Articles

Special Issues November 2019

Click the title above to open the Spectroscopy November 2019 Supplement, X-Ray Spectroscopy Methods & Applications in an interactive PDF format.

Spectroscopy November 2019 X-Ray Spectroscopy Methods & Applications

For the elemental analysis of plant tissue materials, traditionally ICP-OES or ICP-MS are used. The advantages are clear, as the sample prep by digestion is standardized. The instruments deliver the required sensitivity and precision, and the measurement itself is very fast and can be automated. The same is true when analyzing fertilizer samples. However, the infrastructure necessary to operate an ICP instrument is not always available, or possible. An additional drawback of the traditional approaches is the chemical waste, resulting from the acids required for digestion. For fast on-site analysis of plant materials, portable energy-dispersive XRF (EDXRF) is a very suitable tool. For applications in the lab demanding high precision and lower detection limits, EDXRF in the past was limited, as the measurement times needed for the required precision were too long, and detection limits for some critical trace elements were not low enough. Based on new excitation concepts and advanced detector technology, modern EDXRF instruments are suitable to perform plant tissue analysis within a much shorter measurement time. Also, highly accurate analyses of major and minor elements in fertilizers can be done. For some critical trace elements in plant tissue and fertilizers, excellent detection limits can be achieved. This is combined with easy sample preparation and low investment into the infrastructure needed to operate the instrument.

	
	The determination of plant nutrients, trace elements, and heavy metals in plant tissue and in fertilizers is of great importance. High crop yields require a sufficient supply of plant nutrients, such as phosphorus and potassium, and of essential trace elements, such as copper and zinc. At the same time, the content of possibly harmful, heavy metals, such as lead, cadmium, nickel, arsenic, and uranium, must be kept low, in order to avoid accumulation in soil and their entrance into the food chain.

	In December 2018, the European Union decided to introduce limits for toxic contaminants in certain fertilizers, including a 60 mg/kg limit for cadmium. To limit the pick-up of uranium by plants, the German Environment Agency recommends a maximum concentration of 50 mg/kg of phosphate.

	XRF allows determination of potassium and phosphorus concentration in fertilizer. It also is suitable as a fast screening tool to test for trace element content. For example, important trace elements, are cadmium and uranium as possible contaminants of fertilizer.

	Improvements in detector and digital signal processing techniques have opened new possibilities for multi-element analysis using EDXRF instruments. In combination with optimized excitation, high sensitivities and low background levels can be achieved. The instrument setup is described in (1). For the application example described in this article, the following benefits can be achieved:

	The analysis of nutritional elements in plant tissue-this can accomplished with shorter measurement times.

	Lower detection limits for critical trace elements can be realized.

	Major element content in fertilizer material can be determined with high precision, which is a basic requirement for achieving high accuracy.

	The analysis of plant materials for toxic and nutritional elements requires little sample preparation. The sample material was dried and pulverized. From the powdered material, 8 g were taken and pressed into a pellet with a diameter of 40 mm, using a pressure of 15 tons.

	All measurements were made using a Spectro XEPOS simultaneous EDXRF spectrometer (Spectro Analytical Instruments). This instrument has an air-cooled, 50 W end-window X-ray tube. The tube anode consists of a thick, binary-alloy cobalt-palladium construction, combined with an adaptive excitation system. The analyzer includes a high-resolution silicon drift detector (SDD), plus the fixed excitation optics, and a filter changer in front of the X-ray tube. The resolution of the SDD is <130 eV (Mn Kα) at an input rate of 1 million counts per s (cps).

	Table I lists the measurement conditions of the EDXRF instrument.

	The analysis of fertilizer samples also requires drying and pulverization of the samples. The analysis of trace element content can be done from powdered samples. For sampling, 4 g of the fine powder is placed into a disposable sample cup with an outer diameter of 32 mm, which is typically closed with a polypropylene foil of 4 µm thickness at the analysis side. The main components, such as phosphorus pentoxide (P

O), and magnesium oxide (MgO), are ideally analyzed as fused beads. To prepare the fused beads, 1 g of the fine powdered material was mixed with 8 g of Lithium tetraborate and fused to a bead with 40 mm diameter using an automatic fluxer.

	Table II lists the measurement conditions of the EDXRF instrument.

Analysis of Plant Materials for Toxic and Nutritional Elements

	For the analysis of plant tissue samples, the analyzer was calibrated using various well-characterized plant material samples. The calibration is based on the fundamental parameters approach. The validation of the method was done using two reference samples from WEPAL (2) and from NIST (3).

	Table III lists the calibration ranges for some minor and trace elements as well as the correlation coefficients.

	The following example correlation graphs for P (Figure 1) and K (Figure 2) show good agreement between the reference values and the analyzed concentrations.

	To validate the method, two reference samples were analyzed (Analysis Results). These results are displayed in Tables IV and V. Information values from the certificate (Certified Concentration) are printed in italics. The printed error of the analysis results is the statistical error for a 1 sigma confidence interval.

	To test the precision of the EDXRF, the NIST 1570a reference sample was analyzed ten times. The results are displayed in Table VI.

Analysis of Fertilizers for Toxic and Nutritional Elements

	For the analysis of the fused beads, the calibration was done using various well-characterized fertilizer samples. The calibration is based on the fundamental parameters approach.

	Figures 3 and 4 show the correlation graphs for the analysis of P

	To test the precision of the analyzer, one of the fused bead samples was analyzed 10 times. The results are displayed in Table VII.

	For the analysis of the powder samples, a screening method for the analysis of various types of geological samples was applied. This screening method is based on a combination of fundamental parameters for fluorescence and scattering and on a Compton-normalization approach.

	Table VIII shows a summary of some typical fertilizer samples. The results for the major elements were taken from the analysis of the fused beads, those results for the trace elements are taken from the analysis of the powder samples. The printed error is the statistical error for a 1 sigma confidence interval.

