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At present, different types of XRF systems are commercially available. The selection of the most suitable spectrometer is based on the demands for a given purpose. Table 2.1 present a summary of the features and analytical capabilities of XRF systems used in vegetation sample analysis. More detailed information about basic principles and components of XRF spectrometers can be found elsewhere [5].

Table 2.1 Features and analytical capabilities of XRF systems used in vegetation sample analysis.

Feature	WDXRF	2D‐EDXRF	3D‐EDXRF	Portable EDXRF	TXRF	μ‐EDXRF
Elements (L: low Z, M: Mid Z, H: High Z)	L, M, H	L, M, H	L, M, H	M, H	M, H	L, M, H
Amount of sample	few g	few g	few g	n.a.a	few mg/μl	n.a.a
Bulk analysis	Yes	Yes	Yes	Yes	Yes	No
Element distribution information	No	No	No	No	No	Yes
Limits of detection	mg/kg‐μg/kg	mg/kg	mg/kg‐μg/kg	mg/kg	μg/kg‐ng/kg	ng‐pg
Critical moving parts	Crystals	None	Targets	No	Incidence angle	No
Portability (in situ analysis)	No	No	No	Yes	No	No
Capital costs	High	Medium	High	Low	High/medium	Medium
Consumables	Medium	Low	Medium	Low	Low	Low/medium

^a n.a.: not applicable (direct analysis of the solid sample).

WDXRF: Wavelength dispersive XRF, 2D‐EDXRF: Two‐dimensional energy dispersive XRF, 3D‐EDXRF: Three‐dimensional energy dispersive XRF or polarized energy dispersive XRF, TXRF: total reflection XRF, μ‐EDXRF: micro‐energy dispersive XRF.

Usually in WDXRF spectrometers the analysis of different elements is carried out in a sequential way by changing synchronously the orientation of the goniometer‐controlled detection system to catch the discrete wavelengths corresponding to each chemical element. Therefore, they are not usually employed for multi‐elemental analysis of unknown samples and their use in vegetation samples analysis is not very common. Nevertheless, one of the benefits of WDXRF systems is the possibility to accurately determine light elements such as P, S, Cl which can play an important role in vegetation metabolism and are difficult to determine with other atomic spectroscopic techniques. For instance, WDXRF was used by Barua and co‐workers [6] to determine the concentration of P, K, S, Ca, Fe, Mg, Cl, and Na in seeds of chili for nutritional purposes. Other applications include the determination of specific elements (Nd, Pb, Th, and U) in fungi [7] or the combined determination of light and some trace elements in vegetation species collected in mining environments [3]. In general, quantitative analysis by WDXRF is performed by using the empirical calibration method. However, it is sometimes difficult to get sufficient reference materials with matrices similar to the target samples. In those cases, synthetic standards made of spiked cellulose with the elements of interest could provide a good option to simulate the vegetal matrix and to produce suitable calibration curves for quantification purposes [3].

