Hyphenated Techniques in Speciation Analysis

By Joanna Szpunar, Ryszard Lobinski

The Royal Society of Chemistry

Copyright © 2003 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-545-7

Contents

Part I Principles and Fundamentals,
Chapter 1 The Concept of Speciation Analysis and Hyphenated Techniques, 2,
Chapter 2 Element Specific Detection in Chromatography, 14,
Chapter 3 Gas Chromatography with ICP MS Detection, 30,
Chapter 4 Liquid Chromatography with ICP MS Detection, 53,
Chapter 5 Electrophoretic Techniques with Element Selective Detection, 65,
Chapter 6 Electrospray Mass Spectrometry in Elemental Speciation Analysis, 76,
Chapter 7 Quality Control and Assurance in Speciation Analysis, 88,
Chapter 8 Multielement Analysis of Organometallic Species in the Environment, 100,
Chapter 9 Speciation of Organotin Compounds, 111,
Chapter 10 Speciation of Organolead Compounds, 122,
Chapter 11 Speciation of Organomercury Compounds, 129,
Chapter 12 Metal Speciation in Petroleum-Related Samples, 135,
Chapter 13 Speciation of Redox States, 143,
Chapter 14 Speciation of Organoarsenic Compounds in Biological Materials, 149,
Chapter 15 Speciation of Organoselenium Compounds in Biological Materials, 162,
Chapter 16 Speciation of Metal Complexes in Microorganisms, Plants and Food of Plant Origin, 179,
Chapter 17 Speciation of Metal Complexes with Metallothioneins, 189,
Chapter 18 Speciation of Metal Complexes in Human Body Fluids and Tissues, 200,
Chapter 19 Metal Speciation in Pharmacology: Metallodrugs, 209,

CHAPTER 1

The Concept of Speciation Analysis and Hyphenated Techniques

1 Introduction

The analysis of metal(loid) organic compounds has become increasingly important in the last decade because the organic species of some elements have turned out to be much more toxic than their inorganic forms. The origin of metal species can be either anthropogenic input (e.g. as pesticides) or a result of biological transformations of inorganic forms of elements by living organisms. The harmful effects of trace metal species were fully recognised after the death of 50 residents of the Minamata fishing village in Japan who had experienced the biomethylation of mercury in their everyday food. The spillage of tetraalkyl-lead in the Mediteranean due to the M/S Cavteat accident made the analytical community sensitive to organic forms of lead. Extinction of the oyster population in the Arcachon Bay in Southern France stimulated interest in the possible release of butyltins from antifouling paints. The above cases raised awareness of the importance of knowing the concentration of a particular species, defined the suspected analyte and analytical sample and, consequently, stimulated the development of analytical methodology.

It has also been widely recognised in biochemistry that the proper functioning of life is critically dependent on trace elements in a number of different ways. Some metals (e.g. Hg, Pb) and metalloids (As) are highly toxic whereas others (e.g. Mo, Mn, Fe, Co, Cu, Zn), considered essential, are needed for the accomplishment of life processes. A number of other elements (e.g. V, Cr, Ni) are recognised as being beneficial to life. From a chemical point of view the intake, accumulation, transport and storage of essential or toxic metals and metalloids are realised by surrounding the element ion by electron pair donating biological ligands. Sometimes this process is accompanied by the synthesis of specific ligands such as metallothioneins, or by the formation of a metalloid-carbon bond as in the case of selenoamino acids or organoarsenic compounds. Since the evolution of a metal in a living organism happens by its interaction with the highly complex coordinating environment and involves a number of species with different properties, information on the total elemental concentration in a biotissue is not sufficient and may even be misleading in understanding the metabolism, bioavailability and toxicity of a metal or a metalloid.

Classical analytical approaches based on high performance liquid chromatography (HPLC) with ultraviolet (UV) detection, and gas chromatography (GC) with flame ionisation (FID) or electron capture (ECD) detection have become insufficient to efficiently address the increasing challenges of metal species analysis in terms of species-specificity and sensitivity. The origins of modern speciation analyses are closely related to a wider use of element or molecule specific detection in chromatography and electrophoresis, the developments referred to as hyphenated techniques.