	Since the sample preparation, especially when analyzing powder samples, involves only a little effort, an analysis using EDXRF can be done in a smaller lab, which is not equipped with a sample prep infrastructure required for ICP-OES or ICP-MS analyses.

	The determination of the content of major and minor elements can be done with good precision. For labs running batches of high numbers of samples, the relatively long measurement times of XRF are a drawback compared to those using ICP techniques. Therefore, EDXRF is very suitable for labs with low to medium sample throughput.

	The detection limits for some critical trace elements are sufficient for a quick screening analysis, but may not be sufficient for every application. Looking at the maximum tolerable limit of 50 milligrams uranium per kilogram of phosphate, as proposed by the German Federal Institute for Risk Assessment, this means a content of 2.5 to 5 mg/kg of uranium in the fertilizer. Reaching a limit of quantification of 2.5 mg/kg would mean reaching a limit of detection of <1 mg/kg, which is a challenge for XRF.

	The improvements in detector and digital signal processing techniques have opened new possibilities for multi-element analysis of plant tissue and fertilizer samples using energy dispersive XRF. As described the technique enables good precision and low detection limits and can be recommended to be used in labs with low to medium sample throughput.

Certificate of Analysis for Reference Material IPE Sample 154, Raye Grass /Lolium perenne 

(Wageningen Evaluation Programs for Analytical Laboratories, Wageningen, The Netherlands)

Certificate for SRM 1570a - Trace Elements in Spinach Leaves

 (National Institute of Standards and Technology, Washington, D.C., 2002)  .
		 

	R.E. Van Grieken and A.A. Markowicz, Eds., 

 (Marcel Dekker Inc., New York, New York, 2nd Ed.,1982).

	
	J. Willis, C. Feather, and K. Turner, 

 (James Willis Consultants Cape Town, South Africa, 2014).

	Dirk Wissmann is the Product Manager of XRF instruments with Spectro Analytical Instruments, in Kleve, Gemany. Direct correspondence to: 

New excitation developments and advanced detector technology enable the use of EDXRF for multielement analysis of plant material and fertilizers, with improved detection limits and reduced measurement time. These features are combined with easy sample preparation and low cost of investment.

XRF in Agronomy Applications—Analysis of Plant Tissues and Fertilizers

Elemental analysis serves many purposes in agriculture. For example, farmers need to ensure the nutritional value of grass they feed to cattle, and also need to know that dairy sludges used as fertilizer have the right nutrient value and will not harm fresh waters, such as through toxic heavy metals. Traditionally, these analyses are conducted using inductively coupled plasma–optical emission spectroscopy (ICP-OES), but energy-dispersive X-ray fluorescence (EDXRF) offers some potential advantages over ICP-OES for these applications. Karen Daly and Anna Fenelon of the Environment and Land Use Programme at Teagasc, the Agriculture and Food Development Authority of Ireland, have been studying the use of EDXRF in agriculture. They recently spoke to us about their work.

ou recently presented work at the 2019 SciX conference on the use of energy-dispersive X-ray fluorescence (EDXRF) spectroscopy for the elemental characterization of pasture-based agricultural samples (1). Why did you choose EDXRF for this analysis rather than wavelength-dispersive XRF (WDXRF)? 

	We wanted to explore the potential applications of XRF for agricultural and environmental samples, and felt that the EDXRF with a Cartesian geometry and polarized system provided the detection limits and robustness we needed for the sample types that we handle. The WDXRF systems on the market are more expensive, and, for this type of exploratory work in a research environment, EDXRF was the most suitable system for our needs.

What results did you achieve, and how did those results compare in terms of precision and accuracy to those you have obtained using inductively coupled plasma–optical emission spectroscopy (ICP-OES)?

	We have used EDXRF on a range of agricultural and environmental samples, and have been very impressed with how our results compare with conventional wet chemistry methods. We have found that the level of accuracy and precision that we achieve depends on a number of factors, such as sample preparation, calibration methods, and matrix effects. For example, grass samples were analyzed as a pressed powder with excellent agreement with traditional methods; however, we found that soils and skimmed-milk powders and dairy sludges gave us better results when presented as a pressed pellet, sometimes using a binder. Our initial work began with grass samples to see if the technique could capture some of the nutrients and trace elements with similar accuracy and precision that our digestion–ICP-OES method produces, and we found that by developing our own calibrations, empirically, or with matching libraries, the results for elements such as P, K, Ca, S, Mg, Cu, Zn, and Mn were in excellent agreement with digestive methods using ICP-OES. For major elements such percent P, K, Mg, and Ca, EDXRF values were within 0.03, 0.32, 0.02, and 0.06 of ICP-OES values, respectively, given that the database of over 600 samples spanned ranges from 0.1 to 3% across these elements. This was our first study using EDXRF for agricultural samples that we published in 2017 (4). Since then, we have examined the precision and accuracy of EDXRF for other materials, such as soil, milk powder, and dairy waste sludge, and have adapted our sample preparation and calibration methods to suit the sample types, so that we achieve the best agreement with traditional methods.

You have also studied the use of EDXRF to analyzed dairy processing sludge (2). Why does such sludge need to be analyzed, and for which elements? What action might be taken based on these results?

