Editorial. Technical Articles

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1 Winter 2016 Vol.32 No.1 Contents Editorial 1 The 2015 Nobel Prize in Physiology or Medicine Technical Articles 3 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system with ultra-high brilliance microfocus X-ray optics and two-dimensional detector HyPix Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis 17 Sample preparation for X-ray fluorescence analysis V. Fusion bead method part 2: practical applications English version February 21, 2016 Rigaku Corporation Head Office , Matsubara-cho, Akishima-shi, Tokyo , Japan Tokyo Branch , Sendagaya, Shibuya-ku, Tokyo , Japan Tel: Fax: rinttyo@rigaku.co.jp URL: 22 Advantage of handheld Raman spectrometer with 1064 nm excitation in pharmaceutical raw material identification New Products 27 Wavelength dispersive X-ray fluorescence spectrometer ZSX Primus IV 30 Benchtop total reflection XRF spectrometer NANOHUNTER II 33 Automated dislocation evaluation software for X-ray topography images Topography Analysis

2 Editorial The 2015 Nobel Prize in Physiology or Medicine Akihito Yamano* Dr. Satoshi Omura of Kitasato Univerisity and his collaborator, Dr. William C. Campbell of Merck, were awarded the 2015 Nobel Prize in Physiology or Medicine for their discoveries concerning a novel therapy against infections caused by roundworm parasites. What they discovered is the breakthrough medicine ivermectin (1) which cures onchocerciasis (2), an insect-borne disease caused by the parasite Onchocerca volvulus. Onchocerciasis is estimated to affect 18 million people every year primarily in tropical regions such as West and Central Africa. Once infected, intense itching, rash, scarring and visual impairment occur, and in severe cases blindness is resulted. It is estimated nearly 300,000 people lose their eyesight to every year to onchocerciasis. Onchocerciasis is also called river blindness because the blackfly abundant near tropical riversides transmits the disease. The infection cycle begins by the blackfly ingesting microfilaria upon stinging a carrier of onchocerciasis. Microfilaria grows to larvae in the blackfly. When the blackfly stings a healthy person, larvae enter the skin and become an adult in lymphatics after over a year. An adult filaria spread thousands of eggs containing microfilaria every day and numerous microfilariae migrate inside the body through skin, lymph, blood vessel and eventually reaches eye tissue. Mectizan, the Merck brand name of ivermectin, presents striking efficacy when 150 mg per 1 kg of body weight is taken orally once a year. Ivermectin has additional outstanding features. It has fewer side effects than other common antimicrobial drugs and it can be administered in tablet form which eases distribution, particularly in undeveloped areas. Lastly, there have been no reports of resistance to ivermectin since its introduction in Ivermectin is nearly an ideal drug. Ivermectin (Fig. 1(a)) is a dihydro derivative of avermectin (Fig. 1(b)) found by Dr. Omura. These two compounds are very similar to each other. Only the difference is at positions 22 and 23 where these two carbon atoms are fully hydrogenated in ivermectin compared to avermectin. Avermectin is a natural organic compound produced by actinomycetes living in soil. Researchers in Dr. Omura s laboratory at Kitasato Institute carried plastic bags no matter where they went. The particular actinomycetes was found in soil sampled near the scenic golf course Kawana in Shizuoka Prefecture. When the soil was screened back in the laboratory, an unknown actinomyces was identified and names as Streptomyces avermitilis. This sample was sent to Merck, as usual, with data including its antimicrobial activity profile against various microorganisms. When a cultured solution of S. avermitilis was fed to mice infected with parasites, it showed strong antiparasitic activity. The research groups of Dr. Omura and Merck extracted and isolated the active ingredient and named it avermectin. Avermectin was refined to ivermectin through optimization by synthesizing various derivatives and successive studies. The detailed action mechanism of ivermectin is still being elucidated. The targets of ivermectin are the glutamate-gated Cl channels that are invertebratespecific members of the Cys-loop family of ligand-gated ion channels. Glutamate-gated Cl channels are also Fig. 1(a). Ivermectin B 1a. Fig. 1(b). Avermectin B 1a. * Application Laboratories, Rigaku Corporation. Rigaku Journal, 32(1),

3 The 2015 Nobel Prize in Physiology or Medicine present in neurons in mammals however ivermectin cannot cross the blood brain barrier into the mammalian central nervous system where these receptors are located. This model explains why ivermectin paralyzes nematode but is not toxic to mammals. Currently, it is estimated that the number of people spared from onchocerciasis by taking ivermectin is at least 200 million worldwide and may be even 300 million. The reason ivermectin has helped so many people is the groundbreaking decision by Merck to donate Mectizan. Usually endemic diseases occur in economically poor regions and it is not expected that a drug developer could recover its costs from the patients. This is why it is difficult for pharmaceutical companies to develop drugs for endemic diseases no matter how many people are suffering. At the beginning of the ivermectin development, Merck anticipated that governments would purchase the medicine once the development was completed. What is unique in this case, is that Merck, even after they determined that governments in onchocerciasis-prevalent regions had not intention to purchase the drug, continued the development project. This is a rare example where the policy to get the drug to people who need it was given priority over the pursuit of making profit that is essential to viability of a private company. Merck is well known as one of the so-called Mega Pharmas. Not only it is financially sound, but also it has attracted much respect as a good citizen. Merck was selected as the The most respected company in the world in the US business magazine Fortune 7 years in a row since What made the reputation of Merck immovable was the donation of Mectizan. An additional contribution of ivermectin to human society has been an increase in food production. Initially, the contract between Dr. Omura and Merck was limited to the development of veterinary drugs. Thus, ivermectin was first launched as an antiparasitic drug for commercial livestock such as cattle. Ivermectin contributed to an increase in animal food production by restoring the appetites of livestock. It also contributed to an increase in the harvest of crops by enabling people to stay and cultivate fertile riverside areas that were previously avoided for fear of river blindness. Onchocerciasis, which has been a scourge of mankind for centuries in certain areas, is being eliminated country by country. The idealism of Dr. Omura in contributing human to society and the idealism of Merck donating Mectizan, is achieving a triumph that seemed to be absolutely impossible 25 years ago. Considering the direct and indirect impact in the improvement of the life of human beings, the value of the development of ivermectin is immeasurable. Among the Nobel Prize winners of 2015, Dr. Omura has the closest relationship to Rigaku. In a search of the Cambridge Structure Database, 38 structures were found under Dr. Omura as of October, 2015 and at least 13 of them were analyzed by Rigaku single crystal systems such as an AFC5R or Mercury CCD. We are proud to have helped Dr. Omura in his quest to alleviate human pain and suffering. References ( 1 ) A. Crump and S. mura: Proc Jpn Acad Ser B Phys Biol Sci., 87 (2011), ( 2 ) for example. Rigaku Journal, 32(1),

4 Technical articles Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system with ultra-high brilliance microfocus X-ray optics and two-dimensional detector HyPix-3000 Yuji Shiramata* 1. Introduction X-ray diffraction is an analytical method for the characterization of the crystalline structure of a material, where the X-ray intensity (I) variation is recorded as a function of diffraction angle (2θ). The diffraction region where 2θ 10 is called the Small Angle X-ray Scattering (SAXS) area, and the area where 2θ 5 is called the Wide Angle X-ray Scattering (WAXS) or the Wide Angle X-ray Diffraction (WAXD) area. Since the X-ray diffraction method enables evaluation of various physical properties, it is widely applicable to qualitative analysis (crystal phase identification), quantitative analysis, crystal structure analysis, orientation analysis, particle size analysis and so on. In general, with the powder X-ray diffraction method, measurement is performed by irradiating X-rays onto a large area of the sample surface, approx. 10 mm 2 3 mm. On the other hand, in order to perform an analysis of a tiny sample, or to analyze something like a micro area of a rock specimen, it is necessary to narrow the X-ray irradiation field to approx mm 1 mm. In the past, because these measurements used the slit collimation method to form a narrow beam by inserting a slit into the incident optical system, X-ray intensity was low and therefore measurements took a long time. However, with the latest X-ray diffractometers, which take advantage of the remarkable progress in technology for components such as the X-ray sources, optical components, detectors and so on, performing high-sensitivity measurements even for tiny samples has been made possible. In this article, various examples of characterizations realized by the state-of-the-art SmartLab μhr diffractometer system, equipped with cutting-edge technologies, such as the ultra-high brilliance microfocus X-ray source, a magnificent optic system, combined with the multidimensional detector HyPix-3000 are presented. incident optic system (Fig. 1). Since the goniometer holds the sample horizontally, it is easy to measure powders, liquids and bulky specimens without worrying about the sample falling. Moreover, by making use of optional attachments, it is possible to perform a mapping measurement of any location on the sample specified with a camera, or to make in-situ measurements by changing the atmosphere (temperature, humidity and so on) around the sample. In the following section, a detailed explanation is provided in regards to each technology and component that comprise the SmartLab μhr system X-ray source and CMF mirror Sealed-tube and rotating anode generators are well known X-ray sources. In general, higher intensity can be achieved with the rotating anode type, for which higher input power can be set, than with the sealed-tube X-ray source. X-ray focus is categorized into two types: line focus or point focus (Fig. 2). For line focus, X-rays are taken off in the direction orthogonal to the longitudinal direction of the focal profile on an X-ray target. This focus type is employed as the incident X-rays for measurement with para-focusing optics, parallel beam optics and so on. In the case of point focus, X-rays are taken off in the longitudinal direction (end-on) of the focal profile so that the projected cross-section of the X-rays can be arranged to have an equi-dimensional shape. As the X-ray source of SmartLab μhr, the microfocus rotating anode X-ray source RA-Micro7 2. Features of SmartLab μhr 2.1. Horizontal goniometer system SmartLab μhr is a diffractometer system combining the goniometer of the general purpose SmartLab instrument with a microfocus rotating anode X-ray source and a CMF (Confocal Max-Flux) mirror as the * Application Laboratories, Rigaku Corporation. Fig. 1. SmartLab μhr. Rigaku Journal, 32(1),

5 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system with point focus geometry is used, which realizes the world s highest level of power loading density at 18 kw/mm 2 with the electric power supply of 800 W and the focal size of ϕ0.07 mm. The diverging X-ray beam emitted from this X-ray source is condensed and collimated by a focusing optical element, the CMF mirror, (1) (6) and formed into a 1.2 mm 1.2 mm beam that has a divergence angle collimated to approx in both directions orthogonal to the forward direction of the beam, as shown in Fig. 3. By employing a collimator and a pinhole in combination with a CMF mirror, a beam of ϕ0.05 mm 1.2 mm can be easily created. By combining these X-ray source and optical elements, X-rays with significantly higher brilliance and improved collimation compared to the conventional equipment can be obtained. The name MicroMax-007 is given to the X-ray generating system that consists of a combination of the RA-Micro7 microfocus X-ray source and Confocal Max-Flux optics. The results of intensity comparisons between SmartLab (line focus) and SmartLab μhr (point focus) with a ϕ0.05 mm collimator are shown in Fig. 4. This demonstrates that the intensity obtained by SmartLab μhr is about 100 times greater than that obtained from SmartLab with the same power loading Hybrid multi-dimensional pixel detector HyPix-3000 The HyPix-3000 is a hybrid multi-dimensional Fig. 2. Geometrical configuration of take-off directions for point focus and line focus. Fig. 3. Example of incident optical system configuration of SmartLab μhr. Fig. 4. Comparison of intensity between SmartLab and SmartLab μhr. Fig. 5. Comparison of Debye rings. (1) Quartz (particle size: 10 μm). (2) Quartz (particle diameter: 100 μm). (3) Aluminum (rolled plate). Rigaku Journal, 32(1),