2 Speciation Analysis: The Definition

The term speciation was first introduced by biologists to describe the evolution of species. Referring to this evolutionary concept, geochemists and environmental chemists have often applied the word speciation to the transformations taking place during cycling of the elements. An example is the changes that occur between the leaching of trace elements from soil or rock and their subsequent distribution in the aquatic environment. The term speciation has been used since the 50s by aquatic geochemists to distinguish between ‘dissolved metal’ and ‘particulate metal’ in order to improve the understanding of the metal transport in waterways. A simple filtration through a 0.45 fim membrane allowed discrimination between the two different phases. The simultaneous and rapid development of electrochemistry enabled the analyst to distinguish between free and complexed metal species in the dissolved fraction.

This philosophy was developed by two well known works often cited in the context of environmental speciation analysis. One is Tessier’s sequential extraction scheme developed for the determination of the metal partitioning between the various mineral phases of environmental particulate material. The other is Florence’s scheme implying the differentiation between different metal fractions in water using preconcentration with Chelex-100 ion-exchange resin and final detection by electrochemistry or atomic absorption spectrometry (AAS).

The common feature of these works was the impossibility of distinguishing between individual metal-containing species at the molecular level. They dealt with metal fractions and not with metal species. Truly specific information was first acquired by Kolb who coupled chromatography for the separation of the species of interest with atomic spectrometry for the sensitive and selective detection of the metal. This concept was popularized by van Loon in an A-page Analytical Chemistry article in 1979 which initiated an exponential growth in the number of papers on speciation analysis.

In an attempt to end the confusion regarding the usage of the term speciation, an IUPAC Interdivisional Working Party recommended the use of this term to describe the distribution of an element amongst defined chemical species in a system. A species was defined as a form of an element specified as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure. According to the same definition speciation analysis denoted the analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample.

The term speciation is different from fractionation which describes the process of classification of an analyte or a group of analytes from a certain sample according to physical (e.g. size, solubility) or chemical (e.g. bonding, reactivity) properties.

3 Occurrence and Classification of Metal Species

The variety of chemical species of interest in different disciplines is shown schematically in Figure 1.1. Basically they can be divided into exogeneous species, such as environmental contaminants or metallodrugs and products of their degradation, and endogenous species, such as natural metabolites of arsenic or metal complexes with bioligands. From a chemical point of view elemental species can be divided into redox states, organometallic species (containing a covalent carbon-metal bond) and coordination complexes. The latter include simple (e.g. halide) or complex (e.g. citrate, tartrate, oxalate, phytate, amino acids, oligopeptides) ligands, macrocyclic chelating ligands (e.g. porphyrins), or macro-molecules (e.g. polypeptides, proteins, DNA restriction fragments, polysaccharides).

The principal classes of metal species of interest include:

• Volatile organometallics in air and landfill gases Volatile metal(loid) compounds have been identified in a variety of anthropogenic gases, including landfill gas and sewage sludge digester gas. These compounds are non-charged hydrides and/or methylated or alkylated compounds of main-group elements of groups 12 to 17. Compounds such as dimethyl-mercury (Me2Hg), dimethyl selenide (Me2Se), methyl-and butyltin, trimethylstibine (Me3Sb), trimethylbismuthine (Me3Bi), methylated arsines (MexAsHy, x + y = 3), dimethyltelluride (Me2Te), alkylated lead (EtxMeyPb, x + y = 4) have been identified in concentrations ranging from ng m-3 to µg m-3. Carbonyls of Ni, Mo and W have been found in sewage gas and landfill gas. Natural environments, such as hot springs rich in algae can also produce volatile metalloid compounds.

