X-ray fluorescence (XRF) is a useful technique in agricultural science to determine the elemental constituents of plant materials. It allows for quick analysis, high spatial resolution, measurement of multiple elements within a single scan, and requires minimal sample preparation while being nondestructive. In this study, we used wavelength dispersive XRF (WDXRF) to study agricultural diseases. We performed a spectroscopic investigation in agricultural samples to measure the elemental changes caused by infestation. A broad range of major, trace, and heavy elements were detected and quantified in healthy and infested crop samples, and we observed significant changes in the elemental concentrations. We also used laser-induced breakdown spectroscopy (LIBS) in combination with XRF spectrometry. Further improvements will provide exciting perspectives for the application of XRF in the area of plant and agricultural science.

It has been observed that approximately 14 essential elements are needed for the proper growth and development of plants, along with water, hydrogen, oxygen, and carbon dioxide. These nutrient elements are further categorized as macronutrients and micronutrients. Macronutrients, such as calcium, magnesium, nitrogen, potassium, phosphorus, and sulfur, are needed by plants in large amounts (>1000 mg per kg dry weight), whereas micronutrients, including boron, chlorine, copper, iron, manganese, molybdenum, nickel, and zinc, are required by plants in small amounts (<100 mg per kg dry weight) (2). Even though plants require only small amounts of micronutrients, these nutrients are equally important for the proper growth and development of plants.

Trace element detection is important in plant science. The concentration of trace mineral elements in plants, as well as in the soil, can change, and this variability can lead to deficiencies or excess accumulation of these mineral metals (3). Knowledge of the concentrations of trace elements in plants is important for diagnosis of various agricultural crop diseases. Crop disease development depends on three main factors: a susceptible host plant; favorable environmental conditions; and the pathogen involved, sometimes displayed as a “disease triangle” (Figure 1). Environmental conditions, in particular, play a major role in the occurrence and the growth and development of plant diseases; many diseases will not emerge if environmental conditions, such as temperature, relative humidity, soil moisture, soil pH, rain, and water content, are not favorable.

Atomic spectroscopy methods are the first choice for assessing the mineral composition of agricultural crop samples. For most atomic spectroscopic techniques, samples preparation is required, such as sample dissolution using a mixture of strong acids (generally nitric and sulfuric) and oxidants (commonly hydrogen peroxide). In many cases, it is easy to dissolve the samples in the solution. Because of the presence of silicon in plant samples, sample preparation usually requires a mechanisms that ensure complete decomposition, to ensure efficient dissolution of metallic elements into solution. Hydrofluoric acid addition, along with evaporation for achieving dryness, is needed to complete the proper dissolution of the analyte; otherwise, the presence of silicon may result in poor recoveries of some elements due to the binding of analytes with the insoluble residues (4).

Proposed methods found in the literature, from large numbers of acid mixtures used in wet washing procedures, reveal a lack of consensus for achieving the complete dissolution of plant specimens. The choice of decomposition method for a given plant sample material must be evaluated carefully, considering each of the matrices and analytes under study. In reviewing these challenges of sample digestion, analytical methods that do not require matrix destruction can be advantageous for such studies.

Nearly all X-ray fluorescence (XRF) techniques have desirable features for the analysis of plant samples. These features include the capability to carry out direct analysis of solid samples, multielemental analysis, the capability to carry out the qualitative analysis along with quantitative determination, a broad dynamic range, high throughput, and low maintenance and analysis cost. The major drawback of XRF is its limited sensitivity for elements like cadmium and lead. The poor precision and accuracy of XRF compared to other atomic absorption spectroscopic techniques is also a limitation. However, some recent improvements, such as the advancement of digital processing-based spectrometers, along with better detector designs in XRF instrumentation, have increased instrumental sensitivity and improved both the precision and productivity of XRF. These improvements have increased interest in adopting XRF spectroscopy in environmental science for analyzing various plant materials. The use of different quantification strategies employing distinct configurations of XRF instrumentation for the analysis of various vegetation matrices has been discussed.

Identification of Nematode Species in Okra and Papaya

We have collected healthy and nematode-infested okra root samples, which are shown in Figures 2a and 2b, from field sites of Uttar Pradesh, India. We have also collected nematode-infested and healthy papaya root samples, as depicted in Figure 2c. After collection, the samples were washed, and after drying the samples, they were milled into a fine powder for spectroscopic studies.

Cobb’s sieving and decanting methods were used to draw out the body parts of male, female, and juvenile nematodes to identify the species of nematodes in the diseased okra and papaya plant root samples. We adopted the procedure for species identification described by Goodey (1963) (5), and the species was identified on the basis of the characteristics observed in the perineal regions of the female nematodes by employing the method provided by Eisenback and others (1985) (6). Our examinations revealed that the species of root-knot nematode pathogens in the diseased okra and papaya roots was Meloidogyne incognita. Details regarding the sample preparation and identification of nematode species have been described by Sharma and others (7,8).

Collection and Isolation of Rice Samples (Healthy and Infected with False Smut)

A survey was conducted in rice fields suspected of false smut infestation in Uttar Pradesh, India, during the Kharib season of 2018–2020. Healthy rice grains and rice grains infested with false smut disease were collected (Figure 2d). The rice grain samples were carefully separated along with smutty balls from rice spikelets and milled into a fine powder form. The upper surface of smutty balls was sterilized by dipping them in a solution of 1% sodium hypochlorite for approximately 1 min and then cleaning with 70% ethanol solution for about 1 min. More details regarding the isolation of false smut disease pathogens from rice grains has been described by Sharma and others (9).

Study of Root-Knot Nematode–Infected Okra Plant Roots

In the present study, we employed wavelength dispersive X-ray fluorescence (WDXRF) spectroscopy to examine in healthy and nematode-diseased okra root samples. We detected many elements, such as aluminum, calcium, copper, chromium, chlorine, iron, manganese, magnesium, nickel, phosphorus, potassium, rubidium, sodium, silicon, strontium, sulfur, titanium, zinc, and zirconium. We noticed significant differences in the elemental composition of healthy and diseased samples. Figure 3 shows WDXRF spectra of healthy and nematode-infested okra roots in the energy range of 5–10 keV, which shows the relative abundance of the elements chromium, manganese, iron, nickel, zinc, and copper.

Several mineral elements—particularly calcium, chlorine, magnesium, potassium, sodium, and sulfur—were found to have higher concentrations in nematode-infested okra plant samples compared to healthy root samples. Other elements, including copper, phosphorus, manganese, silicon, and zinc, were found to be in lower concentrations in nematode-infected root samples as compared to healthy okra root samples. Some toxic elements, such as aluminum and chromium, were observed in lower concentrations in the infested root samples. No significant differences in the concentration of elements like bromine, rubidium, nickel, and zirconium were observed. Some traces of silver were found only in nematode-infected okra root samples, whereas traces of palladium were found only in nematode-infested root samples.

Our experimental findings were supported by the literature and other published reports. The observation of lower concentrations of iron, phosphorus, and zinc in nematode-infected okra root samples aligns with results reported by Farahat and others (10). Lower concentrations of iron, phosphorus, and zinc also have been observed in the diseased tissues of nematode-infected citrus, eggplant, jasmine, cowpea, and papaya plants (10). Sharma and others have also witnessed the decreasing concentration of phosphorus in nematode-infested okra root samples as compared to root samples from healthy okra plants (11). We also observed a higher concentration of sodium and a lower concentration of phosphorus in the nematode-infested okra root samples, which also aligns with results reported by Sharma and others (11).