6 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system pixel detector with a large effective detection area of approximately 3000 mm 2 which enables measurement in 0D, 1D and 2D modes (7) (9). Thanks to the spatial resolution of the detector, the shape of Debye rings can be directly observed by measurement in 2D mode. Therefore, preferred orientation texture and coarseness of crystallites in a sample can be easily understood. An example of measurement of samples in different conditions is shown in Fig. 5. Uniform Debye rings are observed with a sample that has sufficiently fine grain size (Fig. 5(1)) and, on the other hand, discontinuous Debye rings consisting of dispersed spots are observed with the sample with coarse grain size (Fig. 5(2)). Moreover, with samples having a preferred orientation texture, Debye rings are observed as arcs rather than continuous rings (Fig. 5(3)). The HyPix-3000 (7),(9) is a detector with the many splendid features such as: * Low background * High-speed measurement with zero dead time * Variable measuring mode (0D, 1D and 2D) So far, the exposure method (snapshot) with the detector at a fixed position has been mainly used for measurements using two-dimensional detectors. However, the HyPix-3000 can perform TDI (Time Delay Integration) measurement (10) in 2D mode as well as in 1D mode. As a result, even for measurements that require collection of two-dimensional information over a wide range, the HyPix-3000 enables collecting signals by conducting TDI measurement in 2D mode; therefore, it makes obtaining high-quality data in a short time possible. When 2θ/θ measurement is performed in this measuring mode, it is possible to obtain information from the lattice plane nearly parallel to the sample surface (Fig. 6(1)). With the exposure measurement as (1) 2θ/θ measured on a diffracting plane nearly parallel to the sample surface. (2) Exposure (2θ) measurement. Diffracting planes not parallel to the sample surface are also observed. Fig. 6. Schematic drawing of 2θ/θ and 2θ (exposure) measurement using 2D detector. (2) Exposure measurement at 2θ 24 (incident angle: 12 ). (3) Exposure measurement at 2θ 38 (incident angle: 19 ). (1) TDI measurement. Fig. 7. TDI (2θ/θ) measurement and exposure (2θ) measurement of Au thin film on Si substrate. Rigaku Journal, 32(1),

7 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system schematically shown in Fig. 6(2), since the observed lattice planes satisfying the diffraction conditions are greatly tilted from the sample surface for the diffraction signals at higher or lower 2θ positions, it should be noted that some peaks for samples with preferred orientation textures may be missed. An example is shown in Fig. 7. This sample is Au thin film with (111) preferred orientation texture grown on Si substrate. With TDI (2θ/θ) measurement, the incident angle varies continuously, while the 2θ/θ coupling scan enables observation of a diffracting plane nearly parallel to the surface (Fig. 7(1)). On the other hand, with exposure (2θ) measurement, because a lattice plane that satisfies the diffraction condition for Au111 does not exist for a certain fixed incident angle, no diffraction peaks might be observed in some cases (Fig. 7(2), (3)). Therefore, it should be noted that, with exposure measurement of samples with preferred orientation textures, the diffraction peak might not be observed for some incident angles in exposure (2θ) measurements. As shown above, performing 2D-TDI measurement mode with the HyPix-3000 leads to the same result as measurements using multiple detectors that measure and collect the intensities simultaneously; therefore, it enables measurement in a short time compared to a 0D detector. This feature also impacts the measurement of a limited micro region on a sample surface. For 2D-mode measurement, which generally requires a point-shaped beam, the system combining the SmartLab μhr and HyPix-3000 may be regarded as the best match. 3. Examples of Measurement 3.1. Evaluation of preferred orientation texture of Au thin film by mapping measurement With the regular X-ray diffraction method, averaged information within an area is obtained when a large incident X-ray beam is employed. On the other hand, by limiting the size of the incident X-ray beam, it is possible to obtain local information about the sample. In the presented case, by using a ϕ0.05 mm collimator, mapping measurement on 289 points within the area of 1.7 mm 1.7 mm by 0.1 mm step was performed for the Au thin film sample on Si substrate. Then the variation of the degree of preferred orientation was evaluated. The measurement was performed with an exposure time of 5 seconds per point. During the mapping measurement, it is possible to capture an image of the sample by combining the measuring software SmartLab Guidance with a sample monitoring camera to designate the location of measurement. An example of a two-dimensional diffraction image of Au111 reflection is shown in Fig. 8. From this data, the intensity profile as a function of tilt (β) direction (β I profile: Fig. 9) is derived, and the peak width at each measurement point was calculated. The mapping display of the result is shown in Fig. 10. It is revealed that the sample has a variation of peak width (FWHM) ranging from depending on the location, and that the FWHM is slightly larger at the Fig. 8. Two-dimensional diffraction image of 111 reflection of Au thin film on Si substrate. Fig. 9. β I profile of 111 reflection of Au thin film on Si substrate. Fig. 10. Result of peak width distribution analysis for mapping data of 111 reflection of Au thin film on Si substrate. center of the sample. Therefore, it is understood that the degree of preferred orientation texture for the Au thin film is relatively low in the center part of this sample. For an identical characterization using a 0D detector instead of a 2D detector, it is necessary to perform rocking curve measurements (ω scan) at each point. By combining a high-brilliance X-ray source and a 2D detector, it is possible to obtain information not only on tilt distribution (mosaic spread) but also on 2θ-direction simultaneously; therefore, a lot of information can be obtained by an exposure measurement in a short time even for a micro-area XRD measurement with weak or faint signals. Rigaku Journal, 32(1),

8 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system 3.2. Phase ID analysis of a rock specimen by micro XRD measurement Generally, the definition of a mineral is a naturally formed crystalline substance. A rock is an aggregate of minerals in which the crystallites tend to be small. Therefore, in order to perform a measurement of tiny mineral crystallites contained in rock, an optical system that can selectively measure the designated small area is required. For this measurement, by using a ϕ0.05 mm collimator, black and white areas of natural basalt rock specimen as shown in Fig. 11 are measured in approx. 40 minutes and, based on the resulting two-dimensional diffraction images, crystal phase identification analysis (identification of crystal phase) was performed. In the two-dimensional diffraction image, discontinuous Debye rings composed of sparsely scattered spots are observed, therefore it is understood that the constituent crystallites are considerably coarse. Consequently, in order to increase the number of crystallites contributing to diffraction, the measurement was performed by oscillating the ϕ-axis and the χ-axis (tilt axis) (Figs. 12(1) and 13(1)). For this experiment, TDI measurement in 2D mode as described in section 2.3 was performed and, thanks to the large effective detection area of the HyPix-3000 and as a result of sample oscillation, wide scale twodimensional information was collected in a short time and the necessary data for crystal phase identification was obtained. As a result, it was revealed that the white area was composed mainly of forsterite (Mg-rich (Mg,Fe) 2 SiO 4 ) and the black area was composed mainly of enstatite (Mg-rich (Mg,Fe)SiO 3 ) (Figs. 12(2) and 13(2)). As seen in Fig. 12(1), forsterite which makes up the white area has a considerably large particle crystallite size, therefore only discontinuous Debye rings composed of scattered spots are observed in the two-dimensional diffraction image. However, in the 1D profile derived from wide-scale two-dimensional information (Fig. 12(2)), a substantial number of diffraction peaks were observed and, thus the phase identification was successful. On the other hand, the 1D profile of the black area resembles the diffraction pattern of a powder sample more than the white area and, at the same time, the twodimensional diffraction image is composed of diffraction Fig. 11. Photographs of basalt rock specimen. Fig. 12. (1) Two-dimensional diffraction image of white area by measurement with oscillation. (2) Result of qualitative analysis of white area. Fig. 13. (1) Two-dimensional diffraction image of black area by measurement with oscillation. (2) Result of qualitative analysis of black area. Rigaku Journal, 32(1),

9 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system arcs rather than sparsely scattered spots. This leads to the conclusion that the black area is composed mainly of randomly oriented or weakly textured enstatite crystals with relatively small crystallite sizes. For microarea XRD analysis for small regions not composed of polycrystals but of a single crystal or a small number of crystallites, it is expected that the SmartLab μhr HyPix-3000 system will powerfully assist these measurements and analyses Analysis of textured polypropylene by 2D-WAXS and 2D-SAXS Polypropylene, a thermoplastic polymer produced by polymerizing propylene, is widely used as plastic containers for packaging material and so on. Differences in the heat treatment process may lead to a variety of orientation textures and higher order structure that, as a result, affect the property of the material. For this reason, it is important to characterize this material or its composite substances by two kinds of measurements; namely, the analysis of texture of the molecular structure by WAXS and the analysis of higher order texture and long range order structure by SAXS. Due to the characteristics of organic material, measurement by the transmission method is effective. With SmartLab μhr, measurement of transmission 2D-WAXS and 2D-SAXS by utilizing the large effective area of the HyPix-3000 is available, therefore this requirement can be satisfied. By setting the camera length to 27 mm, exposure measurement of 2D-WAXS for the range of 2θ can be performed. Then, by setting the camera length to 300 mm, measurement of 2D-SAXS for the range of 2θ can be performed. In this section, results of characterization of two kinds of polypropylene are presented. The first one is of polypropylene films with stretching treatment. For uniaxially and biaxially stretched polypropylene films (approx. 20 mm 20 mm 0.1 mm), two-dimensional diffraction images by 2D-WAXS measurement with a camera length of 27 mm and an exposure time of 3 minutes are shown in Fig. 14. The orientation relationship between the directions of the stretching treatment of the sample and the incident X-rays is schematically shown in Fig. 15. A two-dimensional diffraction image of uniaxially stretched film is shown in Fig. 14(1). It is clearly seen that reflections such as 110, 040 and 130 are observed mostly in the TD direction. On the other hand, from the two-dimensional diffraction image of biaxially stretched film (Fig. 14(2)), it can be easily understood that the distribution of 110 reflections shows the maximum of four peaks in the direction of MD and TD, therefore it has a different orientation texture from the uniaxially stretched sample. The degree of orientation texture can be evaluated by calculating the peak width of these peaks in the β-direction of the 110 reflection of each polypropylene film (Fig. 16). The second example is a polypropylene plate. Fig. 14. Result of 2D-WAXS measurement of uniaxially and biaxially stretched polypropylene film. (1) Uniaxially stretched film. (2) Biaxially stretched film. Fig. 15. The orientation relation between the directions of stretching treatment of the sample and incident X-rays. Fig. 16. β I profile of 110 reflection of polypropylene films. The two-dimensional diffraction images of a press molded polypropylene flat plate (approx. 20 mm 20 mm 2 mm) by 2D-SAXS measurement with a camera length of 300 mm and an exposure time of 60 and 90 minutes are shown in Fig. 17. While Debye rings with uniform intensity are observed in the two-dimensional diffraction image of press molded polypropylene flat plate shown in Fig. 17(2), the intensity of Debye rings in the twodimensional diffraction image of polypropylene flat plate without press molding shown in Fig. 17(1) is not uniform and two maxima are observed. By comparing Rigaku Journal, 32(1),