Unlike WDXRF systems, conventional 2D‐EDXRF spectrometers only consist of two basic units, the excitation source and the detection system. Using this configuration, all the X‐rays emitted by the samples are collected at the same time in the detector and thus, 2D‐EDXRF systems can provide simultaneous multi‐elemental information of the sample. For this reason 2D‐EDXRF are widely used in environmental sample analysis, including vegetation samples [8]. Moreover, since the development of lower power ceramic micro‐focus X‐ray tubes and air‐cooled silicon drift detectors, low‐cost benchtop and even portable 2D‐EDXRF systems are commercially available and have been successfully used in the field of vegetation analysis [9, 10]. Nevertheless one of the main drawbacks of 2D‐EDXRF systems is the limited sensitivity for trace and ultra trace elements including some important environmentally‐relevant metals such as Pb and Cd. This is mostly due to the high spectral background arising from the elevated degree of scattering of the X‐ray beam by light organic matrices. This background can be decreased very significantly by using polarized EDXRF systems in which a secondary target is interposed between the X‐ray tube and the sample configuring a Cartesian geometry (tri‐axial, 3D) between source, sample and detector. With this geometry, a significant decrease of the background is achieved because the exciting radiation is polarized when scatter occurs through the right angle and cannot then be scattered a second time into the detector. Therefore, the sensitivity and detection limits for minor and trace elements are improved as compared with those associated with conventional 2D‐EDXRF spectrometers. In Figure 2.2, as an example, spectra obtained in the analysis of a leaf sample using a 2D‐EDXRF spectrometer (W X‐ray tube) and a 3D‐EDXRF spectrometer (W X‐ray tube and Mo secondary target) are displayed. An additional advantage of the use of secondary targets is that they can be used to modify the energies of the beam impinging the samples but in a simpler way. With the right target, we can selectively excite the elements of the sample and, sometimes, act as a near‐monochromatic secondary source of somewhat higher energy than the absorption edges of the analytical lines of the requested analytes.

Figure 2.2 Spectra acquired in the analysis of a leaf sample collected in a contaminated mining area using (a) 2D‐EDXRF system(W X‐ray tube) and (b) 3D‐EDXRF system(W X‐ray tube and Mo secondary target).

Therefore, with the combination of polarization and the use of different secondary targets, a system can be designed to produce a range of excitation conditions adequate for different groups of elements. For example, for the determination of light elements (Na, Mg, Al, Si, P, and S) in spruce needles it was adequate to use a highly oriented pyrolytic graphite (HOPG) crystal as a secondary target [11]. Meanwhile, for determination of elements such as Pb, Fe, Cu, and Zn in different vegetation specimens, a secondary target made of Zr proved to be a good choice [12]. Targets made of pure metals (i.e. Mo, Zr, Co) have proven to be adequate for the excitation of specific elements or a reduced group of neighboring elements. The use of an Al₂O₃ target (Barkla scatter) in a 3D‐EDXRF system was also successful for the determination of low amounts of Cd (<1 mg/kg) in vegetation samples. However, in this application the use of a Gd X‐ray tube and a Ge semiconductor detector was necessary in order to allow the determination of Cd through the Cd‐K lines overcoming in this way the reduced sensitivity and the spectral interferences issues that occur when using L lines for the determination of heavy elements such as Cd [12].

In the last 20 years, technological improvements in XRF instrumentation have allowed an enhancement of analytical capabilities as well as the commercialization of portable systems, opening up interesting applications. In this sense, the development of miniature X‐ray tubes to substitute radioactive isotope sources (i.e. ⁵⁵Fe, ¹⁰⁹Cd, ²⁴¹Am) in portable‐XRF systems (p‐EDXRF) was a successful insight [5]. It is also interesting to highlight the use of source modifiers (primary filters composed of different metal layers) between the X‐ray source and the sample in some hand‐held units to improve limits of detection for elements of interest. In a study by Marguí and co‐workers [13] it was demonstrated that for Ni, Cu, Zn, and Pb determination, the best results, in terms of signal‐to‐noise ratio, were obtained using a filter composed of (25 μm Ti) + (300 μm Al) + (150 μm Cu) meanwhile for Cd determination a filter composed of (25 μm Ti) + (200 μm Al) + (75 μm Cu) was the best choice. p‐EDXRF instrumentation also present low sensitivity for light elements due to the attenuation of low energy fluorescence X‐rays by air. To overcome this problem, some portable instruments are equipped with a partial vacuum device (Analytical Methods Committee, Royal Society of Chemistry 2008) or measurements can be also performed under a helium atmosphere. This last approach was applied for instance in plant nutrient analysis using a portable XRF analyzer [14].