Some nonessential elements, such as silver and palladium, were detected only in infected root samples, which indicates that their presence may be associated with the formation and development of nematodic pathogens on okra roots. Elements like strontium and titanium were also observed in increased concentrations in nematode-infested okra root samples. Heavy elements such as aluminum, chromium, and titanium were also observed in higher concentrations in nematode-diseased root samples of okra plants, and appears to be related to the formation of galls on the surface of roots with the invasion of pathogens. The higher concentration of aluminum can be considered a threat to agricultural crops because aluminum obstructs root growth and disturbs nutritional balance, encouraging the induction of oxidative stress. This stress leads to peroxidation reactions in the cytoplasmic membrane and ultimately leads to cell death (12). The physiological and biochemical functions of chromium in plants is not particularly clear (13). It has been observed that excessive levels of chromium can encourage several detrimental biochemical, morphological, and physiological effects on plant health (13,14). Nevertheless, the overall toxicity of chromium in plants is well documented (13). All these elemental changes are capable of posing serious and toxic effects in plants such as hindering growth mechanisms, disturbing photosynthesis, translocating mineral nutrients, disrupting the water intake capacity, and ultimately declining the overall quality of the okra plant.

Study of Root-Knot Nematode–Infected Papaya Plant Roots

We have also studied root-knot nematode-infected papaya plant root samples employing WDXRF to obtain the elemental variations between healthy and diseased papaya root samples. Using WDXRF, numerous elements, such as aluminum, chlorine, copper, chromium, calcium, magnesium, manganese, iron, potassium, sodium, phosphorus, nickel, silicon, rubidium, sulfur, and zinc were detected in root samples of healthy and diseased papaya plants (8). Figures 4a and 4b show the overlapping WDXRF spectra of healthy and diseased papaya root samples within different energy ranges, indicating the presence of elements such as aluminum, calcium, and potassium. The WDXRF spectra of papaya root samples showed that concentration of mineral elements changed with nematode infestation. It has been documented that the induction of antioxidants, creation of mechanical obstructions, and triggering of defense systems in plant bodies are all associated with good accessibility of mineral nutrients in plants (15). It was observed that the concentrations of one major nutrient, calcium, increased from 2.4 to 3.14% from healthy to diseased root samples, whereas another major element, magnesium, was found to increase from 0.39 in healthy root samples to 0.46% in diseased samples.

The concentration of silicon was found to be elevated from 0.23 to 0.53% from uninfected to nematode-infected papaya root samples. Sharma and others also observed the same elevated concentration patterns of calcium, magnesium, and silicon while performing elemental and molecular studies of nematode-diseased okra root samples (7). It has been observed that the elements calcium, magnesium, and silicon are the major nutrients that are beneficial in maintaining the outer coating of cell membranes and thereby helping plant cells defend against pathogen invasions. The increasing concentrations of calcium, magnesium, and silicon seen in our findings can be correlated with the induction and activation of defense systems in response to the attack of pathogens. In addition to these elements, we also found increasing concentrations of iron, aluminum, and titanium in papaya plants with the arrival of nematodic infection. The particular role of aluminum and chromium in the physiological and biochemical behavior of plants has not been determined yet.

Macroelements, such as potassium and phosphorus, are essential and play various roles, like activating enzymatic systems, inducing protein synthesis, managing photosynthesis processes, moving stomata, maintaining balance between cations and anions, and transferring energy (8). It has been observed from WDXRF analysis of healthy and diseased papaya root samples that the concentrations of these two macroelements decreased with the arrival of nematodes and fungal infections on papaya plants. It is well reported that potassium and phosphorus encourage the thickening of cell walls and thereby induce barriers that hinder the further movement of the attacking pathogens inside the plant body (8).

Decreasing concentrations of some other elements, such as copper, manganese, sodium, and zinc, were observed in the papaya root samples with the arrival of a nematode attack. Similar results were also reported by Sharma and others. Traces of the elements zirconium and molybdenum were found in the diseased papaya root samples only, also similar to observations made by Sharma and others (7).

The relative concentrations of the elements calcium, magnesium, silicon, potassium, sodium, and iron, are shown in Figure 5. Changes in the concentrations of several of these elements can lead to serious consequences for the health and viability of papaya plants.

We have also validated our experimental findings using laser-induced breakdown spectroscopy (LIBS), as shown in Figure 5. For a LIBS method that is free from self-absorption, it has been observed that the intensity of the elemental characteristic line is more closely related to the concentrations of the sample analytes (8). To confirm that there is no self-absorption in the LIBS method, we have verified all the conditions, such as the existence of stoichiometric ablation, local thermal equilibrium, and optical thickness of the plasma. Furthermore, we have calculated the peak area intensity ratios of certain elements like calcium (393.36 nm), potassium (766.48 nm), magnesium (279.55 nm), sodium (588.99 nm), iron (238.40 nm), and silicon (288.15 nm) from the LIBS results of papaya plant samples. These calculated values were further taken as the concentrations of these elements and directly compared with the concentration values of the WDXRF spectra of papaya plants. Given that LIBS and WDXRF follow different criteria, to compare the results of the techniques, we focused only on the trends we observed for each element in the papaya plant samples, which are clearly indicated in Figure 5.

Figures 5a and 5b show bar graphs of the results obtained using LIBS and WDXRF and reveals the patterns of the concentrations of major elements such as calcium, sodium, iron, magnesium, potassium, and silicon in the diseased and healthy papaya root samples. Because the same trends of the element concentrations were observed by both techniques for the analysis of papaya plant samples, we believe that both techniques can be considered proficient methods for assessing the composition of mineral elements in healthy and diseased plant samples, both qualitatively and quantitatively.

Study of Rice Crop Infected by False Smut Disease

WDXRF spectroscopy was also employed for the spectroscopic investigation of rice grains from rice crops infected with false smut disease. The WDXRF spectra of healthy and diseased rice grains were recorded within different energy ranges. Changes in the elemental composition of the rice grains associated with black and yellow smutty fungal spores on the upper and lower surface of the rice spikelets, were investigated. For the sake of simplicity, we have encoded our samples R1, R2, and R3 on the basis of severity of false smut disease, where R1 stands for uninfected rice grain samples, R2 stands for rice grain samples with low levels of infection, and R3 for highly infested rice grain samples.

Using WDXRF spectroscopy, we have quantified the presence of numerous essential and non-essential elements in rice samples, including calcium, aluminum, copper, iron, chlorine, magnesium, manganese, nickel, phosphorus, chromium, potassium, rubidium, silicon, sulfur, and zinc. Figures 6a and 6b show the overlapping WDXRF spectra for the different rice grain samples, indicating the relative presence of phosphorus, silicon, sulfur, chromium, manganese, iron, nickel, copper, and zinc in the various rice grain samples (R1, R2, and R3). We also verified our results using LIBS. The detailed results of similar studies have been discussed by Sharma and others (9).

The trends of relative concentrations of mineral nutrients observed using LIBS and WDXRF were similar. Using LIBS and WDXRF, we have observed the pattern for copper, silicon, and calcium as R1 > R2 > R3, whereas for chromium, iron, and magnesium, the trends was R2 > R1 > R3. Some mineral elements, such as potassium and manganese, followed patterns of R3 > R2 > R1 and R2 > R3 > R1. From these studies, it was concluded that the two techniques are complementary to each other and can be used collectively for investigating elemental changes that may take place in the composition of the plant samples with the association of various plant diseases.

