Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited

Fourier-transform infrared (FT-IR) difference spectroscopy has been proven to be a significant tool in biospectroscopy. In particular, the step-scan technique monitors structural and electronic changes at time resolutions down to a few nanoseconds retaining the multiplex advantage of FT-IR. For the elucidation of the functional mechanisms of proteins, this technique is currently limited to repetitive systems undergoing a rapid photocycle. To overcome this obstacle, we developed a flow-flash experiment in a miniaturised flow channel which was integrated into a step-scan FT-IR spectroscopic setup. As a proof of principle, we studied the rebinding reaction of CO to myoglobin after photodissociation. The use of microfluidics reduced the sample consumption drastically such that a typical step-scan experiment takes only a few 10 ml of a millimolar sample solution, making this method particularly interesting for the investigation of biological samples that are only available in small quantities. Moreover, the flow cell provides the unique opportunity to assess the reaction mechanism of proteins that cycle slowly or react irreversibly. We infer that this novel approach will help in the elucidation of molecular reactions as complex as those of vectorial ion transfer in membrane proteins. The potential application to the oxygen splitting reaction of cytochrome c oxidase is discussed. An erratum to this article can be found at     

电化学原位步进扫描时间分辨显微镜FTIR反射光谱

结合步进扫描傅立叶变换红外光谱仪和红外显微镜，建立了电化学原位步进扫描时间分辨显微镜FTIR反射光谱.采用微电极和利用纳米结构表面的异常红外效应，显著提高表面吸附物种的红外检测灵敏度和红外电极表面对极化电位的响应速度，使广泛应用的外反射型薄层红外电解池的时间常数(τ=R1Cd)从使用常规盘电极的约40 ms降低到0.65 ms.研究了酸性介质中CO在Pt电极上的吸附过程，谱图的时间分辨率达50 μs.     

Selective detection of the structural changes upon photoreactions of several redox cofactors in photosystem II by means of light-induced ATR-FTIR difference spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was applied for the first time to detect the structural changes upon photoreactions of redox cofactors in photosystem II (PSII). The PSII-enriched membranes from spinach were adsorbed on the surface of a silicon prism, and FTIR measurements of various redox cofactors were performed for the same sample but under different conditions by exchanging buffers in a flow cell. Light-induced FTIR difference spectra upon redox reactions of the oxygen-evolving Mn cluster, the primary quinone electron acceptor QA, the redox-active tyrosine YD, the primary electron acceptor pheophytin, and the primary electron donor chlorophyll P680 were successively recorded in buffers including different redox reagents and inhibitors. All of these cofactors remained active in the PSII membranes on the silicon surface, and the resultant spectra were basically identical to those previously recorded by the conventional transmission method. These ATR-FTIR measurements enable accurate comparison between reactions of different active sites in a single PSII sample. The present results demonstrated that the ATR-FTIR spectroscopy is a useful technique for investigation of the reaction mechanism of PSII.     

Preparation of thin-sections of painting fragments: Classical and innovative strategies

For more than a century, the analyses of painting fragments have been carried out mainly through the preparation of thick resin-embedded cross-sections. Taking into account the development of innovative micro-analytical imaging techniques, alternatives to this standard preparation method are considered. Consequently, dedicated efforts are required to develop preparation protocols limiting the risks of chemical interferences (solubilisation, reduction/oxidation or other reactions) which modify the sample during its preparation, as well as the risks of analytical interferences (overlap of detected signals coming from the sample and from materials used in the preparation). This study focuses particularly on the preparation of thin-sections (1–20 μm) for single or combined fourier transform infrared (FTIR) spectroscopy and X-ray 2D micro-analysis. A few strategies specially developed for the μFTIR analysis of painting cross-sections have already been reported and their potential extrapolation to the preparation of thin-sections is discussed. In addition, we propose two new specific methods: (i) the first is based on a free-embedding approach, ensuring a complete chemical and analytical neutrality. It is illustrated through application on polymeric design objects corpus; (ii) the second is based on a barrier coating approach which strengthens the sample and avoids the penetration of the resin into the sample. The barrier coating investigated is a silver chloride salt, an infrared transparent material, which remains malleable and soft after pellet compression, enabling microtoming. This last method was successfully applied to the preparation of a fragment from a gilded Chinese sculpture (15th C.) and was used to unravel a unique complex stratigraphy when combining μFTIR and μXRF.     

Diffuse reflectance Fourier transform IR spectroscopy

Fourier transform infrared (FTIR) spectroscopy has greatly enhanced the utility and range of applications of infrared (IR) spectroscopy in chemistry. Until the development of routine FTIR instrumentation, applications of IR were essentially limited to direct transmission measurements. Newer sampling methods in FTIR spectroscopy can be successful with less than 1% throughput of the infrared radiation and yet yield a spectrum with acceptable signal-to-noise ratio. The decreasing cost of FTIR spectrometers has made a number of such sampling techniques accessible for routine applications without compromises in performance. This article will describe one of these, diffuse reflectance.     

