2D Correlation Spectroscopy Research Articles

2D Correlation Analysis of Spin-Coated Films of Biodegradable P(HB-co-HHx)/PEG Blends

We investigated thermal behavior of spin-coated films of P(HB-co-HHx)/PEG blends by using infraredreflection absorption (IRRAS) spectroscopy and 2D correlation spectroscopy. Based on 2D IRRAS correlation spectra, we could determine the sequence of spectral intensity changes with increasing temperature that PEG band changes first and then a band for crystalline component of P(HB-co-HHx) changes before a band for amorphous component. The intensities of bands for PEG and amorphous P(HB-co-HHx) were changed greatly as PEG weigh % of P(HB-co-HHx)/PEG blends increased. Transition temperatures of P(HB-co-HHx)/PEG blends were successfully determined by 2D gradient mapping method. The transition temperature of spincoated films of 98/2 and 90/10 P(HB-co-HHx)/PEG blends and 80/20 P(HB-co-HHx)/PEG blend determined by 2D gradient map are, respectively, about 137.5 and 132.5 o C. Furthermore, P(HB-co-HHx)/PEG blends show an additional transition temperature that have been interpreted in terms of different lamellar thicknesses in spin coated films.

Study of the pre-melting behavior of polyethylene using Raman spectroscopy combined with two-dimensional (2D) correlation spectroscopy

The thermal behavior of polyethylene (PE) below its melting temperature (Tm) in relation to the density variation was studied by Raman spectroscopy combined with two-dimensional (2D) correlation analysis. The Raman spectra of homo and 1-octene copolymerized PEs were collected by varying the sample temperature from 30 to 100°C, where pre-melting would occur. The spectral variation upon temperature perturbation was dependent on the density. For a deeper understanding of the thermal behaviors, 2D hetero correlation analysis was performed for the temperature-dependent Raman spectra of homo and 1-octene copolymerized PEs. The hetero-sample correlation analysis revealed that even below Tm, co-monomer side chains excluded from the crystalline lattice of the PE underwent partial fusion, indicating that such a conformational defect was closely related to the pre-melting of PE.

Multiple Fluorescence Labeling and Two Dimensional FTIR–13C NMR Heterospectral Correlation Spectroscopy to Characterize Extracellular Polymeric Substances in Biofilms Produced during Composting

Knowledge on the structure and function of extracellular polymeric substances (EPS) in biofilms is essential for understanding biodegradation processes. Herein, a novel method based on multiple fluorescence labeling and two-dimensional (2D) FTIR-(13)C NMR heterospectral correlation spectroscopy was developed to gain insight on the composition, architecture, and function of EPS in biofilms during composting. Compared to other environmental biofilms, biofilms in the thermophilic (>55 °C) and cooling (mature) stage of composting have distinct characteristics. The results of multiple fluorescence labeling demonstrated that biofilms were distributed in clusters during the thermophilic stage (day 14), and dead cells were detected. In the mature stage (day 26), the biofilm formed a continuous layer with a thickness of approximately 20-100 μm around the compost, and recolonization of cells at the surface of the compost was easily observed. Through 2D FTIR-(13)C NMR correlation heterospectral spectroscopy, the following trend in the ease of the degradation of organic compounds was observed: heteropolysaccharides > cellulose > amide I in proteins. And proteins and cellulose showed significantly more degradation than heteropolysaccharides. In summary, the combination of multiple fluorescence labeling and 2D correlation spectroscopy is a promising approach for the characterization of EPS in biofilms.

Heat-induced denaturation/aggregation of porcine plasma and its fractions studied by FTIR spectroscopy

The aim of the present work is the in depth study of the protein aggregation mechanisms of whole porcine plasma and its fractions (serum, albumin and globulins) during heating using FTIR spectroscopy. Also, 2D correlation spectroscopy (2D COS) was used to establish the sequence of events during heat-induced gelation for all fractions. The results indicate that serum albumin quickly aggregates from 70 °C through non-native intramolecular β-sheets while globulins show lower susceptibility to protein aggregation. When found together, the aggregation pattern strongly depends on the composition of the protein mixture. That makes the great difference between plasma (serum albumin + globulins + fibrinogen) and serum (serum albumin + globulins) behavior, with the aggregation degree at the end of the thermal process being enhanced in the presence of fibrinogen – and achieving a similar level to that of serum albumin – while minimized in its absence. Attending on the low content of fibrinogen in plasma, our results suggest a great fibrinogen ability to alter the thermal serum albumin and globulins behavior by modifying the negative interactions established between them when no more proteins are found in the media. Moreover, it is noteworthy the slow plasma aggregation pattern at the beginning of the thermal process relative to serum albumin, this way allowing a higher protein unfolding. This could be related to the high heat-induced gel properties of plasma. Also, 2D COS indicates that the sequence of events is very similar for the all analyzed samples, with α-helix being more thermo-labile than native β-sheet structure.