	Dairy sludge is a waste product derived from processing raw milk into other products, such as cheese and yogurt, and it has enormous potential as a source of nutrients owing to its high P and N content, and as a use for this waste stream so that it can become part of the circular economy in agri-food systems. However, before it is applied to land, farmers and producers need to know the nutrient content of the sludge for the rate of applications to be optimized, and to avoid any environmental losses of N and P that have detrimental effects on fresh waters. In addition, to date, there have been very few studies characterizing this material for heavy metals and other elements that could be toxic to plants. We characterized a number of different sludge types, for 12 elements: P, K, Mg, S, Al, Cu, Fe, Na, Cu, Mn, Zn, and Ni. The range of concentrations of major nutrients such as P, K, Mg, and S were of the order of 15–280 g/kg, and this high concentration range allowed us to use the theoretical approach to calibration, known as the fundamental parameters (FP) approach, which gave us the best agreement with ICP values. The trace elements Cu, Mn, Zn, and Ni were present in mg/kg quantities and the best agreement with ICP-OES values was observed when we applied a matching library to the FP calibrations for these elements.

In your study, what specific data analysis approach did you use for calibrating your results (to convert intensities into concentrations)?

	In our system, we worked with concentrations that were calculated from intensities, and we compared the instrument concentrations with those that we had gathered from ICP-OES analysis. First, we tried correlation and regression models to compare EDXRF with ICP-OES, but found that while there was good correlation for a lot of elements across both methods. We found some bias exhibited for EDXRF in some elements that was not immediately obvious using correlation, until we plotted the data with a 1:1 line of perfect agreement. When we observed the bias, we explored the use of agreement statistics instead, and this provided the best outcome for our data. Agreement statistics were able to tell us how close our EDXRF values were to ICP-OES values and what bias, if any, was there, and the type of bias; that is, whether it was fixed or proportional. From this, we were then able to correct for bias where it existed, and develop our method to account for bias correction.

What results did you see in this study? And what conclusions did you reach about the feasibility of using EDXRF for this type of analysis?

	Our conclusions so far about EDXRF tell us that it is a versatile system for the types of samples we encounter in agricultural research. We have presented grass, soil, milk powders, and dairy waste to EDXRF for a wide range of elements. The instrument we have allows us to create our own bespoke calibrations to improve results where needed, and also to correct for any bias that we suspect might be in the data. We have used empirical calibration and matching libraries to augment the FP approach, and have seen improvement in accuracy and precision with only a few matching library samples. Furthermore, the benchtop EDXRF instrument we used provided reliable and repeatable results, which we looked at in one of our publications (4). One observation on soils, so far, is that for some elements, we found that ICP-OES following aqua regia digestion underestimated element concentrations, so we are now looking at certified reference materials as a way to examine the agreement and accuracy of EDXRF for soil analysis. For the other materials that we have mentioned, we have found that if elements are present in high concentrations, the FP calibrations work well, but trace elements in low concentration often need either an empirical calibration or a few samples as a matching library to bring the values in line with ICP-OES.

If one were to use EDXRF for this type of analysis, would portable approaches be viable?

	We are planning to look at portable XRF to see what these systems can offer us in terms of in situ and on-site analysis of agricultural samples. 

In a previous study, you evaluated the use of EDXRF for the determination of copper, manganese, zinc, and sulfur in grass used for cattle grazing (3). Why is such analysis important? What are the advantages of using EDXRF for this application?

	In agriculture, farmers are interested in the nutrient and trace element status of the grass they grow, so that it meets the dietary requirements of the grazing animals, and if any deficiencies in grass are indicated, they can take action and use feed supplements to avoid any health issues with their animals. The advantages of using EDXRF for this type of analysis is that results can be returned to farmers more rapidly than conventional analytical methods allow, as bottlenecks in commercial laboratories for these types of analysis are often common, and delay the transfer of important results about grass nutrient and trace element status back to farms.

In the study, you compared the theoretical (FP or standardless) approach to calibrating spectroscopic intensities with empirical or standards-based procedures. What did you find?

	We developed empirical calibrations by using an archive of samples with known concentrations as standards, and we used this method for grass, milk powder, and dairy waste sludges. The advantage of having standard samples is that you can improve the calibration if the theoretical approach gives results that are not as accurate as you would like. However, we found that whether you need to calibrate with standards or use the standardless (theoretical) method  depends on the element and type of material or matrix that you are analyzing. From our experience, low concentrations of elements are possibly best determined using empirical standards (or matching libraries) whereas elements with high concentration values in a material can be determined using standardless (FP) calibrations.

What approach did you develop for correcting bias? Were you able to thoroughly evaluate the accuracy of this approach?

	We used Bland-Altman plots to calculate any bias that existed between EDXRF and ICP-OES, and also visualization of the scatter plots with the 1:1 line of perfect agreement to see in which direction the bias was observed, and, whether bias was fixed across the range or proportional to values as they increased. For fixed bias, we simply used the bias value from the Bland-Altman plots as a correction factor. For proportional bias, we used the regression equation (as long as it was a good model) between the methods to predict one value from another and we described this in a paper last year (3).

How do the time and cost involved in EDXRF analysis compare to those for ICP-OES analysis? Are there important tradeoffs?

	We discussed this in one of our papers (4), and estimated that the costs of running acid-digest ICP analysis (including microwave digestors, argon gas, reagents, and certified standards, and so on) outweighs the running cost of most benchtop XRF spectrometers. Furthermore, using EDXRF eliminates the need to dispose of hazardous waste reagents and provides results more rapidly.

Our plans in this area are to try to develop a field method for portable XRF, and to examine the role of EDXRF for characterization of dairy sludges on-site before land application.

	(1) A. Fenelon, M. Croffie, and K. Daly, “The Use of Energy Dispersive X-ray Fluorescence Spectroscopy for Elemental Characterisation of Pasture Based Agricultural Samples,” poster presented at the 2019 SciX conference, October 16, 2019.

