During the last decade, substantial progress has been made in improving the sensitivity of the detection methods of commercially available equipment. The trend is to go for on-line, also called hyphenated, systems. The most convenient method for on-line coup- ling is ICP-MS. It is, unfortunately, the most expensive “detector”
for HPLC and other chromatographic systems, as well as for CE, especially when one considers that this multielement method will be used for the detection of only a single element. For economical reasons, it is certainly worthwhile to consider what other atomic spectrometric methods have to offer.
4.6.1 Atomic absorption spectrometry
The difficulty, if not the impossibility, of making flame atomic absorption spectrometry (AAS) and graphite furnace AAS on-line methods for measuring the elements in the fractions obtained during LC makes them less popular for elemental speciation purposes. They have, however, earned their merits in the field. Graphite furnace AAS has been used for the off-line measurement of elements in the
elution fractions of LC, although insufficient detection limits proved to be a serious drawback in the case of many clinical applications, where the concentrations of the elemental species in the biological fluids and tissues are very low (Zhang & Zhang, 2003).
When species can be converted to hydrides, such as is routinely done for mercury, selenium, arsenic, and antimony, then hydride generation AAS is a very interesting and cheap detection technique.
An on-line method was developed for the speciation of arsenic in human serum, including MMA, DMA, arsenobetaine, and arseno- choline. It has been applied for the speciation of arsenic in persons with abnormally high arsenic concentrations in serum, such as dialysis patients (Zhang et al., 1996, 1998). The method is based on cation exchange LC separation, UV photo-oxidation for sample digestion, and continuous hydride generation AAS for the measure- ment of arsenic in the LC eluent. By developing the technique of argon segmented flow in the post-column eluent, a substantial improvement in chromatographic resolution for the separation of these four arsenic species was obtained. The LC separation, photo- oxidation, hydride generation, and AAS measurement could be completed on-line within 10 min. The response is different for the different species. The detection limits (as arsenic) were 1.0, 1.3, 1.5, and 1.4 μg/l for MMA, DMA, arsenobetaine, and arsenocholine, respectively, in serum. The concentration of the four species was determined in serum samples of six patients with chronic renal insufficiency. Only arsenobetaine and DMA were significantly detected by this method. The main part of arsenic in human serum is arsenobetaine. No MMA, arsenocholine, or inorganic arsenic were detected in these six samples.
AAS with a quartz tube atomizer is a very sensitive, specific, rugged, and comparatively inexpensive detector for GC. GC coupled with AAS has been described as a sensitive instrumentation for mercury speciation (Emteborg et al., 1996). On-line solid-phase extraction coupled to graphite furnace AAS has also been explored.
Cold vapour AAS is the most widely used technique for mea- suring mercury. Direct coupling of solid-phase microextraction and quartz tube AAS has been used for selective and sensitive determination of methylmercury in seafood (Fragueiro et al., 2004).
4.6.2 Atomic fluorescence spectrometry
When species can be converted into hydrides, such as is rou- tinely the case for mercury, selenium, arsenic, and antimony species, then atomic fluorescence spectrometry becomes a very economical elemental detection technique. It is, however, necessary to keep in mind that the conversion of elemental species into hydrides is not occurring to the same extent and at the same rate for all species.
This has been documented, for example, in the case of arsenic. The conversion of methylated arsenic species into methylated hydrides gives a different response than the conversion of inorganic arsenite or arsenate to AsH3 (Zhang et al., 1996).
4.6.3 Atomic emission spectrometry
ICP-AES is the most common technique for emission spectrom- etry. It is sometimes referred to as ICP–optical emission spectrom- etry (OES). This method offers in principle the advantage of being multielemental, although in the case of elemental speciation, it will usually be used as a single-element detector. It is easy to couple on- line with LC because it can accept a continuous flow of eluent. The disadvantages are the overall inefficiency of the nebulizer and the plasma’s sensitivity to organic solvents. The poor tolerance of the plasma source to common mobile phases, such as ion pair reagents, limits the applicability of the technique. The fact that many ion exchange chromatography elutions are not isocratic (i.e. the elution is effected under variable, usually increasing, ionic strength) requires special protocols to circumvent the problem of varying analyte response during the elution (Zhang & Zhang, 2003).
