A new approach to recognizing the correct pattern of cross-peaks from a noisy 2D asynchronous spectrum by detecting intrinsic symmetry via the Kolmogorov-Smirnov test.

The characteristic cluster pattern of cross-peaks in a 2D asynchronous spectrum provides an effective way to reveal the specific physicochemical nature of subtle spectral changes caused by intermolecular interactions. However, the inevitable presence of noise in the 1D spectra used to construct a 2D asynchronous spectrum is significantly amplified, which poses a serious challenge in identifying the correct cluster pattern of the cross-peaks. While mirror symmetry occurs in some types of cross-peaks, it does not occur in other types. The Kolmogorov-Smirnov test provides a statistical means to check whether the mirror symmetry exists or not between a pair of cross-peaks covered by heavy noise. Thus, different types of cross-peak clusters can be distinguished by excavating intrinsic spectral features from the noisy 2D asynchronous spectrum. The effectiveness of this approach in investigating the nature of intermolecular interactions was showcased in both a simulated model system and a real artemisinin/N-methyl pyrrolidone system.

Novel Method for Extracting the Spectrum of a Supramolecular Complex via a Comprehensive Approach Involving Two-Dimensional Correlation Spectroscopy, Genetic Algorithm, and Grid Searching.

A supramolecular complex may be formed by two solutes via a weak intermolecular interaction in a solution. The spectrum of the complex is often inundated by the spectra of the solutes that are not involved in the intermolecular interaction. Herein, a novel spectral analysis approach is proposed to retrieve the spectrum of the supramolecular complex. First, a two-dimensional (2D) asynchronous spectrum is constructed. Then, a genetic algorithm is used to obtain a heuristic spectrum of the supramolecular complex. The heuristic spectrum is a linear combination of the spectrum of the complex and the spectrum of a solute. The coefficients of the linear combination are then obtained, according to which the equilibrium constants are invariant among the sample solutions used to construct the 2D asynchronous spectrum. We have applied the approach to a supramolecular system formed by benzene and I2. In the analysis, several binding models are evaluated, and a benzene molecule interacting with two iodine molecules via halogen bonding turns out to be the only possible model. Hence, the characteristic band of the benzene/I2 supramolecular complex around 1819 cm-1 in the Fourier transform infrared (FTIR) spectrum and the corresponding equilibrium constant are obtained. The above results indicate that the novel approach provides a chance to get new insight into various intermolecular interactions studied by spectroscopy.

Robust Approach to Estimating the Stoichiometric Ratio of Supramolecular Complexes Using the Volume of Cross-Peaks in 2D Asynchronous Spectra and the Jonckheere-Terpstra Test.

Estimation of the stoichiometric ratio of a supramolecular aggregate formed by different compounds is very important in elucidating the structure and function of the aggregate. Many spectroscopic methods used to estimate the stoichiometric ratios of coordination complexes become invalid when characteristic peaks of the aggregate overlap with peaks of compounds that form the aggregate. Previously, we combined the asynchronous orthogonal sample design with Job's method to address the abovementioned problem. However, the interference of noise may lead to incorrect results. Herein, a new method has been developed. In the generation of corresponding Job's curve, the intensity of a cross peak at a single apex is replaced by the volume of the cross peak. Since most noise is canceled in the calculation of the volume of the cross peak, resultant Job's curve is robust to noise. Moreover, the Jonckheere-Terpstra statistical test, a famous nonparametric method to detect whether the data has an upward or downward trend, could further reduce the risk of yielding incorrect results caused by noise. We have applied this approach to two real-world examples (resveratrol/ß-Cyclodextrin (ß-CD) and N, N-diethyl-N'-benzoylthiourea (DEBT)/Cu2+) with satisfactory results. The method described in this paper provides a robust way to measure the stoichiometric ratio in supramolecular systems.

Retrieving Spectra of Pure Components from the DOSY-NMR Experiment via a Comprehensive Approach Involving the 2D Asynchronous Spectrum, 2D Quotient Spectrum, and Genetic Algorithm Refinement.