The main advantage of p‐EDXRF is the possibility to get real‐time information in the field through in‐situ analysis with limited operational costs in comparison to laboratory 2D‐EDXRF units. This fact has led to the widespread adoption of p‐EDXRF systems by governmental agencies, environmental consultancies, and research institutions for multi‐elemental analysis of environmental samples in the last years [15–17]. Despite the fact that handheld systems have been mostly employed for the rapid in‐situ analysis of soils and sediments to facilitate elemental mapping at the field scale, their application for multi‐elemental analysis of plant materials has also been documented. For instance, p‐EDXRF was employed to scan a large set of vegetation matrices (i.e. thatch, deciduous leaves, grasses, tree bark, and herbaceous plants) to study the potential metal contamination of a smelter‐impacted area [18]. Hand‐held units also proved to be very effective in providing useful data for plant nutrition status, crop requirements, and potential deficiencies, leading to more effective fertilization programs. In fact, a recent study demonstrated a similar analytical performance between laboratory 2D‐EDXRF and p‐EDXRF systems with regards to multi‐elemental (P, K, Ca, S, Fe, Mn, and Si) analysis of sugar cane varieties [19].

In addition to the aforementioned XRF configurations, there are other X‐ray systems which have important roles in special applications in the field of vegetation analysis. A good example for that is for instance the use of total reflection X‐ray spectrometry (TXRF) for analysis of vegetal mass‐limited samples. To perform analysis under total reflection conditions, samples must be provided as thin films by deposition of a small amount of sample (few μg or μl) on a reflector. For this reason, TXRF is especially suited when the amount of vegetation sample available to perform the analysis is scarce such as xylem sap [20]. Another interesting application, which has been the topic of research of some scientific contributions, is the use of TXRF for the determination of minor and trace elements in biofilms [21]. In addition to trace metals, carbon content is also important to determine for a better understanding of the biofilm's growth. However, to detect such a low atomic number element, a specially designed TXRF spectrometer with a Cr X‐ray source, a vacuum chamber, and a detector with an ultra‐thin window is required [22]. It is interesting to note that, in the last years, other publications have highlighted the potential of TXRF as a reliable technique for multi‐elemental analysis of other types of samples such as mosses [23] and vegetal foodstuff [24].

In some studies, in addition to elemental determination content in vegetation samples is also of significance to get information about the element distribution and location within vegetal tissues. For this purpose, imaging techniques with an adequate lateral resolution are required, such as μ‐EDXRF. A major advantage of μ‐EDXRF over many other microscope techniques is the possibility of sample analysis with minimal sample preparation. For instance, the possibility of analyzing non‐conductive samples without the need of a vacuum has been exploited in nearly all areas of plant science.

Usually, most of the studies dealing with μ‐EDXRF in plant sciences are using the combination of high‐performance X‐ray micro‐focusing optics with high‐brilliance synchrotron radiation (SR‐μ‐XRF). Using this approach, very fast bulk analysis on small areas (spot smaller than 1 mm) with very low detection limits are assessed, allowing the investigation of different aspects of plant sciences (i.e. plant physiology, morphology, ecology, biochemistry, etc.), even at the cellular level, as it has been reported by Vijayan and colleagues [25]. Analytical methods for elemental mapping in vegetation tissues using laboratory μ‐EDXRF systems have also been described despite the limited resolution and sensibility in comparison with that achievable with SR [26]. Nevertheless, since the development and use of polycapillary focusing optics in laboratory μ‐EDXRF systems, spot sizes less than 25 μm for Mo‐K lines are possible with a reasonable sensibility. This fact has promoted their use in different applications such as the mapping of macro and micro nutrients in biofortified wheat grains [27] as well as in carrot sections grown in soils irrigated with municipal treated wastewater [10]. In a recent contribution, Fittschen and co‐workers [28] used a laboratory‐made μ‐XRF spectrometer with a special sample holder developed by 3D printing for in vivo μ‐XRF measurements in vegetation samples. The spot size was less than 14 μm at Rh Kα line (20.214 keV) and detection limits were similar to those obtained in a previous work performed at a second generation synchrotron facility.