of compounds. the mobile phase. Due to the sensitivity insuf¢cient for environmental analysis, a silica tube furnace electrically heated to the atomising temperature is needed ^ a feature that commercial furnaces do not have.
Suitable for organometals ( those of Pb, Sn, As, Se ). Determination of tributyltin in sea water. Determination of trimethyllead. The outlet of the column can contain Determination of organotin molecules which block nebuliser. Organic compounds in water, sedicompounds in the outlet can undergo ments and shell¢sh tissue. incomplete combustion in the burner Speciation analysis of As, Sb, ( blowpipe? ) and then block. Solvents Se, Hg, Pb, Cr. from the mobile phase can cause plasma extinguishing in ICP. The HPLC ef£uent is volatilised to an Limitations in the use of solvents and Determination of tin species. aerosol in a heated silica capillary and buffer solutions in the eluent ( buffers are Analysis of organolead comenters the furnace through a vitreous highly volatile ). Only well-separated spe- pounds. graphite tube. cies can be distinguished. Very simple coupling. The outlet of the column can contain Determination of inorganic molecules which block nebuliser. Organic Se and Cr compounds in compounds in the outlet can undergo water. incomplete combustion in the burner and then block. Solvents from the mobile phase can cause plasma extinguishing in ICP. From the technical point of view the couOnly several applications of pling is quite dif¢cult. The £ow rate of these techniques are mobile phase in CZE is usually not comreported in the literature due patible with the £ow rate of sample in AAS to the connection problems. spectrometry. Coupling relatively simple. This technique does not identify species Determination, with a very that do not give rise to volatile derivatives high sensitivity, of As, Se, Sn, during reduction Bi, Te, Sb, Pb and Hg species. The ef£uent from GC can be introduced Speciation analysis of orgadirectly into the £ame without extinguishnolead in rainwater, snow, ing it. harbour, river, lake and tap water. Speciation of Hg, Se, Sn. As for MIP, GC ef£uent is usually transSpeciation of Sn, Pb, Hg. ported to the plasma via heated line which Determination of Sb, Se, Sn, is either glass-lined or has the analytical Te, Hg, Pb, Bi in natural column passing through the centre. waters, land¢ll and fermentation gas after purge and trap preconcentration. Large plasma gas £ow rate causes dilution Low detection limits because the sensitiv- Speciation of Hg. of the analytes. ity of an atomic emission spectrometer does not overcome the dilution with the plasma gas; large dead volume; poor excitation / ionisation of non-metals.
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Table 5 ( continued ) Technique
Comments on the coupling device
Possible problems
Examples of application
LC^ICP-MS
The necessity to use nebuliser spray chamber ( plasma technique is sensitive to organic solvents present in LC ef£uents ). Different sample introduction techniques are investigated ( glass frit nebulisers, thermospray vaporisers, ultrasonic nebulisers ). As above.
Low ef¢ciency of sample nebulisation, which has a major effect on sensitivity. Instability of the plasma to organic vapours. Deposition of the carbon on the sampling cone and torch.
Speciation of organoarsenic and organochromium. Determination of tributyltin in sea water. Determination of organolead.
As above
Speciation of organoarsenic compounds. Determination of Cr( III ) and Cr(VI ). Determination of metalloprotein. Analysis of organotin compounds in marine mussel samples.
LC^ICP-AES
AAS: atomic absorption spectromety; EF AAS: electrothermal atomic absorption spectrometry ( graphite furnace ); FAAS: £ame atomic absorption spectrometry; MIP: microwave-induced plasma; ICP^AES: inductively coupled plasma atomic emission spectrometry; ICP^MS: inductively coupled plasma mass spectrometry.