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

 206–210 (2019). https://doi.org/10.1016/j.psep.2019.05.026

(11), 1661–1673 (2018). https://doi.org/10.1177/0003702818787165

(1), 1–11 (2017). DOI: 10.1515/ijafr-2017-0001

 is a research officer for soil and catchment science in the Environment and Land Use Programme, at Teagasc, the Agriculture and Food Development Authority of Ireland, in Johnstown Castle, in Wexford, Ireland. Her work on nutrient cycling in soil focusses on soil phosphorus cycling in agricultural soils and the mechanisms of uptake and release in different soil types. Her research also includes the application of soil spectroscopy for the prediction of soil properties. 

is a research technologist at Teagasc. Her  work focuses  on  developing   methods  for agricultural analysis using spectroscopy  techniques. Direct correspondence to: SpectroscopyEdit@mmhgroup.com

EDXRF offers potential advantages over ICP-OES for elemental analysis in agriculture. Karen Daly and Anna Fenelon of the Agriculture and Food Development Authority of Ireland spoke to us about their work investigating agricultural applications of this technique.

Applying EDXRF to Agricultural Analysis

Gallstones and kidney stones are very painful, and also can lead to cancer. The accumulation of heavy and toxic elements has been associated with the formation of such stones. Wavelength dispersive X-ray fluorescence (WDXRF) spectroscopy can be a useful technique for analyzing the types and concentrations of such elements in these stones as a means of better understanding their causes and development, as well as potential ways to avoid them. Vivek K. Singh, an assistant professor of physics at Shri Mata Vaishno Devi University in Katra, India, has been studying the use of WDXRF for such analyses, and he recently spoke to us about this work.

You recently conducted a study of the use of wavelength dispersive X-ray fluorescence (WDXRF) spectroscopy for the detection and quantification of heavy metals in gallstones (1). First, why is such determination important? And what does information about the presence of heavy metals reveal? 

	Stone formations (such as gallstones and kidney stones) inside the human body are very painful diseases that also involve time and cost. Accumulation of heavy and toxic elements inside the various organs is one of the main causes of stone formation, which also leads to gallbladder cancer if it remains untreated. Detection and quantification of these elements within stones is important so as to define etiopathogenic factors that, in turn, are useful to identify preventive and therapeutic strategies. This is why we have started to investigate and quantify a wide range of heavy and toxic elements in different types of stones, employing advanced elemental analysis technology such as WDXRF. These elements were found responsible for the formation and growth of gallstones. Most importantly, these studies provide information about the dietary habits to be followed by concerned patients, which could be very beneficial to prevent stone formation and their recurrence.

Why did you want to evaluate the use of WDXRF for this type of analysis? Specifically, what advantages does it have compared to energy-dispersive XRF (EDXRF), as well as other elemental spectroscopy techniques, such as inductively coupled plasma–mass spectrometry (ICP-MS) or laser-induced breakdown spectroscopy (LIBS)? 

	WDXRF spectroscopy is a modern technology that is capable of measuring all elements from beryllium (Be) to uranium (U) simultaneously in a concentration range from a few ppm to 100%, and can provide reliable and quality data. Compared with atomic absorption (AA) spectroscopy and ICP-MS, WDXRF is very fast and versatile. Techniques such as AA and ICP-MS are very time consuming, and can be hard to use because of sample preparation protocols in liquid form, which is very difficult in the case of gallstones and kidney stones. Also, the spatial distributions of the heavy elements are lost within the stones during sample dissolution. On the other hand, using micro-XRF, spatial information can also be obtained within the stones, which will be very useful to elucidate their growth and development processes. Although AA and ICP-MS are still very sensitive elemental analysis techniques, XRF has now become an advanced technology for bioscientists for multielemental research to analyze biomaterials, given its nondestructive nature. Compared with EDXRF, WDRXF provides high resolution to reduce spectral overlaps and thus more complex biosamples, such as stones, can be analyzed accurately. LIBS is a very strong competitor of XRF for elemental analysis. When using LIBS for the quantitative analysis of biological samples such as kidney stones, certified reference materials (CRMs) are required in order to obtain calibration curves, which are very difficult to obtain for heterogeneous kidney stones. In the present study using WDXRF, a specialized standardless software was used to quantify the elements. The EDXRF system and software can be used together to get very good results with biological samples, and we have used them together to analyze different kinds of gallstones.

You also used Fourier-transform infrared (FT-IR) spectroscopy in this study. What was the role of FT-IR? How did you accurately interpret or make assignments for the infrared spectra? 

	Before an elemental study is performed, it is very important to classify stones, because the elemental composition of stones also depends on their types. To classify stones into different categories, we have used FT-IR spectroscopy, which is a very sensitive and powerful analytical tool to study molecular composition of complex samples. In this study, gallstones were categorized into cholesterol and pigmented types based on their relative cholesterol and bilirubin content. The presence of chemical constituents (cholesterol and bilirubin) of stones was confirmed with the presence of vibrational frequencies observed in the fingerprint region. With the help of literature values, accurate absorption peaks were identified and assignments were made in their respective infrared spectra.

What results did you achieve? What did this study show in terms of the feasibility or advantages of WDXRF for such analysis? 

	Using WDXRF, we achieved very accurate and reliable quantitative data on heavy and carcinogenic elements present in gallstones that were collected from patients of different age groups and with different dietary habits. We found that the stones containing higher amounts of phosphorous also contain higher amount of calcium. The presence of other elements and their potential role was also discussed in the present study. Also, the data obtained in the present work using WDXRF is well correlated with other technique like AA and LIBS, which indicates the feasibility of its routine application to analyze stone samples in the field of gastroenterology and urology.

How does this study help us understand the etiopathogenesis (or cause) of gallstones? Are there many possibly pathways in which the body can form these stones? 

	There are several factors, such as age, sex, obesity, high blood pressure, chronic diarrhea, urinary tract infections, metabolic abnormalities, diabetes and liver disease, intestinal hypomotality and many more, that influence the formation of stones inside various organs of the human body. Besides these, nutritional imbalances in trace and essential minerals, including the quality of water intake, are the main cause for stone formation. However, mineral water containing calcium and magnesium is beneficial for calcium oxalate kidney stone patients. A large number of studies have indicated the role of heavy and toxic elements on stone formation, growth, and development. With this study, we were able to measure trace and heavy elements in stones, which further indicated the potential role of these elements in the cause of stone formation.