10 Micro-area X-ray diffraction measurement by SmartLab μhr diffractometer system Exposure time: 60 minutes Exposure time: 90 minutes Fig. 17. Result of 2D-SAXS measurement of polypropylene flat plates. (1) Polypropylene flat plate. (2) Polypropylene flat plate (press molded). Fig. 18. β I profile of polypropylene flat plates for longrange order peak. intensity distribution profiles in the β-direction (Fig. 18). Additionally, as shown in Fig. 19, the length for long-range order structure can be calculated from peak positions in the 2θ-profile derived from the twodimensional diffraction image, and the variation of their interplanar distances can be evaluated by calculating the peak width. The full width at half maxima (FWHM) of the peak for the press molded sample and the variation of their interplanar distances are found to be smaller than the one for the non-pressed sample 4. Conclusion The SmartLab μhr is equipped with state-of-art device technology and functional components, such as a high-brilliance X-ray source, a CMF mirror (producing a high-quality incident X-ray beam), a versatile goniometer system (horizontal sample holding, controlling software and so on), and the multidimensional detector HyPix By making the best of these assets, measurements that previously could only be performed at a synchrotron radiation facility can now be done in the home lab, along with complex evaluation methods that were difficult with conventional equipment (11). With this system, the time required for material characterization should be considerably shortened, leading to a wider range of possibilities for new material development. References Fig θ I profile of polypropylene flat plates. these two-dimensional diffraction images, the apparent difference in preferred orientation texture of long-range order structure within surface planes can be confirmed. Also, quantitative evaluation of the degree of preferred orientation texture of long-range order structure within surface planes can be evaluated as well from their ( 1 ) L. Jiang, B. Verman, B. Kim, Y. Pulatonov, Z. Al-Mosheky, R. Smith and N. Grupid, Rigaku Journal (English version), 18 (2001), No. 2, ( 2 ) L. Jiang, Z. Al-Mosheky and N. Grupid: Powder Diffraction, 17 (2002), ( 3 ) B. Verman, L. Jian and B. Kim: Rigaku Journal (English version), 19 (2002), No. 1, ( 4 ) A. M. Z. Slawin and J. D. Woollins: Rigaku Journal (English version), 22 (2005), No. 1, ( 5 ) K. Shimizu and K. Omote: Rigaku Journal (English version), 24 (2008), No. 1, 1 9. ( 6 ) Y. Ito: Rigaku Journal, 25 (2009), No. 1, 1 6. (7) Rigaku Journal (English version), 30 (2014), No. 2, ( 8 ) P. Maj, R.Szczygiel, P. Grybo, T. Taguchi and Y. Nakaye: J. Instrum., 10 (2015), C ( 9 ) A. Ohbuchi: Rigaku Journal (English version), 31 (2015), No. 1, 4 9. (10) S. Kobayashi and K. Inaba: Rigaku Journal (English version), 28 (2012), No. 1, (11) P. Zaumseil: J. Appl. Cryst., 48 (2015), Rigaku Journal, 32(1),

11 Technical articles Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis Akihito Yamano* 1. Introduction The previous series have discussed single crystal X-ray analysis of small molecules. This series will discuss structure analysis of proteins using X-ray diffraction. The explanation will focus in particular on differences between structure analysis of proteins and that of small molecules. In structure analysis of proteins or small molecules, the purpose of single crystal X-ray analysis is to determine molecular structure and the basic principle is the same. However, there are considerable differences in the method pursuing each stage of structure analysis. These differences can be attributed primarily to differences in the size of the molecules. Whereas small molecules normally have a molecular weight ranging from a few hundred to thousand daltons, while that of proteins ranges from a few thousand to million daltons. There also are large differences in the components of the molecules and their chemical degrees of freedom. The atomic species comprising small molecules, and their framework, are diverse and there are no limitations in principle, however proteins are basically made up of 20 amino acids of known structure. Furthermore, the proteins of any living organisms on the earth are all comprised of L-amino acids alone, with just a few exceptions, and thus their absolute configurations are known. Also, an α-helix, which is one distinctive structure of proteins, is always a right-handed helix when tracing from the N-terminus to the C-terminus. Despite the large amount of structure-related information which exists beforehand as described above, it is not unusual for structure analysis of a new protein to take a few years, including the stages of expression and purification of the target molecule. Why is this troublesome task carried out? The reason is that various benefits can be obtained once the structure of a protein has been determined. 2. What is learned in structure analysis of a protein? Although it is not clear how long it will take to reach the final goal, humanity seems to be moving toward a complete understanding of the phenomenon of life at the atomic level. What is important in understanding the phenomenon of life is to understand metabolism and biological reactions at the atomic level, and this * Application Laboratory, Rigaku Corporation. cannot be achieved without elucidating the atomic-level structure of the proteins which are the key players in living organisms. Once a specific metabolic pathway has been determined, the reaction can be controlled by creating compounds which act on the involved proteins. For example, if research elucidates the structure of a protein which is the key to the life cycle of a virus, then it is possible to create an anti-viral drug. The method of developing drugs based on the three-dimensional structure of the target protein, obtained typically via X-ray structure analysis, is called Structure Based Drug Design (SBDD). An example of a remarkable success in the early stages of SBDD was the development of drugs that block the HIV protease. In the final stage of replicating itself, HIV protease cuts a protein produced all at once by the host cell to create its components. Since HIV protease governs this critical reaction, when the protease is blocked, HIV cannot to replicate itself and eventually perishes, even if it succeeds infecting to a host. Today, SBDD has progressed further, and numerous drugs with higher specificity have been appeared. These drugs block a DNA RNA reverse transcriptase specific only to retroviruses such as HIV, and the integrase which catalyzes the integration of HIV DNA into the host's DNA. A more familiar example is the case of headache medications. Figure 1 shows the mechanism whereby a headache occurs when a person drinks excessively. The ingested alcohol is broken down into acetaldehyde by alcohol dehydrogenase. Acetaldehyde is converted to acetyl-coa by acetaldehyde dehydrogenase, which then enters the TCA cycle. The TCA cycle is the metabolic pathway plays the central role in the glycolytic pathway, and this pathway produces ATPs, one of the main energy carriers inside the body. There is no problem when all acetaldehyde are broken down by acetaldehyde dehydrogenase, however when the breakdown cannot keep up with the speed of alcohol ingestion, a headache will occur. Acetaldehyde has the effect of relaxing the smooth muscle of blood vessels, and inflammation occurs when blood vessels expand, then bradykinin is produced. Bradykinin activates phospholipase A 2, and the activated phospholipase A 2 breaks down fatty acids, and turns them into arachidonic acid. Arachidonic acid is then converted into prostaglandin (PGE 2 ) by cyclooxygenase. This PGE 2 is an algesic substance resulting headache. If a molecule which inhibits phospholipase A 2 or cyclooxygenase is Rigaku Journal, 32(1),

12 Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis Fig. 1. Mechanism of headache occurrence after excessive ingestion of alcohol. If the activity of phospholipase A 2 or cyclooxygenase is inhibited, the pain producing substance will no longer be produced. Thus a pain-relieving efficacy can be expected. 3. What is the difference between protein structure analysis and small molecule structure analysis? Structure analysis of proteins and small molecules is the same in terms of the purpose, basic principle, and analysis procedure, but there are major differences in the specifics of each stage of analysis. The following section provides a review of the small molecule analysis procedure. Fig. 2. Cocrystal structure of cyclooxygenase and the cyclooxygenase inhibitor ibuprofen. Some commercially available pain relievers contain ibuprofen as their effective ingredient. designed, it can be a pain killer. In the initial stage of searching for drug candidate, scaffolds generally include compounds selected using techniques such as computerbased docking calculation referencing to compound libraries; comparison with existing drugs; and natural materials contained in medicinal herbs and the like. X-ray structure analysis is performed by bonding these compounds to target proteins, and then the structure is optimized to improve bonding strength. Figure 2 shows the cocrystal structure of cyclooxygenase and the painreliever ibuprofen in the PDB (Protein Data Bank). Ibuprofen is already commercially available as the painrelieving ingredient in cold medications, and it is known to bond efficiently to the active sites of cyclooxygenase Procedure for small molecule structure analysis Small molecule compounds are ordinarily prepared through chemical synthesis. Then the compound is crystallized. Crystallization is performed mainly by using a technique called the batch method. The compound is dissolved in a solvent, and then the solvent is gradually evaporated, thereby producing a supersaturated solution. Whether or not crystallization succeeds depends on the type of solvent, the concentration speed, and the temperature. When crystals are obtained successfully, then diffraction intensity is measured using an X-ray diffractometer. With semiconductor detectors such as the PILATUS 200K, measurement is typically completed in a few minutes to a few hours because the PILATUS 200K has a short readout time of 7 ms, and it enables true shutterless data collection. In order to calculate electron density, it is necessary to estimate initial phases. The direct method is mostly used in small molecule structural analysis. The theory of the direct method is difficult to understand, but it has been implemented as a program, and thus executing it is extremely easy. By simply providing reflection intensity and crystallographic information, the program calculates initial phases. A wide variety of such programs are available, and can be obtained either free or for Rigaku Journal, 32(1),