When diffusion coefficients of different components in a mixture are similar, NMR spectra of pure individual components are difficult to be obtained via a diffusion-ordered spectroscopy (DOSY) experiment. Two-dimensional correlation spectroscopy (2D-COS) is used to analyze the data from the DOSY experiment. Through the properties of the systematic absence of cross-peak (SACP) in the 2D asynchronous spectra, spectra of pure components can be obtained even if their diffusion coefficients are similar. However, fluctuations in peak-position and peak-width are often unavoidable in NMR spectra, which makes SACPs unrecognizable. To address the problem, a 2D quotient spectrum is used to identify the masked SACPs. However, undesirable interference peaks due to the fluctuations in peak-position and peak-width are still present when we extract a spectrum of a component by slicing the 2D asynchronous spectrum across the SACP. A genetic algorithm (GA) is used to select a suitable subset of spectra where the diversities of peak-position and peak-width are significantly reduced. Then, we used the selected spectra to construct a refined 2D asynchronous spectrum so that the spectra of pure components with significant attenuated interference can be obtained. The above approach has been proven to be effective on a model system and a real-world example, demonstrating that 2D-COS possesses a bright perspective in the analysis of the bilinear data from DOSY experiments.

Deprotonation from an OH on myo-Inositol Promoted by µ2-Bridges with Possible Regioselectivity/Chiral Selectivity.

Single-crystal structures of myo-inositol complexes with erbium ([Er2(C6H11O6)2(H2O)5Cl2]Cl2(H2O)4, denoted ErI hereafter) and strontium (Sr(C6H12O6)2(H2O)2Cl2, denoted SrI hereafter) are described. In ErI, deprotonation occurs on an OH of myo-inositol, although the complex is synthesized in an acidic solution, and the pKa values of all of the OHs in myo-inositol are larger than 12. The deprotonated OH is involved in a µ2-bridge. The polarization from two Er3+ ions activates the chemically relatively inert OH and promotes deprotonation. In the stable conformation of myo-inositol, there are five equatorial OHs and one axial OH. The deprotonation occurs on the only axial OH, suggesting that the deprotonation possesses characteristics of regioselectivity/chiral selectivity. Two Er3+ ions in the µ2-bridge are stabilized by five-membered rings formed by chelating Er3+ with an O-C-C-O moiety. As revealed by the X-ray crystallography study, the absolute values of the O-C-C-O torsion angles decrease from ∼60 to ∼45° upon chelating. Since the O-C-C-O moiety is within a six-membered ring, the variation of the torsion angle may exert distortion of the chair conformation. Quantum chemistry calculation results indicate that an axial OH flanked by two equatorial OHs (double ax-eq motif) is favorable for the formation of a µ2-bridge, accounting for the selectivity. The double ax-eq motif may be used in a rational design of high-performance catalysts where deprotonation with high regioselectivity/chiral selectivity is carried out.

Unexpected Deprotonation from a Chemically Inert OH Group Promoted by Metal Ions in Lanthanide-Erythritol Complexes.

Single-crystal structures of five lanthanide-erythritol complexes are reported. The analysis of the chemical compositions and scrutinization of structural features in the single-crystal data of the complexes led us to find that unexpected deprotonation occurs on the OH group of erythritol of three complexes. Considering these complexes were prepared in acidic environments, where spontaneous ionization on an OH group is suppressed, we suggest metal ions play an important role in promoting the proton transfer. To find out why the chemically inert OH is activated, the single-crystal structures of 63 rare-earth complexes containing organic ligands with multiple hydroxyl groups (OLMHs) were surveyed. The formation of µ2-bridges turns out to be directly relevant to the occurrence of deprotonation. When an OH group from an OLMH molecule participates in the formation of a µ2-bridge, the polarization ability of the metal ions becomes strong enough to promote the deprotonation on the OH group. The above structural characteristics may be useful in the rational design of catalysts that can activate the chemically inert OH group and promote the relevant chemical conversions.

Sample-Sample Correlation Asynchronous Spectroscopic Method Coupled with Multivariate Curve Resolution-Alternating Least Squares To Analyze Challenging Bilinear Data.