具有辐射聚合能力的HfO2/SiO2溶胶-凝胶玻璃的制备及其X射线光刻性能研究

采用溶胶-凝胶方法制备了一种新颖的具有辐射聚合能力的HfO2/SiO2凝胶薄膜. 并采用X射线作曝光光源对薄膜进行了曝光, 通过FTIR的测试, 分析了薄膜曝光前后的结构变化. 结果表明, 该材料具有良好的辐射聚合能力. 采用XPS分析了薄膜的成分, 并证实了Hf元素的存在. 用椭偏仪测试了薄膜的折射率, 结果证实, 加入HfO2提高了体系的折射率. 利用其辐射聚合能力, 采用X射线通过掩模板进行曝光, 利用曝光部分与未曝光部分在溶剂中的溶解度差, 在薄膜上制备了高为0.8 μm、周期为1 μm的衍射光栅, 进一步证实了材料具有良好的辐射聚合能力.     

Infrared emission spectroscopy of liquid samples with multivariate calibration as a model for process analytical applications

Infrared emission spectroscopy and multivariate calibration are used to provide a method for the quantitative analysis of liquid samples. Differing forms of the data including second derivative and interferogram representation are used for prediction of sample composition, thickness and temperature. Comparisons are made with transmission measurements of the same samples. In some situations emission measurements may be the preferred method of analysis.     

FT-Raman and FTIR spectroscopic studies of N-octadecanoyl-L-alanine amphiphiles

FTIR spectroscopy is used to compare the difference in molecular structure between Langmuir-Blodgett (LB) films (transferred at the surface pressure 40 mN/m with the vertical method and 0 mN/m with the horizontal method) and bulk sample of N-Octadecanoyl-L-alanine amphiphiles. The bulk sample possesses a very similar microstructure (intermolecular hydrogen-bonding interaction and triclinic chain packing) to the well-ordered LB films. Much information on molecular structure of the bulk sample is obtained using FT-Raman spectroscopy, and several weak Raman scattering peaks are assigned.     

Multi-frequency S2FTIR PA spectral depth profiling by use of sinusoidal phase modulation and harmonic demodulation up to 2250 Hz (10th harmonic)

Multi-frequency phase modulation for step-scan Fourier transform infrared photoacoustic spectroscopy (S²FTIR PAS) is demonstrated using a single frequency sinusoidal modulation of the interferometer movable mirror with modulation amplitude 5.2 λ_He-Ne. A digital signal processing (DSP) lock-in amplifier is used to demodulate the photoacoustic response at the fundamental phase modulation frequency and its harmonic frequencies. Because the phase modulation is sinusoidal and of the appropriate amplitude, all the even and odd harmonics (up to the 10th harmonic) can be detected with good signal-to-noise ratio (SNR). Applications of this multi-frequency phase modulation approach for photoacoustic depth profiling are demonstrated by the study of a simple two-layered polymer sample. The sampling depth multiplexing advantage (as compared to that obtained with other types of phase modulation) is clearly demonstrated with the analysis of the phase and magnitude of signals at the fundamental frequency and all the harmonic frequencies. This is the first report of using the sinusoidal phase modulation method to easily obtain multi-frequency and high-frequency light modulation for FTIR PAS depth profiling. Although these data have been obtained sequentially, it would be possible (with considerable economy of time) to obtain them simultaneously by use of the internal digital signal processing capability of the instrument.     

Infrared spectromicroscopy of biochemistry in functional single cells

Over the years Fourier-Transform Infrared (FTIR) spectroscopy has been widely employed in the structural and functional characterization of biomolecules. The introduction of infrared (IR) microscopes and of synchrotron light sources has created expectations that FTIR could become a generally viable technique to study both structure and reactivity in vivo, inside single cells, by performing measurements that up to a few years ago were the preserve of in vitro experiments on purified macromolecules. In this review we present the state-of-the-art in the application of FTIR spectromicroscopy as a technique for the study of structure and dynamics in single cells, we discuss the performance requirements for this application and review developments in sample handling methods.     

Characterization of Enzymatically Induced Aggregation of Casein Micelles in Natural Concentration by in Situ Static Light Scattering and Ultra Low Shear Viscosimetry

The aggregation of casein micelles in undiluted skim milk after the addition of chymosin was studied by static light scattering and ultra low shear viscometry. The static light scattering measurements were made with two different sample thicknesses, 72 and 16 μm. The scattering data were analyzed by indirect Fourier transformation and by the polydispersity inversion technique which led to pair distance distribution functions and size distribution function, respectively. The minimum scattering angle was 1 degrees, which allows for the determination of particle sizes up to a maximum diameter of 12 μm. The fractal dimension determined from double logarithmic plots of intensity versus scattering vector resulted in values between 1.9 and 2.0. The influence of multiple scattering was determined by comparison of the measurements with the different sample thicknesses. The measurements show no significant influence of multiple scattering when the transmission is above 0.85. Due to the very complex and porous structure of the casein aggregates the Rayleigh-Debye-Gans scattering theory has been used in the data analysis. Measurements with a new instrument using ultra low shear showed good agreement with theory. Copyright 1999 Academic Press.     