	In our study, elemental concentrations were found in varying concentrations in gallstone samples,  including a very rare element, palladium.

	These differences and occurrence of palladium may be due to differences in patients’ geographical locations, diets, and exposure to environmental pollution. Addition of heavy and toxic metals were found in the gallstones of patients who chew or smoke tobacco. Also, the data obtained from WDXRF were found to be in good correlation with the results obtained from AA spectroscopy. Therefore, it has been concluded that smoking and chewing tobacco should be avoided, particularly by patients of gallstone disease, to prevent gallbladder cancer. Further, this kind of systematic study would be useful to formulate future strategies for the treatment and prevention of these stones.

In a previous study, you used a combination of FT-IR spectroscopy and XRF spectroscopy for the analysis of kidney stones (2). What conclusions did you make from the analysis results in terms of the concentrations of the elements you analyzed and the specific etiopathogenesis of the stones? 

	To analyze kidney stones, we used a combination of FT-IR and WDXRF spectroscopy. To analyze the molecular constituents of kidney stones and their classification, we used FT-IR spectroscopy. The advantage of using WDXRF is its nondestructive capability. Also, the elements could be quantified using the WDXRF standardless software. Using FT-IR spectroscopy, kidney stones were categorized as oxalate or struvite types, based on their major chemical constituents, calcium oxalate and magnesium ammonium phosphate. Using WDXRF, numerous elements including heavy and toxic elements were quantified in oxalate- and struvite-type kidney stones. Our results revealed that the presence and relative concentrations of trace and heavy elements vary, depending on the stone type. The presence of heavy and toxic metals in kidney stones was found to be related to patients’ consumption of meat products, chewing tobacco, herbal supplements, and nephrotoxin products, such as aspirin, non-steroidal anti-inflammatory drugs, and antibiotics. In conclusion, the differences in the elemental concentrations of different stones may be correlated with geographical, environmental, and dietary differences.

How will this work help in understanding the causes of kidney stone development, or in guiding the treatment or diet or other means to prevent the formation of these stones? 

	Based on our experimental results, it appears that dietary habits are one of the main causes of kidney stone formation and their development. Oxalate stones are rich is oxalate and calcium, whereas struvite stones are rich in magnesium and phosphorous. Thus, after getting signs of stone formation inside the kidneys, patients should take precautions. Patients of hypercalciuria should avoid calcium supplementation and oxalate-rich foods, such as spinach, cauliflower, milk products, chocolate, and tomatoes. Salt intake should also be reduced, because high sodium intake promotes crystallization of calcium salts by reducing concentrations of citrate. According to our study, positive correlations have been found between the occurrence of heavy and toxic elements in the stones of patients who chew tobacco or consume nephrotoxins and herbal supplements. Thus, intake of these products, including alcoholic beverages, should be reduced or avoided by kidney disease patients. Because of the inhibition action of potassium and magnesium, potassium-magnesium citrate also raises urinary citrate, and diminishes stone formation and recurrence.

	Also, one should consult a nephrologist for further necessary steps based on one’s medical history after getting symptoms of stone formation, such as urinary blockage, urinary tract infections, or blood in the urine. Despite many medical advances, the role of many heavy and toxic elements are still unclear and poorly understood in nephrology and urology. More research is still needed to explore the role of heavy elements in kidney stone formation.

In an earlier study, you analyzed heterogenous gallstones using both LIBS and WDXRF (3). What new insights did that study reveal? 

	Because of the unique and complementary capabilities of LIBS and WDXRF, we have used both these techniques to study the heterogeneous nature of cholesterol- and pigment-type gallstones. Pigment gallstones have been found to be richer in heavy and toxic elements than cholesterol stones, which indicated the difference in formation mechanism of the two types of stones. Using WDXRF, we have detected ruthenium in some gallstones, which is very rare in gallstones. This may be due to the geographical region of the patients involved. These were the preliminary reports on the joint use of LIBS and WDXRF, and more studies are being performed on large number of stone samples. Further studies on gallstones and kidneys stones using LIBS and WDXRF are in progress by our group, in order to obtain further information to provide treatment insights and comfort for patients. I would like to thank Dr. Pradeep K. Rai, a senior nephrologist at the Opal Hospital, in Varanasi, India, for his expertise and help in current research activity.

	(1) B.B.S.Jaswal, P.K. Rai, T. Singh, V. Zorba, and V.K. Singh, 

(3), 178–187 (2019). DOI: 10.1002/xrs.3010

	(2) V.K. Singh,B.B.S. Jaswal, J.Sharmaa, and P.K. Raib,

	(3).  B.B.S. Jaswal, V. Kumar, J. Sharma, P.K. Rai, M.A. Gondal, B. Gondal, and V.K. Singh,

 is an assistant professor of physics at Shri Mata Vaishno Devi University in Katra, India. In the last decade, Singh has worked extensively on applications of XRF, LIBS, TOF-SIMS, and FT-IR spectroscopy for the study of biological specimens. His current research interests include multitechnique spectroscopy studies of biological, organic, medicinal plant, agricultural, and food samples, as well as trace and heavy metal determination in biological, food, and medicinal plant samples. He has published more than 70 research papers and review articles and several book chapters. Singh is also the recipient of the prestigious Raman Fellowship awarded by the University Grants Commission of the Government of India for post-doctoral research at Lawrence Berkeley National Laboratory in Berkeley, California, in 2015–2016. Direct correspondence about this interview to 

The accumulation of heavy and toxic elements in gallstones and kidney stones can be studied using WDXRF spectroscopy. Here, Vivek K. Singh of Shri Mata Vaishno Devi University in India talks about his work using this technique to understand the formation and content of these stones in the body.