An approach to construct a secondary asynchronous spectrum via sample-sample correlation (SASS) is proposed to analyze bilinear data from hyphenated spectroscopic experiments. In SASS, bilinear data is used to construct a series of two-dimensional (2D) sample-sample correlation spectra. Then a vertical slice is extracted from each 2D sample-sample correlation spectrum so that a secondary 2D asynchronous spectrum is constructed via these slices. The advantage of SASS is demonstrated by a model system with the following challenging situations: (1) Temporal profiles of different components severely overlap, making spectra of pure components difficult to directly obtain from either original bilinear data or multivariate curve resolution-alternating least squares (MCR-ALS) with non-negativity and unimodality constraints. (2) Every peak in the spectra of the eluted samples contains contributions from at least two components. Hence, two-dimensional correlation spectroscopy (2D-COS) and n-dimensional (nD) asynchronous spectroscopic method developed in our previous work, which previously worked so well, are now invalid. SASS managed to reveal different groups of systematic absences of cross peaks (SACPs) that reflect the lack of spectral contributions of different components at different regions in the second asynchronous spectrum. Spectra of different components can still be faithfully retrieved via MCR-ALS calculation using constraints revealed by different groups of SACPs. The results demonstrate that implicit but intrinsic information revealed by SASS is indispensable in solving challenging bilinear data as the model system. We applied SASS on two real-world examples from thermogravimetry-Fourier transform infrared spectroscopy (TG-FT-IR) experiments of mixtures (H2O/HOD/D2O and H2O/isopropanol/pyridine). FT-IR spectra of different components were successfully recovered. Moreover, FT-IR spectrum of HOD, which is difficult to obtain, was successfully extracted. SASS can be applied in the analysis of gaseous mixtures from TG-FT-IR experiment and a combination of quantum cascade lasers with substrate-integrated hollow waveguides in environmental monitoring and biomedical diagnosis. Furthermore, SASS is also useful in various advanced hyphenated spectroscopic experiments.

Two-dimensional correlation analysis of highly spatially resolved simultaneous IR and Raman spectral imaging of bioplastics composite using optical photothermal Infrared and Raman spectroscopy.

Optical photothermal infrared (O-PTIR) and Raman spectroscopy and imaging was used to explore the spatial distributions of molecular constituents of a laminate sample consisting of the bioplastics, polyhydroxyalkanoate (PHA) and polylactic acid (PLA), near the interfacial boundary. Highly spatially resolved simultaneous IR and Raman spectra were sequentially collected at 100 nm increments along a line traversing the interface. The set of spectra were subjected to 2D-COS analysis to extract the detailed nature of the spatial distribution of the laminate constituents. It was revealed that the laminate is not a simple binary system of two non-interacting polymers, but consists of different constituents with more complex spatial distributions. Some portion of PLA seems to penetrate into the PHA layer. The crystallinity of PHA near the interface is reduced compared to the rest of the PHA layer. The result suggests the existence of some partial molecular mixing even for these seemingly immiscible polymer pairs. The mixing probably occurs at the segmental level confined to only several hundred nanometers of space at the interface. Such partial mixing may explain the high compatibility between the two bioplastics.

Investigation of the Phase Transition Mechanism in LiFePO4 Cathode Using In Situ Raman Spectroscopy and 2D Correlation Spectroscopy during Initial Cycle.

The phase transition of the LiFePO4 and FePO4 in Li-ion cell during charging-discharging processes in the first and second cycles is elucidated by Raman spectroscopy in real time. In situ Raman spectroscopy showed the sudden phase transition between LiFePO4 and FePO4. Principal component analysis (PCA) results also indicated that the structural changes and electrochemical performance (charge-discharge curve) are correlated with each other. Phase transition between LiFePO4 and FePO4 principally appeared in the second charging process compared with that in the first charging process. 2D correlation spectra provided the phase transition mechanism of LiFePO4 cathode which occurred during the charging-discharging processes in the first and second cycles. PCA and 2D correlation spectroscopy are very helpful methods to understand in situ Raman spectra for the Li-ion battery.

Novel Method of Constructing Two-Dimensional Correlation Spectroscopy without Subtracting a Reference Spectrum.