Preparation of bone powder for FTIR-ATR analysis: The particle size effect

Fourier transform infrared (FTIR) spectroscopy using attenuated total reflection (ATR) is commonly used for the examination of bone. During sample preparation bone is commonly ground, changing the particle size distribution. Although previous studies have examined changes in crystallinity caused by the intensity of grinding using FTIR, the effect of sample preparation (i.e. particle size and bone tissue type) on the FTIR data is still unknown.This study reports on the bone powder particle size effects on mid-IR spectra and within sample variation (i.e. periosteal, mesosteal, trabecular) using FTIR-ATR. Twenty-four archaeological human and faunal bone samples (5 heated and 19 unheated) of different chronological age (Neolithic to post-Medieval) and origin (Belgium, Britain, Denmark, Greece) were ground using either (1) a ball-mill grinder, or (2) an agate pestle and mortar, and split into grain fractions (>500 μm, 250–500 μm, 125–250 μm, 63–125 μm, and 20–63 μm).Bone powder particle size has a strong but predictable effect on the infrared splitting factor (IRSF), carbonate/phosphate (C/P) ratio, and amide/phosphate (Am/P) values. The absorbance and positions of the main peaks, the 2nd derivative components of the phosphate and carbonate bands, as well as the full width at half maximum (FWHM) of the 1010 cm⁻¹ phosphate peak are particle size dependent. This is likely to be because of the impact of the particle size on the short- and long-range crystal order, as well as the contact between the sample and the prism, and hence the penetration depth of the IR light. Variations can be also observed between periosteal, cortical and trabecular areas of bone. We therefore propose a standard preparation method for bone powder for FTIR-ATR analysis that significantly improves accuracy, consistency, reliability, replicability and comparability of the data, enabling systematic evaluation of bone in archaeological, anthropological, paleontological, forensic and biomedical studies.     

低温合成樟脑磺酸掺杂聚苯胺微管的电化学电容行为

在低温条件下合成了长约为2-3 μm, 外径约为300-400 nm 的樟脑磺酸掺杂聚苯胺微管. 扫描电镜(SEM)和透射电镜(TEM)显示, 生成的聚苯胺微管管径受樟脑磺酸浓度的影响, 高浓度的掺杂剂有利于管状聚苯胺的形成. 采用交流阻抗、循环伏安、恒流充放电等测试技术对不同产物的电化学电容行为进行了研究, 结果表明, 苯胺单体与樟脑磺酸的摩尔比为1:1时所得掺杂态聚苯胺电极具有较好的循环稳定性, 单电极比电容达到522 F·g-1.     

Size-fractionated sampling and chemical analysis by total-reflection X-ray fluorescence spectrometry of PMx in ambient air and emissions

PM 10 and PM 2.5 (PMx) have been recently introduced as new air quality standards in the EU (Council Directive 1999/30/EC) for particulate matter. Different estimates and measurements showed that the limit values for PM 10 will be exceeded at different locations in Europe, and thus measures will have to be taken to reduce PMx mass concentrations. Source apportionment has to be carried out, demanding comparable methods for ambient air and emission sampling and chemical analysis. Therefore, a special ambient-air sampler and a specially designed emission sampler have been developed. Total-reflection X-ray fluorescence analysis (TXRF) was used for multi-element analyses as a fast method with low detection limits. For ambient air measurements, a sampling unit was built, impacting particle size classes 10–2.5 μm and 2.5–1.0 μm directly onto TXRF sample carriers. An electrostatic precipitator (ESP) was used as back-up filter to also collect particles <1 μm directly onto the TXRF sample carriers. Air quality is affected by natural and anthropogenic sources, and the emissions of particles <10 μm and <2.5 μm, respectively, have to be determined to quantify their contributions to the so-called coarse (10–2.5 μm) and fine (<2.5 μm) particle modes in ambient air. For this, an in-stack particle sampling system was developed, according to the new ambient air quality standards and in view of subsequent analysis by TXRF. These newly developed samplers, in combination with TXRF analyses, were employed in field campaigns to prove the feasibility and capabilities of the approach. Ambient air data show the quantification of a wide spectrum of elements. From those concentrations, PMx ratios were calculated as an indicator for different sources of elements. Results useful for source apportionment are also the elemental day/night ratios calculated to determine local contributions to PMx mass concentrations. With regard to the emission measurements, results of mass and elemental concentrations obtained in two different processes (steel industry) show that the new PM 10/PM 2.5 cascade impactor and measurements with TXRF give characteristic fingerprints for different sources. Size-fractionated ambient air and emission sampling, together with multi-element analysis, prove to be a useful approach to derive information for source–receptor modeling, a method necessary to set up effective abatement strategies to reduce PMx mass concentrations.     