13 Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis a comparatively low price. When phases have been determined, it is possible to calculate electron density. With the direct method, normalized structure factors represented as E are used, and thus a list of peak positions in the E-map is output when the calculation is finished. A molecular model is built assuming that atoms are present at the peak positions in the E-map. In general, precision of initial phases is relatively low, and therefore at the beginning, parts having higher electron density such as metal atoms, five-membered rings and six-membered rings are readily observed. Even if a part of the molecule is not observed, one should proceed to the least-squares refinement, and calculate a difference Fourier map taking the residual Fo Fc as coefficients. At this time, the phase precision is already improved over the initial phase, and thus the remaining structure which could not be observed at the initial stage appears. Each stage of single crystal X-ray analysis can be regarded as a step of improving phases to approach the true phases Protein structure analysis In protein structure analysis, the basic stages of analysis are the same as in small molecule structure analysis. However, there are major differences in the execution of each stage Sample acquisition Small molecules are primarily prepared using chemical synthesis. Proteins, on the other hand, are produced through overexpression using genetic engineering techniques, and purified using techniques such as solid metal ion affinity chromatography, affinity chromatography using antigen-antibody reactions, and gel filtration chromatography in which molecules are separated by size. To make separation and purification easier using these liquid chromatography methods, the mainstream approach is to express as a chimeric protein linked beforehand with another protein, or as protein with histidine chain tags on the N-terminal and/ or C-terminal. An expression system most suited to the purpose and conditions is chosen from various options such as E. coli, insect cells, and human cells Crystallization A variety of methods have been devised for protein crystallization, ranging from conventional methods to the latest techniques using electronic elements and magnetic fields. More than enough research results have been reported on crystallization techniques themselves. However, the most important point for protein crystallization is not the crystallization technique itself; rather, it is preparation of easily crystallized protein. At the stage where protein is overexpressed, various constructs are prepared incorporating modifications at sites expected to be crucial for crystallization, or modifications for limiting relative movement between domains. Then the construct most suitable for crystallization is selected. The most popular crystallization method is the vapor diffusion technique (Fig. 3(a)). First a precipitant is added to the same buffer solution used to dissolve the protein. A sufficient amount of reservoir solution is added to the crystallization well. The same amount of reservoir solution is added to the drop of protein solution on a cover slip, and it is turned upside down and shielded. The concentration of precipitant in the crystallization drop is nearly one-half of that of reservoir, and therefore water migrates to reservoir until it becomes at the same concentration as the reservoir solution. Concentration and super saturation occur due to this process, and crystal is obtained. At present, progress is being made with the hardware and software needed for data collection and phase determination, and, as in the case of small molecule structure analysis, if a good crystal can be obtained, then it is possible to achieve the final structure with Fig. 3. (a) Set-up for crystallization using the hanging drop vapor diffusion method. (b) Heavy atom soaking method. Rigaku Journal, 32(1),

14 Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis a far greater probability than before. Therefore, it is no exaggeration to say that the major determinant of success in protein structure analysis is how biochemical experiments are carried out Data collection In small molecule structure analysis, data collection is often done at low temperature for reasons such as preventing evaporation of crystallization solvent, suppressing thermal vibration of atoms, and suppressing damage to crystals. In protein structure analysis, on the other hand, measurement is almost always performed at a low temperature around 100 K. This is because protein crystals contain typically 50% solvent, that is water, and they are also more easily damaged by X-rays. A cryoprotectant must be added to perform low-temperature measurement. The problem is mother liquor for crystallization picked up with a crystal by a loop. The mother liquor is buffer solution whose main component is water, therefore when it is frozen as it is, it forms hexagonal ice and swells to degrade the crystal unless it is frozen extremely quickly. Initially, it was thought that crystals are damaged during freezing because the water inside protein crystals freezes and forms ice, however water molecules inside crystals are stabilized by the protein itself, and therefore ice formation due to freezing is expected to be limited. Ordinary freezing is performed with a cold nitrogen gas stream of a low-temperature system. The low-temperature gas stream is first blocked with a card or similar item, and after setting the crystal in place, the card is quickly removed, thereby increasing the freezing speed. This is called the flash freezing method. There is also a method in which the loop used to scoop up the crystal is quickly dipped into low-temperature liquid such as liquid nitrogen contained in a Dewar. Compared with gas, a liquid medium is known to have better heat conduction, but if freezing is not done with a great care, it may have the opposite effect to what was intended. The most common cause of failure is the nitrogen gas layer present on top of liquid nitrogen. Since the gas layer has a comparatively mild temperature gradient, it impedes rapid freezing of the crystal. This can be avoided in various ways, such as filling the Dewar up to the rim with liquid nitrogen, or blowing away the gas layer using some methods (1) Phase determination In small molecule structure analysis, initial phases are determined through calculation, almost without exception. In protein structure analysis, on the other hand, it is not practical yet to determine phases using the direct method, but usually determined experimentally. A typical technique is the multiple isomorphous replacement (MIR) method. In the MIR method, the phase of the original (native) protein is derived by using the slight discrepancy in phase which arises when heavy atoms are introduced. Theoretically, it is possible to calculate the phase of the native crystal if there are a total of three sets of data: for the native crystal, and a minimum of two different types of heavy atom derivatives. There are a number of methods for preparing heavy atom derivative crystals, but the classical method involves soaking the native crystal in heavy atom solution. This is called the soaking method (Fig. 3(b)). At first glance this looks easy, but there are many parameters to be explored such as the heavy atom type, concentration, and soaking time. When conditions are good, heavy atoms bond at specific sites of the protein molecule while crystallinity is maintained. To determine whether or not heavy atoms have been Fig. 4. Principle of phase determination using the multiple isomorphous replacement method. Blue, red and green are obtained, respectively, from the native crystal, first heavy atom derivative, and second heavy atom derivative. Rigaku Journal, 32(1),

15 Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis Fig. 5. Principle of determining structure of heavy atoms using the direct method. When the native protein structure is subtracted from that of the heavy atom derivative, only the structure of heavy atoms remains. The structure of the heavy atoms alone is comparatively simple, therefore it is possible to identify the positions of heavy atoms using the direct method. Fig. 6. Left: Electron density diagram after solvent flattening. Right: Electron density with molecular model created using automatic model building program. introduced successfully, there is no other way but actually measuring data, and therefore a large amount of work is necessary to obtain even one derivative. Figure 4 shows the principle of phase determination using the MIR method. The structure factors can be thought of as waves, and thus it is convenient to think on the Gaussian plane. Information from the native crystal is indicated in blue. The reflection intensity of the native crystal can be determined through data collection, but the phase cannot be measured directly. This corresponds to a situation where the lengths of the structure factor vectors are known, but their directions are unknown. It is here that heavy atom derivatives come into play. First, there is a need to find the structure factors of heavy atoms alone. If the isomorphism of the heavy atom derivative is sufficiently high, then the residual subtracting the intensity of the native crystal Fp from that of heavy atom derivative Fph 1 can be regarded as the intensity of heavy atoms alone. In real space, this indicates that it is enough to determine the structure of the heavy atoms only (Fig. 5). The structure of the heavy atoms alone is simple, therefore it should be possible to determine the structure somehow. Previously, positions of the heavy atoms were determined using the Patterson method, but today it is typically done using the direct method. Once the positions of the heavy atoms are determined, then it is possible to calculate the portion of the structure factors attributable to the heavy atoms. Since structure factors are expressed as vectors, they can be written into the diagram shown as Fh 1 in Fig. 4. However, only the reflection intensity is known for the protein part of the heavy atom derivative, therefore a circle is drawn with radius equal to the amplitude of the structure factor whose center is the end point of the structure factor of the heavy atom (red circle in Fig. 4). At the intersections of the red and blue circles from the derivate and native crystal, the vector relations are satisfied for the heavy atom derivative, native crystal, and heavy atom only. However, there are still two intersection points, hence two possibilities for the phase angle remain. Thus a second heavy atom derivative is prepared, and the same procedure is repeated. For the second derivative, the positions of the heavy atom is determined and the portion of the structure factor attributable to the heavy atom is calculated (Fh 2 in Fig. 4). When a circle is drawn with radius equal to the amplitude of the second heavy atom derivative centered at the end point of Fh 2, then in this case again there are two intersections, however only one of them is consistent with one of the Rigaku Journal, 32(1),

16 Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis Table 1. Summary of small molecule structure analysis and protein structure analysis. Sample preparation Crystallization Data gathering Phase determination Model building Structure refinement Evaluation/Utilization of results Small molecule structure analysis Chemical synthesis. Batch method dissolving in organic solvent and concentrating, or diffusing poor solvent. Mo or Cu radiation is used depending on the magnitude of absorption or necessity of absolute structure determination. There are cases where the oscillation angle during measurement is large, and cases where it is small. Calculation by computer using statistical techniques. Direct method. Building is carried out based on expected structure, chemical knowledge, bonding distance and electron density. At present, this is always done using the least-squares method. Shelxl is the de facto standard. Utilization of molecular structure, absolute structure and crystal packing, etc. Protein structure analysis Biochemical expression and refinement using genetic engineering. Vapor diffusion method. Concentration occurs by adding precipitant to protein dissolved in buffer solution with optimal ph for the individual proteins. No need to determine absolute structure. Cu radiation is always used with the laboratory system to raise the intensity and to resolve neighboring reflections originated from long lattices. The oscillation angle is normally 1 or less. In general, the direct method cannot be used. Determination is done experimentally by the MIR method etc. Components are limited to 20 types of amino acids, and thus the basic approach is to fit amino acid chains. There are multiple automatic building programs. The post popular technique is the SA method. The leastsquares method is also used. Refmac, Shelxl, etc. Primary the molecular structure. It is used for drug development and understanding biological reactions. two intersections obtained with the first derivative (thick arrow in Fig. 4). This makes it possible to determine which of the two phase angle possibilities is correct, and thus to determine the phase of the protein crystal Molecular model building If phases can be calculated, so can the electron density. The electron density calculated with the initial phases usually has unrealistic natures such as negative electron density. In order to eliminate these regions, a procedure called solvent flattening is performed in the solvent region. Phases can be improved by carrying out a Fourier transform of the electron density map modified in real space via solvent flattening. The electron density map after solvent flattening is shown in Fig. 6. When high-quality electron density map to this extent has been obtained, fitting of amino acid chains is performed while looking at the shape of the electron density. Good clues are provided by amino acid side chains with distinctive shape such as tryptophan, tyrosine, and phenylalanine. Even with a novel protein, it is common for the amino acid sequence to be known prior to structure analysis. Therefore, if there are some sites where the allocation is certain, the remaining fitting can be proceeded according to the amino acid sequence. At present, multiple model building software packages are available (Buccaneer (2), ARP/wARP (3), Solve/Resolve (4),(5) ), and these build a molecular model automatically referring to the given amino acid sequence. The advent of molecular model building programs has dramatically reduced the time needed for protein structure analysis. Protein may be better suited to automation because the number of building components is limited to 20 amino acids Structure refinement When the molecular model is finished, the next step is structure refinement. The simulated annealing (SA) method is a characteristic technique for protein structure analysis. In the SA method, energy is first minimized, and then the molecule temperature is virtually increased by connecting a heat bath. After that, gradual cooling is performed by separating the heat bath. This method has a larger convergence radius than the least-squares method, and it is thought that it converges on the correct structure by overcoming higher energy barriers. CNS/ CNX is a typical program for the SA method. The leastsquares method is also used to refine protein structure. The CCP4 basic software package for protein structure analysis includes software for the least-squares method called Refmac (6). SHELXL (the standard refinement program for small molecule structure analysis) is also has a capability of protein structure refinement. With either the SA method or least-squares method, the number of reflections to parameter ratio is lower in protein structure analysis, and hence refinement is executed while providing constraints such as amino acid structure retains its shape. In protein structure analysis, water molecules behaving as part of the protein are occasionally found. There also are water molecules creating a hydrogen bond network covering the molecular surface and contributing to solubilization. Normally, water in the first layer is localized, and this too is incorporated into the molecular model. The final crystallographic R value is generally in the range 10 30%. Rigaku Journal, 32(1),

