REVIEW OF SCIENTIFIC INSTRUMENTS 86, 035103 (2015)

A fluorescence XAFS measurement instrument in the soft x-ray region toward observation under operando conditions M. Honda,a) Y. Baba, I. Shimoyama, and T. Sekiguchi Quantum Beam Science Center, Japan Atomic Energy Agency (JAEA), 2-4 Shirakata-Shirane, Tokai-mura, Naka-gun, Ibaraki 319-1195, Japan

(Received 31 October 2014; accepted 15 February 2015; published online 5 March 2015) X-ray absorption fine structure (XAFS) measurements are widely used for the analysis of electronic structure. Generally, XAFS in the soft X-ray region is measured under vacuum, but chemical structures under vacuum are typically different from those under operando conditions, where chemical species exhibit their function. Here, we developed an XAFS measurement instrument, as a step toward operando fluorescent, which yields XAFS measurement using synchrotron radiation in the soft X-ray region. We applied this method to analyze the local electronic structure of the sulfur atoms in L-cysteine in different pH solutions. In water at pH 7, the hydrogen atom does not dissociate from the thiol (-SH) group in L-cysteine, which forms a structure surrounded by and interacting with water molecules. The XAFS spectrum of L-cysteine in solution was altered by changing the pH. At pH 9, the hydrogen atom dissociated and a thiolate anion was formed. Although the -SH group was oxidized to SO42− when L-cysteine was adsorbed on a metal surface and dried, no oxidation was observed in solution. This may be because the water molecules were densely packed and protected the -SH group from oxidation. Our results show that this instrument aimed toward operando fluorescence XAFS measurements in the soft X-ray region is useful for structural analysis of sulfur atoms in organic molecules in air and in solution. The instrument will be applied to the structural analysis of materials containing elements that have absorption edges in soft X-ray region, such as phosphorus and alkali metals (potassium and cesium). It will be also particularly useful for the analysis of samples that are difficult to handle under vacuum and materials that have specific functions in solution. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4913653]

I. INTRODUCTION

To solve global energy and environmental problems, which have become more diverse in recent years, the development of solar cells, new catalyst materials, and adsorption materials is required. This has stimulated interest in the structure and behavior of the organic molecules in solution, as well as interest in interfaces between solids and organic molecules. For example, understanding of the bonding states and reaction mechanisms at metal-molecule interfaces is desirable for developing medical materials in which biomolecules are adsorbed on a solid surface. In particular, it is important to understand structures and reaction mechanisms at interfaces between metals and amino acids in materials that are widely used in medical applications. Amino acids are amphoteric molecules that have an amino group and a carboxyl group and adopt different structures depending on the pH of the solution. The development of a method that enables in situ structural analysis of amino acids at the solid-liquid interface as well as in solution is an important step in elucidating the mechanisms of chemical reactions. Structures and reactions at the interfaces between metal surfaces and organic molecules have been extensively studied. For example, the adsorption structure of organic molecules

a)Author to whom correspondence should be addressed. Electronic mail:

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containing sulfur, such as thiol on metals, has been widely studied in self-assembled films.1–3 Furthermore, studies on the interfaces between solid surfaces and amino acids have been reported in relation to transistors,4 corrosion prevention,5 blue luminescence emission,6 and various other topics. The amino acid L-cysteine contains a thiol group,7 which strongly adsorbs on metals. The adsorption structure of L-cysteine has been reported previously.8,9 In those reports, the samples were removed from solution and then measured under ultrahigh vacuum after drying. However, biomolecules, such as amino acids, form a wide variety of structures that exhibit their properties only when in solution and these structures induce a wide diversity of functions. Up to now, there have been few reports on the direct observation of the changes in the molecular structure of biomolecules in solution using soft X-rays. While the soft X-ray range extends down to 200 eV or below and is served by grating monochromators below roughly 2000 eV; here, we consider soft X-rays in the 1800–3000 eV range (hereinafter, it is to be called as soft X-ray) served by crystal monochromators. Although X-ray absorption spectroscopy is a powerful tool for determining molecular structures under atmospheric pressure and in solution, there has been little research applying this technique to biomolecules. This is because organic molecules consist of light elements, such as carbon, nitrogen, oxygen, phosphorous, and sulfur, and Xrays around the absorption edges of these light elements are absorbed by air and water. For example, when soft X-rays at the K absorption edge of sulfur atoms (2.4 keV) are transmitted