Correlation chemical shift imaging with low‐power adiabatic pulses and constant‐density spiral trajectories

In this work we introduce the concept of correlation chemical shift imaging (CCSI). Novel CCSI pulse sequences are demonstrated on clinical scanners for two-dimensional Correlation Spectroscopy (COSY) and Total Correlation Spectroscopy (TOCSY) imaging experiments. To date there has been limited progress reported towards a feasible and robust multivoxel 2D COSY. Localized 2D TOCSY imaging is shown for the first time in this work. Excitation with adiabatic GOIA-W(16,4) pulses (Gradient Offset Independent Adiabaticity Wurst modulation) provides minimal chemical shift displacement error, reduced lipid contamination from subcutaneous fat, uniform optimal flip angles, and efficient mixing for coupled spins, while enabling short repetition times due to low power requirements. Constant-density spiral readout trajectories are used to acquire simultaneously two spatial dimensions and f(2) frequency dimension in (k(x),k(y),t(2)) space in order to speed up data collection, while f(1) frequency dimension is encoded by consecutive time increments of t(1) in (k(x),k(y),t(1),t(2)) space. The efficient spiral sampling of the k-space enables the acquisition of a single-slice 2D COSY dataset with an 8 × 8 matrix in 8:32 min on 3 T clinical scanners, which makes it feasible for in vivo studies on human subjects. Here we present the first results obtained on phantoms, human volunteers and patients with brain tumors. The patient data obtained by us represent the first clinical demonstration of a feasible and robust multivoxel 2D COSY. Compared to the 2D J-resolved method, 2D COSY and TOCSY provide increased spectral dispersion which scales up with increasing main magnetic field strength and may have improved ability to unambiguously identify overlapping metabolites. It is expected that the new developments presented in this work will facilitate in vivo application of 2D chemical shift correlation MRS in basic science and clinical studies.

Hydrogen Bonding in Fluorinated Amides: FTIR, Two Dimensional Correlation Spectroscopy and DFT Calculations

AbstractFluorinated macromers with amidic functional groups are used as additives in several high tech applications. We show here how aggregation phenomena related to hydrogen bonding are one of the key factor determining their chemical/physical and macroscopic properties. IR spectra are analyzed depending on different external parameters such as the concentration of amide groups and temperature. The experimental findings have been interpreted by means of DFT (Density Functional Theory) calculations on suitable molecular models. Moreover, 2D correlation spectroscopy has been applied to different sets of data, considering concentration and temperature as perturbing variables. The two dimensional correlation approaches confirmed the computational results and give an overall interpretation of the effects due to concentration and temperature.

Fast high-resolution 2D correlation spectroscopy in inhomogeneous fields via Hadamard intermolecular multiple quantum coherences technique

Recently, a method based on intermolecular multiple quantum coherences (iMQCs) has been proposed to obtain high-resolution 2D COSY spectra in inhomogeneous fields via 3D acquisitions. However, the very long acquisition time prevents its practical application. To overcome this shortage, the Hadamard technique was applied for the iMQC method in this paper. For the new pulse sequence, the direct frequency-domain excitation is used in the first indirect detection dimension, so the 3D acquisition was replaced by an array of 2D acquisitions. The acquisition time can be reduced to 10 min. The resulting spectra retain useful structural information including chemical shifts and multiplet patterns of J coupling even when the inhomogeneous line broadening leads to overlap of neighboring diagonal resonances in the conventional COSY spectrum. The experimental results are consistent with the theoretical predictions and computer simulations. The new sequence may provide a time-efficient way for the studies of chemical solution in inhomogeneous fields.

