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....