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through 3 cm of air, the X-ray intensity is attenuated to 35%. Similarly, at the K absorption edge of carbon, X-rays are attenuated to just 5% by only 5 mm of air. This attenuation is even more pronounced in solution: X-rays at 2.4 keV are attenuated by 50% after transmission through just 20 µm of water.10 For this reason, conventional X-ray absorption spectroscopy measurements have been limited to vacuum conditions. However, the behavior of chemical species in a vacuum may not be the same as that in air or in solution and there is a need for measurement under operando conditions, that is, under the conditions where chemical species exhibit their function. In the present research, we developed a fluorescence Xray absorption fine structure (XAFS) measurement instrument for performing X-ray absorption spectroscopy in air and in solution for elements in the third row of the periodic table, such as sulfur and phosphorous, which are important in organic molecules. We applied this instrument to the in situ observation of the structural changes of L-cysteine molecules in different pH solutions. It has previously been shown that the molecular structure of biomolecules in solution using soft X-rays.11 The electronic structure of amino acids using XAFS measurements in solution in the soft X-ray region has previously been reported by Yagi et al.12 They successfully performed fluorescence XAFS measurements of L-cysteine adsorbed on the surface of transition metals (Ni, Cu) and found that L-cysteine molecules adsorbed on these transition metals form thiolate ions in solution. Furthermore, they observed that the sulfur atom in L-cysteine interacts with 3 or 4 water molecules in solution, and that the S-C bonds are dissociated and water molecules are released after drying the sample. In the other reports, the effect of water molecules on the structure of thiol groups has been reported for L-cysteine in aqueous solution based on theoretical calculations.13 It has been shown that the watersulfur hydrogen bond of aqueous L-cysteine is so weak that the water molecules do not induce structural changes in L-cysteine molecules,13,14 but this is still under debate. The apparatus used by Yagi et al. held the substrate surface in a vertical position for reasons related to the sample holding mechanism. In contrast, one aim of our research was

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to adapt fluorescence XAFS for in situ observations of solidliquid interface reactions. In addition, two papers outlining the design and operation of related in-situ cells have recently reported.15,16 In these reports, there are instruments that can detect liquid-phase reactions using fluorescence and inelastic scattering at the C, N and O, K edges. Such in situ observations are a step toward measurements under operando conditions. To this end, measurements must be made over a large area uniformly and sensitively. Thus, we developed a fluorescence XAFS instrument that meets these requirements (Fig. 1). Since the sample surface faces downward in this instrument, a uniform surface can be maintained even for solution samples. Furthermore, because our instrument employs radiation that spreads out horizontally, a wide area can be uniformly illuminated by radiation at a grazing incidence to the surface, giving high detection sensitivity. We have previously discussed the interfacial bonding of metal and sulfur in detail based on XAFS data collected under vacuum. For an Au/Si substrate, we reported that when the Si(111) surface was completely coated with Au, L-cysteine adsorbed onto the metal surface via only the sulfur atom. In contrast, when only one-third of the surface was covered with Au, L-cysteine molecules adsorbed to the metal in two different arrangements, via the sulfur atom and via the carboxyl group.17 We also found that an anomalous chemical bond was formed between the metal and sulfur.18 In the present research, we measured the fluorescence XAFS spectra of L-cysteine film and L-cysteine in aqueous solution and compared the results with spectra acquired under vacuum, in air, and in solution. We also performed fluorescence XAFS measurements of L-cysteine in aqueous solutions at different pH levels and report the in situ observation of the structural changes in L-cysteine. II. METHODS

A fluorescence XAFS instrument for measurements in air and in solution was setup in the biology end station19 of the BL27A soft X-ray beamline at the Photon Factory of the High Energy Research Organization (KEK-PF). An overview of the experimental instrument is shown in Fig. 1. In this

FIG. 1. Schematic diagram of soft X-ray XAFS instrument (left) and photograph of the instrument (right) at the KEK-PF BL27A end station.