17 Introduction to single crystal X-ray analysis IX. Protein structure analysis and small molecule structure analysis 4. Conclusion Table 1 summarizes each stage of protein structure analysis and small molecule structure analysis. The steps are common, but the way those steps are executed are markedly different. Difficulties in small molecule structure analysis are almost always attributable to crystallographic problems besides obtaining crystals. The most common are cases where analysis is performed without noticing twinning. On the other hand, the difficulty of protein structure analysis is primarily due to the difficulty of experiments. References ( 1 ) M. Warkentin, V. Berejnov, N. S. Husseini and R. E. Thorne: J. Appl. Crystallogr., 39 (2006), ( 2 ) M. D. Winn et al.: Acta Cryst. D67 (2011), ( 3 ) G. Langer, S. X. Cohen, V. S. Lamzin and A. Perrakis: Nature Protocols, 3 (2008), ( 4 ) T. C. Terwilliger: Acta Cryst., D59 (2003), ( 5 ) T. C. Terwilliger: Acta Cryst., D59 (2003), ( 6 ) G. N. Murshudov, A. A. Vagin and E. J. Dodson: Acta Cryst., D53 (1997), Rigaku Journal, 32(1),

18 Technical articles Sample preparation for X-ray fluorescence analysis V. Fusion bead method part 2: practical applications Mitsuru Watanabe* 1. Introduction The general preparation method of fusion bead, equipment, reagents and other important considerations were described in the previous article Sample preparation for X-ray fluorescence analysis IV Fusion bead method part 1 basic principles (1). In this article, the preparation methods of various applications such as ferroalloy, sulfide and carbide are described. Conventionally, these samples had been prepared as fusion beads after they were oxidized completely by mixing with a strong acid followed by drying. However, raising the temperature of the strong acids such as nitric acid degrades the working environment and the surrounding equipment corrodes due to its oxidizing power. Therefore, it has been necessary to work in a well-ventilated environment. In the preparation method described here, oxidation reaction progresses slowly in the platinum crucible by oxidizing agent or oxidation catalyst without the use of strong acids. Thus it is possible to prepare fusion beads in a short time in a conventional environment. It should be noted that the described procedure assumes that grain size of the powder samples and the drying temperature of the samples and reagents are those for common fusion bead preparation methods. If the grain size of the sample is coarse and the fusion bead is prepared without spreading the flux on the bottom of the platinum crucible, the sample will be oxidized insufficiently and form an alloy with the platinum in the crucible and cause irreversible damage. In this case, recasting of crucible may be required. For this reason, it is necessary to perform sample preparation carefully. Other additional analytical considerations regarding measurement of fusion bead are described at the end. Fig. 1. The fusion bead preparation procedure of ferrosilicon. * SBU WDX, X-ray Instrument Division, Rigaku Corporation. Rigaku Journal, 32(1),

19 Sample preparation for X-ray fluorescence analysis V. Fusion bead method part 2: practical applications 2. Sample preparation 2.1. Ferroalloy Ferroalloy is an iron alloy with other elements and is one of the materials used in the steelmaking process. The steel s properties such as strength, toughness, heat resistance and corrosion are improved by adding specific elements to the steel. It is also used for the purposes of desulfurization and deoxidation in the refining process. Ferrosilicon which is one of the ferroalloys is an iron alloy with silicon and the concentration range of silicon can vary by as much as 15 90%. Because ferrosilicon is a metal, without oxidization agent it forms an alloy with the platinum crucible during the fusion process and causes irreversible damage. The fusion bead preparation procedure of ferrosilicon is shown in Fig g of lithium tetraborate is transferred to the platinum crucible and spread on the bottom of the crucible in advance g of sample dried at 105 C for 2 hours and g of mixed oxidizing agent (Li 2 CO 3, Na 2 CO 3, KNO 3 1 : 1 : 1) are mixed well and Fig. 2. Sample setting for ferrosilicon fusion bead. placed onto the flux.platinum crucible with the powders is shown in Fig. 2. In this case, it is recommended that the flux is in powder instead of a granular form to avoid the mixture of sample and oxidizing agent coming in contact with the platinum crucible. A platinum crucible is placed in the electric furnace and the sample is oxidized at 500 C for 1 hour, at 550 C for 30 min. and at 600 C for 30 min. successively. After oxidation, the fusion bead is fused at 1200 C for 7 min. (stationary for 2 min. and swinging for 5 min.) and cooled for 5 min. (passive for 2 min. and active for 3 min.) with the fusion machine (2). It is also possible to prepare fusion beads for ferromanganese, silicomanganese and graphite spheroidizing agent (magnesium ferrosilicon) used in ductile iron with this procedure Copper ore (Sulfide) Copper ore is a raw material for copper and contains sulfides such as chalcopyrite (CuFeS 2 ) and chalcocite (Cu 2 S). It is possible to oxidize the sample using oxidizing agent such as nitric acid followed by drying prior to fusion. However, it is easier to perform oxidization during the pre-heating stage prior to fusion. The fusion bead preparation procedure for copper ores is shown in Fig g of dried sample at 105 C for 2 hours and g of mixed flux (lithium tetraborate : lithium Fig. 3. Fusion bead preparation procedure for copper ores. Rigaku Journal, 32(1),

20 Sample preparation for X-ray fluorescence analysis V. Fusion bead method part 2: practical applications metaborate 35.3 : 64.7) are mixed well. After mixing, it is transferred to a platinum crucible, 0.9 ml of 20% (w/v) lithium nitrate solution and 2 ml of distilled water are added such that sample is completely immersed. It is heated at 800 C for 10 min. in the fusion machine to oxidize the sulfur in the sample and then cooled. After cooling, 20 μl of 20% (w/v) lithium bromide solution is added, fused at 1050 C for 7 min (stationary for 2 min. and swinging for 5 min.). Finally perform cooling for 5 min. (passive cooling 2 min. and active cooling 3 min.) to obtain a fusion bead (3) Silicon carbide Silicon carbide has high hardness property next to diamond and boron carbide, and is superior in wear resistance. It begins to oxidize at over 800 C in the atmosphere, but silicon carbide forms a passivation layer of silicon dioxide on the surface, which acts as a barrier to prevent further oxidation. It also has good chemical stability and thermal conductivity. That is why it is used in many materials such as abrasion materials, cutting tools, heat resistance parts, fireproof materials, heating element and substrate for semiconductors. It is difficult to pulverize these powders finely with typical vessels due to high hardness and wear resistance of silicon carbide. Analysis error increases due to the influence of the grain size and mineralogical effects and the contamination from a vessel increases. It takes a long time for the oxidation reaction in the general procedure of ceramics fusion bead due to heat resistance, oxidation resistance and chemical stability. The fusion bead procedure described in JIS R2011 Annex 3 (Methods for X-ray fluorescence spectrometric analysis of refractory which contains carbon and silicon carbide) is shown in Fig. 4 (4) g of dried sample at 105 C for 2 hours and g of lithium tetraborate are mixed well. After charging them into the platinum crucible, heated at 840 C for 30 hours to oxidize gently and warmed up to 1150 C at the end. Once cooled, it is added to 50 μl of 50% (w/v) lithium iodide solution as a releasing agent, fused at 1150 C for 5 min. (stationary for 2 min. and swinging for 3 min.) and cooled for 5 min. (passive for 2 min. and active for 3 min.) However, in this procedure it takes more than 30 hours to prepare the bead because no acid and oxidizing agent are used. The procedure which can prepare the bead more quickly with oxidizing agent is shown in Fig g of lithium tetraborate is transferred to the platinum crucible and spread on the bottom of the crucible in advance g of dried sample at 105 C for 2 hours and g of oxidizing agents (lithium Fig. 4. The fusion bead preparation procedure of silicon carbide (JIS R2011 Annex 3). Rigaku Journal, 32(1),

21 Sample preparation for X-ray fluorescence analysis V. Fusion bead method part 2: practical applications Fig. 5. Fusion bead preparation procedure of silicon carbide. carbonate, vanadium oxide and strontium nitrate are mixed in equal amounts) are mixed well and then placed onto the flux. This mixture must not contact the crucible directly. After oxidizing at 800 C for 20 min., fuse at 1200 C for 7 min. (still 2 min. and swinging 5 min.), and then cool for 5 minutes (passive 2 min. and active 3 min.). Fusion beads can be prepared in about 40 min. with this procedure, including weighing and mixing (5). 3. Analytical considerations 3.1. Dilution ratio If analysis is carried out in accordance with a specific standard method, the dilution ratio (flux weight/sample weight) should be as prescribed by the standard method. If not, the dilution ratio should be chosen based on solubility of the sample in the flux. As described in the previous article, the X-ray intensity of the fusion bead decreases with dilution. It can be expected that decreasing the dilution ratio (increasing sample ratio) improves the analytical precision. The correlation between the dilution ratio and the X-ray intensity is shown in Fig. 6. X-ray intensity is the theoretical intensity which is calculated by FP method for 1.72 g/cm 3 density and 40 kv excitation voltage. The relative intensity assumes pressed pellet sample to be 100. Table 1 shows that Ti-Kα intensity for the fusion bead with dilution ratio 10 is half of pressed sample intensity. As the wavelength of the analytical line increases, the X-ray intensity decreases due to absorption effect by the flux. Fig. 6. Correlation between the sample weight fraction in the fusion bead and relative intensity. (Sample: Portland cement) Dilution ratio Table 1. Dilution ratio and relative intensity. Relative intensity Si-Kα Ca-Kα Ti-Kα Fe-Kα Relative intensity to pressed pellet sample as 100. Rigaku Journal, 32(1),