In this study, we propose a new approach to generate two-dimension spectra to enhance the intensity of cross peaks relevant to intermolecular interaction. We investigate intermolecular interaction between two solutes (denoted as P and Q, where P has a characteristic peak at XP) dissolved in the same solvent via the near diagonal cross peaks around the coordinate (XP, XP) in a two-dimensional (2D) asynchronous spectrum of generalized spectroscopy. Because of physical constrains in many cases, the variation ranges of the initial concentrations of P or Q must be kept very narrow, leading to very weak cross peak intensities. The weak cross peaks vulnerable to noise bring about difficulty in the investigation of subtle intermolecular interaction. Herein, we propose a new of way constructing a 2D asynchronous spectrum without the subtraction of the average spectrum often used as a reference spectrum. Mathematical analysis and computer simulation demonstrate that the near diagonal cross peaks around the coordinate (XP, XP) in the 2D asynchronous spectrum using the new approach possess two characteristics: (1) they can still reflect an intermolecular interaction reliably; 2) the absolute intensities of the cross peaks are significantly stronger than those generated by the conventional method. We incorporate the novel method with the DAOSD (double asynchronous orthogonal sample design scheme) approach and applied the modified DAOSD approach to study hydrogen bonding behavior in diethyl either/methanol/THF system. The new approach made the weak cross peaks, which are not observable in 2D asynchronous spectrum generated using conventional approach, become observable. The appearance of the cross peak demonstrate that When a small amount of THF is introduced into diethyl solution containing low amount of methanol, THF breaks the methanol-diethyl ether complex and forms methanol-THF complex via new hydrogen bond. This process takes place in spite of the fact that the content of diethyl ether is overwhelmingly larger than that of THF. The above result demonstrates that the new approach described in this article is applicable to enhance intensity of cross peaks in real chemical systems.

Two-Dimensional Correlation Spectroscopy for Multimodal Analysis of FT-IR, Raman, and MALDI-TOF MS Hyperspectral Images with Hamster Brain Tissue.

Hyperspectral imaging (HSI) techniques are useful for obtaining very detailed structural and compositional information from biomedical, pharmaceutical, or clinical samples, among others. The informative value of these methods can be further increased through the application of different HSI techniques and joint analysis of the data. However, interpretation and understanding of multimodal HSI have been impeded by difficulties in registration of the different HSI data sets and by the lack of integrative analysis concepts. Here, we introduce two-dimensional correlation spectroscopy (2DCOS) as a novel technique for jointly analyzing HSI data which allows one to obtain deeper insights into the chemistry of complex samples by decrypting auto- and heterospectral correlations that may exist between features of the different HSI data. The general workflow of 2DCOS analysis is demonstrated by HSI examples acquired from cryo-sections of hamster brain tissue using Fourier-transform infrared (FT-IR) microspectroscopy, confocal Raman microspectroscopy (CRM), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Multimodal hyperspectral image analysis by 2DCOS opens up new opportunities for spectral band assignments and thus the interpretation of structure-spectra and composition-spectra relationships. We foresee wide application potential for describing complex samples in various fields ranging from biomedicine to industrial applications.

Chloroform induces outstanding crystallization of poly(hydroxybutyrate) (PHB) vesicles within bacteria.

Poly[(R)-3-hydroxyalkanoate]s or PHAs are aliphatic polyesters produced by numerous microorganisms. They are accumulated as energy and carbon reserve in the form of small intracellular vesicles. Poly[(R)-3-hydroxybutyrate] (PHB) is the most ubiquitous and simplest PHA. An atomic force microscope coupled with a tunable infrared laser (AFM-IR) was used to record highly spatially resolved infrared spectra of commercial purified PHB and native PHB within bacteria. For the first time, the crystallinity degree of native PHB within vesicle has been directly evaluated in situ without alteration due to the measure or extraction and purification steps of the polymer: native PHB is in crystalline state at 15% whereas crystallinity degree reaches 57% in commercial PHB. Chloroform addition on native PHB induces crystallization of the polymer within bacteria up to 60%. This possibility of probing and changing the physical state of polymer in situ could open alternative ways of production for PHB and others biopolymers. Graphical abstract An atomic force microscope coupled with a tunable infrared laser (AFM-IR) has been used to record local infrared spectra of biopolymer PHB within bacteria. Deconvolution of those spectra has allowed to determine in situ the crystallinity degree of native PHB.