Studying Gallstones and Kidney Stones with WDXRF

Over the past decade, total-reflection X-Ray fluorescence analysis (TXRF) has become a valuable analytical technique for surface analysis and bulk trace element analysis. The composition of thin layered systems of low surface and interface roughness can be measured using an X-ray standing wave (XSW) field by tuning the angle of incidence across the critical angle of total external reflection. This technique has been referred to as the grazing-incidence XRF technique (GIXRF). TXRF and GIXRF have been employed for many applications in nanotechnology, and even  biotechnology. Using these techniques, information may be obtained for elemental mass depositions and related chemical binding states, which can be revealed when using tunable excitation radiation. We were recently able to interview Burkhard Beckhoff of the Physikalisch-Technische Bundesanstalt (PTB), in Berlin, Germany regarding his recent work in this field.

ou have said that TXRF, and specifically GIXRF, is a powerful technique for surface analysis. Would you describe in detail what specifically GIXRF is, and how it is useful for surface analysis? . 

	GIXRF analysis is X-ray fluorescence analysis under varying grazing incidence (GI) conditions of the incident X-ray beam with respect to the sample surface. The variation of the angle of incidence results in different excitation conditions allowing for depth profiling in a controlled manner. In case of very flat samples, the nano-scaled depth modulation of the excitation conditions is given by the strength of the X-ray standing wave (XSW) field, which depends on the spatial distribution of the elements to be analyzed. In the case of rough samples, line intensity ratios of matrix elements can be used for depth profiling at the micro-scale. Compared to TXRF, that is XRF under total-reflection conditions, the angles of incidence can be about six times larger. However, the detection sensitivity of GIXRF is about one to two orders of magnitude smaller at the surface as compared to TXRF analysis.

Would you explain for our readers what TXRF chemical traceability is, based on calibration using one- or multielemental solutions? How is this calibration procedure conducted and what are its limitations? 

	In general, chemical traceability in TXRF is established by means of a well known amount of substance of an element, which is added to the sample of interest as a standard. For example, gallium (Ga) can be used as an internal standard for conventional TXRF. The reliability of the TXRF quantification based on an internal standard strongly depends on having a very similar spatial distribution of this standard as compared to the element(s) analyzed. In addition, the linear response of TXRF is only valid below a certain value of the total mass deposition of the sample, being in the nanogram range. Otherwise, ideal TXRF analytical conditions are not  valid, as can be easily revealed by an angular scan in GIXRF mode.

The GIXRF technique has been described in the literature (1), and multiple applications of TXRF and GIXRF have been demonstrated in bio- and nanotechnologies (2–7). What are some of the most important applications of GIXRF or general TXRF from those you have described?

	In view of TXRF’s high detection sensitivity ranging between femtogram (fg) and picogram (pg) for absolute masses, or expressed as mass depositions in the 10

, typical applications range from drinking water analysis and wafer surface contamination control, which are well in line with the linear TXRF quantification regime; to the geological, biological, pharmaceutical, industrial, archaeometric, and forensic fields, where the elemental mass depositions may be higher.

	Ideal applications of GIXRF range from biomedical and environmental sciences (such as size-fractionated aerosols deposited on substrates), over nano- and microlayered advanced materials (such as solid state batteries and photovoltaics) to nanoscaled 3D patterns such as test structures in nanoelectronics.

What are some of the key details of the data analysis tools that have been developed to analyze TXRF and GIXRF data? For example, your modeling approach of reference-free GIXRF-XRR data (7)? 

	For surface contamination or nanolayer analysis, both these methods require reliable knowledge on the depth dependent intensity of the X-ray standing wave (XSW) field arising when the incident and reï¬ected beams interfere with each other. There are public domain XSW software and related library data of optical constants or complex refraction indexes. Moving on from 2D nanolayers to 3D nanostructures GIXRF has the potential to reveal analytical information on depth profiles or cap layers of 3D structures while the method of XRR provides complementary dimensional information. The transition from 2D to 3D objects requires the solution of 3D Maxwell equations by means of appropriate solvers, going well beyond the conventional matrix XSW algorithms used in TXRF and GIXRF.

Would you briefly describe your work and its importance as related to liquid-metal jet X-ray sources (8)? What is the significance of this work, and what are the technical challenges? 

	Liquid-metal jet X-ray sources are among the most promising concepts for high-brilliance and high-flux sources for XRF laboratory applications. High power induced radiation damage of the anode is avoided by the continuous provision of new liquid material. Due to the lack of certified reference materials at the nanoscale (see http://www.nano-refmat.bam.de/en/), the alternate route to employ calibrated instrumentation is getting more relevant. For this purpose, we determined the absolute emission intensity of a liquid-metal jet for the first time. The technical challenges involve the stability of such sources and their application for combined in-situ XRD and GIXRF investigations of chalcopyrite photovoltaics.

What measurement improvements in physical or chemical measurements, speed of analysis, detection limit, or sample handling have you been able to achieve in your research? . 

	Apart from having arranged for instrumentation allowing for sample sizes from 5 mm up to 300 mm wafers, we arranged for calibrated wavelength-dispersive spectrometers in the soft and hard X-ray ranges. These spectrometers enable novel concepts to determine atomic fundamental parameters, such as fluorescence yields and ionization cross sections needed for improved traceability in X-ray spectrometry. Currently, we are building an even more efficient soft X-ray spectrometer arrangement, allowing for the simultaneous recording of X-ray emission in two orthogonal polarization directions. This instrument is expected to be used by both the German and US metrology institutes for high-resolution X-ray emission spectroscopy.

What do you anticipate is your next area of research in this field; what would be your next steps to advance this work? 