• Anthropogenic organometallic contaminants Organotin compounds, e.g. butyl- and phenyltins, are used in antifouling paints whereas tetraalkyllead compounds are still added in some countries as antiknock additives to petrol. The latter are being replaced by organomanganese additives, e.g. methylcyclopentadienylmanganese tricarbonyl. These tin and lead species, usually toxic, find their way into the environment raising the need for monitoring of both native species and products of their degradation.

• Natural organometallics in shale oils, natural gas and condensate Porphyr ins, ubiquitously present in natural energy resources, such as shale oil or coal, have the facility of binding many metals, e.g. V, Ni, Fe, Ga, Ti in thermally stable complexes. Their fate becomes of concern during burning and processing of these materials. Speciation of mercury and arsenic, which undergo alkylation reactions in natural gas, is of concern during processing oof gas and gas condensates

• Biosynthesized molecules with ‘true’ metal(metalloid) – carbon bonds This category includes selenoamino acids and their higher analogues: selenopeptides and selenoproteins. They can further coordinate metals with 5-affinity using the Se atom as the coordination center. Another important class includes organoarsenic compounds: methylarsonic acids, quaternary com pounds (e.g. arsenobetaine) and arsinoylriboside derivatives (arseno-sugars).

• Complexes with biosynthesized macrocyclic chelating agents The most important group is the analogues of tetrapyrrole which in their deprotonated forms can tightly bind even relatively labile divalent metal cations. The best known compounds of this group include chlorophyll and products of its degradation, cobalamins (the coenzymatically active forms of vitamin B12), and porphyrins including the heme group found in hemoglobin, myoglobin, cytochromes and peroxidases.

• Complexes with nucleobases, oligo- and polynucleotides, and -nucleosides Heterocyclic nucleobases, alone or as constituents of nucleosides or nucleotides, offer several different coordination sites for metal ions. Of particular interest is the coordination of metal ions e.g. CrO4- or inert metal complexes to DNA because of their specificity with regard to certain base-pair sequences in the double helix.

• Complexes with amino acids, oligopeptides and polypeptides (proteins) Metal complexes with proteins, including enzymes, are carriers of bio chemical function. Whereas the carboxamide function of peptide bonds -C(=O)-N(-H)- is only a poor metal coordination site, peptides contain several functional groups in the side chains that are particularly well suited for metal coordination. They include especially cysteine (-CH2SH) and methionine – CH2CH2SCH3, which bind metals with sulfur affinity (Cd, Cu, Zn), and histidine of which both nitrogen atoms become available for coordination after metal-induced deprotonation (e.g. Cu, Zn in superoxide dismutase). Peptide-complexed metal ions are known to perform a wide variety of specific functions (regulatory, storage, catalytic, transport) associated with life processes. The greatest interest has been attracted by essential elements associated with ferritin (Fe, Cu, Zn), β-amylase (Cu), alcohol dehydrogenase (Zn), carbonic anhydrase (Cu, Zn) and other proteins. Homeostatic control, metabolism and detoxification of toxic elements (e.g. Cd, Hg) by their interaction with metallothioneins (MTs) have been in the focus of ecotoxicology and clinical chemistry. Detoxification mechanisms of plants exposed to heavy metals involve the synthesis of small thiol peptides (phytochelatines) able to chelate heavy metals due to the high cysteine content in the molecule.

• Complexes with other biomacromolecules (polysaccharides, glycoproteins) Relatively little is known about the relevance of metal coordination to lipids and carbohydrates, although the potentially negatively charged oxygen functions can bind cations electrostatically and even undergo chelate coordination via polyhydroxy groups. The complexation of divalent cations with the carboxylic acid groups of uronic acids from plant cell wall polysaccharides (pectins) is well established.

