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     

A new accessory for the detection of trace components by FT-IR spectroscopy

We have developed a specific compartment to increase the sensitivity of a routine FT-IR spectrometer. A high performance MCT detector has been included in the system. The SNR of the spectrometer has been improved by a factor of 10.     

Solvent-dependent photoacidity state of pyranine monitored by transient mid-infrared spectroscopy.

We investigate with femtosecond mid-infrared spectroscopy the vibrational-mode characteristics of the electronic states involved in the excited-state dynamics of pyranine (HPTS) that ultimately lead to efficient proton (deuteron) transfer in H2O (D2O). We also study the methoxy derivative of pyranine (MPTS), which is similar in electronic structure but does not have the photoacidity property. We compare the observed vibrational band patterns of MPTS and HPTS after electronic excitation in the solvents: deuterated dimethylsulfoxide, deuterated methanol and H2O/D2O, from which we conclude that for MPTS and HPTS photoacids the first excited singlet state appears to have charge-transfer (CT) properties in water within our time resolution (150 fs), whereas in aprotic dimethylsulfoxide the photoacid appears to be in a non-polar electronic excited state, and in methanol (less polar and less acidic than water) the behaviour is intermediate between these two extremes. For the fingerprint vibrations we do not observe dynamics on a time scale of a few picoseconds, and with our results obtained on the O-H stretching vibration we argue that the dynamic behaviour observed in previous UV/Vis pump-probe studies is likely to be related to solvation dynamics.     

Application of Fourier-transform infrared (FT-IR) transmission spectroscopy for the estimation of roxithromycin in pharmaceutical formulations

A quick and reliable analytical method for the quantitative assessment of roxithromycin in pharmaceutical formulations was developed using Fourier-transform infrared (FT-IR) transmission spectroscopy for routine quality control. The sample preparation was avoided except grinding for pellet formation and eliminated the use of toxic solvents. For the determination of roxithromycin, conventional KBr was used in the form of pellets for acquisition of the FT-IR spectra of standards and samples. The calibration model was developed based on simple Beer's law using the FT-IR carbonyl region (CO) from 1765 to 1705 cm⁻¹. The excellent coefficient of determination (R²) was achieved (0.9992) with 0.01 mg standard error of calibration. This work clearly revealed the capability of the transmission FT-IR spectroscopy and extended its application for determining the exact quantity of roxithromycin to control the processing formulation and quality of finished product with the analysis time of less than 3 min using the neat solid samples.     

Single-molecule fluorescence spectroscopy of protein folding.

Single-molecule spectroscopy is an important new approach for studying the intrinsically heterogeneous process of protein folding. This Review illustrates how different single-molecule fluorescence techniques have improved our understanding of mechanistic aspects in protein folding, exemplified by a series of recent experiments on a small protein.     

Reversible Carbene Formation in the Ionic Liquid 1‐Ethyl‐3‐Methylimidazolium Acetate by Vaporization and Condensation

The role of N‐heterocyclic carbenes in the chemistry of ionic liquids based on imidazolium salts has long been discussed. Here, we present experimental evidence that 1‐ethyl‐3‐methylimidazolium‐2‐ylidene (EMIm) can coexist with its protonated imidazolium cation (EMImH⁺) at low temperatures. If the vapor of the ionic liquid [EMImH⁺][AcO^?] is trapped in solid argon or nitrogen at 9 K, only acetic acid (AcOH) and the carbene, but no ionic species, are found by IR spectroscopy. This indicates that during the evaporation of [EMImH⁺][AcO^?] proton transfer occurs to form the neutral species. If the vapor of [EMImH⁺][AcO^?] is trapped at 9 K as film in the absence of a host matrix, a solid consisting of EMImH⁺, EMIm, AcO^?, and AcOH is formed. During warming to room temperature the proton transfer in the solid to form back the IL [EMImH⁺][AcO^?] can be monitored by IR spectroscopy. This clearly demonstrates that evaporation and condensation of the IL [EMImH⁺][AcO^?] results in a double proton transfer, and the carbene EMIm is only metastable even at low temperatures.     

Ethane cation decomposition characterization by EBMI spectroscopy: gas‐phase dissociative recombination as a source of secondary products

The decomposition products of the d₆‐ethane cation following charge‐transfer ionization with Ar⁺, under conditions of varying ionization electron current, have been isolated in solid argon matrices at 18 K and examined using Fourier transform infrared spectroscopy. Gas samples containing 1 : 1600 d₆‐ethane : Ar were subjected to electron bombardment by using either a high (pin) or a low (plate) ionization density anode configuration with ionization currents between 20 and 150 μA. Under high ionization density conditions, the observed major products were d₄‐ethene (C₂D₄) and d₂‐acetylene (C₂D₂), with smaller yields of C₂D₅, C₂D₃, and C₂D. The yield of each dehydrogenation product was enhanced with increased current. Analogous experiments employing the low ionization density plate anode resulted in reduced C₂D₆ destruction and the formation of only C₂D₄ and C₂D₂. The results suggest the onset of dissociative recombination processes under high ion density conditions. In this context, the results can be interpreted as a dissociative recombination of primary ion products, which gives rise to further dehydrogenation, and appearance of additional neutral radical products. Copyright © 2012 John Wiley & Sons, Ltd.     