As mentioned previously, it is documented in the literature that the induction of some mechanical obstructions, production of anti-oxidant compounds, and triggering of defense systems in the plant body all are correlated with good accessibility of the important mineral nutrients in the plant body (9). In our study, we have observed that some major elements, such as potassium, phosphorus, and sulfur, were present in higher concentrations in the diseased rice grain samples, and it is feasible to relate these increases with the production, induction, and activation of various defense systems that hinder the further movement of the pathogens into the plants body. Sieverding and others have also reported increased concentrations of potassium and phosphorus in corn crop plants following inoculation with 

 (16). The concentration values of the nutrient element sulfur increased from 0.14 in uninfected rice grains to 0.2% in diseased rice grain samples.

We also noticed significant increases in the concentrations of chlorine and manganese in diseased rice grain samples. The increased level of chlorine can be related with the initiation and activation of defense responses against fungal spores (15). Identical increasing trends were also seen and reported by other researchers (7,17). In one study, where an elevated concentration of manganese was observed in the barley plant samples, the author claimed that such elevation is because of the stimulation of manganese content with the association of microbes present in the rhizosphere of soil (18). The same patterns corresponding to elements such as chlorine, manganese, potassium, phosphorus, nickel, and sulfur has been seen and reported by Singh and others in studies of nematode-infested wheat grains by WDXRF spectroscopy (17).

In our studies, we also observed lower concentrations of aluminum, calcium, copper, iron, magnesium, silicon, and zinc with the infestation of black and yellow smutty fungal spores in rice. The decreased level of magnesium was also seen and reported by Bateman and others while doing investigations of beans from plants infected with the fungus 

 (19). Because major elements such as calcium and magnesium are related with the maintenance of outer cell walls, the decreasing levels of these elements may be associated with the weakening and diminishing of cell walls, leading to escape of sugars and amino acids from plant tissues and thereby attracting the attention of several fungal species. Imbalances in the contents of the macroelements such as calcium, potassium, and magnesium, were also reported with the occurrence of green stem and foliar retention (GSFR) disease on the plant samples (9).

Rice plants are well-known for their capacity to accumulate high levels of silicon from the soil (20). In our findings, we noticed a decrease in the content of silicon with the association of smutty balls in the rice crop plant. The content of silicon falls from 1.27% in uninfected rice grains to 0.45% in diseased rice grain samples with the occurrence of fungal disease. The role of silicon is similar to that of calcium and magnesium: It is known for maintaining the strength of the outer cell walls, so a decreased level of silicon indicates the diminishing of the outer layer of cell walls (21).

We also saw lower concentrations of aluminum, copper, iron, and zinc in diseased rice grain samples with the arrival of fungal diseases on rice crop plants. The particular role of aluminum and chromium elements in the physiological and biological behavior of plants has not been determined yet. In our findings, we have observed that the level of aluminum decreases from 0.03 to 0.01% with the occurrence of fungal disease in rice crop plants.

The element chromium is known for its toxicity and causes various detrimental effects on the physiological behavior of a plant system, such as disturbing the uptake capacity of mineral nutrients and water intake, inducing oxidative stresses, interfering the photosynthesis mechanism, and causing plant cell death (9). These toxic effects of chromium begin with the association of 0.0005% of chromium in the agronomic plants (9). The concentration of chromium in our results rose from 0.0011 in uninfected rice grains to 0.0046% in rice grain samples infected with fungal disease. Similar results for chromium were observed by Singh and others (17) in spectroscopic investigations of nematode- and fungal-infested wheat seeds and by Sharma and others (7) in nematode- and fungal-infested okra root samples.

It is well-documented in the literature that the pathogens started developing strategies to minimize its defense systems and make them totally depriving of basic nutrients after intruding the host body whereas the host body in response makes some changes in its internal composition to restrict further movement of the pathogens into the plant body (9). Thus, the decreasing and increasing contents of some nutrient elements with the occurrence of smutty fungal spores on the surface of rice spikelets due to the arrival of false smut disease can be considered the consequences and responses of the host body with the attack of pathogens.

In conclusion, we present a summary of the plant species, disease, and elements detected in healthy and diseased plant samples in Table I. Elements whose concentrations increased and decreased because of the disease are listed in Table I. Table I indicates the observable changes in the concentrations of elements, related to the diseases that occurred in the plant samples.

In the present work, we explored potential applications of WDXRF for analyzing important agricultural crops, which provided valuable information regarding their elemental composition. This information is then used to characterize their behavior after infestation by plant diseases such as nematodes and false smut disease.

However, elemental imaging techniques, such as scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), field emission (FE)-SEM, and micro-XRF, would provide additional information regarding the role of heavy and toxic elements in these plant diseases. These methods would enable a useful visualization and mapping of the distribution of organic matter as well as the changes in the mineral elements on a micrometer scale. Also, more exhaustive work on the multivariate analysis of WDXRF data will provide more information related to these kinds of diseases (nematodes, false smut and other bacterial and fungal diseases) in agricultural crops.

Ms. Neha Sharma (Senior Research Fellow, SRF) is thankful to DST, Government of India for INSPIRE Fellowship (IF160893). Author Vivek K. Singh is thankful to Shri Mata Vaishno Devi University, India, for all kinds of support to carry out this research.

Encyclopaedia of Life Support Systems (EOLSS)

(2) L. Trevizan, D. Santos, R. Samad, N. Vieira, C. Nomura, L. Nunes, L. Rufini, and F. Krug, 

(3) G. Dalcorso, A.Manara, S. Piasentin, and A. Furini, 

(4) E. Margui, M. Hidalgo, and I. Queralt, 

(5) J.B. Godey, Laboratory Methods for Work with Soil and Plant Nematodes (Technical Bulletin Ministry of Agriculture, London, United Kingdom, 1963). 

(7) N. Sharma, Y. Khajuria, J. Sharma, D.K. Tripathi, D.K. Chauhan, V.K. Singh, V. Kumar, and V.K. Singh, 

(8) N. Sharma, Y. Khajuria, V.K. Singh, S. Kumar, Y. Lee, P.K. Rai, and V.K. Singh, 

(9) N. Sharma, Kamni, V.K. Singh, S. Kumar, Y. Lee, P.K. Rai, and V.K. Singh, 

(10) A.A. Farahat, A. Al-Sayed, and N.A. Mahfoud, 

(12) H. Brhane, E. Fikru, T. Haileselassie, and K. Tesfaye, 

(13) L. Reale, F. Ferranti, S. Mantilacci, M. Corboli, S. Aversa, F. Landucci, C. Baldisserotto, L. Ferroni, S. Pancaldi, and R. Venanzoni, 

(16) E. Sieverding, G.A. Silva, R. Berndt, and F. Oehl, 

(17) V.K. Singh, A. Devi, S. Pathania, V. Kumar, D.K. Tripathi, S. Sharma, D.K. Chauhan, et al, 

(20) E. Takahashi, J.F. Ma, and Y. Miyake, 

(21) D. Bhaduri, R. Rakshit, and K. Chakraborty, 

 are with the School of Physics at Shri Mata Vaishno Devi University, in Katra, India. 

 is with the Department of Plant Pathology in the College of Agriculture at Banda University of Agriculture and Technology, in Banda, Uttar Pradesh, India. Direct correspondence to: 

Articles

In these studies, wavelength dispersive X-ray fluorescence (WDXRF) was used to examine differences in the elemental composition of agricultural samples, comparing healthy and diseased samples of okra, papaya, and rice. Both the mineral nutrient profiles (macro and micronutrients) and toxic metals were examined, revealing common patterns.