• Exogeneous species: metallodrugs Platinum (cisplatin, carboplatin), Ru3+ (fac-RuCl3(NH) 3)3) and gold (auranofin) compounds are well-known in cancer therapy whereas some other gold compounds (aurithiomalate, aurothioglucose) are important antiarthritic drugs. A wide range of Tc compounds (e.g. Tc-labelled antibodies, Tc-mercaptoacetyl glycine complex) are used for diagnostic imaging of renal, cardiac and cerebral functions and of various forms of cancer. Gadolinium (III) polyaminopo-lycarboxylic crown complexes are widely used as magnetic resonance imaging contrast reagents. Some vanadium compounds are antidiabetic agents. The analytical challenges include both the identification of products of metallodrug metabolism and the understanding of the binding of metallodrugs to transport proteins and DNA fragments.

4 The Concept of Hyphenated Techniques

Interest in species-selective instrumental analysis was raised amongst the analytical community by gas chromatographers who first appreciated the advantages of an element selective detector in the mid 60s. As shown in Figure 1.2,it allows the elimination of the non-specific background and thus a considerable increase in the signal-to-background ratio. Peaks in the chromatograms are due to metal-containing species only and can be identified on the basis of the retention time. The detection limits can be decreased to femtogram levels in the case of gas chromatography, and subpicogram levels in liquid chromatography. The sensitivity offers a considerable advantage over classical biochemical separation/purification protocols prior to structural analysis by X-ray diffraction, nuclear magnetic resonance (NMR), Mossbauer spectroscopy, of electronic, vibrational or circular dichroism spectroscopy which require fairly large amounts of well purified analyte compounds, typically in the milligram range.

The term hyphenated techniques, introduced by Hirschfeld, refers to an online combination of a chromatographic (later also electrophoretic) separation technique with a sensitive and element-specific detector (usually an atomic absorption, emission or mass spectrometer). The approach gained the particular attention in the speciation analysis of environmental organometallic pollutants (organotin, organolead, organomercury) and redox states, and has been the subject of a number of status papers, edited works, and fundamental and comprehensive reviews. The lattest were directed to the separation component of the hyphenated techniques (gas chromatography (GC), supercritical fluid (SFC)or liquid chromatography (LC)), the detection component (inductively coupled plasma (ICP) atomic emission spectroscopy (AES) and ICP mass spectrometry (MS)), or a particular coupling such as GC-ICP MS, HPLC-ICP MS or capillary zone eletrophoresis (CZE)-ICP MS.

The various possibilities for the on-line coupling of a separation technique with an element (moiety, species) specific detector for bioinorganic speciation analysis include different types of HPLC or electrophoresis for separation, and atomic spectrometry (or molecular MS) for detection. The hyphenated techniques available for species-selective analysis are schematically shown in Figure 1.3. The presence of a metal incorporated or bound to an organic molecule in a sample is considered to be the prerequisite for using an element-specific detector. Nevertheless, some reports have indicated the possibility of employing a coupled technique for the analysis of a metal-free compound, provided that the latter is derivatised on-column or post-column by saturating the metal binding sites with a metal.

5 The Choice of a Hyphenated Technique

The choice of hyphenated technique depends primarily on the research objective. The separation component of the coupled system becomes of particular importance when the target species have similar physicochemical properties. It may even be necessary to combine two or more separation mechanisms in series to assure that a unique species arrives at the detector at a given time. The choice of detector component becomes crucial when the concentration of analyte species in the sample is very small and low limits of detection are required. An important problem is often the interface between chromatography and spectrometry as the separation conditions may be not compatible in terms of flow rate and mobile phase composition with those required by the detector.

Usually, chromatography and spectrometry can be coupled on-line. However, when a polyacrylamide gel electrophoresis (PAGE) technique is used, off-line detection of metal species carried out directly in the gel or after extraction (blotting) of proteins from the gel is necessary. Also, the preference for a highly sensitive discrete atomisation technique such as electrothermal atomic absorption spectroscopy (ETAAS) or electrothermal vapourisation (ETV) ICP MS may be the reason for choosing an off-line method of coupling.

Table 1 summarizes the selection of a hyphenated technique as a function of the application objective.

(Continues…)Excerpted from Hyphenated Techniques in Speciation Analysis by Joanna Szpunar, Ryszard Lobinski. Copyright © 2003 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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