X-Ray Fluorescence in Biological Sciences

One more difference between the techniques is that with an EDXRF system, the full spectrum is obtained virtually at once. So, a range of elements belonging to the periodic table can be determined simultaneously. With an WDXRF system, the spectrum has been procured by a series of discrete step, which is time‐consuming, and also expensive due to the restricted number of detectors.

XRF is a bulk technique with the analysis range varying from several millimeters to several centimeters. Inhomogeneous samples compacted into a pellet form and thereby make it little time consuming. Also it requires a large amount of sample material for the analysis. Many advancements have been made in the field of X‐ray optics that gave rise to originate to narrow X‐ray beams (1 mm to 10 μm). Such developments allow even a solo microscopic particle to be discretely analyzed for an explicit elemental image of high spatial resolution.

XRF [7] is based on an energy‐dispersive detection system. For the generation of precise and accurate elemental images consisting of thousands of pixels, a fast acquisition is needed at each and every pixel position. When using a simple WDXRF spectrometer, the scanning procedure is time consuming and does not support the imaging applications as provided by XRF. XRF has broad range of research applications including geology, mineralogy, gemology, archaeology, motor engineering, electronics, pharmaceutics, environmental studies, and biomedicine.

Owing to advancements in XRF spectrometry, EDXRF systems are used in combination with scanning electron microscopy () to determine elemental constituents at small scales. Importantly, XRF is used with synchrotron radiation sources (SRXRF) which is very similar to μ‐XRF that covers numerous applications.

There exist many analytical techniques such as XRF, μ‐XRF, SRXRF, total reflection X‐ray spectroscopy (TXRF), atomic absorption spectrometry (AAS), laser‐induced breakdown spectroscopy (LIBS), laser ablation inductively coupled plasma mass spectroscopy (LA‐ICP‐MS), inductively coupled plasma optical emission spectrometry (ICP‐OES), time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS), PIXE, etc. which are currently being used for elemental analysis of materials, including biological samples. Some important parameters that distinguish the analytical capabilities of the techniques such as elemental range, imaging possibility, depth resolution, and instrumental effort are summarized in Table 1.1[3,8–11].

All the techniques have their own advantages and different analytical capabilities that can be used to analyze different kinds of materials. Also, the instrumental efforts of these techniques are also different and thus, some techniques require more or less effort on the part of the operator. PIXE and synchrotron radiation‐XRF require high instrumental effort [3, 8, 9]. Additionally, complex sample handling is necessary in ultrahigh vacuum for Auger electron spectrometry, transmission electron microscopy (TEM), X‐ray photoelectron spectroscopy (XPS), and secondary ion mass spectrometry (SIMS) [3, 8]. On the other hand, a complex laser interaction with the samples occurs in LA‐ICP‐MS. However, a few methods with a restricted spatial resolution, such as conventional XRF or atomic emission spectroscopy (AES), are also available and are often used for elemental analysis in comparison to above techniques.

The methods discussed above produce similar information about the sample compositions and in most cases they provide complementary information. The utility of these techniques depends on their analytical performance and availability particularly their costs.

Figure 1.1shows a concise visual reference for comparing analytical techniques used for materials characterization, elemental analysis, evaluation surface analysis, and purity surveys etc. in terms of their detection limits and analytical resolutions.

ICP spectroscopy is the most popular analytical method to detect and quantify the chemical elements present in a given sample. This is based on the ionization of samples by extremely hot plasma, usually made from argon gas [4]. In the next Sections 1.3.2.1and 1.3.3, we have briefly discussed the working principles and analytical capabilities of two forms of ICP spectroscopy such as ICP‐MS and ICP‐AES.

ICP‐MS uses inductively coupled plasma to ionize the atoms [3, 4]. It is used to atomize the sample, creating polyatomic ions, which are then detected. The ions from the plasma are separated through a series of cones into a mass spectrometer, usually a quadrupole. The extraction of ions is based on their mass‐to‐charge ratio. An ionic signal is received by a detector which is correlated to the concentrations of atoms in the sample. These concentrations are obtained by drawing the calibration curves using some certified reference materials (CRMs).

It has the capability to investigate several metals as well as non‐metals in the liquefied samples at milligram to nanogram levels per liter. It is also used for isotopic analysis of the elements. This technique has been recognized as one of the most important analytical techniques in a variety of industries for the monitoring of impurities in semiconductor manufacturing, environmental monitoring, geochemical analysis, mining and metallurgy, pharmaceutical industries, and biomedical analysis. ICP‐MS has a good precision, sensitivity, and greater speed. The main disadvantage of ICP‐MS is that it produces many interfering species like component gases of air, argon from the plasma, and contamination from glassware and cones used.

Table 1.1 Overview of some analytical techniques including AAS, ICP, LIBS, XRF, μ‐XRF, SR‐XRF, and TOF‐SIMS [3,8–11].

Figure 1.1 A concise visual reference of most of the ring analytical techniques to compare the detection limits and analytical resolutions for materials characterization.