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beamline, X-rays from a bending magnet are focused by a horizontal mirror, and the X-ray beam is deflected upward at an angle of 1.6◦. As a result, a wide area of the horizontally placed sample is uniformly illuminated from lower side at a grazing angle of 1.6◦ to the surface. A two-crystal InSb (111) monochromator is used for tuning the X-ray energy. The energy range of the incident X-rays is in the soft X-ray region from 1800 to 6000 eV. The fluorescence XAFS measurement instrument consists of (1) a glovebox into which X-rays are introduced, (2) a sample holder, and (3) a fluorescence X-ray detector. Component (1) is an acrylic vacuum glovebox equipped with a gas inlet line and evacuation line and is designed to obtain a low vacuum through evacuation by a scroll pump (ISP-90, Anest Iwata Co., Ltd.). The connection part of this glovebox is equipped with KaptonR film window. The KaptonR polyimide film thickness is 25 µm. The diameter of this window size is 10 mm. Gas replacement can be performed easily by evacuating for a fixed period of time using the scroll pump and then flushing the box with He to allow measurement under atmospheric pressure. Component (2) is a sample holder mechanism with a remote controlled stage (Sigma Koki Co., Ltd.). This allows the sample position to be adjusted in the x (beam direction), y (horizontal direction perpendicular to the beam), z (vertical direction), and θ (X-ray angle of incidence) directions by remote control during X-ray irradiation. Component (3) is an X-ray detector. A Peltier-cooled Si-PIN X-ray detector (X123, Amptek Co., Ltd.) was used that fits in the limited space of the small glove box. The energy resolution (FWHM) of the detector was 145 eV at 5.9 keV.20 As shown in Fig. 2, in this geometry, the detector is far from any secularly reflected xrays from the sample. Any high-angle diffuse scattering from the sample is expected to be weak, even with the predominant s-polarization used. In addition, experimental geometry shown in Fig. 2, elastic scattering from the sample is not suppressed; however, at this BL-27 beamline, approximately 10% of vertical polarization ingredients exist. During XAFS measurements, the spectrum is obtained by varying the incident Xray energy, with the Si-PIN semiconductor detector in multichannel scalar mode. The X-ray fluorescence from the sample

FIG. 2. Experimental arrangement of the fluorescence XAFS instrument around the sample. Linearly polarized soft X-rays with the electric field vector E in the horizontal plane were incident on the horizontal sample at an angle θ with respect to the surface. The sample could be rotated around the y-axis within θ◦ ± 6◦.

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was detected and its intensity was plotted for each incident Xray energy. The X-ray intensity was normalized with respect to the current in an aluminum mesh placed in front of the sample in the vacuum chamber. During the measurements, glovebox was further shielded by acrylic hutch in order to protect radiation exposure to the operator. All the XAFS measurements were conducted by remote control. Two samples were used: (a) L-cysteine in aqueous solution and (b) L-cysteine on a metal substrate. For sample (a), L-cysteine was dissolved in pure water to give a 0.5 mol dm−3 aqueous solution (pH 7). For sample (b), a gold metal substrate was immersed in the 0.5 mol dm−3 L-cysteine solution for 24 h and then rinsed with water. Furthermore, to investigate the changes in the L-cysteine molecular structure in solutions at different pH levels, standard buffer solutions of pH 4.01 (phthalate pH standard solution) and pH 9.18 (tetraborate pH standard solution) containing L-cysteine were used.

III. RESULTS AND DISCUSSION

First, we investigated the structural differences between L-cysteine dissolved in water and adsorbed on the gold surface. Figure 3 shows the XAFS spectra of (a) L-cysteine in water and (b) L-cysteine on the gold substrate measured in a He atmosphere. At first, no radiation damage for the samples was confirmed in this energy region before and after XAFS measurement. In both spectra, a main peak (labeled B) is observed near 2470 eV, and the structure (labeled A) is seen as a shoulder of peak B. A broad peak (labeled C) is observed near 2476 eV in only the spectrum of aqueous L-cysteine (a). First, we assign structures A and B. Sample (a) was the L-cysteine in water at pH 7, so we suppose that the thiol group was not dissociated because L-cysteine maintains its amphoteric state

FIG. 3. Comparison of S K -edge XAFS spectra for (a) L-cysteine in H2O and (b) L-cysteine on an Au substrate. Molecular structures inferred from the XAFS spectra for (I) L-cysteine, (II) L-cysteine in H2O, and (III) L-cysteine on an Au substrate are shown at the top right.

