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4 Total Reflection X‐Ray Fluorescence and it’s Suitability for Biological Samples

       N.L. Mishra1,2,* and Sangita Dhara1,2

       1 Fuel Chemistry Division, Bhabha Atomic Research Centre, Mumbai, India

       2 Homi Bhabha National Institute, Mumbai, India

4.1 Introduction

Determination of different elements in ultra‐trace and trace amounts in biological systems is of great importance. These elements may be essential, which are required in certain amounts for smooth biological functioning or may be non‐essential and responsible for certain types of diseases if present in body. The essential elements may be a part of some enzymes and affect the chemical interactions in biological systems [1]. Some elements such as cadmium (Cd), nickel (Ni), arsenic (As), beryllium (Be), and chromium (Cr) are carcinogenic and have toxic effects on human and animal health, whereas accumulation of some elements in trace amounts such as zinc (Zn), strontium (Sr), and lead (Pb) in cartilages affects the progression of arthritis [2–3]. Several types of minerals and trace essential elements such as Zn, Fe, Cu, etc. also act as catalytic or structural components of large biochemical molecules and are thus essential trace elements, whereas some metallic elements are used in dental and medical materials and devices [4]. In addition, some toxic elements get introduced into the biological system from contaminated food, polluted air or water and cause harmful effects to human beings or animals. Determinations of such elements are very important to correlate them with the observed biological diseases or effects. Some of the elements, present in drinking water, vegetables, and food products, are essential for human and animals, whereas some may be toxic to biological systems if taken in more than the specified quantities. The extent of toxicity and dietary requirement of these elements, for inherent dangers and good progression of human life, determines the specifications of the elements in various biological systems. In view of the role played by these elements in biological processes, the determination of concentration of these elements in above materials is very important. The classical methods used for such determinations are mostly destructive and damage the cell tissue samples. Moreover, they require a comparatively large amount of sample and are thus not well suited for analysis of biological samples which are normally available only in very small quantities. This is because getting large amount of such samples, even in mg levels, is difficult in most cases and destruction of the sample inhibits the study of distribution of trace elements and other properties in the biological cells. Moreover, the samples once used, especially in inductively coupled plasma with optical emission spectroscopy (ICP‐OES) and inductively coupled plasma mass spectrometric (ICP‐MS) analysis, are completely consumed and not available for analysis again, if required, for re‐checking in case of any doubt in the results. Therefore, a rapid and non‐destructive/non‐consumptive method of elemental analysis of biological samples is required. X‐ray fluorescence (XRF) analysis provides useful elemental information about specimens without causing specimen damage with simple sample preparations. The determination of the required elements in biological systems can be done in a non‐destructive manner using XRF [5, 6].

4.2 Advantages and Limitations of conventional XRF for Elemental Determinations in Biological Systems

The theory of XRF has been already described in earlier chapters of this book and elsewhere in literature [5, 6]. However, for the sake of continuity a brief description of it is being given in this chapter. When high energy X‐rays (energy >1 keV) fall on a sample, they cause ejection of the core‐shell electrons depending on their binding energies. Thus, electron vacancies are created and the atoms become unstable. The atom stabilizes itself by filling the vacancies by transition of electrons from outer shells to the vacant positions.However, the outer shell electrons moving to inner shell to fill the vacancies created by incident X‐rays leave vacancies in outer shells which in turn are filled further from next outer shell electrons. This activity starts a chain reaction to fill the vacancies, thus created by incident X‐rays and electrons moving to inner shells from outer shells. This process results in emission of photons having energies equal to the difference in binding energies of the two shells involved in such electron transitions. The photon energies emitted due to such transitions are in the X‐ray energy region and have systematic nomenclature. The energies of these X‐rays are characteristic of the the particular element involved and hence these are called characteristic X‐rays. The energies of the characteristic X‐rays are related to the atomic number of the element by Moseley's Law [5]. The intensities of the elemental X‐ray lines, thus emitted, are proportional to the elemental concentrations in ideal situations and give information about the concentration of the elements present in the samples. After excitation of the samples with the X‐rays, the samples do not get destroyed and remain in same form. For this reason, XRF analysis is considered a non‐destructive and non‐consumptive analytical technique, though it requires some additional sample preparation steps involving pelletization, dissolution, or bead making. The sample specimen after XRF analysis remains available for further investigation and can be used for further studies by other methods or can be reused for repeating XRF measurements, in case of some doubt in analysis. In addition to the elemental analysis, XRF can be used to find out the distribution of the elements in samples such as bones, hairs, nails, and cancerous and normal tissues using a very small X‐ray beam spot of size of a few μm (the technique is then called μ‐XRF). The distribution of elements in the body parts such as bones, hairs, nails, etc. during treatment through medicines can be studied using μ‐XRF. The above description shows that XRF analysis is very simple technique and can produce the several insights in biological samples as elaborate above. However, it has some limitations as well. It's first limitation is its inability to detect lower concentrations of elements in the ppb level.This is due to high background resulting from scattering of the X‐rays penetrating deep into the sample and emitted electrons loosing their energies in form of X‐rays coming as spectral background. Due to this limitation, XRF requires a comparatively larger amount of sample during analysis, which is not feasible always especially in case of precious, toxic, or scarce samples, such as forensic and biological samples. In addition, the XRF analysis gives inaccurate results in absence of matrix matching standards due to severe matrix effects resulting from deep penetration of X‐rays into the samples. These two limitations, i.e. higher detection limit and severe matrix effects, limit the applicability of XRF in studying the trace and ultra‐trace elements in biological samples.

4.3 Factors Limiting the Application of XRF for Biological Sample Analysis

      The main reason for the comparatively higher (poorer) detection limits observed in XRF, as stated above, is due to the higher spectral background in XRF. In energy dispersive X‐ray fluorescence (EDXRF), the X‐ray beam is made to fall on the sample at an angle of about 45°. The emitted X‐rays are detected by a detector which is also placed at an angle of 45° from the sample surface. This geometry, having