	I expect three different developments gaining speed in applied X-ray spectrometry. The correlation of materials functionality with the underlying chemical and physical properties is calling for steadily improved in-situ and operando metrology. This involves the characterization of next generation batteries and photovoltaics. Second, the inspection of nanostructures will require combined analytical and dimensional information. Here, grazing emission XRF will complement GIXRF schemes, in particular for laboratory applications. Third, quality management procedures used in industrial manufacturing and R&D call for improved ISO regulation. The German mirror committee to ISO TC201 (surface chemical analysis) is expecting to receive proposals for improved TXRF regulation, and later on, for GIXRF applications.

	(1) R. Unterumsberger, B. Pollakowski, M. Müller, and B. Beckhoff, 

	(2)   B. Pollakowski-Herrmann, A. Hornemann, A.M. Giovannozzi, F. Green, P. Gunning, C. Portesi, A. Rossi, C. Seim, R. Steven, B. Tyler, and B. Beckhoff

	(3)   A.M. Giovannozzi, A. Hornemann, B. Pollakowski-Herrmann, F.M. Green, P. Gunning, T.L. Salter, R.T. Steven, J. Bunch, C. Portesi, B.J. Tyler, and B. Beckhoff, 

	(4)   P.M. Dietrich, C. Streeck, S. Glamsch, C. Ehlert, A. Lippitz, A. Nutsch, N. Kulak, B. Beckhoff, and W.E.S. Unger, 

	(5)   T. Fischer, P.M. Dietrich, C. Streeck, S. Ray, A. Nutsch, A. Shard, B. Beckhoff, W.E. Unger, and K. Rurack, 

	(6) F. Reinhardt, S.H. Nowak, B. Beckhoff, J.C. Dousse, and M. Schoengen, 

	(7) V. Soltwisch, P. Hönicke, Y. Kayser, J. Eilbracht, J. Probst, F. Scholze, and B. Beckhoff, 

	(8) M. Wansleben, C. Zech, C. Streeck, J. Weser, C. Genzel, B. Beckhoff, and R. Mainz, 

Burkhard Beckhoff is affiliated with X-ray spectroscopy at the German National Metrology institute Physikalisch-Technische Bundesanstalt (PTB), where he is currently working as a researcher and head of PTB’s X-Ray Spectrometry group. Beckhoff  has authored and co-authored several national and international publications and also working as a reviewer for reputed professional technical journals. Beckhoff has an active role with different scientific societies and academies around the world. He has received several awards for contributions to analytical science. His major research interests involve quantitative X-ray spectroscopic measurement techniques using synchrotron radiation for their development towards laboratory applications. He is coordinator of the European research project AEROMET on aerosol metrology and organizer of the European E-MRS symposia ALTECH on nanomaterials characterization.

Total-reflection X-Ray fluorescence analysis (TXRF) and grazing-incidence XRF (GIXRF) may be applied for surface and bulk trace element analysis of thin-layered systems. Burkhard Beckhoff of the Physikalisch-Technische Bundesanstalt (PTB) in Germany spoke to us about his recent work in this field.

Total-Reflection X-Ray Fluorescence Analysis (TXRF)as a Powerful and Cost Competitive Technique for Surface Chemical and Trace Element Analyses

Serial femtosecond crystallography is a promising new technique for protein structure determination, where a liquid stream containing protein crystals is intersected with a high-intensity X-ray free electron laser beam that is 10

 times brighter than traditional synchrotron X-ray sources. While the crystals diffract and immediately after are destroyed by the intense X-ray free electron lasers (XFELs) beam, the resulting diffraction patterns can be recorded with state-of-the-art detectors. Powerful new data analysis methods have been developed, allowing a team to analyze these diffraction patterns and obtain electron density maps and detailed structural information of proteins. The method is specifically appealing for hard-to-crystallize proteins, such as membrane proteins. The technique is able to yield high-resolution structural information from small micro- or nanocrystals, thus reducing the contribution of crystal defects and avoiding tedious (if not impossible) growth of large crystals as is required in traditional synchrotron based crystallography. We were recently able to interview Prof. Alexandra Ros, a FACSS Innovation Award winner from the 2018 SciX conference regarding her research work on this subject.

You have stated that all currently available 

X-ray free electron lasers (XFELs) are pulsed and since the protein suspensions are typically injected in a continuous flow, sample injected in-between the XFEL’s pulses is wasted. As an example, you have stated that the traditional approach can lead to up to 99 percent of sample wasted and would require about 1 gram of purified protein for a full data set recording. How is what you have accomplished in your current approach, different from what others have done previously?

	There are indeed other approaches to reducing sample waste, which all come at a price, such as either being too slow for very high XFEL repetition rates, not being compatible with time-resolved crystallography, not being compatible with many classes of proteins, or introducing large background. We base our technique on a very successful method of sample delivery to an XFEL beam accomplished with a so-called gas dynamic virtual nozzle forming a jet of the sample stream. Protein crystals suspended in solution can be jetted with this approach and it is useful for a large variety of proteins that can be grown in solutions of relatively low viscosity. Our method is fully compatible with the injection through a GDVN and delivers droplets of crystal laden sample intersected by an immiscible oil phase. The droplets reach the XFEL beam only when it fires, so oil is wasted but not precious sample. Our method thus requires little to no adjustment compared to non-droplet approaches for the crystallographer and additionally can be adapted to time-resolved crystallography techniques, where sample requirements is a limiting factor. Also, our approach is compatible with all current operating XFEL facilities.

Would you explain for our readers what developments make the high-intensity XFEL beam a billion times brighter than traditional synchrotron X-ray sources. How did the combination of serial femtosecond crystallography and high-intensity X-ray free electron laser take place? Why is this an optimum approach to take in solving the protein sampling problem?