Investigation on the Behavior of Noise in Asynchronous Spectra in Generalized Two-Dimensional (2D) Correlation Spectroscopy and Application of Butterworth Filter in the Improvement of Signal-to-Noise Ratio of 2D Asynchronous Spectra.

The behavior of noise in asynchronous spectrum in generalized two-dimensional (2D) correlation spectroscopy is investigated. Mathematical analysis on the noise of 2D spectra and computer simulation on a model system show that the fluctuation of noise in a 2D asynchronous spectrum can be characterized by the standard deviation of noise in 1D spectra. Furthermore, a new approach to improve a signal-to-noise ratio of 2D asynchronous spectrum by a Butterworth filter is developed. A strategy to determine the optimal conditions is proposed. Computer simulation on a model system indicates that the noise of 2D asynchronous spectrum can be significantly suppressed using the Butterworth filtering. In addition, we have tested the approach to a real chemical system where interaction between berberine and ß-cyclodextrin is investigated using 2D UV-vis asynchronous spectra. When artificial noise is added, cross peaks that reflect intermolecular interaction between berberine and ß-cyclodextrin are completely masked by noise. After the method described in this article is utilized, noise is effectively suppressed, and cross peaks are faithfully recovered. The above result demonstrates that the approach described in this article is applicable in real chemical systems.

The dependence of signal-to-noise ratio on number of scans in covariance spectroscopy.

The dependence of signal-to-noise ratio on the number of scans in covariance spectroscopy has been systematically analyzed for the first time with the intriguing relationship of SNRcov∝n/2, which is different from that in FT2D spectrum with SNRFT∝n. This relationship guarantees the signal-to-noise ratio when increasing the number of scans.

Using 2D Correlation Analysis to Enhance Spectral Information Available from Highly Spatially Resolved AFM-IR Spectra.

The recent combination of atomic force microscopy and infrared spectroscopy (AFM-IR) has led to the ability to obtain IR spectra with nanoscale spatial resolution, nearly two orders-of-magnitude better than conventional Fourier transform infrared (FT-IR) microspectroscopy. This advanced methodology can lead to significantly sharper spectral features than are typically seen in conventional IR spectra of inhomogeneous materials, where a wider range of molecular environments are coaveraged by the larger sample cross section being probed. In this work, two-dimensional (2D) correlation analysis is used to examine position sensitive spectral variations in datasets of closely spaced AFM-IR spectra. This analysis can reveal new key insights, providing a better understanding of the new spectral information that was previously hidden under broader overlapped spectral features. Two examples of the utility of this new approach are presented. Two-dimensional correlation analysis of a set of AFM-IR spectra were collected at 200-nm increments along a line through a nucleation site generated by remelting a small spot on a thin film of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). There are two different crystalline carbonyl band components near 1720 cm-1 that sequentially disappear before a band at 1740 cm-1 due to more disordered material appears. In the second example, 2D correlation analysis of a series of AFM-IR spectra spaced every 1 micrometer of a thin cross section of a bone sample measured outward from an osteon center of bone growth. There are many changes in the amide I and phosphate band contours, suggesting changes in the bone structure are occurring as the bone matures.

Enhanced Spectral Resolution and Two-Dimensional Correlation Spectroscopy (2D-COS).

The apparent enhancement of spectral resolution is one of the attractive features of two-dimensional correlation spectroscopy (2D-COS). Highly overlapped adjacent bands often encountered in one-dimensional spectra may be effectively differentiated and identified by spreading peaks along the second dimension. This differentiating feature or selectivity is especially prominent in asynchronous spectra, where even a slight difference in the variation patterns of overlapped bands in response to a given perturbation results in the generation of cross-peaks. While cross-peaks in asynchronous spectra can identify signals originating from different moieties or bands, they do not effectively specify which regions of spectra actually share the same molecular origin. Overreliance on asynchronous spectra alone risks the potential false negative assessment or lack of sufficient specificity, leading to the failure of classifying signals into a reasonable set of component groups. The combined use of synchronous and asynchronous spectra coupled with the scaling techniques, elimination of anti-correlated negative synchronous peaks, and a robust line shape narrowing method provides a means to achieve both selectivity and specificity for resolution-enhancement of 2D-COS.