Multivariate control charts based on net analyte signal (NAS) for characterization of the polymorphic composition of Piroxicam using near infrared spectroscopy

Near infrared spectroscopic and multivariate statistical control charts based on the net analyte signal (NAS) were applied to the polymorphic characterization of Piroxicam samples. Three different polymorphic forms (I, II and III) were studied, using X-ray powder diffraction (XRPD) and scanning electron microscopy as reference techniques. Samples containing form I were considered inside the quality specifications and forms II and III were impurities. Three control charts were developed: the NAS chart that corresponds to the analyte of interest (polymorphic form I), the interference chart that corresponds to the contribution of other compounds in the sample and the residual chart that corresponds to nonsystematic variation. From the limits estimated for each chart using samples inside the quality specifications, it was possible to identify samples that did not present polymorphic form I. The use of multivariate control charts provides a rapid evaluation of purity and the polymorphic composition of pharmaceutical formulations based on Piroxicam.     

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.     

Infrared spectroscopy studies of the cyclic anhydride as the intermediate for the ester crosslinking of cotton cellulose by polycarboxylic acids. I. Identification of the cyclic anhydride intermediate

The mechanism of esterification of cotton cellulose by a polycarboxylic acid was investigated using Fourier transform infrared spectroscopy (FT-IR). The infrared spectroscopic data indicate that a polycarboxylic acid esterifies with cotton cellulose through the formation of an acid anhydride intermediate. A five-member cyclic anhydride intermediate was identified in the cotton fabric treated with poly(maleic acid). The five-member cyclic anhydride is a reactive intermediate and readily esterifies when reaction sites are available. We also found that those polycarboxylic acids, which form five-member cyclic anhydride intermediates, crosslink cotton cellulose more effectively than those polycarboxylic acids which form six-member cyclic anhydride intermediates. © 1993 John Wiley & Sons, Inc.     

Probing the water coordination of protein-targeted MRI contrast agents by pulsed ENDOR spectroscopy.

A novel methodology based on electron-nuclear double resonance (ENDOR) spectroscopy is used for the direct determination of the water coordination number (q) of gadolinium-based magnetic resonance imaging (MRI) contrast agents. Proton ENDOR spectra can be obtained at approximately physiological concentrations for metal complexes in frozen aqueous solutions either in the presence or absence of protein targets. It is shown that, depending on the structure of the co-ligand, the water hydration number of a complex in aqueous solution can be significantly different to when the complex is noncovalently bound to a protein. From the ENDOR spectra of the exchangeable protons, precise information on the metal-proton distance can be derived as well. These essential parameters directly correlate with the efficacy of MRI contrast agents and should therefore aid the development of novel, highly efficient compounds targeted to various proteins.     

Direct orthogonal signal correction as data pretreatment in the classification of clinical lots of creams from near infrared spectroscopy data

Direct orthogonal signal correction (DOSC) is applied to correct for major variance sources such as temperature effects, time influences and instrumental differences in near infrared (NIR) data. The samples analysed are creams containing different concentrations of an active drug. The final aim is to classify the samples according to their concentration of active compound. Having performed DOSC on the data, it is not necessary anymore to apply sophisticated chemometric techniques to correct for temperature or time effects and to attribute the samples to their respective concentration classes. Moreover, the application of DOSC on the NIR spectra recorded on two different instruments shows that this method can be considered as a valuable alternative for the standardisation in classification applications. Since the applied algorithm tends to overfit, in a second part of this paper, a comparison is made with an algorithm designed by Westerhuis, which should overcome this problem. Although the calibration set results show that the overfitting has been partially corrected for by the latter algorithm, the test set results did not improve significantly.     

Surface- and resonance-enhanced micro-Raman spectroscopy of xanthene dyes: from the ensemble to single molecules.

Surface-enhanced resonance Raman scattering (SERRS) spectra of various rhodamine dyes, of pyronine G and thiopyronine adsorbed on isolated silver clusters were recorded at the ensemble level and at the single-molecule level with a high-resolution confocal laser microscope equipped with a spectrograph and a CCD-detector. Comparing single-molecule spectra with ensemble spectra, various inhomogeneous spectral features, such as line splitting, spectral wandering, spectral diffusion and abrupt spectral jumps between different metastable spectral states, are revealed positions and the relative intensities of the vibronic bands. Resonance enhancement is investigated with respect to single-molecule surface-enhanced Raman scattering (SERS) spectroscopy and is found to be responsible for approximately three orders of magnitude in sensitivity. A significant influence of the substituents on the single-molecule SERRS sensitivity is found, showing that various chemical effects are responsible for surface enhancement in addition to the electromagnetic enhancement effect.     