Application of Wavelength Dispersive X-ray Fluorescence to Agricultural Disease Research

Lamellar structures are common in many polymeric materials and biological tissues. These structures are characterized by alternating layers of different compositions or densities that act as electron density gratings that diffract X-rays, giving rise to reflections at small scattering angles. This paper discusses the characteristics of these small-angle X-ray scattering patterns, relate these parameters to the features of the lamellar structure, and describe the analyses of these features to investigate deformations at the microstructural length scales. Structural models generated using known principles of lamellar assembly and evolution can be refined by comparing the simulated and the observed diffraction patterns. The method is illustrated using data from semicrystalline polymers where the contrast arises from the electron density differences between the crystalline lamellae and the amorphous segments. Use of these data to study the deformation of lamellae under strain is presented.

The fundamental structural unit in many crystallizable polymers is the lamella, consisting of tightly packed polymer chains. Lamellar structures also occur in block copolymers, in amphiphilic molecules such as surfactants and lipids, and in the smectic and discotic phases of liquid crystals. Lamellae also occur in biological structures—for example, lipids are packed as a liquid crystalline bilayer in cell membranes, on skin, and in the myelin sheath around nerve fibers, and collagen fibrils are organized into lamellar stacks in the cornea. This article discusses the basic characteristics of lamellar structures, and describes how disorders in these structures manifest in small-angle X-ray scattering (SAXS) patterns. We use the data obtained during deformation to illustrate the use of SAXS patterns.

Lamellar phases are 3D layered structures. The scattering pattern in the lamellar stacks arises from the differences in electron density between the lamellae and the interlamellar segments. Lamellar structures have 1D quasi-long-range order normal to the surface of the lamellae; because of this reduced dimensionality, they have unique physical properties and challenging theoretical descriptions. The methods used to investigate lamellar structures are quite different from those used for studying the more common 3D structures, and the more recent 2D structures, such as graphene and ultrathin layers of transition metal chalcogenides and MXenes (1,2).

Characteristics of the SAXS Patterns from Lamellar Structures Lamellae arranged in a stack form a diffraction grating. In oriented materials that are discussed here, the stacks are preferentially oriented (lamellae normal along the 

-axis in Figure 1), giving rise to a diffraction pattern that typically consists two spots symmetrically arranged along 

 (Figure 1a). The position of the spot from the beam center, defined by the scattering angle 2θ or the scattering vector q (4π sinθ/λ), is related to the spacing L

 between the sheets by Bragg’s law, λ = 2L

Figure 1 shows the different classes of SAXS patterns that are typically observed for oriented specimens. The patterns have been shown to arise from different combinations of rotations and displacement of the layers and stacks (3,4). The two-point pattern arises from lamellae whose surface is perpendicular to the orientation direction (Figure 1a). The limited lateral size of the lamellar stack spreads the reflections along a layer line; imperfect orientation adds some rotation, giving the appearance of a banana pattern. If the layers rotate about their lattice point, like the slats in a venetian blind, then the reflections can spread even more along a layer line if the rotation is continuous. A four-point pattern results when this rotation angle has a preferred value (Figure 1b–1d). The angle that the lamellae normal makes with the orientation direction (

) and tilt angle, ф, determines the azimuthal separation of the reflection in the four-point pattern (Figure 1b). In addition to the tilt of the lamellae, the stack can also rotate (rotation angle α). Depending on the sense of ф and α, either eyebrow (Figure 1c; ф and α in the same direction) or butterfly (Figure 1d; ф and α in opposite directions) patterns are produced.

 from the position of the spots along 2θ, ф from the position of the spot along the azimuth, α from the tilt of the reflection from the layer line, can characterize the essential features of SAXS patterns. Additional parameters are required to fully account of the diffuse nature of the reflections. The spread or the width of the reflection in the radial (2θ) and the azimuthal (

) directions may correspond the height (h) and the width (w), respectively, of the lamellar stacks.

The apparent stack rotation, which splits the lamellar reflection, would move the reflections along a circular arc (5,6). But the trajectory of the reflection in all instances (Figures 2b–d) is not a circular arc. Neither is it a line perpendicular to 

, which could be because of either the spread the reflections along a layer line because of limited stack width, or to the rotation around the center of mass of each lamella at a constant L

. Instead, these reflections move along an elliptical trajectory (6,7). Such elliptical trajectory is predicted by the affine deformation of the lattice, which forms the basis for the lamellar stack. It is possible to obtain an elliptical trajectory by a combination of a range of whole-body, uncorrelated rotations of the stacks, and layer line spread (8). Although one ellipse may be sufficient for two-point patterns, two ellipses are required to describe the four-point pattern, one for each pair of reflections as shown in Figures 1c and 1d; this is further discussed later. Changes in the ellipticity are related to changes in the arrangement of the lamellae. Elliptical features are also common in mesostructured silica (9), laponite clay gels (10), block copolymers (11), and in diffuse scattering from oriented polymer chains (12–14).

The patterns are not always simple two- or four-point patterns as indicated in Figure 1. Different patterns may coexist when the polymer undergoes melting or as it becomes deformed (15). Figure 2 shows the changes observed during deformation of nylon fibers (16). In this experiment, the drawn fiber has the four-point pattern. As the fiber is strained by external load, the four-point pattern progressively changes to a two-point pattern (Figures 2a–2d). At various stages during mechanical loading, the four-point and the two-point pattern coexist. This can be clearly seen in the horizontal 1D traces through the lamellar peaks (Figures 2e and 2f). When the load is released, the two-point pattern reverts back to the four-point patterns (Figures 2d and 2g). The two-point pattern arises from the untilted, metastable state of the lamellae. The four-point pattern arises from tilted lamellae, which are the stable structures in relaxed polymers. Under strain, the lamellae flip, not gradually but suddenly, to the untilted state, giving rise to the two-point pattern (Figure 2h). Upon removing the load, the untitled lamellae flip back to the relaxed tilted state. Similar observations have been made in polyethylene terephthalate (PET) fibers (17,18). It was observed that, during cold drawing, the 2-D SAXS pattern transformed from a four-point pattern to the coexistence of two- and four-point patterns. The disappearance of lamellar peaks in final stages of drawing indicated the destruction of periodic lamellar structures caused by the surface fragmentation of crystalline layers (18).

SAXS Patterns and Mechanics of Deformation

The example discussed above reveals that detailed study of the SAXS pattern during polymer deformation, or in general during any structural changes (including melting), relates to changes at the lamellar length scale, and thus can be useful in understanding the linkages between the crystalline and amorphous chain segments. Accordingly, the influence of micromechanical deformations on the bulk mechanical properties of the material can be understood. Important elements of lamellar deformation are chain slip that causes lamellar tilt (ф), and slippage of the lamellae past each other, or interlamellar shear that results in stack rotation (α) without full body rotation (3,4). The two subclasses of the four-point pattern, the eyebrow and butterfly pattern, can be explained by invoking chain and lamellar slip; the first will cause lamellar tilt that will move the reflections off the 

-axis, and the second will cause stack rotation that will rotate the reflections. If the tilt and the rotation are in the same direction, the result is the eyebrow pattern (Figure 3c), and if they are in the opposite direction, then it is the butterfly pattern (Figure 3d) (19).