A new direct Fourier transform infrared analysis of free fatty acids in edible oils using spectral reconstitution

A new transmission-based Fourier transform infrared (FTIR) spectroscopic method for the direct determination of free fatty acids (FFA) in edible oils has been developed using the developed spectral reconstitution (SR) technique. Conventional neat-oil and SR calibrations were devised by spiking hexanoic acid into FFA-free canola oil and measuring the response to added FFA at 1,712 cm(-1) referenced to a baseline at 1,600 cm(-1)(1,712 cm(-1)/1,600 cm(-1)). To compensate for the known oil dependency of such calibration equations resulting from variation of the triacylglycerol ester (C═O) absorption with differences in oil saponification number (SN), a correction equation was devised by recording the spectra of blends of two FFA-free oils (canola and coconut) differing substantially in SN and correlating the intensity of the ester (C═O) absorption at the FFA measurement location with the intensity of the first overtone of this vibration, measured at 3,471 cm(-1)/3,427 cm(-1). Further examination of the spectra of the oil blends by generalized 2D correlation spectroscopy revealed an additional strong correlation with an absorption in the near-infrared (NIR) combination band region, which led to the development of a second correction equation based on the absorbance at 4,258 cm(-1)/4,235 cm(-1). The NIR-based correction equation yielded superior results and was shown to completely eliminate biases due to variations in oil SN, thereby making a single FFA calibration generally applicable to oils, regardless of SN. FTIR methodology incorporating this correction equation and employing the SR technique has been automated.

Noise Reduction Methods Applied to Two-Dimensional Correlation Spectroscopy (2D-COS) Reveal Complementary Benefits of Pre- and Post-treatment

Two-dimensional correlation spectroscopy (2D-COS) is a powerful spectral analysis technique widely used in many fields of spectroscopy because it can reveal spectral information in complex systems that is not readily evident in the original spectral data alone. However, noise may severely distort the information and thus limit the technique's usefulness. Consequently, noise reduction is often performed before implementing 2D-COS. In general, this is implemented using one-dimensional (1D) methods applied to the individual input spectra, but, because 2D-COS is based on sets of successive spectra and produces 2D outputs, there is also scope for the utilization of 2D noise-reduction methods. Furthermore, 2D noise reduction can be applied either to the original set of spectra before performing 2D-COS ("pretreatment") or on the 2D-COS output ("post-treatment"). Very little work has been done on post-treatment; hence, the relative advantages of these two approaches are unclear. In this work we compare the noise-reduction performance on 2D-COS of pretreatment and post-treatment using 1D (wavelets) and 2D algorithms (wavelets, matrix maximum entropy). The 2D methods generally outperformed the 1D method in pretreatment noise reduction. 2D post-treatment in some cases was superior to pretreatment and, unexpectedly, also provided correlation coefficient maps that were similar to 2D correlation spectroscopy maps but with apparent better contrast.

Comparison of chemometric methods in the analysis of pharmaceuticals with hyperspectral Raman imaging

AbstractChemical imaging method of vibrational spectroscopy, which provides both spectral and spatial information, creates a three‐dimensional (3D) dataset with a huge amount of data. When the components of the sample are unknown or their reference spectra are not available, the classical least squares (CLS) method cannot be applied to create visualized distribution maps. Raman image datasets can be evaluated even in such cases using multivariate (chemometric) methods for extracting the needed hidden information. The capability of chemometrics‐assisted Raman mapping is evaluated through the analysis of pharmaceutical tablets (considered as unknown) with the aim of estimating the pure component spectra based on the collected Raman image. Six chemometric methods, namely, principal component analysis (PCA), maximum autocorrelation factors (MAF), sample–sample 2D correlation spectroscopy (SS2D), self‐modeling mixture analysis (SMMA), multivariate curve resolution–alternating least squares (MCR‐ALS), and positive matrix factorization (PMF), were compared. SMMA was found to be the best choice to determine the number of components. MCR‐ALS and PMF provided the pure component spectra with the highest quality. MCR‐ALS was found to be superior to PMF in the estimation of Raman scores (which correspond to the concentrations) and yielded almost the same results as CLS (using the real reference spectra). Thus, the combination of Raman mapping and chemometrics could be successfully used to characterize unknown pharmaceuticals, identify their ingredients, and obtain information about their structures. This may be useful in the struggles against illegal and counterfeit products and also in the field of pharmaceutical industry when contaminants are to be identified. Copyright © 2011 John Wiley & Sons, Ltd.