22 Sample preparation for X-ray fluorescence analysis V. Fusion bead method part 2: practical applications Fig. 7. Correlation between bead thickness and relative intensity. Table 2. Saturation thickness for different analytical lines. (Unit: mm) Analytical line Saturation thickness Fe-Kα 0.65 Zn-Kα 1.15 Sr-Kα Selecting the analytical line Bead thickness can influence X-ray intensity for analytical lines with shorter wavelengths since the fusion bead is composed of light elements such as lithium, boron and oxygen. The correlation between bead thickness and relative K-line intensity for Fe, Zn and Sr is shown in Fig. 7. The thickness where X-ray intensity saturates is shown in Table 2. X-ray intensity is the theoretical intensity and is calculated by FP method for 1.72 g/cm 3 density and 40 kv excitation voltage. Defining saturation thickness as being over 99% of relative intensity, saturation occurs at 0.65 mm for Fe-Kα, 1.15 mm for Zn-Kα and 2.8 mm for Sr-Kα. Thickness of fusion beads depend on sample and flux amount as well as shape of the platinum crucible, but typically is a few millimeters in most cases. Therefore, when selecting analytical lines having shorter wavelengths than the Zn-Kα, it is necessary to prepare fusion beads with consistent thickness. It should be noted that thickness can vary depending on the degree of damage to the platinum crucible and amount of releasing agent added. If fusion bead thickness is less than saturation thickness and it is not possible to prepare fusion beads with consistent thickness, analytical line with longer wavelength should be selected. As an example, the correlation between fusion bead thickness and X-ray intensity for zirconium oxide sample (dilution ratio 10) is shown in Fig. 8. The saturation thickness for Zr-Kα is about 3.8 mm whereas Zr-Lα saturate at about 30 μm. As described above, it is desirable to select Zr-Lα when the fusion beads cannot be prepared with constant thickness. However, because X-ray intensity for L-line of the Fig. 8. Correlation between bead thickness and relative intensity (Zr-Kα and Zr-Lα). fusion bead is lower than K line, the measurement time has to be adjusted to obtain required analytical precision. In addition, it should be noted that surface contamination and deliquescent of the fusion bead has a larger influence on X-ray intensities of longer analytical line wavelengths. 4. Summary Fusion bead method allows accurate analysis by reducing matrix effects due to dilution and eliminating errors caused by inhomogeneity such as grain-size and mineralogical effects and therefore has been commonly applied for oxides samples such as refractories and cement raw materials. By using preparation methods described in this article, it is possible to rapidly fuse non-oxides such as ferroalloys, sulfides and carbides which have previously been considered to be difficult to analyze by fusion bead method. Fusion preparation methods described here are significantly simpler compared to conventional methods using acids. However, irreversible damage to the platinum crucible can occur if sample is insufficiently oxidized due to coarse grain size and comes in contact with the platinum crucible during the fusion process. In such case, recasting of the crucible may be required. It is therefore important to prepare samples carefully. Nevertheless, analytical accuracy can be improved for a variety of samples broadening possible applications by the fusion bead method. References ( 1 ) M. Watanabe: Rigaku Journal (English version), 31 (2015), No. 2, ( 2 ) M. Watanabe, H. Inoue and Y. Yamada, M. Feeney, L. Oelofse and Y. Kataoka: Adv. in X-ray Anal., 56 (2013), ( 3 ) H. Homma, H. Inoue, M. Feeney, L. Oelofse and Y. Kataoka: Adv. in X-ray Anal., 55 (2012), ( 4 ) JIS R 2011:2007 Appendix 3 Methods for X-ray fluorescence spectrometric analysis of refractory which contains carbon and silicon carbide. ( 5 ) M. Watanabe, Y. Yamada, H. Inoue and Y. Kataoka: Adv. in X-ray Chem. Anal. Japan, 44 (2013), Rigaku Journal, 32(1),

23 Technical articles Advantage of handheld Raman spectrometer with 1064 nm excitation in pharmaceutical raw material identification Taro Nogami * and Fumihito Muta * 1. Introduction Quality control is a top priority for the pharmaceutical industry. RMID (raw material identification) is an important part of quality control. Since health authorities of many countries including Japan joined PIC/S (1) recently, quick and reliable RMID (2) is becoming more important. In the field of RMID, IR (infrared) and NIR (near infrared) spectrometry were used earlier than Raman spectroscopy. However, these analytical methods have limitations in the types of materials they can analyze. Raman spectroscopy can solve the many limitations or problems that were encountered in the past and is now widely recognized as a useful method for material identification. One of the important advantages of Raman spectroscopy over IR and NIR spectroscopy is the fact that water does not cause interference, allowing measurement of moist samples and even aqueous solutions. Another advantage is that the Raman spectroscopy enables direct material identification through a glass bottle or a transparent bag, thus minimizing the risk of contamination. In addition, Raman spectroscopy has an advantage over NIR spectroscopy regarding the ease of material identification. This advantage is due to the abundant, yet sharp and well resolved, spectral peaks in the Raman spectra. These sharp and abundant peaks in Raman allow for material identification without the need to build time consuming chemometric models as is required in NIR spectroscopy. Finally, inorganic materials are normally easier to analyze by Raman spectroscopy than by NIR spectroscopy which shows very weak peaks for inorganic materials. plant origin materials, cell culture media and many cellulosic materials are difficult to analyze by the shorter wavelengths. However, Progeny, which has 1064 nm laser, can analyze these materials. Highly fluorescent glass bottles are also troublesome for the shorter wavelength excitation. This problem also can be minimized by the 1064 nm excitation. The second advantage of the Progeny is an adjustable nose cone. The nose cone, which contacts the sample, has a feature as shown in Fig. 2. Since there is a position scale and click stop for the nose cone, the position can be selected easily depending on sample conditions. Figure 3 (3) shows an analysis of liquid contained in a glass bottle taken through the glass. By shifting the nose Fig. 1. Handheld Raman spectrometer Progeny. 2. Handheld Raman spectrometer Figure 1 shows Rigaku s new handheld Raman spectrometer, Progeny. It has several important features for pharmaceutical applications. First of all, it utilizes 1064 nm excitation wavelength. Existing handheld Raman spectrometers traditionally use shorter excitation wavelengths, such as 532 nm and 785 nm. One of the major limitations of existing handheld Raman spectrometers using shorter wavelengths is fluorescence interference. For some types of materials, high fluorescence background creates detector saturation or causes incorrect material identification. For instance, pigments, many colored excipients, animal/ * Portable Analyzer Division, Rigaku Corporation. Fig. 2. Adjustable focal point nose cone. Rigaku Journal, 32(1),

24 Advantage of handheld Raman spectrometer with 1064 nm excitation in pharmaceutical raw material identification Fig. 3. Analysis of liquid contained in a glass bottle. Fig. 5. Screen of positive verification result. Fig. 4. Spectra of methanol in the glass bottle with different focal point settings. cone position towards the chassis of Progeny, the position of the focal point is shifted away from the nose cone, and placed inside of the glass bottle. Figure 4 (3) shows spectra of methanol in the glass bottle with different focal point settings. When the focal point is at the top of the nose cone, mainly the surface of the glass is analyzed. Stretching vibration peaks of Si O at 1092 cm 1 and Si O Si at 550 cm 1 are observed. Both peaks are Raman peaks of the glass, and are marked by in Fig. 4. However, by shifting the focal point further from the nose cone, these peaks become lower, and the stretching vibration peak of C O at 1036 cm 1 and deformation vibration peak of CH 3 at 1458 cm 1 become higher. They are Raman peaks of methanol, and are marked by. By setting the focal point 5 mm away, the peaks of the glass can be eliminated. This focal point adjustment capability, removes the need to open bottles or packages before making a measurement. This function enables RMID in a warehouse, a receiving dock and any location, without sampling and without a contamination risk. The smart-phone inspired interface provides intuitive operation and decreases the learning time. A user with administrator access can adapt Progeny into any existing workflow by optimizing analysis conditions and libraries. Operators simply use the Fig. 6. Screen of negative verification result. analysis conditions prepared by the administrator, and obtain material identification results easily and quickly. Figures 5 and 6 are examples of material verification results. Figure 5 shows the screen of positive verification result (PASS) of lactose. It proves the tested material is really lactose. Figure 6 shows negative verification result (FAIL) of lactose. It means the tested material is not lactose. The bottom part of the screen shows that the material is actually sucrose. 3. Advantage of 1064 nm excitation for complex or difficult samples 3.1. Cellulosic materials Cellulosic materials are important excipients and Rigaku Journal, 32(1),

25 Advantage of handheld Raman spectrometer with 1064 nm excitation in pharmaceutical raw material identification Fig. 9. Raman spectra of Shin-Etsu AQOAT. Fig. 7. Raman spectra of microcrystalline cellulose. Fig. 8. Raman spectra of ethyl cellulose. widely used for tablets. However, inspections of cellulosic materials by traditional handheld Raman spectrometers with 532 nm or 785 nm excitation sometimes give us uncertain result because of featureless fluorescence background. Progeny with 1064 nm excitation is more suitable to identification of cellulosic materials. The following are comparisons of Progeny's spectrum excited at 1064 nm and a spectrum excited at 785 nm using a traditional handheld Raman spectrometer. Figure 7 shows Raman spectra of microcrystalline cellulose (4) that is widely used in tableting as a filler, disintegrant and dry binder. The spectrum of 785 nm excitation has some deformations at the slopes of fluorescence background, but the spectrum of 1064 nm excitation does not have this problem. Figure 8 shows Raman spectra of ethyl cellulose. The spectrum of 785 nm excitation has deformation by the influence of fluorescence in the high wavenumber region. Figure 9 shows Raman spectra of Shin-Etsu AQOAT (hypromellose acetate succinate). The spectrum of 785 nm excitation has very high fluorescence, and Raman peaks are almost unobservable. Table 1 shows library searching to identify six different cellulosic excipients. Each excipient in a plastic bag was tested six times and compared to a usermade library. After each test, a sample bag is removed from the nose cone, then replaced, for making the reproducibility condition severe. The large numerical characters are the numbers of times identified. The small numerical characters are cc values which show accuracy of identification. 1.0 means perfect match with the library item, and 0.97, 0.98 or 0.99 means almost perfect match with the library item. Though large particle sizes of some cellulosic excipients are detrimental to reproducibility, it can be seen from the table that all materials are identified correctly, with a high cc, every time. In other words, specificity of the analytical method in this material category is proven. These correct identifications are possible only with 1064 nm excitation, as far as cellulosic excipients and other fluorescent materials are concerned Inorganic materials Some of the pharmaceutical inorganic materials do not have a problem with fluorescence background, but other pharmaceutical inorganic materials have high fluorescence background if they are excited at 532 nm or 785 nm. Typical example of later case is talc, which is used for baby powder and others. Spectral comparison of talc between a traditional handheld Raman spectrometer with 785 nm excitation and Progeny with 1064 nm excitation is shown in Fig Nutritional plants and herbal plants Materials taken from plants are highly fluorescent. They are typical examples of materials which can be analyzed only with 1064 nm excitation. In this section, analyses of materials in herbal medicines with Progeny are described. When analyzing materials in herbal medicines with Raman spectroscopy, we must pay attention not only to excitation wavelength but also to particle size, homogeneity and color of materials. Sometimes, particle size of herbal material is not small enough, and/or homogeneity is not good. Intentional defocusing of laser beam by utilizing the adjustable nose cone is Rigaku Journal, 32(1),