Spectral biodiagnostics of tissues with fiber optics

The synergy of novel MIR-fibers with FTIR-spectroscopy opens a new horizon in applications of spectroscopy in 4 − 16 μm range in vivo medical diagnostics. The innovated bare core and core-clad MIR-fibers are produced by hot extrusion method from the crystals of silver halides solid solutions. These non-toxic and non-hygroscopic fibers are characterized by low optical losses: 0.1 − 0.5 dB/m nearby 10μm, and high flexibility: R_bending > 10–100 fiber diameters. Some prototypes of accessories for FTIR-spectrometers, based on commercially available MIR-fibers for several areas of promising applications are developed. Non-invasive medical diagnostics in vivo: cancer detection and glucose content measurements in whole human blood is possible. The new family of evanescent fiber sensors is started for the direct in-process applications. Fiberoptic Evanescent Wave FTIR Spectroscopy (FEWS) for cancer diagnosis in human organs is exemplified in this paper, and their great potentiality is demonstrated.     

On-line infrared detection in aqueous micro-volume systems

Mid infrared spectroscopy is a non-destructive technique that can provide detailed information on important, molecule-specific features such as the conformation and functional groups of a large range of compounds. Infrared spectroscopy is now an established and frequently used technique for qualitative analysis, i.e. the identification of chemical constituents in a sample. In addition, its use for quantitative purposes has grown dramatically in recent years. It is important to realise that the analytical problem defines the mode of operation and implementation of the FTIR technique. This Highlight article focuses on the advantages and scope of on-line FTIR detection strategies. However, in common with all techniques, on-line FTIR detection has a number of potential shortcomings, which are also discussed.     

Morphology of a PA / PTFE blend studied by Raman imaging

Raman spectroscopy has been employed for morphological studies on a polymer blend of polyamide-6,6, polytetrafluoroethylene and silicone oil used as a commercial friction bearing. New information about dimensions and distribution of polytetrafluoroethylene clusters in this composite material was revealed by Raman imaging. It was found that the applied processing conditions result in the formation of clusters, which are between 8 μm and 20 μm in diameter and distributed randomly over the whole material. The Raman results were confirmed by SEM images and SEM / EDX elemental mappings. The Raman imaging method has proved to be a good analytical tool for polymer analyses due to the speed of spectra acquisition and the easy sample preparation.     

Application of dynamic 2D FTIR to cellulose

Cellulose, the dominant polymer in the biosphere, is a homopolysaccharide composed of (1,4)-β-d-glucopyranose. Interactions between and within the cellulose polymer chains are mainly determined by inter- and intramolecular hydrogen bonds, which are therefore mainly responsible for mechanical properties of cellulosic materials. The coupling of dynamic mechanical analysis (DMA) and 2D step-scan Fourier transform infrared (FTIR) spectroscopy, is shown to be a very promising way of investigating these submolecular interactions in cellulosic materials. The broad and unstructured band in the OH-stretching vibration region (3100 and 3700 cm⁻¹) of the cellulose vibrational spectra, which contains information about the intra- and intermolecular hydrogen bonds, can be unraveled by this new technique. In the experiments reported here, cellulose sheets have been stretched sinusoidally at low strains while being irradiated with polarized infrared light. For the obtained dynamic IR signals (the in-phase and the out-of-phase responses of the sample), the dynamic IR cross-correlation was defined. It consists of two terms which are referred to as the synchronous and the asynchronous 2D infrared correlation intensities. In the 2D spectra, obtained by DMA–FTIR, several distinct peaks are observed in the OH-range between 3700 and 3100 cm⁻¹ which may be related to specific interactions.     

Classification of the biological material with use of FTIR spectroscopy and statistical analysis

Rapid detection and discrimination of dangerous biological materials such as bacteria and their spores has become a security aim of considerable importance. Various analytical methods, including FTIR spectroscopy combined with statistical analysis have been used to identify vegetative bacteria, bacterial spores and background interferants. The present work discusses the application of FTIR technique performed in reflectance mode using Horizontal Attenuated Total Reflectance accessory (HATR) to the discrimination of biological materials. In comparison with transmission technique the HATR is more rapid and do not require the sample destruction, simultaneously giving similar absorbance bands. HATR-FTIR results combined with statistical analysis PCA and HCA demonstrate that this combination provides novel and accurate microbial identification technique.