Structural analysis of photodegraded lime wood by means of FT-IR and 2D IR correlation spectroscopy

In this study the weathering behavior of lime wood (Tillia cordata Mill.) has been examined using FT-IR and 2D IR correlation spectroscopy, which evidenced chemical changes induced by exposure to weathering conditions. It was showed that lignin is most sensitive component to the photodegradation processes as indicated by considerable decreases in the intensities of the characteristic aromatic lignin band at 1505cm−1 and other associated bands.By 2D correlation spectroscopy has been demonstrated that the moment of CO from carboxyl and acetyl groups in hemicelluloses is changing first, followed by the CC of aromatic skeletal, CO in non-conjugated ketones, carboxyl groups and lactones, absorbed O–H and conjugated C–O groups in quinones.The carbonyl formation corresponded well with lignin degradation, indicating a close relationship between them. Comparing the rate of carbonyl formation and lignin decay clearly showed that the former is remarkably higher than the latter, indicating the formation of carbonyl bands at 1738cm−1 probably resulted from not only lignin oxidation but also from reactions occurring in other components of the wood. Quinine formation is combined with the decay of aromatic structures and the formation of conjugated carbonyl groups.

Adsorption behavior of human serum albumin on ATR crystal studied by in situ ATR/FTIR spectroscopy and two-dimensional correlation analysis

The time-dependent adsorption behavior of human serum albumin (HSA) onto an ATR (ZnSe) crystal was investigated by two-dimensional (2D) correlation analysis and in situ ATR-FTIR spectroscopy following the secondary structural changes in the amide I region. The two major advantages of the generalized 2D correlation spectroscopy were first tested. New extra bands have been resolved by 2D correlation analysis, but they are either artifacts or a result of uncertainty on band position in generalized 2D correlation spectroscopy. The sequence of the intensity variations of the three sub-bands under the amide I band profile deduced from the 'sequential order' rules is contradictory to the experimental observation, which supports our argument on the 'sequential order' rules in generalized 2D correlation spectroscopy (H. Huang, Anal. Chem., 2007, 79, 8281-8292). Subsequent detailed analysis on the in situ ATR-IR spectra shows that the adsorption process of HSA on the ATR (ZnSe) crystal in aqueous solutions can be divided into three stages: no obvious conformational transitions in the first 25 min of adsorption of HSA molecules; large structural rearrangement from α-helix to random coil and short extended chain structures in a fully cooperative way from 25 to 50 min of adsorption; and further slight conformational transformation of short extended chain and turn structures into random coil with no sequential order after 50 min of adsorption.

Mechanism in Brill transition of polyamide 66 studied by two-dimensional correlation infrared spectroscopy

The Brill transition of polyamide 66 was investigated by temperature-dependent infrared spectroscopy combined with moving-window two-dimensional (MW2D) correlation spectroscopy. The temperature range of the Brill transition determined by MW2D correlation spectroscopy was 90–170 °C. We employed generalized 2D correlation spectroscopy to study the sequential order of polyamide 66 chains with linear increment of temperature. The movement of the methylene segments near to NH is earlier than those on the C O sides. At the same time, the methylene which is close to NH varies before the inner methylene. Three kinds of NH groups in polyamide 66 were found. The sequential order of their motions is as follows. The free hydrogen-bonded NH groups change first, and then the disordered hydrogen-bonded NH groups. Finally, the ordered hydrogen-bonded NH groups start to change. We also found that the changes of the ordered hydrogen-bonded NH groups follow with the methylene groups.

Ubiquinol formation in isolated photosynthetic reaction centres monitored by time-resolved differential FTIR in combination with 2D correlation spectroscopy and multivariate curve resolution

