an inert atmosphere (nitrogen or argon) (Dyg et al., 1994;
Christensen et al., 1999). The shorter the time between collection and analysis, the better. Anyway, the reverse — namely, the oxidation of CrIII to CrVI — is most unlikely under usual conditions of storage and sample treatment.
The rule of thumb is that when no data are available from reliable studies by other research groups, the effect of sampling and storage conditions on the stability of the species in the matrix should be studied. Many species are thermodynamically unstable. The simple act of sampling and storing the species may alter them. The information is then irreversibly lost.
anticoagulant is added, normal blood withdrawn from the circulation forms a clot due to the polymerization of fibrinogen to fibrin; this process normally requires 5–15 min at room temperature. On standing, the clot retracts (packed cells expressing serum, which differs from plasma, in that it contains no fibrinogen). After centrifugation, serum may be decanted or drawn off with a pipette (Versieck & Cornelis, 1989). Centrifugation should be performed within 1 h after sampling the blood.
For speciation or fractionation purposes, it is preferable to collect the blood without anticoagulant. It is also inadvisable to add any preservative. First of all, both anticoagulant and preservative may contain impurities (e.g. a mercury compound). Secondly, both anticoagulant and preservatives may contain substances that are liable to break up the bonds between the elemental species and the serum matrix. Most anticoagulants are either polyanions (e.g.
heparin) or metal chelators and therefore have a high affinity for metal species. As a rule, neither heparinized samples nor ethylene- diaminetetraacetic acid (EDTA), citrate, or any other anticoagulant- doped samples may be used.
The spontaneous clotting of the blood lasts for about 15–30 min at room temperature. As mentioned, centrifugation should be com- pleted within 1 h. Haemolysed samples should never be considered for speciation purposes. The distribution of the trace element species between serum and packed cells is of a totally different nature and may also differ by several orders of magnitude. The concentrations in these two phases are controlled by different mechanisms.
Packed cells have to be lysed before any speciation study can be envisaged. This can be done by mixing one part of packed cells with one part of cold toluene and 40 parts of ice-cold water. The lysate is centrifuged at 15 000 × g at 40 °C and is then filtered through a 0.45-μm filter.
Urine will usually show a deposit some time after collection.
Substances that are dissolved at body temperature have a tendency to precipitate at lower temperatures. It is a matter of concern to find out if the species to be studied are also present in this precipitate.
Addition of preservatives or acidification (lowering of pH) cannot be considered as a general rule, because both steps are liable to alter
the species. Here again, a preliminary detailed study of the influence of a possible additive on the nature of the species is essential.
4.3.2 Preliminary treatment of tissues and plants
Sampling of tissues should be done according to a strict proto- col whereby contamination is excluded and species preservation guaranteed. Tools made of stainless steel are the preferred material for removal of the samples from the organism; however, with stainless steel containing 8–20% chromium, 8–12% nickel, and minor concentrations of cobalt and manganese, the risk of con- taminating the samples by contact with it cannot be neglected. Much better materials for knives from the point of view of eliminating contamination are polyethylene, polypropylene, Teflon, and quartz (Versieck et al., 1982). The listing of materials is not exhaustive, and the choice will be made on the basis of experimental data proving the validity of the sampling procedure. Chemical speciation of a trace element in tissues begins with the separation of the soluble species from those bound to insoluble compounds. The tissue is commonly subjected to a very harsh ultrasonic homogenization in isotonic phosphate-buffered (pH 7.35) saline solution using short bursts (10 s) in an ice-water bath. The homogenization can be considered sufficient when the power output on the homogenizer display decreases drastically. The homogenate is then centrifuged at 4 °C at 15 300 × g for 1 h. The supernatant is removed with a pipette. Soluble compounds trapped inside the precipitate are removed by washing 3 times with an equal volume of phosphate- buffered saline solution, followed by centrifugation of the suspended precipitate. The joined supernatants are further treated according to and hair, show high mechanical resistance due to the presence of fibrous, insoluble, structural proteins. Speciation of elements bound to insoluble compounds cannot be pursued any further.
In the case of tissue, the distribution of the trace elements can also be approached in a cytological way. This refers to the subcellular-level distribution of the trace elements between cytosol, mitochondria, and nucleus. An overview of the overall procedure is concentration can be measured in each step of these separations, in order to make up the balance, as a first check of the validity of the given in Figure 1 (Cornelis et al., 1998). The total trace element the procedures described in section 4.4. Some tissues, such as lung
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results. Chemical speciation of the cytosol can be done according to the procedure outlined in the following section.
4.3.3 Choice between low molecular mass and high molecular mass compounds
Once the preliminary sample preparation described in the pre- vious paragraphs is finished, the analyst has to opt for either low molecular mass or high molecular mass compounds. This is because a good separation within the low molecular mass group can be achieved only in the absence of high molecular mass compounds.