cially those used for the determination of organic compounds, were used a long time ago, but without the name `speciation'. In the 1950s, when speciation analysis was connected only with biological cycles of metals in the hydrosphere, two forms of metals were distinguished: metals in soluble forms and metals adsorbed on suspended matter. It was enough to ¢lter the aqueous sample through a ¢lter with pore size 0.45 Wm to obtain the separation of the two phases. Later, owing to the development of electrochemical methods, the differentiation of miscellaneous existing forms of metals in soluble form ^ free metal ion and complex ions ^ became possible. Parallel investigations of the possible equilibrium state between ions and ligands ( organic and inorganic, also ) permitted the conclusion that in a water environment a very wide range of metal forms can appear. Now, speciation analysis includes not only metals, but also other elements in different types of samples [ 1,10 ]. In 1976 a detailed scheme for an analytical procedure for speciation of trace metals in water was proposed [ 20 ]. Seven possible chemical and physical forms presented in water were emphasised: õ õ õ
free metal ions; labile metals coupled with organic complexes; metals permanently coupled with organic complexes;
õ õ
õ õ
labile metals coupled with inorganic complexes; metals permanently coupled with inorganic complexes; metals absorbed in organic matter; metals absorbed in inorganic matter.
Now, according to this scheme, the determinations are performed of trace metal contents in different types of waters, with the application of measurements in the laboratory and in situ. Basic types of speciation analysis in the area of chemical analysis are shown in Fig. 1.
6. Application of different analytical techniques to speciation studies The successful approach for speciation analysis depends on two factors: selectivity ( to be sure of determining the proper species ) and sensitivity ( to match the analyte's level in the sample ). The breakthrough in terms of these two key issues was achieved using on-line coupling of chromatographic or electrophoresis technique with an element-selective detector ( atomic absorption, emission, £uorescence, or mass spectrometry, inductively coupled plasma, or microwaveinduced plasma ). The development of speciation techniques, especially the chromatographic ones, was the
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most important factor in the sudden evolution of speciation studies. Speciation analysis now uses the following separation techniques: gas chromatography ( GC ), supercritical £uid chromatography (SFC ), liquid chromatography ( partition, ionexchange, gel, af¢nity ), capillary zone electrophoresis ( CZE ), and ¢eld-£ow fractionation ( FFF ) [ 10,21,22 ]. For volatile, thermally stable species of various elements in a large variety of biological and environmental matrices GC-related analysis is the universally accepted approach. To change polar species into volatile ones which may be separated by GC, a derivatisation step is required. The widespread derivatisation procedures involve formation of volatile hydrides by borohydride reduction ( e.g., As, Sb, Se, Ge, Sn ). Another possibility is ethylation by sodium tetraethyl borate ( e.g., Sn ) or derivatisation by Grignard reagents ( e.g., Sn, Hg, Pb ) [ 10,21 ]. For non-volatile analytes various modes of liquid chromatography can be used, e.g., normal and reversed-phase liquid chromatography, ionexchange chromatography, size-exclusion chromatography. There are hardly limits to the application of other techniques such as SFC or electrophoresis, with special emphasis on CZE and FFF. There is no space to present here all the possible applications of the techniques applied to speciation analysis, so in Table 4 only selected examples of the application of the various analytical methods used in metal speciation analysis are shown. Excellent comprehensive reviews of the state of the art of analytical techniques employed for elemental speciation studies have been presented in the literature by several authors [ 10,21,23^25 ]. Coupled techniques play a particular role in speciation analysis. Chromatographic separation methods are hyphenated with extremely sensitive and selective detectors. The absolute detection limits of, e.g., LC^ICP^MS are in the sub-nanogram to picogram range. Absolute detection limits in picograms, even to the sub-pg range, are gained when GC, SFC and CE are coupled to ICP^MS [ 26 ]. Thus, coupled techniques have gained an important place in different areas of speciation analysis [ 26^ 30 ]. Speciation studies are contributing signi¢cantly to the progress of the life sciences, as is well re£ected by the continuously growing number of papers that has been produced in recent years. Only selected applications of coupled techniques applied to speciation analysis of metals described in the literature are presented in Table 5.
