through the formation of volatile chelates, such as acetonates, tri- fluoroacetonates, and dithiocarbamates. There is also mention of the derivatization of selenoaminoacids, selenomethionine, and organic arsenicals with a variety of reagents (Bouyssiere et al., 2003).
Frequently, the derivatives are concentrated by cryotrapping or extraction into an organic solvent prior to injection on a GC column.
(Harrington et al., 1996), selenium species (Montes-Bayon et al., 2000), arsenic species (Ali & Jain, 2004; B’Hymer & Caruso, 2004), mercury species (Harrington, 2000), elemental species bound to proteins (Templeton, 2005), elemental species bound to humic acids (Heumann, 2005), etc. LC will surely remain the major separa- tion technique in the foreseeable future. Electronic databanks (e.g.
Web of Science) prove to be very helpful in putting together a procedure that is ideally suited for the combination of matrix, analyte, and the available infrastructure of the laboratory.
4.4.2 Gas chromatography
Only volatile and thermally stable species qualify for separation by GC. Very few compounds fulfil these requirements, but for- tunately the analyst can resort to chemical reactions that transform non-volatile compounds into volatile, thermally stable compounds.
This process is referred to as “derivatization” (García Alonso &
Naturally occurring volatile species include dimethylmercury [(CH3)2Hg)], dimethylselenium [(CH3)2Se], tetramethyltin [(CH3)4Sn], trimethylantimony [(CH3)3Sb], trimethylbismuth [(CH3)3Bi], methylated arsines, tetraalkylated lead compounds in sewage sludge, and many more gases from municipal waste disposal sites. This list is not exhaustive. Very interesting research on these compounds has been done by Feldmann (1997), who described innovative ways to convert non-volatile species into volatile species by derivatization techniques. Various separation schemes have been developed. Most common is the cryogenic trapping and sequential thermal desorption from packed columns. This method is not very selective, but unstable compounds can be preserved for a long time before analysis. Next comes GC on packed columns, offering an improved separation of the analytes through interaction with the column, combined with separation on the basis of their volatility (Szpunar et al., 1996). GC with capillary columns offers a much improved resolution. Their very small loading capacity forms the limiting factor for their exploitation.
The most common detector for this type of speciation is induc- tively coupled plasma mass spectrometry (ICP-MS), followed by inductively coupled plasma atomic emission spectrometry (ICP- AES). It is also possible to do isotope dilution measurements and Encinar, 2003) (see section 4.3.8 above).
isotopic ratio patterns when a high-resolution ICP-MS is available as the elemental detector.
4.4.3 Capillary electrophoresis
The principle of separation by CE is based on differences in the electrically driven mobility of charged analytes, similar to conven- tional electrophoresis. A high-voltage electrical field (typically 20–
30 kV) is applied along an open tube column with small internal diameter (Michalke, 2003).
This technique can be used as a primary or as a secondary separation technique, for example after HPLC, when it is referred to as a two-dimensional technique. Taking into account the very small loading capacity of CE, the two-dimensional approach will yield far more interesting results, bringing the high resolution of CE to its full potential. The system is often connected to ultraviolet (UV) detec- tion for molecular information, but also to ICP-MS or ES-MS for either elemental or molecular information.
There exist different separation modes in CE: capillary zone electrophoresis, with separation on the basis of the charge/mass ratio; capillary isoelectric focusing, based on the isoelectric point;
capillary isotachophoresis, based on analyte conductivity; and micel- lar electrokinetic capillary chromatography, based on hydropho- bicity.
CE analysis offers high resolution and high speed, and it is easily adaptable for automation and quantitative analysis. It has been successfully used for the speciation of many compounds (Alvarez- Llamas et al., 2005), among others CrIII/CrVI (Jung et al., 1997), selenium and arsenic compounds (Sun et al., 2004), selenium in human milk (Michalke, 2000), and copper, cadmium, and zinc in metallothionein (Profrock et al., 2003).
4.4.4 Gel electrophoresis
The field covered by GE for elemental speciation consists of charged macromolecules to which a metal or semi-metal is bound, covalently or not. These macromolecules can be proteins, humic acids, or DNA. There are practical limitations due to the small
amount of material that can be brought onto the gel, and, conse- quently, the limit of detection of the species. For protein separation, its resolution is unsurpassable (Chéry, 2003).
The first prerequisite during the separation procedure is again the preservation of the elemental species. This is not evident, con- sidering the nature of the many reagents needed to operate GE. For example, the contamination of the samples with platinum due to the release of platinum ions from the platinum electrodes during electro- phoresis completely falsifies the results when pursuing platinum speciation. Substitution of platinum by silver solves this problem when searching for platinum species (Lustig et al., 1999). Other critical parameters are the choice of buffer and pH.
When the metal is covalently bound, such as copper in caerulo- plasmin, denaturing conditions can be used during electrophoresis.
This is not the case for more weakly bound elements, for which non- denaturing conditions or native electrophoresis should be applied, in order to prevent the loss of the basic structure of the complex and stripping of the metal. Another factor that may even jeopardize the stability of strongly bound elements is oxidation of residues of proteins, as has been documented for selenoproteins (Chéry et al., 2001, 2005).
The method can be hyphenated with powerful detection meth- ods, such as laser ablation dynamic reaction cell ICP-MS for ele- mental detection (Chassaigne et al., 2004) or matrix-assisted laser desorption ionization MS for molecular detection. A more tedious way, but reliable for quantitative measurements, consists of cutting out zones of separated proteins in the gel and measuring the element off-line with ICP-MS, by using electrothermal vaporization as the sample introduction system (Chéry et al., 2002).
4.5 Sequential extraction schemes for the fractionation