	High intensity XFELs are linear accelerators, creating an electron beam which is “wiggled” by undulators.  During their path through the long, linear undulators a self-amplification process is induced, which leads to exponential growth and eventually high intensity X-ray lasing. The XFEL approach requires its own sample delivery, which is typically not practiced at Synchrotron sources. Because the XFEL beam is so intense, it destroys every crystal it hits, but, a diffraction pattern is still recorded. This was termed the “diffract before destroy” principle. Thousands of diffraction patters are required to obtain information about protein structures, such that the sample needs to be replenished and delivered to the pulsed XFEL beam quickly. An established way to do this is via the flow focusing technology (also known as gas dynamic virtual nozzle (GDVN) technology), and our droplet generation method was thus explicitly developed to be compatible with it.

You have stated that while the protein crystals diffract and immediately after are destroyed by the intense XFEL beam, the resulting diffraction patterns can be recorded with state-of-the-art detectors. Would you explain the unique characteristics of these detection systems and how they differ from previous instruments?

	Each experiment end station has a detector that is adapted to a particular set of scientific experiments. Some detectors are commercial X-ray cameras, others are specifically developed by researchers to meet specific requirements, for example in time resolution or sensitivity. At the newly opened European XFEL for example a completely new detector was developed, which allows us to record diffraction images at megahertz (10

 Hz, MHz) frequencies, as the pulses are delivered in bunches spaced less than a microsecond. While the bunches repeat at 10 Hz, an extremely fast detector is needed.

What are some of the key details of the new powerful data analysis tools that have been developed to analyze the diffraction patterns and electron density maps to derive more detailed structural information of proteins?

	In contrast to synchrotrons, where diffraction is recorded in a series of rotations on the same crystal, diffraction recorded from XFELs renders partial reflections – one from each crystal, and requires a sophisticated, novel data analysis method. Thousands of partial diffractions are typically recorded for a full data set and the new data analysis methods require Monte Carlo methods to obtain structure factors and determine the underlying structure.

In your work you interleaved sample-laden liquid “slugs” within sacrificial liquid “slugs”, to create a fast-moving liquid microjet having the sample present only during exposure to the femtosecond laser pulses (10

 second in duration). What main challenges did you face in developing this method or approach?

	There are several challenges. The major challenge stems from the synchronization with the XFEL pulses. The droplets are currently generated upstream of the nozzle, and while they are generated at the repetition frequency of the XFEL, they might lag in phase. The adjustment of the phase was our major concern and we propose to solve it with electrical stimulation of the droplet release. We have developed 3D-printed devices that allow the integration of the microfluidic components as well electrical triggering facilitated through conductive device components as well as hard and software components for synchronization. Another important factor that is easily underestimated is the requirement for any samples delivery – droplet-based or not - to be compatible with high pressures, very similar to typical HPLC pressures. Therefore, all newly developed components must withstand these high pressures and be compatible with the workflow of an XFEL experiment.

What measurement improvements in structural elucidation, speed of analysis, detection limit, or sample handling have you been able to achieve with your new method?

	One can probably mention two areas, where serial crystallography with XFELs has a major impact: the ability to determine structures of membrane proteins with high resolution. Membrane proteins can be composed of several tens of different protein units and contain co-factors, which makes crystallization of crystals with little defects extremely difficult, but is required for traditional crystallography approaches. Serial femtosecond crystallography (SFX) with XFELs does not require the growth of large crystals, in fact protein crystals may be only 10 micrometers (µm) in size or smaller. The second area relates to time resolved crystallography, where we aim in capturing snapshots of structural changes, for example during light induced or substrate induced reactions. There have been at first, impressive demonstrations for light induced reactions with photosystems for example, but also with other proteins such as cytochrome C oxidase recently. The ultimate goal of the time resolved studies is to obtain a molecular movie, which allows us to follow the entire reaction path. 

What do you anticipate is your next area of research in this field; what would be your next steps in this work?

	One of our major efforts right now is the demonstration of the sample saving approach at XFELs for a relevant protein. We have chosen to structurally characterize an enzyme which is believed to be a target for novel antibiotics. This will however depend on beam time allocation, which is granted through a committee of experts at each XFEL facility. The next step for us will then be the integration of droplet generators within one 3-D printed form piece with the sample delivery nozzles to reduce the footprint of the entire assembly, but also to eliminate perturbing factors influencing droplet generation and triggering. Eventually we would like to combine the sample saving approach with time resolved crystallography, where the sample needs are aggravated, since every time point requires a large amount of protein crystals. This is a major limitation in time-resolved SFX with XFELs, which we hope to overcome with the integration approaches.

	(1) C. Kupitz et al., Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser, 

	(2) A. Ros, and B.G. Abdallah, Methods, systems and apparatus for microfluidic crystallization based on gradient mixing. U.S. Patent Application 15/510,

	(3) A. Ros, and B.G. Abdallah, Methods, systems and apparatus for microfluidic crystallization based on gradient mixing. U.S. Patent Application 10/166,

Alexandra Ros is an associate professor in the Arizona State University School of Molecular Sciences, and faculty member of the Center for Applied Structural Discovery (CASD) at the Biodesign Institute. Dr. Ros received her Diploma in Chemistry from the Ruprecht-Karls University in Heidelberg, Germany, and her PhD from the Swiss Federal Institute of Technology, Lausanne, Switzerland. Her current research interests include migration mechanisms in the micro- and nanoenvironment, single cell analysis, surface design and developing microfluidic tools for crystallography.

Serial femtosecond crystallography is a promising new technique for protein structure determination, where a liquid stream containing protein crystals is intersected with a high-intensity X-ray free electron laser beam that is 109 times brighter than traditional synchrotron X-ray sources. We were recently able to interview Prof. Alexandra Ros, a FACSS Innovation Award winner from the 2018 SciX conference regarding her research work on this subject.