7. Conclusions There is no doubt that speciation analysis now offers a great challenge for analysts. The proper approach for the sequential extraction and application of appropriate analytical techniques and instruments can encourage wider use of speciation analysis in the laboratory. Elemental speciation information is crucial today because the toxicity and biological activity of many elements depend not only on their quantities, but also on their oxidation states and / or chemical forms. Thus speciation analysis can increase the information capacity of collected results.
References [ 1 ] D.M. Templeton, F. Ariese, R. Cornelis, L.-G. Danielsson, H. Huntan, H.P. van Leeuwen, Pure Appl. Chem. ( submitted ). [ 2 ] B.A.N. Popp, F.J. Sansone, T.M. Rust, D.A. Merritt, Anal. Chem. 67 ( 1995 ) 405. [ 3 ] M. Potin-Gautier, Analusis 25 ( 1997 ) M22. [ 4 ] G.A. Pedersen, E.M. Larsen, Fresenius' J. Anal. Chem. 358 ( 1997 ) 591. [ 5 ] I. Havezov, Fresenius' J. Anal. Chem. 355 ( 1996 ) 452. [ 6 ] L. Ebdon, S.J. Hill, C. Rivas, Trends Anal. Chem. 17 ( 1998 ) 277. [ 7 ] R. Jobinèski, Appl. Spectrosc. 51 ( 1997 ) 260A. [ 8 ] Ph. Quevauviller, O.F.X. Donard, in S. Caroli ( Editor ), Element Speciation in Bioinorganic Chemistry, Wiley, New York, 1996, 331. [ 9 ] S.J. de Mora, ( Editor ) Tributyltin: Case Study of an Environmental Contaminant, Cambridge University Press, Cambridge, 1996. [ 10 ] A. Ure, Ph. Quevauviller, H. Muntau, B. Griepink, Int. J. Environ. Anal. Chem. 51 ( 1993 ) 129. [ 11 ] A. Tessier, P.G.C. Campbell, N. Bisson, Anal. Chem. 51 ( 1979 ) 844. [ 12 ] U. Forstner, Int. J. Environ. Anal. Chem. 51 ( 1993 ) 5. [ 13 ] Ph. Quevauviller, Trends Anal. Chem. 17 ( 1998 ) 289. [ 14 ] M. Vidal, G. Rauret, Int. J. Environ. Anal. Chem. 51 ( 1993 ) 85. [ 15 ] C.M. Davidson, A.L. Duncan, D. Littlejohn, A.M. Ure, L.M. Garden, Anal. Chim. Acta 363 ( 1998 ) 45. [ 16 ] Ph. Quevauviller, G. Rauret, J.F. Lopez-Sanchez, R. Rubio, A. Ure, H. Muntan, Sci. Total Environ. 205 ( 1997 ) 223. [ 17 ] Ph. Quevauviller, Spectrochim. Acta Part B 53 ( 1998 ) 1261. [ 18 ] Ph. Quevauviller, Method Performance Studies for Speciation Analysis, Royal Society of Chemistry, Cambridge, 1998. [ 19 ] R. Jobinèski, Z. Marczenko, Spectrochemical Trace Analysis for Metals and Metalloids, Elsevier, Amsterdam, 1996. [ 20 ] G.E. Batley, T.M. Florence, Anal. Lett. 9 ( 1976 ) 379.
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[ 21 ] S. Caroli, in S. Caroli ( Editor ), Element Speciation in Bioinorganic Chemistry, Wiley, New York, 1996, p. 1. [ 22 ] R. Jobinèski, F.C. Adams, Spectrochim. Acta Part B. 52 ( 1997 ) 1865. [ 23 ] J.A.C. Broekaert, Mikrochim. Acta 120 ( 1995 ) 21. [ 24 ] R. Jobinèski, F.C. Adams, Trends Anal. Chem. 12 ( 1993 ) 41. [ 25 ] Y.K. Chau, Analyst ( London ) 117 ( 1992 ) 571.
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