of the remaining sample solution after matrix separation with a suitable internal standard.4 Aliquoting a small volume (2–10 μl) of the above solution on the center of a sample support and drying it under an IR lamp to produce a thin film specimen.
5 Loading of the specimen in the TXRF spectrometer to measure its spectra.
6 Processing of the TXRF spectra obtained: identification of the peaks, deconvolution of the interfering peaks, determining the intensity of the X‐ray lines, and conversion of the intensities to the concentration.
Figure 4.4 A schematic depiction of TXRF instrumentation for trace element analysis.
Figure 4.5 Schematic flow chart of sample preparation in TXRF analysis.
The above steps are shown in Figure 4.5. An aliquot of 2–10 μl of a sample and internal standard, mixed with each other thoroughly, is placed on a sample support and dried so that a thin film of the sample is formed. For trace element determinations in large matrix sample, the matrix separation is necessary because if the matrix is not separated, even 2 μl solutions shall form a thick deposit and produce matrix effects. In addition, the spectral background shall be very high. After selective separation of matrix, even a concentrated solution becomes very dilute, having analytes only predominantly in the solution. Normally, solvent extraction or anion exchange resin is used for such extraction. However, for biological samples, since the matrix is organic tissues made of low Z elements, the matrix separation is not required in most cases and the samples can be measured directly without any processing. This is a great advantage of TXRF for biological samples, as the possibility of samples being contaminated, or analyte being lost during complicated sample processing, as in other conventional techniques of analysis, is negligible. The X‐ray beams generally used for sample excitation in TXRF analysis are Rh Kα (20.216 keV), Mo Kα (17.479 keV), W Lα (8.396 keV) and W Lβ (9.671 keV). For low atomic number (Z) element analysis, low energy X‐rays, e.g. Rh Lα (2.698 keV), Mo Lα (2.295 keV), Αl Kα (1.487 keV), Cr Kα (5.415 keV) and Sc Kα (4.090 keV) etc. are preferred. The TXRF spectra are measured by keeping the samples in an ambient air atmosphere. However, for low Z elemental determinations, vacuum atmosphere sample chambers are required to avoid absorption of low Z elemental X‐ray lines in air atmosphere, and reduce the background due to scattering from the atmospheric air component atoms in the sample chamber and X‐ray beam path. A beam path of helium atmosphere between the detector and samples is also useful, especially to avoid interference of Ar Kα with the analytes as Ar is present (about 1%) in atmosphere. Using different sample excitation X‐ray sources and sample atmospheres (air, helium, or vacuum), elements from C to U can be determined by TXRF with different detection limits. Since the fluorescence yield of different elemental X‐ray lines are different, a correction for the sensitivity factors is required. The sensitivity values are defined as the ratio of intensity of a particular X‐ray line obtained from the TXRF spectrum and its concentration (or amount) deposited on the support. It may change with instrumental parameters such as X‐ray tube voltage and current, area of the sample exposed to the exciting X‐rays, as well as the area of the sample seen by the detector's solid angle on the sample. It is very difficult to make the sample area excited constant in each TXRF measurement and hence the sensitivity values of the elemental X‐ray lines measured changes with different specimens, even if the TXRF instrumental parameters are same. However, the change in the sensitivity values, due to the above reasons, is by a fixed factor for all the elements present in a particular sample during one measurement. This factor may change from one specimen to another. However, the ratio of sensitivity values of elemental X‐ray lines with respect to a particular elemental X‐ray line shall remain constant for all the specimens of a sample, if instrumental conditions are same. Due to this reason, a known amount of an internal standard, of an element which is not present in the sample, is added and the elemental X‐ray line`s sensitivities are determined with respect to this elemental X‐ray line intensity. The elemental X‐ray line intensities are normalized with the respective relative sensitivity values and the analyte concentrations are determined using the formula:
(4.3)
Where, C x and CIS are the TXRF‐determined concentration of analyte x and actual concentration of the internal standard (IS) added, respectively. N x and N IS are the area of the analyte (x) X‐ray line peak and area of internal standard X‐ray line peak (in counts) chosen for analysis, respectively. RS x is the relative sensitivity of the analyte X‐ray line with respect to the internal standard X‐ray line. Since the relative sensitivities are the ratio of the elements sensitivity (Sx) and the sensitivity of the internal standard (SIS), the relative sensitivity of the internal standard is one. This equation holds good as long as the instrumental parameters are the same and the matrix effects are negligible. Once the matrix effect becomes appreciable, the intensity of the X‐ray lines coming from the sample varies not only with the sensitivity but also due to the effect of the matrix on the analyte lines. The analysis results shall be erroneous in that case. From the above discussion, it is clear that this methodology of sample analysis is very simple and no matrix matched standards are required [8, 10].
4.5 Suitability of TXRF for Elemental Analysis in Biological Samples
Biological samples, e.g. blood, serum, urine, plasma, tissues, etc. contain more than 80% water and the residues left behind by the removal of water have mainly low Z elements like C, H, N, O, etc. and hence do not pose the risk of appreciable matrix effects. These samples can be analyzed by TXRF for trace elements using simple sample processing and mixing with a suitable internal standard. Other biological samples, e.g. dental plaque, hair, nails, bones, etc. can be analyzed either directly with a very small amount of sample giving no appreciable matrix effect, or after sample processing. As stated in the beginning of this chapter, the trace elements present in biological samples play an essential role in human health and their concentration level may be an indicator of good health or disease involved or a risk of certain diseases. The role of trace elements in biological progress in animals and plants has been investigated in detail and reported in several literatures. Some of the elements may enter into the biological system due to pollution or the working/living atmosphere of the human being. Also the determination of concentration of certain metals in parts of the body acts as a bio‐marker or indictor of some serious disease or disorder. Moreover, during treatment of cancer some carrier elements of medicines, e.g. platinum, may enter into the body system and their presence must be assessed in order to study the action mechanism of platinum‐containing drugs in the body. For these reasons, the determination of the trace elements in biological systems is very important. The techniques which can normally be used for such determinations are ICP‐MS, ICP‐OES, atomic absorption spectrometry with either flame (FAAS) or electro thermal atomization (ETAAS), etc. In order to avoid severe interferences, these techniques generally require the digestion of the sample and matrix separation. In addition, the matrix removal makes the solution used for analysis less viscous and free flowing during atomization for ICP‐OES and ICP‐MS analysis. Further, the sample requirement for ICP‐OES and ICP‐MS is a few ml of solution, which requires at least a few mg of biological sample. These samples can also be analyzed by TXRF with almost similar detection limits as by other techniques. However, TXRF has some advantages over these techniques, e.g. the sample requirement in TXRF is very small, all metals