An elegant way to do this is through centrifugal ultrafiltration. The solution is held in a semipermeable membrane cone and subjected to a centrifugal force, typically at 800 × g. Those compounds with an effective radius smaller than the pore size of the membrane are pushed through it, whereas the other compounds are retained on the inner side of the membrane. The separation is characterized by the cut-off of the membrane, this being the maximum molar mass of the compound able to pass through the pores. If no precipitation or adsorption takes place, the concentrations of the compounds in the ultrafiltrate will equal those in the original sample. There are some difficulties that may arise during ultrafiltration, related to the ther- modynamic and kinetic stability of the chemical bond between a low molecular mass compound of a trace element and a protein. Indeed, a weak and easily dissociative chemical bond may break up during ultrafiltration, freeing more low molecular mass compounds than originally present.
The thermodynamic and kinetic aspects of centrifugal filtration have been discussed in detail by Whitlam & Brown (1981). By ultrafiltering only part of the sample (e.g. 10%), possible dissoci- ation of the protein–low molecular mass complexes is suppressed. It is advisable to work under a nitrogen blanket to create an inert envi- ronment, preferably in a cold room or with the equipment cooled down to a fixed temperature below the ambient temperature of the workplace.
4.3.4 Desalting
Desalting of the sample is necessary whenever the ionic strength of the solution does not fit that of the chromatographic separation.
Urine samples have an electrolyte composition that is very variable.
Supernatant of tissue samples may also display a salt content that is too high. Desalting of the solutions allows the analyst to work in controlled conditions. Gel filtration columns with a fractionation range of 1–5 kilodaltons are commercially available. If no over- loading of the column occurs, the sample protein fraction can be collected in the first fractions of the eluate, as low molecular mass compounds have a longer retention time. Eluents for desalting are first degassed and filtered through a Millex-HV13 filter with a pore size of 0.45 μm before use. Phosphate-buffered saline can serve as an eluent during the desalting run.
4.3.5 Sample cleanup
Most biological (clinical and food) samples have a fairly com- plex matrix due to the presence of amino acids, lipids, hydrocarbons, polysaccharides, etc. Cleanup procedures are therefore necessary to remove all those compounds that are of no interest for the purpose of the analysis and whose presence may even compromise the detection sensitivity of the analytical method (MuĖoz-Olivas &
Cámara, 2003).
One of the fastest methods to eliminate lipids is a mixture of chloroform and ethanol in such proportions that a miscible system is formed with the water suspension of the sample. Dilution with chloroform and water then separates the homogenate into two layers, the chloroform layer containing all the lipids and the ethanol–water layer all the non-lipids.
Some analyses require an extraction step in order to isolate and also enrich the analyte. The different types of extraction procedures will be described in the following section. It may be necessary to purify the extract. Solid-phase extraction with C18 cartridges pro- vides a very useful cleanup of the sample, ensuring the stability of the compounds. This has been illustrated in the case of selenium speciation (Wrobel et al., 2003).
4.3.6 Extraction procedures
Many species need to be purified from major matrix constituents prior to further separation and measurement. There exists a broad choice of extraction procedures, varying from simple
aqueous or solvent extraction to more complex methods, such as enzymatic extraction, solid-phase extraction, solid-phase micro- extraction, steam distillation, supercritical fluid extraction, liquid–
gas extraction (purge and trap), accelerated solvent extraction, and microwave-assisted extraction (MuĖoz-Olivas & Cámara, 2003).
Extensive research has been done on the extraction of various species of arsenic, copper, lead, mercury, selenium, tin, and zinc in biological matrices. A synopsis of a number of examples of ana- lytical procedures, including sample pretreatment for elemental speciation purposes, has recently been published by MuĖoz-Olivas
& Cámara (2003).
4.3.7 Preconcentration of the species
When the concentration of the analyte is very low, it is neces- sary to include a preconcentration step. This will often be followed by chromatographic separation. There are four main strategies described in the literature whose choice is determined by the chemi- cal characteristics of the species: amalgam formation, cold trap, high-temperature trap, and active charcoal retention.
In the case of mercury compounds, amalgam formation on a gold trap is most effective and widely applied. The cold trap method is used for derivatized species — i.e. elemental species that have been transformed into volatile compounds, such as tin, lead, and mercury species (Szpunar et al., 1996). The high-temperature trap has been used for arsenic in biological material (Ceulemans et al., 1993). Active charcoal retention has been employed for trapping non-polar volatile chelates (Heisterkamp & Adams, 1999).
4.3.8 Derivatization
To derivatize means to convert a chemical compound into a derivative — i.e. a new compound derived from the original one, for the purpose of identification. Derivatization of non-volatile organometallic species into volatile compounds, which are then separated with gas chromatography (GC), is common practice (Bouyssiere et al., 2003). The volatile derivatives need to retain the original moiety and be non-polar, volatile, and thermally stable.
Such derivatizations have been accomplished by hydride generation, with tetraalkyl(aryl) borates, with Grignard reagents, and even
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.