DISCUSSION:

There are several methods that can be use to analyze Selenium. The choice of method will depend largely on the laboratory facilities and the technical expertise available to the investigators, as well as, on the particular object of their study. The most widely used and generally more practical end-determinations currently available are discussed below.

A widely used method for the determination of Selenium in foods and other biological materials, especially in small-scale studies, is spectrofluorimetry. It is based on the reaction of Selenites with diamines to produce a piazoselenol that is fluorescent. It is a highly sensitive method and can measure selenium concentrations down to monogram quantities in many different biological matrices. It also has the advantage of requiring only small sample sizes. However, the method is somewhat cumbersome and requires careful supervision and has to a great extent been replaced by techniques such as hydride generation atomic absorption spectrophotometry (HGAAS) ⁹². 2.7.2.2 Atomic Absorption Spectophotometry

Atomic absorption spectrophotometry (ASS) in one of its various modes is the most commonly used technique for the determination of a wide range of trace elements, including Selenium, in biological materials. It has replaced the formerly, almost universally, used flame atomic emission spectrophotometry (FAES), except for the determination of alkali metals. Its popularity has largely been due to the relatively low cost of the instrumentation required and, possibly more, to the fact that it is easily learned by investigators who have not been formally trained as analysts.

Graphite furnace or electrothermal atomic absorption spectrophotometry (GFAAS/ETAAS) is a rapid and efficient method and allows determination of Selenium level in biological materials down to the microgram per gram range with ease⁴⁶. Problems can be experienced because of matrix interferences, particularly from the presence of phosphates, as well as from excessive volatilization. The use of hydride generation atomic absorption spectrophotometry will help to avoid such problems⁴⁷. An efficient form of background correction, such as Zeeman mode, is essential, especially with Graphite furnace atomic absorption spectrophotometry, for overcoming spectral interference^46,47.

A significant enhancement of analytical capabilities was achieved in the late 1980s with the replacement of the acetylene or other gas flame by a plasma discharge for atomization-excitation on flame atomic emission spectrophotometry and related instruments. The plasma consists of ions, electrons, and neutral particles formed from argon gas and operate at far higher temperatures than does the gas flames of flame atomic emission spectrophotometry. Inductively coupled plasma atomic/optical emission spectrophotometry allows simultaneous multi elements per sample. Early versions of Inductively coupled plasma atomic emission spectrophotometry lacked sufficient sensitivity to determine concentrations at the very low levels found in some biological samples ⁹².

2.7.2.4 Inductively Coupled Plasma Mass Spectrometry

The problem of relatively low sensitivity has been overcome by the coupling of Inductively coupled plasma with the techniques of mass spectrometry (MS) in inductively coupled plasma mass spectrometry (ICP-MS). This is a very powerful tool for trace and ultra-trace element analysis, capable, for example, of determining selenium down to levels of 10 pg/g, compared to microgram per gram achieved using graphite furnace atomic absorption spectrophotometry.

Unfortunately, the equipment is expensive and requires for its operation a level of technical expertise often unavailable in many research laboratories in developing countries. However, spare capacity makes it possible for large commercial and other laboratories that do possess inductively coupled plasma mass spectrometry to undertake analyses, usually for a fee, to meet the needs of less well-endowed establishments.

In inductively coupled plasma mass spectrometry the ions produced by the plasma are passed through a series of apertures (comes) into high-vacuum mass analyzer. The isotopes of the elements are identified by their mass/charge ratio and the intensity of a specific peak in the mass

spectrum is proportional to the amount of the element in the original sample. Several different methods of analysis can be employed, depending on the particular model of the instrument. The quaripole mass analyzer (PQ) is capable handling high-resolution mass analyzer (HRMA) provides higher sensitivities a higher mass resolution, but with a smaller throughput than PQ University Missouri Research Reactor Centre, 2004. A problem encountered in the determination of selenium using earlier versions of quaripole instruments was that the dimer from the ICP’s argon plasma, ⁴⁰Ar2, has the same mass as the most abundant selenium isotope, ⁸⁰Se, thus precluding measurement of this isotope¹²⁴.

2.7.2.5 Other methods

Several other well-tried instrumental methods of analysis for Selenium are available. They include instrumental neutron activation analysis (INAA), which has the advantage of requiring only minimal sample preparation. It is a powerful analytical technique, which has been shown to be especially useful for determining low levels of Selenium, for example, in human breast milk⁹². However, it requires access to sophisticated equipment, including a nuclear reactor.

Nevertheless, when these are available, even on a commercial basis, instrumental neutron activation analysis is recommended as a valuable quality control reference method against which routine laboratory methods can be evaluated (World Health Organization, 1987).

Other instrumental techniques, normally available only in well-endowed laboratories, include isotope dilution mass spectrometry (IDMS) and X-ray fluorescence, Hybrid systems, in which two or more analytical techniques are interfaced to enhance analytical capabilities, are also in use. In several of these, high-performance liquid chromatography (HPLC) is joined to atomic absorption spectrometry, Inductive couple plasma, mass spectrometry, and other procedures, for

performance liquid chromatography-inductive couple plasma-mass spectrometry. These are powerful analytical tools, especially useful in the determination of different chemical species of elements ^92,93.