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in neutral solution. If the SH bonds are not dissociated, the main peak corresponds to the transition from the S 1s orbital of the thiol group (R-CH2-SH) to an unoccupied σ∗ orbital of the sulfur atom. Because L-cysteine (structure II, Fig. 3) has a molecular structure of R(COO−)-CH2-SH structure, the resonant excitations from the S 1s orbital to the σ∗ orbital localized at the S-C bond (hereinafter denoted S 1s → σ∗ (SC)) and to the σ∗ orbital localized at the S-H bond (hereinafter denoted S 1s → σ∗ (S-H)) must be considered. By comparison with the XAFS spectrum reference values for alkane thiol molecules such as CH3-SH21 in the gas phase, structure A is probably attributable to the S 1s → σ∗ (S-C) transition, and peak B is assigned to the S 1s → σ∗ (S-H) transition. In contrast, if we assume that the hydrogen dissociates, the peak should shift to the low energy side because peak B would disappear. However, the XAFS spectrum of sample (a) shows that the σ∗ (S-H) resonance peak still remains as peak B, and the spectral structure is similar to that for CH3-SH.21 The result indicates that hydrogen surely does not dissociate in water at pH 7. The data suggest that water molecules are not chemically bonded to L-cysteine but coordinated to L-cysteine to form a complex. The possible structure of L-cysteine in water at pH 7 is shown in Fig. 3 (structure II). Next, we will discuss spectrum (b). When L-cysteine adsorbs on a gold metal surface, the hydrogen dissociates from the thiol group (-SH) and the L-cysteine binds to the gold substrate via the sulfur atoms, as shown in structure III of Fig. 3.7 As a result, peak B is thought to arise from the Au-S bonding. However, it should be noted that the spectral shape obtained by the fluorescence XAFS instrument for Lcysteine on the gold substrate does not accurately reflect the real absorption spectrum. This is because the density of S atoms in L-cysteine film is 2.07 g/cm3, whereas the density of Au in the substrate is 19.28 g/cm3; thus, the signal from the substrate is much greater. The fluorescence quantum yield also differs between sulfur and gold.22 In addition, S Kα X-rays have an energy of 2309 eV, which is close to the energy of Au Mα1 X-rays (2123 eV). The fluorescence X-rays measured by the detector, therefore, have a poor signal to background ratio because the background emissions from the Au are high. However, the peak energy and spectral shape matched previous results obtained for a sample in a vacuum using the total electron yield method.18 Our results suggest that this fluorescence XAFS instrument can correctly measure organic molecules adsorb on a heavy metal surface. Next, we will discuss the broad peak C around 2476 eV observed in XAFS spectrum of sample (a). The energy of peak C is approximately 6 eV higher than that of structure A or peak B. From the results of theoretical calculations for thiol molecules (CH2SH, C2H5SH, C6H5SH), this peak is thought to be resonant absorption from the S 1s orbital to the 2e− orbital.21 We found that the sulfur in L-cysteine is not oxidized in water because the resonance peaks for oxidized sulfur such as sulfate located at higher energy were not observed in the wide-scan spectrum shown in the lower side of Fig. 3. Later, we will discuss this point in detail compared with the XAFS spectrum for dried sample. To ascertain the influence of pH on the L-cysteine molecular structure, the S K-edge fluorescence XAFS spectra of

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FIG. 4. Comparison of S K -edge XAFS spectra for L-cysteine in pH 4 (black) and L-cysteine in pH 9 (red) solutions.

L-cysteine molecules were measured in aqueous solution at different pH levels. The results are shown in Fig. 4. XAFS data processing was simply smooth. Although peaks A and B were not separated because the resolution was poor, the position of the peak energy for pH 9 was lower than that for pH 4. The peak is expected to shift toward the low energy side when the intensity of peak A becomes larger, but toward the high energy side when the intensity of peak B becomes larger. Thus, peak A is larger at pH 9 and peak B is larger at pH 4. The results in Fig. 4 therefore show that the S-C bond was dominant under basic conditions, while the ratio of S-H relatively increased under acidic conditions. Figure 5 shows the plausible structure of L-cysteine molecules in solutions at different pH levels. Because amino acids are amphoteric, the structures of amino group and carboxyl group change between pH 4 and 9. As the isoelectric point, where the L-cysteine molecule has an amphoteric structure is around pH 5, L-cysteine molecules should be deprotonated at pH 9. This structural change would lower the S 1s → σ∗ (S-H) resonant absorption intensity, which is consistent with the peak shifting toward the lower energy side in Fig. 4. The result indicates that the structural changes of Lcysteine in solution according to pH induce the observed spectral changes of S K-edge XAFS. Thus, it is demonstrated that present fluorescence XAFS measurement instrument is useful for observing changes in the molecular structural of amino acids. To investigate differences between the structure of Lcysteine molecules in solution and solid L-cysteine molecules, XAFS spectrum for L-cysteine in water was compared with a