26 Advantage of handheld Raman spectrometer with 1064 nm excitation in pharmaceutical raw material identification Table 1. Library searching of cellulosic excipients. Real sample Library HPMC Shin-Etsu AQOAT Ethyl cellulose Hydroxypropyl cellulose Low-substituted hydroxypropyl cellulose Microcrystalline cellulose HPMC Shin-Etsu AQOAT Ethyl cellulose Hydroxypropyl cellulose Low-substituted hydroxypropyl cellulose Microcrystalline cellulose 6 1.0, 1.0, 1.0, 0.99, 0.99, , 0.98, 0.98, 0.98, 0.97, , 1.0, 1.0, 1.0, 1.0, , 0.99, 0.99, 0.99, 0.99, , 0.99, 0.99, 0.99, 1.0, , 1.0, 1.0, 1.0, 1.0, 1.0 Fig. 10. Raman spectra of talc. Fig. 12. Raman spectra of cinnamomi cortex. Fig. 11. Raman spectra of poria. Fig. 13. Raman spectra of glycerol in amber color glass bottle. Rigaku Journal, 32(1),

27 Advantage of handheld Raman spectrometer with 1064 nm excitation in pharmaceutical raw material identification often necessary. Another point is color of materials. When material color is dark brown, it absorbs laser light drastically, and rapid local temperature increase easily occurs. Further defocusing of the laser beam or reduction of laser power may be necessary. Figure 11 shows Raman spectra of poria known as an herb for sedative effect and diuretic effect. The spectrum of 785 nm excitation has extremely high fluorescence, and Raman peaks are almost unobservable, whereas the 1064 nm excitation Raman spectrum collected with Progeny shows clear Raman peaks. Figure 12 shows Raman spectra of cinnamomi cortex known as an herb for warming one s body. The Raman spectrum is collected with the Progeny and Raman peaks are seen. Fluorescence intensity was too high to prevent saturation of signal, when 785 nm excitation was used. 3.4 Test of liquid in amber color glass bottle Figure 13 shows Raman spectra of glycerol in an amber-color glass bottle. Glycerol itself does not have a fluorescence problem. However, the amber-color glass bottle emits fluorescence depending on excitation wavelength. The fluorescence from the glass bottle can be reduced by shifting the focal point to inside of the bottle, but the fluorescence of the bottle is still a problem if excitation wavelength is 532 nm or 785 nm. Progeny does not have this problem. Since many pharmaceutical liquid materials are kept in similar types of bottles to prevent the influence of room light, this is also an important advantage of Progeny s 1064 nm excitation. 4. Other important matters in RMID In this report, advantages of 1064 nm excitation in RMID (raw material identification) were highlighted. However, when analytical instruments are used in the pharmaceutical industry or other GMP settings regulatory compliance is also very important in RMID. Progeny s software is truly compliant with 21 CFR part 11. An on-board digital camera and barcode reader provide error free data tracking. All data, spectral processing and library searches are stored together to meet required audit trails. Systems and procedures for IQ/OQ/PQ are well established. Also, customer support for method validation and other regulatory matters are now ready. 5. Conclusion Fluorescence interference can be now minimized by the new technology incorporated in Progeny. By this progress, the applicability of handheld Raman spectrometers to different types of samples and sample conditions can be expanded. In the past, handheld Raman could not be used for some categories of samples. So, complementary analytical methods including laboratory level analytical methods were necessary. Now, Progeny can cover many types of samples by itself, and it increases total sample throughput. The authors believe the new technology and the new instrument will contribute to inspection of samples in all containers in the future. References ( 1 ) PIC/S GMP Guide, Annex 8. ( 2 ) T. Nogami and F. Muta: Rigaku Journal (English version), 29 (2013), No. 2, ( 3 ) H. Hisada, T. Nogami and F. Muta: Journal of pharmaceutical machinery and Engineering, 24, (2015), No. 5, ( 4 ) M. Szyma ska-chargot, J. Cybulska and A. Zdunek: Sensors, 11 (2011), Rigaku Journal, 32(1),

28 New products Wavelength dispersive X-ray fluorescence spectrometer ZSX Primus IV 1. Introduction X-ray fluorescence spectrometry is one of the common instrumental analysis techniques for routine quality control. This is due to high precision and easy sample preparation compared to other instrumental analytical methods. It is also a powerful analytical tool in the field of research and development for the analysis of advanced materials and products with the recent improvement of data processing of fundamental parameter method. Rigaku has released a new high power sequential type Wavelength Dispersive X-ray Fluorescence (WDXRF) spectrometer ZSX Primus IV with tube above optics to the ZSX Primus family which meets wide variety of recent application needs. The advantages of tube above optics have been recognized in the fields more widely due to its safe measurement and easy sample preparation for pressed pellets without using binder and protect film. The new ZSX Primus IV has been developed as a successor of ZSX Primus II with higher performances and many additional software features. The following are the major features of the ZSX Primus IV: (1) Automatic quant application setup function (2) Improved precision, sensitivity and analysis throughput (3) Intuitive ZSX Guidance software with intelligent functionality The new ZSX Guidance software of ZSX Primus IV is an advanced software package assuring better analytical results and easy operation from novice to skilled operators. 2. Automatic quant application setup function 2.1. Availability of Automatic quant application setting function There are cases that application parameter settings for quant application are not simple and not easy for novice operators such as line overlap and matrix correction. This function provides accurate settings for individual analytical parameters with integration of Rigaku s intelligent expertise of X-ray fluorescence analysis to the software. The followings are the common mistakes in quant parameter settings. Selected analytical line is seriously overlapped with a co-existing element. Background measuring angle is interfered with an interfering line. Matrix correction is not properly set. Overlap correction is not properly set. The element of an interfering line is not included in the elements of measuring lines. The Automatic quant application setup function provides both novice and skilled users the capability to set measuring conditions and various correction parameters for quantitative analysis automatically avoiding the mistakes above. Figure 1 shows setting parameters in the new ZSX Guidance for the quant application setup. In the conventional software, quant parameters are determined by manual measurement of spectrum of each line and study of calibration considering matrix and overlap correction which often requires extensive XRF knowledge. As shown in the Fig. 1, the key parameters Rigaku Journal, 32(1),

29 Wavelength dispersive X-ray fluorescence spectrometer ZSX Primus IV Fig. 1. Setting parameters in the new ZSX Guidance for quant application setup. of measuring conditions, necessary corrections are properly determined by using the data of spectra, peak intensities composition of the standards by the software. This function greatly releases novice and skilled operators from complicated procedure of quant application setup Automatic quant application setting function operation The operation of the automatic setup is very simple as shown below. (1) Check Set the analytical conditions in automatic in Create a New Application window (2) Input sample information, standard sample compositions (3) Select the menu of Run Standards SQX in the quant application flowbar (Fig. 3). All specified standards are measured with Rigaku semi-quant software of SQX. After the SQX analysis of the standards, the software setup quant parameters by itself as described below. (1) Selects optimum analytical lines, determines measuring conditions including peak and background angles using with the qualitative spectra of all standards measured considering line overlap and precision. (2) When undefined elements are detected in the SQX analysis, the elements are added to measuring elements considering the influence to the original analytes. (3) Line overlap corrections and matrix corrections are registered with using the composition information and SQX analysis results. (4) Net intensities for elements of the standards obtained in SQX analysis are registered for tentative evaluation of calibrations. Fig. 2. Initiate Automatic quant setting. Fig. 3. SQX measurements of standards. 3. Example of the automatic quant application setting The application example to zirconia refractory application is explained below. A qualitative spectrum of the Hf-Lα and Hf-Lβ 1 lines from hafnium oxide (1.59 mass%) is shown in Fig. 4. Although the Lα line or Lβ 1 line is generally used for hafnium analysis and the intensity of Lα and Lβ 1 are nearly equal under normal conditions, this spectrum shows that the Lα line intensity is much stronger than the Lβ 1 line. Figure 5 (a) and (b) are the pulse height distribution curves of Hf-Lα and Hf-Lβ 1 of a zirconia refractory sample. Pulse height value (energy) of around 200 is Rigaku Journal, 32(1),

30 Wavelength dispersive X-ray fluorescence spectrometer ZSX Primus IV Fig. 4. Qualitative chart of Hf-Lα and Hf-Lβ 1. Analyzing crystal: LiF (200), Detector: SC Fig. 5. PH distribution curves of Hf-Lα and Hf-Lβ 1. the center of the first order line of the objective pulses, and two times higher pulse height value of around 400 is the second order line (which is an interference line). The Hf-Lα line should be on the around 200 of the pulse height value in the Fig. 5(a), however, due to the very high intensity of the second order line of the Zr-Kα 1 (Zr-Kα 1-2nd from the major component of zirconium), a tail is overlapping into the first order line region. It is obvious that the most of intensity counted under the ordinary pulse height condition is Zr-Kα 1-2nd. On the other hand, Fig. 5(b) clearly shows the separation of the first order line of Hf-Lβ 1 from the Zr-Kβ 1-2nd (which is the second order line from the major component of zirconium). Therefore, the measurement with an ordinary pulse height condition of can be made without the interference of the second order line. The ZSX Guidance software automatically judges the interference situation and selects the proper Hf-Lβ 1 line for the hafnium analysis. It is no longer necessary for an analyst to check manually. The Run Standards SQX runs the full range qualitative analyses, even if the major component is not preset to zirconium, for example, and the software checks the interference of all elements detected in SQX and select optimum analysis lines. Only a portion of the useful features of the Automatic quant application setup function is described above. The software includes variety of advanced database based on accumulated XRF expertise and experiences of Rigaku. The burden placed on the analysts is drastically reduced with this software and at the same time the reliability of analysis results can be improved. 4. Summary The new Rigaku wavelength dispersive X-ray spectrometer with tube above optics, ZSX Primus IV, which now incorporates ZSX Guidance software can be used with ease by even a XRF beginner. Moreover, the adoption of newly developed hardware also provides advanced performance in sensitivity, precision, and throughput. The ZSX Primus IV is the optimal spectrometer not only for process control but also for R&D. Rigaku Journal, 32(1),

31 New products Benchtop total reflection XRF spectrometer NANOHUNTER II 1. Introduction X-ray fluorescence spectrometry (XRF) is well known as an analysis method with high precision by means of non-destructive and no contact analysis, and is therefore widely used for process and quality control at production sites. In particular, total reflection X-ray fluorescence spectrometry (TXRF) developed in the 1970s is a special energy dispersive type XRF which excites elements only on the surface of sample. TXRF has been one of the standard methods for wafer contamination analysis. However, TXRF has recently been gaining attention as a new technique for analysis of environmental samples. Benchtop TXRF spectrometers are manufactured by a few companies in the world, but no models other than the original NANOHUNTER has the highly advanced functions such as auto alignment of the optical system. The new TXRF spectrometer NANOHUNTER II, which is improved by the employment of newly developed components, is introduced here. 2. Features of the instrument 2.1. Realization of significantly higher sensitivity The previous product employed a 50 W X-ray tube and composed the parallel optics with a multi-layer mirror and the Silicon Drift Detector (SDD). The new model is equipped with a high power 600 W X-ray source whose robustness has been proved in X-ray diffractometers. The focusing optics combines a Fig. 1. Spectral chart of Se containing water solution. (Blue: Se10 ppb, Red: Glass substrate) Rigaku Journal, 32(1),

