The detection of weak absorption changes induced by an external excitation is often hindered by intense background absorptions as well as by noise. Modulation spectroscopy is an adequate tool to be applied in such a case, provided the system may be periodically stimulated, leading to a periodic reversible or pseudoreversible response. In modulated excitation ͑ME͒ Fourier transform infrared spectroscopy the phase sensitive detection ͑PSD͒ used for the demodulation of the periodic system response is generally performed during data acquisition, i.e., applied to the intensity of the interferogram. This leads to a number of problems in quantitative analysis and the requirement of optional equipment. In this article, a method is presented to perform an off-line vector PSD of conventional time-resolved spectra after data acquisition. A detailed mathematical analysis of PSD applied to the spectral intensity, the interferogram intensity, and to time-resolved spectra is presented. It is shown, that vector PSD applied to a set of time-resolved spectra is straightforward and avoids any additional mathematical corrections. Furthermore, it will be shown how ME spectroscopy can be used for experimental separation of overlapping bands and a detailed description for the determination of absolute modulation amplitudes and phase lags is given.

High-temperature synthesized monodisperse superparamagnetic iron oxide nanoparticles are obtained with a strongly bound ligand shell of oleic acid and its decomposition products. Most applications require a stable presentation of a defined surface chemistry; therefore, the native shell has to be completely exchanged for dispersants with irreversible affinity to the nanoparticle surface. We evaluate by attenuated total reflectance−Fourier transform infrared spectroscopy (ATR−FTIR) and thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) the limitations of commonly used approaches. A mechanism and multiple exchange scheme that attains the goal of complete and irreversible ligand replacement on monodisperse nanoparticles of various sizes is presented. The obtained hydrophobic nanoparticles are ideally suited for magnetically controlled drug delivery and membrane applications and for the investigation of fundamental interfacial properties of ultrasmall core–shell architectures.

Even though the structures of cytochrome c oxidase (CcO) from different sources have been determined by X-ray crystallography in both the reduced and oxidized redox states, information about redox-induced structure-function relationships is still very limited. In the current work, redox-dependent structural changes are determined for CcO reconstituted in a protein-tethered bilayer lipid membrane by surface-enhanced infrared absorption spectroscopy in the ATR mode. Significantly, the redox changes in the enzyme are attained by direct wiring of CcO to a gold electrode, ensuring that sequential intra-protein electron transfer occurs by a directed pathway that is natural to the system. The characteristics of CcO were observed to be dramatically altered after the reconstituted enzyme was allowed to turn over in the presence of O 2. The data suggest that the enzyme is initially in an "inactive" state, but that direct electron transfer in the presence of O 2 converts the enzyme to an "activated" form which returns to the inactive conformation when the enzyme remains idle under anaerobic conditions. Potentiometric titrations are performed and reduced-minus-fully oxidized and oxidized-minus-fully reduced absorbance spectra are recorded at decreasing and increasing potentials, respectively, applied to the electrode in a regular succession. The two sets of difference spectra show mirror symmetry, however, they markedly differ from those measured in the presence of redox mediators. Plots of band area of individual bands obtained by Fourier self deconvolution vs. applied potential show a sigmoid dependence as expected for a redox process. However, the sigmoid curves do not coincide but are displaced depending on the direction of the potential change. In other words, these curves show hysteresis, which is an indication of cooperativity and non-equilibrium states for electron transfer and/or conformational changes of the protein. This is discussed in terms of known concepts of molecular hysteresis. © 2010 The Royal Society of Chemistry

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