4.6.4 Inductively coupled plasma mass spectrometry
ICP-MS is based on the measurement of m/z ratios. It offers extremely low detection limits for nearly all elements. This is due to the very high degree of atomization in the plasma at about 7000 K.
This extreme temperature makes it far superior as an atomization source than the graphite furnace for AAS with temperatures at only 2000 °C. There exist problems of spectral interference. For instance, when measuring 52Cr, mass 52 will experience interference from the isobars of 40Ar12C+ and35Cl16OH+, because the resolution is limited toǻm/m = 1 (Vanhaecke & Köllensperger, 2003).
Today there exist two major tools to reduce these interferences to a negligible level. The first is the dynamic reaction cell, which allows chemical reactions in a collision cell so that the interfering isobars are neutralized or the analyte is transformed into another, heavier polyatomic compound. Another very reliable, but very expensive, tool to eliminate isobaric interferences is high-resolution ICP-MS with ǻm/m from 1/4000 to 1/10 000 (Houk, 2003).
HPLC works well on-line with ICP-MS. Similar difficulties due to the influence of the eluent on the plasma can be anticipated and need careful consideration, as mentioned in the previous section on ICP-AES.
An alternative way for sample introduction is solid sampling electrothermal vaporization, followed by ICP-MS detection. An interesting application is the direct determination of methylmercury and inorganic mercury in fish tissue with non-specific isotope dilution (Gelaude et al., 2002).
4.6.5 Plasma source time-of-flight mass spectrometry
Plasma source time-of-flight MS is a powerful tool for elemen- tal speciation analysis through the use of a modulated or pulsed ionization source that provides both atomic and molecular fragmen- tation information (Leach et al., 2003). Its use has been documented for the analysis of, among others, organotin compounds and the oxidation states of various elements.
4.6.6 Glow discharge plasmas as tunable sources for elemental speciation
Glow discharge plasmas offer a number of interesting possibil- ities as speciation detectors for gaseous and liquid sample analysis (Marcus, 2003). The plasma works at sufficiently low temperatures (kinetic temperatures in the range of 100–500 K) so as not to induce dissociation in molecular species. Detection can be achieved by OES or MS. The technique has been successfully applied for the speciation of, for example, organotin compounds.
4.6.7 Electrospray mass spectrometry
ES-MS offers soft ionization of metal-containing species fol- lowed by tandem MS for the precise determination of the molecular mass of the original species and that of the individual fragments.
This method is ideal for obtaining structural molecular information about the species. An extensive sample cleanup is needed in order to obtain high sensitivities (Chassaigne, 2003).
The method allows the coupling of HPLC on-line, on condition of using a suitable eluent. This method has been successfully applied for the speciation of organo-arsenicals and selenium species, identi- fication of metallothioneins, etc.
4.6.8 Electrochemical methods
Electrochemical methods are based on the measurement of elec- trical signals associated with the molecular properties or interfacial processes of chemical species (Town et al., 2003). Owing to the direct transformation of the desired chemical information (concen- tration, activity) into an electrical signal (potential, current, resistance, capacity) by the methods themselves, they are easy and cheap. The two major difficulties in the application of electro- analytical techniques to complex real-world samples have been the lack of selectivity of electrochemistry and the susceptibility of the electrode surface to fouling by surface-active materials in the sample. A variety of electroanalytical techniques that differ in the mode of excitation signal–response characteristics are currently being used: potentiometry, fixed-potential methods, amperometry, various forms of voltammetry and electrochemical detection in LC, and flow-injection analysis. These methods have been applied for the quantification of various oxidation states of an element (FeIII/FeII, CrVI/CrIII, TlIII/TlI, SnIV/SnII, MnIV/MnII, SbV/SbIII, AsV/AsIII, and SeVI/SeIV), its organometallic species, or metal com- plexes in equilibrium with each other (e.g. butyltin species in surface water from a harbour by adsorptive stripping voltammetry with tropolone). The ideal is to perform in situ measurements with minimal sample perturbation. Despite many difficulties, well known to specialists in the field, the sensitivity of electroanalytical methods makes them very powerful tools for many applications.