Infrared spectroscopy studies of the cyclic anhydride as the intermediate for the ester crosslinking of cotton cellulose by polycarboxylic acids. II. Comparison of different polycarboxylic acids

Polycarboxylic acids have been used as crosslinking agents for cotton cellulose. In our previous research, we used Fourier transform infrared (FT-IR) spectroscopy to investigate the formation of five-membered cyclic anhydride intermediates on cotton fabric by different polycarboxylic acids. In this research, we found that those polycarboxylic acids capable of forming both five- and six-membered cyclic anhydrides form only five-membered cyclic anhydrides. We compared the effectiveness of the polycarboxylic acids with different molecular structures for esterifying cellulose. Those polycarboxylic acids, which have their carboxyl groups bonded to the adjacent carbons of their molecular backbones and are capable of forming five-membered cyclic anhydrides, are more effective for esterifying cellulose than those polycarboxylic acids having their carboxyl groups bonded to the alternative carbons. The only six-membered cyclic anhydride identified is the anhydride formed on the cotton fabric treated with poly(acrylic acid). © 1996 John Wiley & Sons, Inc.     

Torsional barriers in aromatic molecular clusters as probe of the electronic properties of the chromophore.

We present a computer program that is capable of fitting n-fold torsional barriers Vn (n = 2-6) and torsional constants F simultaneously to high- and low-resolution spectroscopic data of different isotopomeric internal rotors. The program has been utilized to fit independently barriers and torsional constants for both electronic states of several aromatic clusters. The constant F of the ammonia moiety in the phenol-ammonia cluster is shown to decrease upon electronic excitation, thus imaging the formation of a hydrogen-bonded complex between the phenoxy radical and the NH4 radical in the excited state. In contrast, for the naphthol-ammonia 1:1 clusters no change of F of ammonia could be found. For phenol-methanol cluster we found a decrease of F upon excitation which points to a stronger hydrogen bond between phenol and methanol in the excited state. A strong reduction of the torsional barrier upon excitation points to the formation of a methoxonium radical in a similar photoreaction as in phenol-ammonia cluster. For the phenol-water system we postulate the same mechanism, a photoreaction, which leads to a translocated hydrogen atom in the S1 state what can be deduced from the change of the torsional constant upon electronic excitation.     

Comparison of the photophysical parameters for three perylene bisimide derivatives by single-molecule spectroscopy.

Characterization of the photophysical parameters for three perylene bisimide derivatives is presented. We exploited time-resolved and steady-state spectroscopy on both ensembles and single molecules under ambient as well as cryogenic (1.4 K) conditions. The finding is that these chromophores show extraordinary high fluorescence-emission rates, low intersystem crossing yields to the triplet state, and relatively short triplet lifetimes.     

Electron-bombardment matrix isolation and PEPICO studies as complementary techniques in ion chemistry: an FTIR study of the decomposition of the vinyl fluoride cation

FTIR spectra have been obtained for matrices formed following electron bombardment of gas mixtures containing varying amounts of vinyl fluoride (VF) in Ar (1:400 to 1:25 600; VF/Ar). The major matrix‐isolated products are a π‐complex of HF/C₂H₂, fluoroacetylene (HC≡CF) and two isomers of C₂H₂F^?. These products correspond well with the products of photoionization of VF near 15.8 eV. These observations support the dominant mechanism of ionization in the EB‐MI environment as charge transfer of the substrate molecule to Ar^?+. Some differences are noted between the observed EB‐MI products and the results from PEPICO studies, primarily in that the EB‐MI products are observed as neutralized forms. The close correlation in EB‐MI and photoionization results allows the EB‐MI technique to be utilized as an ion structural analysis tool in complement to PEPICO studies, and allows the use of PEPICO studies to help predict and elucidate high‐pressure chemistry mechanisms through EB‐MI studies. The differences in the EB‐MI results and ions observed using the PEPICO technique are rationalized in terms of the differences in the experimental techniques. Using VF as the test system, reagent partial pressure conditions that best complement PEPICO studies are determined. Although the major results are observed for all VF partial pressures, dilute samples give rise to further ionization of the primary products, and more concentrated samples give rise to radical—radical reaction chemistry. As a result, a nominal range of 1:3200 (VF/Ar) is demonstrated to provide the best correlation with the gas‐phase PEPICO measurements. Copyright © 2011 John Wiley & Sons, Ltd.