32 Benchtop total reflection XRF spectrometer NANOHUNTER II newly developed multi-layer mirror and incident slit. Moreover, employment of a new large diameter SDD system realizes 100 times higher sensitivity compared with the former model. For example, Fig. 1 shows the spectral chart of a dropped and dried 10 μl water solution sample containing ultra-trace amount of selenium (10 ppb) on a glass substrate. The Se-Kα peak can be clearly seen for the sample which contains Se one order of magnitude less than the amount in effluent regulated by the Ministry of the Environment (Government of Japan). The previous model was equipped with two X-ray tubes of Mo target and Cu target selectable for better excitation efficiency, but the new model is equipped with the Mo target tube only as the difference of excitation efficiency between Mo and Cu targets can be covered by the more than 10 times higher X-ray tube output, and the compatible peak spectra can be observed in the element range measured with Cu target in the previous model. LLD (Lower Limit of Detection) for each element is shown in Fig. 2 below. 1 ppb (μg/kg) of LLDs are achieved for various elements. As a result of improved sensitivities, analysis with Fig. 2. LLD for each element. 1) lower LLD or 2) shorter measurement time can be performed Capability of high energy excitation (about 30 kev) Standard excitation employs a multi-layer monochromator which monochromates the Mo-Kα line (about 17.4 kev) to irradiate the sample. Conventionally, this multi-layer is designed to construct the alternate lamination of two element layers, namely, a heavy element to be a reflection layer and a light element works as a spacer. A multi-layer monochrometer is placed after X-ray tube to reflect only the Mo-Kα line (17.4 kev) to irradiate the sample. In common multi-layer monochrometers, heavy element layer (reflection layer) and light element layer (spacer layer) are coated alternatively. The new multilayer monochrometer has been specially designed to reflect X-rays with two wavelengths of Mo-Kα line and higher energy with about 30 kev. The higher energy X-rays can effectively excite K line fluorescent X-rays of heavy elements such as elements from Ru to Sn. The measurement of K lines for these elements is very effective and good spectra with high peak to background ratio can be obtained since there are very few line overlaps. It also gives extremely low background in this energy range due to the principle of total reflection, whereas background is very high in conventional X-ray fluorescence spectrometry. For example, Cd, which is the critical element for environmental analysis, used to be analyzed with L-line spectrum excited by the Mo characteristic X-rays in the past (Cu in case of former model). There were many occasions that the Cd-L line at about 3.1 kev cannot be easily detected due to overlap by the potassium peak (about 3.3 kev) common in environmental samples and the Ar peak at about 3.0 kev existing in the air. In contrast, the Cd-K line at about 23 kev has few overlapping spectra nearby and therefore allows easy detection of ultra-trace amounts. Figure 3 shows the spectrum of 10 μl water with 1 ppm Cd dropped and dried on a slide glass. The Cd Fig. 3. Kα-line spectrum of 1 ppm Cd. Rigaku Journal, 32(1),

33 Benchtop total reflection XRF spectrometer NANOHUNTER II Fig. 4. Spectra from Ni thin film (25 nm) with glass substrate. peak is clearly detected due to the low BG unique to TXRF instrument Variable incident angle The incident angle auto adjustment was one of the essential optical adjustment functions to the original TXRF spectrometer. The function continues to be available on NANOHUNTER II. The variable incident angle function also continues to be available which allows measurement with the optimal incident angle for any sample substrate which can have varying critical angles. Programmable driving of incident is a new feature which enables control of divergence angle. Figure 4 shows the spectra of vapor-deposited Ni thin film (25 nm equivalent) on a glass substrate excited with different incident angles. When the incident angle is low, almost no peaks from the substrate is observed and the peak from the surface layer Ni is prominent. On the other hand, the peak intensity from the surface layer Ni decreases and the intensity of the substrate component increases for high incident angle. Only the elements located near the surface are excited efficiently in TXRF condition, and the information of deeper part can be obtained in high incident angle. 3. Application: Analysis of river water The elemental analysis result of river water is shown below as an example. 10 μl sample added with Co as an internal standard element is dropped on an optical slide glass and dried. Measurement of the river water specimen resulted in detection of S, Cl, K, Ca, Fe, Br and Sr. Table 1 shows the quantification result of the detected elements. It can be seen that the Table 1. Result of quantitative analysis of river water (ppm). Z TXRF ICP-AES (A) ICP-AES (B) S Cl (IC) K Ca Fe * * Br (IC) Sr *Suspended solid is not included. results are comparable to inductively-coupled plasma atomic emission spectrometry ICP-AES (partially by ion chromatography IC) measurement results at two laboratories (A, B). 4. Summary The benchtop TXRF NANOHUNTER II is the superior instrument for material surface analysis of materials requiring high sensitivity. Although the most common applications are qualitative and quantitative analysis of trace element in liquids by means of dropping and drying, high sensitivity surface analysis of solid substances is also possible and therefore it is expected that it will be utilized by a wide range of users. Moreover, as it is also applicable as a grazing incidence X-ray fluorescence (GIXRF) instrument, this spectrometer can be a new evaluation apparatus which provides not only elemental analysis of sample surface but also elemental information for depth direction. Rigaku Journal, 32(1),

34 New products Automated dislocation evaluation software for X-ray topography images Topography Analysis 1. Introduction X-ray topography is a powerful technique for evaluating crystal defects such as dislocations, stacking faults, scratches, and so on. High performance electronics devices such as microprocessors, solidstate memories, imaging processors are fabricated on dislocation-free Si single crystal wafers. However, device fabrication processes often induce dislocations in the Si wafers that can affect the device s performance. Because X-ray topography can evaluate these crystal defects efficiently, it plays an important role in the Si industry. Recently, composite semiconductors for example, SiC-based materials have been highlighted due to their higher band gap energies and higher breakdown voltages, features superior to those of Si for achieving higher-efficiency power devices. These devices are sought to improve the utilization efficiency of electric energy and reduce carbon dioxide emissions in order to prevent global warming. Although a lot of effort has been devoted to developing growth technology for achieving lowdislocation density crystals, even the highest quality crystals still have many dislocations, in the range of hundreds to thousands per cm 2. These dislocations can degrade the device s performance. Therefore, quantitative measurement of the dislocation density is crucial for controlling fabrication yields and improving device reliabilities. Recently, a high-resolution and high-speed X-ray topography measurement instrument called XRTmicron has been released that allows users to acquire high-quality topography digital images (1). New software that analyzes the obtained digital topography images to count dislocations and identify their types has been developed. In this note, the operation of the software will be introduced. 2. Dislocation of SiC crystal Figure 1 shows a typical transmission topography image of a (0001) SiC wafer taken using the reflection. Three types of dislocations can be recognized Fig. 1. Transmission topography image taken by reflection for a SiC wafer. Threading screw, threading edge, and basal plane dislocations are clearly recognizable. Rigaku Journal, 32(1),

35 Automated dislocation evaluation software for X-ray topography images Fig. 2. Comparison of synchrotron reflection, laboratory reflection, and laboratory transmission topography images of the same area. TSDs are detected identically in the synchrotron and laboratory reflection topography images. The transmission topography image includes several BPDs inside the wafer and the TSDs correspond well with the other experiments. in this image. Large strong dots indicate threading screw dislocations (TSD), which are directed along the c-axis and are accompanied by large strain fields around the dislocation core. Its Burgers vector is nc 0001 (n 1, 2). Basal plane dislocations (BPD) are extended in the (0001) plane and appear as long lines in this topography image. Weak dots and short lines are classified as threading edge dislocations (TED), which are elongated along the c-axis as were the TSDs. However, TED accompanies a smaller strain field due to its smaller Burgers vector a/ compared to that of TSD, making weak dots in the topography image. We have confirmed the identification of the above described three types of dislocations by comparing the topography images with those taken at a synchrotron facility. Synchrotron topography is known as the standard method for identification of dislocations (2). Figure 2 shows the comparison of topography images taken by synchrotron and laboratory sources, respectively. Although the dislocation image taken at the synchrotron is much clearer than that taken with the laboratory system, almost identical TSD results are obtained at the synchrotron as with laboratory Cu reflection topography. In the transmission topography image, there are a lot of BPDs and some TSDs that are unclear or missing. However, more than 80% of TSDs can be detected using transmission geometry. In addition, clearly detected BPDs are known to affect device performance; therefore, reliable quantitative detection of BPDs is a crucial function. 3. Dislocation analysis software for SiC crystals Rigaku s new X-ray topography system enables us to acquire high-resolution digital image data, which is of sufficient quality to identify dislocation types TSD, TED, and BPD in SiC crystals,. Then, automated identification software was developed. It can not only identify dislocation types, but also count the numbers of TSDs, TEDs, and BPDs. However, counting BPDs is not Fig. 3. Software display for the detection of dislocations for (a) TEDs, (b) TSDs, and (c) BPDs. Rigaku Journal, 32(1),

36 Automated dislocation evaluation software for X-ray topography images in the image, the user can control the threshold of the lower detection limit for each dislocation type. Fig. 4. Flow chart demonstrating an automated topography measurement and analysis (left) and the results of wafer mapping for TSD and BPD (right). appropriate for transmission topography, because BPDs are connected, making networks. Therefore, a function measuring the lengths of BPDs has also been developed. How the software operates is shown in Fig. 3(a), (b), and (c), which identify TEDs, TSDs, and BPDs, respectively. The resultant dislocation density is indicated as number/area in units of cm 2. The length of a BPD is converted to a dislocation density by calculating length/ (area thickness) in units of cm/cm 3. The software contains a field where the wafer thickness can be entered by the user. In order to avoid misdetection due to noise 4. Whole wafer mapping of the dislocations For quality and yields control for SiC devices, dislocation densities should be one of the key parameters. Therefore, wafer mapping of dislocation densities is strongly recommended. Combination of a topography measurement system and the dislocation analysis software can achieve automated wafer mapping of dislocations. The user can select the size of the unit area, make a whole wafer map as shown in Fig. 4 and compare device performance at each point. X-ray topography has long been used for evaluating the crystal quality of wafers. However, very few specialists can interpret the observed topography images, so they were never used for quality and production control. Rigaku s automated topography measurement and analysis system makes it possible to utilize this analysis for the quality control of production wafers. The developed dislocation analysis software can be easily modified for other crystals, such as Si, GaN, Al 2 O 3, others. Those software enhancements will be released in the near future. References ( 1 ) K. Omote: Rigaku Journal (English version), 29 (2013), 1 8. ( 2 ) H. Tsuchida, I. Kamarta and M. Nagano: J. Cryst. Growth, 310 (2008), Rigaku Journal, 32(1),