Two-dimensional correlation analysis was carried out in combination with multivariate curve resolution-alternating least squares (MCR-ALS) to analyse time-resolved infrared (IR) difference spectra probing photoinduced ubiquinol formation in detergent-isolated reaction centres from Rhodobacter sphaeroides. The dynamic 2D IR correlation spectra have not only allowed the determination of the concomitance or non-concomitance of different chemical events through known marker bands but also have helped identify new vibrational bands related to the complex series of photochemical and redox reactions. In particular, a strong positive band located at 1565 cm⁻¹ was found to be synchronous with the process of ubiquinol formation. In addition, a tailored MCR-ALS analysis was performed using a priori chemical knowledge of the system, in particular including the pure spectrum of one species obtained from an external measurement. Enhancing the MCR-ALS performance in this way in time-dependent processes is relevant, especially when other essential pieces of information, such as kinetic models, are unavailable. The results give evidence of four independent spectral contributions. Three of them show marker bands for a monoelectronic reduction of the primary quinone QA (QA⁻/QA transition, first contribution), for a monoelectronic reduction of a secondary quinone QB (QB⁻/QB transition, second contribution) and for ubiquinol formation (third contribution). The results obtained also confirm that a key rate-limiting factor is the slow ubiquinone and ubiquinol exchange among micelles, which strongly influences the kinetic profiles of the involved species.

Using Two-Dimensional Correlations of 13C NMR and FTIR To Investigate Changes in the Chemical Composition of Dissolved Organic Matter along an Estuarine Transect

Applying two-dimensional correlation spectroscopy to (13)C NMR and FTIR spectra of the high molecular-weight dissolved organic matter (HMW-DOM) isolated along an Elizabeth River/Chesapeake Bay salinity transect shows that HMW-DOM consists of three major components that have different biogeochemical reactivities. The first appears to be a heteropolysaccharide (HPS) component and its contribution to carbon increases as we approach the marine offshore. The second appears to be composed of carboxyl-rich compounds (CRC); its carbon percentage decreases. The third component contains the major functional group of amide/amino sugar (AMS) and its carbon percentage stays almost constant along the salinity transect. It seems that the HPS and CRC are present in many aquatic environments at different relative ratios. The 2D-correlation maps reveal that each of these components is composed of dynamic mixtures of compounds that share similar backbone structures but have significant functional group differences. Two-dimensional (2D) correlation spectroscopy is a powerful new biogeochemical tool to track the changes in complex organic matter as a function of space, time, or environmental effects.

FTIR 2D correlation spectroscopy of α1 and α2 fractions of an alkali-pretreated gelatin

An alkali-pretreated gelatin (pI ~ 4.9) was fractionated by means of alcohol coacervation and semi-preparative gel chromatography. The thermal responses of the isolated α fractions, the coacervate and the total gelatin were investigated by 2D-correlation FTIR spectroscopy in the amide I band region (1600–1700 cm − 1 ). The gelation temperature was the same for all examined samples (24.5 °C) while the melting temperature of the α 2 fraction was lower (30 °C) than that of the other samples (32.5 °C). The 2D COS plots indicate that on cooling (gelation) the core sequence of conformational changes is the same for all samples. On heating, however, the α 2 fraction deviates from the α 1-containing samples and shows an earlier disappearance of the triple helix signal in the event sequence. The lower melting temperature (less thermostable gelatin gel) of the α 2 fraction thus results from a different conformational cascade of the α 2 chains upon melting. In all samples the initial conformational changes take place in the β-turns, providing further evidence for the models proposed previously.

Time-dependent adsorption behavior of β-lactoglobulin on ZnSe crystal surface studied by 2D correlation ATR/FTIR spectroscopy

The time-dependent adsorption behavior of β-Lactoglobulin (β-Lg) on ATR crystal (ZnSe) surface was studied by two-dimensional (2D) correlation ATR/FTIR spectroscopy. More bands were resolved by 2D correlation spectroscopy compared to the results from second derivative (SD) and Fourier self-deconvolution (FSD) analyses, but some of the new bands resolved may originate from bandwidth changes, wavenumber shifts, etc. The integrated/overall sequential order of the intensity changes of the four sub-bands in amide I region obtained from 2D correlation spectroscopy was not consistent with the experimental observation. Adsorption-induced conformational changes did not occur until 10 min of adsorption of β-Lg molecules on the ZnSe crystal surface. The relative contents of the low-wavenumber component of the antiparallel β-strands (1627cm(-1)) and random segments with α-helix (1651cm(-1)) changed prior to β-turns (1666cm(-1)) and the high-wavenumber component of the antiparallel β-strands (1684cm(-1)). More specifically, from about 10 to 15min of adsorption, the loss content of the low-wavenumber component of the antiparallel β-strands (1627cm(-1)) was simultaneously transformed into random segments (1651cm(-1)). After 20 min of adsorption, the content of β-turns (1666cm(-1)) started to decrease, and the loss of β-turns (1666cm(-1)) was also transformed into antiparallel β-strands (high-wavenumber component at 1684cm(-1)) in a cooperative way as the β-Lg molecules become more extended.