Both chain slip and lamellar shear act on opposing faces of the amorphous layer, and bring about net shear in the interlamellar amorphous layer (net shear angle Ψ = ф-α). When ф and α have the same sense, the amorphous segments deform less. Thus, the eyebrow pattern that occurs under these conditions signifies smaller amorphous deformation. Conversely, when ф and α have the opposite sense, the amorphous segments deform more. Thus, the butterfly pattern that occurs under these conditions signify larger deformation in the amorphous phase. If the stack rotation occurs without an interlamellar shear (for example, by stack rotation), then the shear is at both the stem and the fold surfaces of the lamellae. We find that, in low octene content polymer where strain hardening is observed, a butterfly pattern emerges when the load is removed, indicating large interlamellar shear. In high octene content polymers, where little strain hardening is observed, less interlamellar shear might explain the observed eyebrow patterns in the relaxed samples.

The parameters that describe the SAXS lamellar patterns, L

, ф, α, h, and w, can be determined by profile fitting the reflections. This may be done as a sequence of 1D scans obtained by stepping across the pattern (20), or by fitting the entire 2D pattern in a single analysis. Because the reflections fall on an ellipse, such 2D profile fitting can be efficiently done in elliptical coordinates (7). Parameters of appropriately chosen functions can be used to determine the structure characteristics, such as lamellar spacing. However, functional analysis may not reveal new or unknown features present in the structure because it essentially parameterizes the patterns according to the features identified by the chosen functions. Modeling addresses this drawback, and provides an alternative to functional analysis. If a reasonable model for the structure can be assumed, then the simulated pattern compared with the observed pattern to iteratively refined to the observed pattern to arrive at the most plausible structure (Figure 3).

In one recent attempt, structural representations of the lamellar assembly were generated using Mathematica (9). In building this model, the lamellar stacks are rep- resented by parallelograms representing tilted and sheared lamellae. Assembly of many hundreds of non-overlapping parallelograms were used to represent the bulk structure. The shape of the parallelogram is defined by ф, and its tilt by α. To mimic the disorder in the system, the height, width, shear and tilt angles (h, w, ф, and α) for each lamellar stack were selected at random from normal distributions with preselected means and standard deviation. Once the space is filled with lamellar stacks, individual lamellae are inserted within each stack. Each is filled with two to six lamellae, represented by dark and white bands corresponding to the electron densities of the crystalline and amorphous layers. The lamellar spacing (L

), stack length (h), stack width (w), the tilt angle (ф) because of chain slip and the stack rotation angle due to lamellar shear (α) were varied. Additionally, “waviness” was introduced to mimic the stack disorder, and reproduce the breadth of the reflections in the observed patterns. The square of the amplitude of the 2D Fourier transform of the generated test objects represents the diffraction pattern.

Figure 3 shows the basic lamellar stack, assembly of lamellar stacks, and a comparison of the simulated and the observed diffraction patterns. Three representative models of the lamellar stacks corresponding to three distinctive classes dis- cussed earlier were used (Figure 1). The observed patterns shown on the left half of the figure in column 3 were obtained by Stribeck and coworkers from poly(ethylene-co-1-octene)s (21). There is a remarkable degree of agreement between the observed and calculated diffraction patterns showing that simulations can give insight into the structures that give rise to the diffraction patterns. However, it is necessary to realize that such lamellar models are only one possible solution, not necessarily the correct one. Other independent evidence is required to validate the structural models. These validated models will be useful to understand the micromechanical deformation of the lamellar stacks by following the lamellar parameters discussed earlier. With the current computing power, these tools can be used to derive possible morphologies of materials as they are being deformed under mechanical load in real time at X-ray sources.

2D SAXS pattern provide a rapid and rigorous means for quantifying the dynamic changes in the lamellar structures during transformation such as during strain or melting. The features that are important to quantify are L, ф, α, h, and w, and the spread in these parameters. These parameters can be determined by 2D functional analysis of the pattern. Alternatively, details of the structure that gives rise to the various features of the SAXS pat- terns can be built into a structural model, and validated by comparing the simulated pattern with the observed pattern.

(1) A. VahidMohammadi, J. Rosen, and Y. Gogotsi, 

(2) E. Lhuillier, S. Pedetti, S. Ithurria, B. Nadal, H. Heuclin, B. Dubertret, 

(5) H.H. Song, A.S. Argon, and R.E. Cohen, 

(6) N.S. Murthy, D.T. Grubb, and K. Zero, 

(7) N.S. Murthy, K. Zero, and D.T. Grubb, 

(8) D. Grubb, N.S. Murthy, and O. Francescangeli, 

(9) R.C. Hayward, P.C.A. Alberius, E.J. Kramer, and B.F. Chmelka, 

(10) B.J. Lemaire, P. Panine, J.C.P. Gabriel, and P. Davidson, 

(12) K. Hahn, J. Kerth, R. Zolk, D. Schwahn, T. Springer, and J. Kugler, 

(13) G. Hadziioannou, L.H. Wang, R.S. Stein, and R.S. Porter, 

(14) P. Young, R.S. Stein, T. Kyu, and J.S. Lin, 

(15) H. Koerner, J.J. Kelley, and R.A. Vaia, 

(18) Y. Liu, L. Yin, H. Zhao, G. Song, F. Tang, L. Wang, H. Shao, and Y. Zhang, 

(19) D.T. Grubb, W.J. Kennedy, H. Koerner, and N.S. Murthy, 

(20) N.S. Murthy, C. Bednarczyk, R.A.F. Moore, and D.T. Grubb, 

(21) R. Androsch, N. Stribeck, T. Lüpke, and S.S. Funari, 

 is with the Department of Chemistry and Chemical Biology at Rutgers University, in Piscataway, New Jersey. 

 are with the Materials and Manufacturing Directorate at the Air Force Research Laboratory of the Wright-Patterson Air Force Base, in Dayton, Ohio. 

 is with the Department of Materials Science and Engineering, at Cornell University, in Ithaca, New York. Direct correspondence to: 

Lamellar structures, which are common in many polymeric materials and biological tissues, can diffract X-rays and give rise to reflections at small scattering angles. Analysis of these scattering features can be used investigate the deformation of lamellar structures at the microstructural length.

Small-Angle X-ray Scattering from Lamellar Structures

X-ray fluorescence (XRF) analysis is used in environmental, geological, and biomedical applications for the elemental speciation of samples. Chemical or physical traceability chains are used for the quantification of the absolute elemental content within a sample; they also assess, in a reliable manner, the chemical composition of a sample to possibly meet regulatory requirements. The physical traceability chain relies on absolute knowledge of the X-ray spectral distribution used for the excitation of the XRF instrument and is currently used at synchrotron radiation facilities. We discuss the transfer of the physical traceability chain to laboratory-based X-ray sources, which are often polychromatic, with the view to generate wider application of quantitative X-ray fluorescence analysis. Furthermore, the combination with micro-focusing optics enables the extension of these capabilities to spatially resolved measurements such that samples can be investigated in more detail in cases where localized measurements are required of individual structures or of cells stemming from organic tissue.