FIG. 5. L-Cysteine structures under various pH conditions.

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FIG. 6. S K -edge XAFS spectra for (a) L-cysteine in H2O and (b) L-cysteine recrystallized from water and dried.

sample that was first dissolved in water and then recrystallized by evaporation and dried (Fig. 6). In the recrystallized L-cysteine, peak D was observed on the high energy side (2484 eV), 14 eV from the main peak. Peak E was observed on the higher energy side (2489 eV), 19 eV from the main peak. XAFS analysis of the S K-edge absorption spectra of Na2SO4 suggests that these peaks (D, E) originate from the resonant excitations from S 1s to d-type shape resonance in SO42−.23 This may be because the sample was oxidized during the recrystallization process. In contrast, for the structure of L-cysteine in water, as previously discussed with respect to Figure 3, the resonance peaks of SO42− (peaks D and E) are not observed although the resonance absorption peak from the S 1s orbital to the 2e− orbital is seen as peak C at the lower energy side. These results confirm that the sulfur atoms are not oxidized to SO42− in water at pH 7. We expect that L-cysteine molecules in pH 7 water are surrounded by H2O, and the -SH group should form a hydrate with the water molecules without dissociating. However, when L-cysteine is recrystallized and dried (sample in air), -SH dissociates in the course of water evaporation, and the sample is oxidized by the oxygen in air. This suggests that in water, -SH group should form a hydrate without oxidization, owing to the close packing of water molecules around L-cysteine molecules, even though the concentration of oxygen atoms in water is much higher than that in air. In conclusion, we have developed a soft X-ray fluorescence XAFS instrument that can be applied to in situ structural analysis as a step toward observations under operando condition. Using this instrument, we have shown the structures of L-cysteine molecules in solution on the basis of S K-edge XAFS spectra around 2.4 keV. Similarly, this instrument can be applied to materials containing the other elements that have absorption edges in this energy region, such as period 3 elements (Si, P, etc.), 4d transition metals (Y, Zr, Nb, Mo, etc.), actinide elements (U, etc.), and so forth.

IV. CONCLUSIONS

Vacuum conditions have previously been required for XAFS measurements in the soft X-ray region. In this study, we succeeded in measuring the local electronic structure at the

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sulfur atom in L-cysteine in air and in solution by fluorescence XAFS measurements. The results showed that H2O molecules are coordinated to the -SH group in solution. Furthermore, the XAFS spectra for solution are different at different pH levels. At pH 4, the local structure at the sulfur atom in Lcysteine adopts an R-S-H configuration without dissociation of the H from the -SH group. At pH 9, the L-cysteine molecules form an R-S- structure after the dissociation of the hydrogen from the -SH groups. When L-cysteine dissolved in water was recrystallized by evaporation and dried, SH groups were oxidized to SO42−. Although the density of the water molecules is high in solution compared with in air, we found that there was no oxidized species in water. This suggests that in water, the -SH group forms a hydrate without oxidization, as a result of the close packing of water molecules around Lcysteine molecules. ACKNOWLEDGMENTS

This research was performed using the BL27A biology end station at the KEK-PF. We wish to thank Katsumi Kobayashi and Noriko Usami, who have previously performed research and development of BL-27, for their tremendous efforts in building the instrument. We also wish to thank Dr. Norie Hirao for kind support. Part of this research project was supported by a JSPS Grant-in-Aid for Young Scientists (B) 25790059. 1G.

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A fluorescence XAFS measurement instrument in the soft X-ray region toward observation under operando conditions.

X-ray absorption fine structure (XAFS) measurements are widely used for the analysis of electronic structure. Generally, XAFS in the soft X-ray region...
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