Photo-Cross-Linked Poly(dl-lactide)-Based Networks. Structural Characterization by HR-MAS NMR Spectroscopy and Hydrolytic Degradation Behavior

To date, biodegradable networks and particularly their kinetic chain lengths have been characterized by analysis of their degradation products in solution. We characterize the network itself by NMR analysis in the solvent-swollen state under magic angle spinning conditions. The networks were prepared by photoinitiated cross-linking of poly(dl-lactide)−dimethacrylate macromers (5 kg/mol) in the presence of an unreactive diluent. Using diffusion filtering and 2D correlation spectroscopy techniques, all network components are identified. By quantification of network-bound photoinitiator fragments, an average kinetic chain length of 9 ± 2 methacrylate units is determined. The PDLLA macromer solution was also used with a dye to prepare computer-designed structures by stereolithography. For these networks structures, the average kinetic chain length is 24 ± 4 methacrylate units. In all cases the calculated molecular weights of the polymethacrylate chains after degradation are maximally 8.8 kg/mol, which is far below the threshold for renal clearance. Upon incubation in phosphate buffered saline at 37 °C, the networks show a similar mass loss profile in time as linear high-molecular-weight PDLLA (HMW PDLLA). The mechanical properties are preserved longer for the PDLLA networks than for HMW PDLLA. The initial tensile strength of 47 ± 2 MPa does not decrease significantly for the first 15 weeks, while HMW PDLLA lost 85 ± 5% of its strength within 5 weeks. The physical properties, kinetic chain length, and degradation profile of these photo-cross-linked PDLLA networks make them most suited materials for orthopedic applications and use in (bone) tissue engineering.

Epoxy curing reaction studied by using two‐dimensional correlation infrared and near‐infrared spectroscopy

AbstractThe isothermal curing process of bisphenol A epoxy resin with polyamine reagent (1,6‐diaminohexane) was monitored in situ by using temperature‐controlled Fourier‐transform infrared (FTIR) and Fourier‐transform near infrared (FTNIR) spectroscopy to elucidate the relative changes in functional groups during the curing reaction. It was shown that generalized two‐dimensional correlation spectroscopy can provide new information about the mechanisms and kinetics of the curing process, and the band assignments for complex NIR spectrum associated with this system. The sequential order of relative changes in functional groups during the curing process was examined by generalized 2D correlation spectroscopy and NIR‐IR hetero‐correlation spectroscopy, and the details of the complex epoxy curing reaction involving both primary and secondary amino group were revealed. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Structural analysis of some soluble elastins by means of FT‐IR and 2D IR correlation spectroscopy

Fourier transform infrared (FT-IR) spectroscopy combined with 2D correlation spectroscopy has been used to offer some information about stability and structure of some soluble elastins. Temperature has been chosen as the perturbation to monitor the infrared behavior of various soluble elastins, namely, α-elastin p, α-elastin, and k-elastin. In the 3800-2700 cm⁻¹ region, the H-containing groups were analyzed. The bonded hydroxyls are found to decrease prior to the NH-related hydrogen bonds and also to the conformational reorganization of hydrocarbon chains. The transition temperatures were evaluated and they were found to agree with those obtained from DSC data. The FTIR spectra and their 2nd derivatives denote that α- elastins exhibited amide-I, -II and -III bands at 1656, 1539 and 1236 cm⁻¹, respectively, while in k-elastin these bands were found at 1652 cm⁻¹ for amide I, 1540 cm⁻¹ for amide II and 1248 cm⁻¹ for amide III. The macroscopic IR finger-print method, which combines: general IR spectra, secondary derivative spectra, and 2D-IR correlation spectra, is useful to discriminate different elastins. Thus using the differences of the position and intensity of the bands from "fingerprint region" of studied elastins, which include the peaks assigned to C==O, C--C groups from α-helix, β-turn, and the peaks assigned to the amide groups, it is possible to identify and discriminate elastins from each others. Furthermore, the pattern of 2D-IR correlation spectra under thermal perturbation, allow their direct identification and discrimination.