Quantitative X-ray fluorescence (XRF) analysis provides traceable access to the absolute elemental composition of a sample in terms of elemental mass deposition (number of atoms per unit area) of the different chemical elements detected. One way to quantify the elemental composition of a sample in absolute terms is using calibration samples to account for unknown instrumental sensitivity. The peculiarity of this approach is that the selected calibration or reference samples need to be representative of the sample under investigation in terms of elemental composition and concentration (sample matrix) and to some extent the morphology as well, to ensure comparability. Alternatively, an internal standard mixture with a predetermined composition can be added to the sample of interest, but care has to be taken to ensure homogeneous intermixing and to use nontoxic and nonvolatile elements that do not have characteristic X-ray lines in the vicinity of the elements that are being quantified. The reference-free approach described here is based on a physical traceability chain and relies solely on the use of calibrated instrumentation.

In the reference-free XRF quantification scheme (1), (radiometrically) calibrated instrumentation and knowledge of atomic fundamental parameters (FPs) involved in the production of X-ray fluorescence is used to derive the elemental mass deposition from the mea- sured count rate of the characteristic lines of one element. The atomic FPs, such as photoionization cross-sections and fluorescence yields, are element-dependent and also largely energy-dependent, but can be taken from literature databases (2) or determined by means of dedicated experiments (3,4). To excite the XRF, tunable monochromatic synchrotron radiation is used, of which the spectral distribution is well-known through the characterization of the beamlines used, whereas the use of radiometrically calibrated photodiodes provides access to the photon flux incident on the sample. For detection of the fluorescence radiation, a radiometrically calibrated silicon drift detector (SDD) and calibrated apertures are used, such that the detection efficiency and the solid angle of detection are accurately known. Following a deconvolution of the measured XRF spectra and using only physical contributions to describe possible background radiation underlying the characteristic X-ray lines, solving the Sherman equation (5) makes it possible to obtain an elemental mass deposition from the modeled count rate.

Reference-Free Quantification in Conjunction with Polychromatic X-ray Radiation

The use of monochromatic X-ray radiation has several characteristics, which distinguishes its use cases from those of polychromatic radiation. For example, the tunability of monochromatized synchrotron radiation may be used to selectively induce or suppress individual electronic transitions. Conversely, polychromatic radiation can be used to ensure simultaneous and efficient excitation of a multitude of different chemical elements. Exemplary fluorescence radiation intensities are shown in Figure 1 for a thin copper foil excited by calculable dipole-white light (dashed blue line) and excited by monochromatized synchrotron radiation (solid red line). Even though the peak intensity is far lower for the polychromatic excitation condition (mainly because of the utilization of a small aperture), the total intensity exceeds that of monochromatic excitation by almost an order of magnitude.

Although these differences are important from a practical viewpoint, the physical principles on which a quantitative, reference-free analysis is based on are identical. In practice, a description of the polychromatic excitation spectrum from a dedicated dipole-white light beamline can be derived from ab initio calculations (based on Maxwell’s equations) quite accurately (6). A reference-free quantification utilizing such calculable polychromatic X-ray radiation should produce quantitative results with roughly the same accuracy as a reference-free quantification based on monochromatic X-ray radiation. Additionally, it should reveal how the reference-free approach could be transferred to more commonly available X-ray radiation sources, such as laboratory setups relying on X-ray tubes.

Toward Reference-Free Quantification with Microfocused Polychromatic Radiation

In conjunction with microfocusing optics, a transition from quantitative elemental analysis towards spatially resolved (quantitative) elemental analysis can be realized. Combined with polychromatic X-ray sources, achromatic focusing optics such as mono- or polycapillary lenses are required. Depending on the source used, either full lenses are needed for point-to-point focusing or half-lenses are required to condense a parallel X-ray beam originating from a collimated source. Although transmission losses in the optics are unavoidable, the gain offered in terms of photons on the sample per unit time and per unit area is a major benefit with respect to simpler pinhole arrangements. Further benefits of poly- and monocapillary optics are their small dimensions and short working distances (typically of the order of millimeters), such that compact setups can be realized, and their high flexibility with respect to the X-ray source in terms of spatial and energetic acceptance. The achromatic character of poly- and monocapillary optics make them usable in conjunction with polychromatic laboratory-based sources, which is greatly beneficial in terms of accessibility and flexibility compared to synchrotron radiation–based sources, for which beamtime can be costly or limited.

In reference-free quantification using polychromatic X-ray sources, the downside of using focusing optics is that the determination of their energy-dependent transmission characteristics is rather complex. Indeed, the modulation in terms of spectral distribution and intensity of the X-ray radiation incident on the sample needs to be measured using adequate detection schemes such that a correct model can be implemented in the quantification scheme. This type of measurement can be done using either a photodiode at a beamline offering tunable monochromatic radiation or using an energy-dispersive detector in conjunction with polychromatic radiation. In both cases, the detectors are positioned directly in the incident X-ray beam and measurements with and without the optics are performed. When using laboratory instrumentation, the second type of measurement can be performed (7), but the high flux at synchrotron radiation facilities usually limits the characterization of the optics to the first type of measurement. For the setup presented, this part of the characterization still needs to be realized.

Experiments were carried out at the dipole-white light beamline (6) in the laboratory of the Physikalisch-Technische Bundesanstalt (8). This beamline provides a polychromatic excitation spectrum corresponding to bending magnet radiation with a critical energy of 2.5 keV originating from a 1.3 Tesla dipole magnet. A 1.4 mm in diameter pinhole was used to spatially confine the bending magnet radiation and a 4 μm thick gold filter was used to eliminate visible light while ensuring good excitation conditions for mid-Z elements.

A monocapillary with a platinum coating and a gold beamstop was used. The optics used had an entrance aperture of approximately 1.5 mm in diameter, an exit aperture of about 0.8 mm in diameter, and a working distance of approximately 14 mm. The monocapillary optics were mounted on a five-axis piezo manipulator installed in an ultrahigh vacuum instrument, which can be used as a mobile end station at different synchrotron radiation beamlines and is similar to the one described in the literature (9). The required degrees of freedom for alignment of the optics are three translational and two rotational stages. The accuracy provided by piezo motors is beneficial for the rotational alignment of the optics because minor misalignments can severely impede the transmission of the X-ray radiation. The rotational alignment is performed with respect to two orthogonal axes, which are perpendicular to the incident X-ray beam. The translational alignment is first realized with respect to the two same axes such that the entrance of the optics is positioned centrally in the axis of the synchrotron radiation beam. Finally, the third translational axis makes it possible to adjust the distance between the optics and the sample to the working distance of the optics. The criteria for optimization are minimization of the spot size and enhancement of the transmission. The spot size can be qualitatively monitored using a phosphor screen and a video camera system and quantitatively characterized using a knife-edge scan that allows for investigating the beam profile downstream of the capillary (Figure 2).

The comparison of fluorescence line intensities from a sample detected with and without the monocapillary optics showed a reduction of the incident flux on the sample to approximately 3% for the aluminum K line and to approximately 2% for the whole spectrum. Note that in the case of measuring with monocapillary optics, a large but not determined part of the incident radiation is blocked by the housing and the central beam stop of the optics. For the polychromatic radiation used, this reduction corresponds to the convolution of the energy-dependent transmission and photoionization cross-section factors such that no direct information on the optics can be deduced without knowledge of the incident and transmitted spectral distributions.