Two-Dimensional Correlation Spectroscopy (2D-COS) Analysis of Evolving Hyperspectral Images.

The evolutionary behavior is examined for heterogeneously distributed hyperspectral images of a simulated biological tissue sample comprising lipid-like and protein-like components during the aging process. Taking a simple planar average of a spectral image loses useful information about the spatially resolved nature of the data. In contrast, multivariate curve resolution (MCR) analysis of a spectral image at a given stage of aging produces a set of loadings of major component groups. Each loading represents the combined spectral contributions of a mixture of similar but not identical constituents (i.e., lipid-like and protein-like components). Temporal analysis of individual component groups using two-dimensional correlation spectroscopy (2D-COS) and MCR provides much-streamlined results without interferences from the overlapped contributions. Grouping of data into separate components also allows for the effective comparison of the parallel processes of lipid oxidation and protein denaturation involving a number of constituents using the heterocomponent 2D-COS analysis. The complex interplays of lipid constituents and protein secondary structures during the tissue aging process are unambiguously highlighted. The possibility of extending this approach to a much more general form of applications using a moving window analysis is also discussed.

Novel Developments and Progress in Two-Dimensional Correlation Spectroscopy (2D-COS).

This first of the two-part series of the comprehensive survey review on the progress of the two-dimensional correlation spectroscopy (2D-COS) field during the period 2021-2022, covers books, reviews, tutorials, novel concepts and theories, and patent applications that appeared in the last two years, as well as some inappropriate use or citations of 2D-COS. The overall trend clearly shows that 2D-COS is continually growing and evolving with notable new developments. The technique is well recognized as a powerful analytical tool that provides deep insights into systems in many science fields.

Diverse Applications of Two-Dimensional Correlation Spectroscopy (2D-COS).

This second of the two-part series of a comprehensive survey review provides the diverse applications of two-dimensional correlation spectroscopy (2D-COS) covering different probes, perturbations, and systems in the last two years. Infrared spectroscopy has maintained its top popularity in 2D-COS over the past two years. Fluorescence spectroscopy is the second most frequently used analytical method, which has been heavily applied to the analysis of heavy metal binding, environmental, and solution systems. Various other analytical methods including laser-induced breakdown spectroscopy, dynamic mechanical analysis, differential scanning calorimetry, capillary electrophoresis, seismologic, and so on, have also been reported. In the last two years, concentration, composition, and pH are the main effects of perturbation used in the 2D-COS fields, as well as temperature. Environmental science is especially heavily studied using 2D-COS. This comprehensive survey review shows that 2D-COS undergoes continuous evolution and growth, marked by novel developments and successful applications across diverse scientific fields.

Dispersion Analysis of Perpendicular Modes Using a Hybrid Two-Trace Two-Dimensional (2T2D) Smart Error Sum.

Using linear dichroism theory, one would assume that a z-cut of a uniaxial crystal is equivalent to an x-cut to determine the perpendicular component of the dielectric tensor and the corresponding oscillator parameters. However, Fresnel's equations show that the effect of interfaces in the form of the continuity relations of the different components of the electric field must be considered. A consequence of the continuity relations is that perpendicular modes increase less significantly in strength with increasing angle of incidence than expected. This is a consequence of the fact that it is the inverse of the perpendicular component of the dielectric function that increasingly becomes important with a growing angle of incidence. An inverse dielectric function, however, has typically much smaller values than the dielectric function. An additional consequence is that perpendicular modes are blueshifted and coupled in such a way that oscillator strength is transferred to the higher wavenumber mode. Thus, the spectral signatures of perpendicular modes are often weak and masked by the parallel modes when two modes overlap. Accordingly, to enable dispersion analysis, it is suggested to use a hybrid of the conventional residual sum of squares and the two-trace two-dimensional (2T2D) smart error sum, which can correct systematic multiplicable errors in the experimental spectrum. As demonstrated for fresnoite (Ba2TiSi2O8), this is an important step toward determining the perpendicular component of the dielectric tensor and the corresponding oscillator parameters using dispersion analysis, since asynchronous 2T2D correlation spectra are, in particular, sensitive to perpendicular modes.