The alignment of the monocapillary optics results in a spot size of about 24 μm x 3 μm horizontally and vertically, respectively. With this beam spot, the element distribution of a tin–silver solder bump array with underlying copper vias was measured by scanning the lateral motor axes. A sample area of about 0.25 mm x 0.5 mm was investigated. This area includes about 50 solder bump structures in a periodic arrangement. The different horizontal and vertical spot size was caused presumably because of non-perfect alignment or different horizontal and vertical divergences of the dipole radiation, and results in an asymmetric shape of the tin–silver structures in the spatial element distribution. To consider the different horizontal and vertical spot size in the characterization of the structures, a maximum entropy deconvolution of the measured element distribution and the spot size was performed. Results are shown in Figure 3 for the tin fluorescence on the left, and the copper fluorescence on the right.

A line cut (subset of the element distribution) of the lower line is shown in Figure 4. From raster scans and closer inspection via cuts in the spatial distribution, a quality inspection can be conducted in terms of similarity of the different solder bumps or as a void inspection. Using intensity variations and intensity ratios as fingerprints, possible defects and inhomogeneities can be detected. In the case of possible defects, larger variations than for perfectly homogeneous samples must be expected.

To summarize, the route toward reference-free polychromatic micro-focused XRF experiments has been described. In the present work, a single-bounce monocapillary-focusing optics was used in conjunction with the undispersed radiation from a bending magnet. But this approach is also realized in combination with laboratory-based X-ray sources. This way, the route toward reliable, traceable quantification schemes can be opened up for more commonly available instrumentation. The effort for calibration and characterization is considerable, but access to absolute elemental mass depositions while using polychromatic and micro-focused X-ray radiation provides more accurate information about the local stoichiometry. In addition, access to absolute elemental mass depositions while using polychromatic and micro-focused X-ray radiation leads to improved detection sensitivity as compared to monochromatic excitation. This information can be relevant in geological or biological applications as well as for technologically relevant samples where absolute information about the elemental composition can be correlated with the functionality of a material.

The project is supported by the EMPIR program co-financed by the participating states and from the European Union’s Horizon 2020 research and innovation program through grant agreements 20FUN06 MEMQuD (Memristive Devices as Quantum Standard for Nanometrology) and 19ENV08 AEROMET II (Advanced Aerosol Metrology for Atmospheric Science and Air Quality). Weblinks of the European Research projects can be found here: memqud.inrim.it and aerometprojectii.com.

(2) T. Schoonjans, A. Brunetti, B. Golosio, M. Sanchez del Rio, V. A. Solé, C. Ferrero, and L. Vincze, 

(3) P. Hönicke, M. Kolbe, M. Müller, M. Mantler, M. Krämer, and B. Beckhoff, 

(4) R. Unterumsberger, P. Hönicke, J. L. Colaux, C. Jeynes, M. Wansleben, M. Müller and Burkhard Beckhoff, 

(6) R. Thornagel , R. Klein, and G. Ulm, 

(7) Y. Kayser, W. Błachucki, J.-Cl. Dousse, J. Hoszowska, M. Neff, and V. Romano, 

(8) B. Beckhoff, A. Gottwald, R. Klein, M. Krumrey, R. Müller, M. Richter, F. Scholze, R. Thornagel, and G. Ulm, 

(9) J. Lubeck, B. Beckhoff, R. Fliegauf, I. Holfelder, P. Hönicke, M. Müller, B. Pollakowski, F. Reinhardt, and J. Weser, 

 are with the Physikalisch-Technische Bundesanstalt, in Berlin, Germany. Direct correspondence to: 

In X-ray fluorescence (XRF) analysis, physical traceability chains are used to quantify the absolute elemental content in a sample. The physical traceability chain relies on absolute knowledge of the X-ray spectral distribution used for the excitation of the instrument and is currently used at synchrotron radiation facilities. Here, we discuss the transfer of the physical traceability chain to laboratory-based X-ray sources, which are often polychromatic, with the view to generate wider application of quantitative XRF analysis.

Polychromatic and Microfocused X-ray Radiation for Traceable Quantitative X-ray Fluorescence Analysis

Energy dispersive X-ray fluorescence (XRF) has many applications in geosciences and geology, but for soil scientists, the technique is not as widely used as conventional methods involving acid digestion and inductively coupled plasma–optical emission spectrometry (ICP-OES) for elemental analysis. In the agrifood sector, soil sampling and analysis is a prerequisite for accurate fertilizer management, essentially to ensure that applications meet the nutritional demands of crops and grazing animals, but also to monitor the accumulation of heavy metals in soils receiving bio-solids and other recycled products. At Teagasc, our laboratories have a long history of soil analysis using traditional methods, but have recently looked to energy dispersive X-ray fluorescence (EDXRF) for soils, crops, and fertilizer products. This article describes EDXRF analysis of soils with variable textures (clay and sandy) and compared the percent recovery of elements as a measure of accuracy. Particle-size effects were observed in sandy soils, which compromised the accuracy of results. To improve accuracy, matching libraries using certified reference materials (CRM) were applied; however, this did not improve results when elements were present in low concentrations, indicating that the method is potentially more reliable for contaminated soils.

Regular soil sampling and analysis is required to maintain soil fertility and animal health. Often, this includes multi-elemental analysis, including major nutrients, trace elements, and heavy metals, especially in areas of soil contamination and for remediation work. Conventional methods for total element concentrations in soil require acid digestion and combustion that generate hazardous waste and disposal costs, adding to the overall costs of analysis. Optical sensing and X-ray spectrometry have grown in popularity as both a surrogate for acid digestion methods and a “green chemistry” alternative that avoids chemical waste. At Teagasc, we have examined the potential for energy-dispersive X-ray fluorescence (EDXRF) spectroscopy to quantify element concentrations across a range of sample types, namely soils (1), milk powder (2), and grass (3,4), as well as recycled products from waste streams of the dairy industry (5).

In this article, we describe the applications of benchtop EDXRF in soil analysis to determine elements relevant in agriculture. Typically, soil is a complex material and extremely heterogeneous, and we rarely handle the perfect homogenous sample. The soil matrix depends on the sample’s spatial occurrence in the landscape, the interactions with climatic conditions, degree of weathering, parent material, and management. Despite these factors, the basic soil matrix is always a mix of sand, silt, clay, organic matter, water, and air. Soil samples presenting in our laboratories can be expected to span the full range of texture classes from sandy to clay to silt-based textures, with a range of organic matter and distribution of elements (including nutrients, trace elements, and heavy metals).

Our laboratory is equipped with a benchtop Rigaku NEX CG EDXRF system that is fitted with a Pd X-ray tube and silicon drift detector. This system uses secondary targets in Cartesian geometry for indirect excitation, to improve the sensitivity of trace element analysis by reducing the background intensity. Rapid quantification of element concentration can be achieved with the fundamental parameters (FP) program; however, improving the accuracy of the system allows us to adjust FP results with matching libraries comprised of samples with known values. Typically, we check the reliability of our EDXRF system by running a library calibration of pure elements on a weekly basis to check for instrument drift.

Soils that are presented in our laboratories for extraction and analysis are typically dried and sieved to 2 mm before being suspended in an acid mix. The first step in evaluating EDXRF as a potential tool was to examine the effect of sample preparation on the percent (%) recoveries of a range of elements. The options for preparing soil samples include loose powder (2 mm sieved), pressed pellet, or a pressed pellet with a binder added (Figure 1).

The soils in this study were known samples from an interlaboratory exchange program. The laboratories at Teagasc participates in the International Soil-Analytical Exchange (ISE) program, where registered laboratories receive and analyze samples as part of an interlaboratory comparison to test analytical proficiency. The samples originate from the Wageningen Evaluating Programs for Analytical Laboratories (WEPAL) and are accompanied by a database of elemental concentration and texture class. For this study, we used four WEPAL samples with different soil textures (sand, sandy clay, and clay), prepared them as loose powder (LP), pressed pellets (PP), and pellets pressed with binder (PPB) before scanning. With the EDXRF system, preparing samples as loose powder requires filling specialist plastic sample cups with 8 g of soil, whereas making pressed pellets requires 15 metric tons pressure applied to the same weight of soil using a manual hydraulic press. The third approach involved mixing 8 g soil with a binder (Fluxana Cereox wax binder) before pressing with 20 tons pressure to create the pellet.

Each of the four WEPAL samples were prepared using three different sample preparation methods outlined above, and scanned using the Rigaku NEX CG system. Element concentrations were determined using fundamental parameters (FP), and used to calculate percent recoveries as a benchmark or measure of accuracy. Recoveries (expressed as a %) are used here to compare EDXRF results with “true totals” provided by ISE laboratories based on total acid digestion using hydrofluoric acid, and other acid mixtures of HNO

 and other chemicals, including (but not limited to) HCl, HClO

% recovery = (EDXRF concentration/ISE value) * 100 [1]

Taking a 100% recovery as a bench- mark and allowing a deviation of ±20%, we saw a wide range of recoveries from 87 to 708% across a range of elements, soil textures, and samples types. Element concentrations of K, Fe and Zn, Al, Pb, Mn, Ca, and Ni fell with the acceptable range (±20%), regardless of sample preparation (LP, PP, or PPB) for all of the ISE samples, except the sand sample. From this, we concluded that soils with clay or silt clay texture do not require high-pressure sample preparation methods, and can be analyzed as loose powders. However, the same cannot be said for sandy soils. In our study, the highest recovery of 708% was recorded for S in the sandy soil when prepared as a pressed pellet, and recoveries of >120% were recorded for Cu, Ca, Cr, Mg, and P, irrespective of sample preparation in the sand sample. Large-particle-size fractions in soils, such as sand (quartz), reduce the intensities reaching the detector, thereby limiting the accuracy of EDXRF for these particular soil textures.

In our study, particle size effects were not removed after pelletizing samples. Furthermore, preparing sandy soils as pressed pellets resulted in surface irregularities and heterogeneity, which could have an impact on accuracy of EDXRF element concentrations. Although preparing sandy soils as loose powders did not meet percent recovery thresholds of 100±20%, they did not deviate more than five times above the benchmark as the PP (Figure 1). A visualization of the results is shown in Figure 2. As a general rule for the clay and silt clay textured soils in this study, preparing soils as PPB is the best suited sample preparation method and provided the best accuracy as measured by the percent recoveries.

Once a method of preparing samples was established, we then examined elements that required some correction factors or calibration in XRF to bring the values closer to the true results. The elements examined in this instance were Cr, Cu, S, P, and Mg. As there were no standards for soil samples to evaluate the best sample preparation method for agricultural soils, we used certified reference materials (CRMs). In this case, CRMs are soils with certified values of elements and information around texture class. The accuracy of EDXRF was expressed as the percent recovery—that is, the ability of EDXRF to fully “recover” or determine the element concentration compared to the certified or actual reference concentration in the CRM. The EDXRF system allows us to enter results of CRM, and stores them as matching libraries that can be used to augment or adjust concentrations generated from the built-in FP algorithm. We sourced four CRMs, one of which was a contaminated soil with high concentrations of trace elements; the other CRM were selected to represent low and moderate concentrations of elements.

For this study, 20 ISE samples from WEPAL prepared as pressed pellets with binder were measured on the EDXRF instrument and concentrations generated with and without a matching library were compared for each of the elements—P, S, Cr, Cu, and Mg. Without any correction with matching libraries, the concentrations exceeded the 120% recovery limit when compared with true values, especially at low concentrations of these elements. In our study, the lower the element concentration in soil, the greater the difference was observed from the boundary values for percent recoveries (Figure 3).

At low element concentrations, the intensities of an element line can be obscured by background intensities, especially for soils with “trace” amount of elements present. When the matching library was used to adjust FP values, the concentrations came closer to the boundary recovery value, possibly due to the matching library reducing the effect of background intensities on the signal reaching the detector. When the matching library was used to adjust the FP derived values, the effect was to bring percent recoveries closer to the acceptable boundary value. Similarly, for elements Zn, Pb, Mn, and Ni when present in low concentrations in samples, the percent recoveries when compared to true values, were greater than 120%. However, when concentrations were moderate to high, the percent recoveries fell within the acceptable range (Figure 4). Thus, when determining major plant nutrients and heavy metals in soil, EDXRF is concentration dependent, while at low concentration the values are overestimated; at high concentrations in contaminated soils, the method is more reliable.

Energy dispersive XRF was evaluated in this study as an alternative method, and while some soil types lend themselves to this technique in terms of minimum sample preparation (such as clay and loam soils), sandy soils presented difficulties due to particle size effects. In our study, sandy soils required sample preparation using a hydraulic press with a wax binder to achieve recoveries closer to acceptable levels. Soils with clay or silt-clay texture did not require high pressure sample preparation methods, and can be analyzed as loose powders. In this study, EDXRF was also concentration dependent, and at low concentration the values are overestimated; however, at high concentrations in contaminated soils, EDXRF was shown to be more suitable and reliable for analysis.

(1) M. Croffie, P.N. Williams, O. Fenton, A. Fenelon, K. Metzger, and K. Daly, 

(2) W. McCarthy, K. Daly, A. Fenelon, C. O’Connor, N. McCarthy, S Hogan, J.T. Tobin, and T. O’Callaghan, 

(5) K. Daly, O. Fenton, S.M. Ashekuzzaman, and A. Fenelon. 

 are with the Environment, Soils and Land Use Department of the Johnstown Castle Research Centre of Teagasc–The Agriculture and Food Development Authority, in Wexford, Ireland. 

 is a lecturer at The Institute for Global Food Security, School of Biological Sciences, at Queen’s University Belfast, in Northern Ireland. Direct correspondence to: 

In the agrifood sector, soil sampling and analysis is a prerequisite for accurate fertilizer management and to monitor the accumulation of heavy metals in soils. In this study, energy dispersive X-ray fluorescence (EDXRF) was used to analyze soils with variable textures (clay and sandy) and the percent recovery of elements was compared, as a measure of accuracy.

X-ray Spectroscopy Methods & Applications for Today's Spectroscopists

Application of Wavelength Dispersive X-ray Fluorescence to Agricultural Disease Research

Small-Angle X-ray Scattering from Lamellar Structures

Polychromatic and Microfocused X-ray Radiation for Traceable Quantitative X-ray Fluorescence Analysis

Energy Dispersive XRF in Soil Analysis for the Agrifood Sector