Concatenated Two-Dimensional Correlation Analysis: A New Possibility for Generalized Two-Dimensional Correlation Spectroscopy and Its Application to the Examination of Process Reversibility LIPING ZHANG, ISAO NODA, and YUQING WU* State Key Lab for Supramolecular Structure and Material, Jilin University, No. 2699, Qianjin Street, Changchun, 130012 P. R. China (L.Z., Y.W.); The Procter & Gamble Company, 8611 Beckett Road, West Chester, Ohio 45069 (I.N.); and Jilin Business and Technology College, No. 4728, Xi’an Road, Changchun, 130061, P. R. China (L.Z.) We propose a new application of generalized two-dimensional (2D) correlation spectroscopy called ‘‘concatenated’’ 2D correlation analysis, which is useful in identifying the presence of strict similarity or very subtle difference between two spectral data sets having a similar origin. This approach is very efficient and can offer many potential applications. In this study, the detailed examination of process reversibility is explored. Two forms of concatenation, horizontal and vertical concatenation of data matrices, are introduced and the latter is discussed in detail. Concate- nated 2D correlation analysis allows one to investigate directly the correlation between two independent but related spectral data sets. It can extract more detailed information, such as the comparison of effects of two different perturbations or different systems. We describe the principle of the ‘‘mirror-image concatenation’’ in 2D correlation analysis, which is applied to demonstrate its reliability and efficiency on three spectral models: a synthetic simulation data set; experimental Fourier transform infrared (FT-IR) spectra of the thermally induced unfolding–refolding transition of bovine pancreatic ribonuclease A (RNase A) in aqueous solution; and a set of FT-IR spectra of traditional Chinese medicines (TCM) of similar origin. The concatenated 2D correlation analysis shows its power in revealing the irreversibility of the thermally induced conformation transition of RNase A as well as the comparison of different species of TCM. Index Headings: Two-dimensional correlation spectroscopy; 2D correla- tion; Vertical concatenation; Process reversibility; Traditional Chinese medicine. INTRODUCTION Since the introduction of the basic concept by Noda in 1986, 1 two-dimensional (2D) correlation spectroscopy has become a popular tool applicable to a variety of analytical science problems. 2–14 It enhances spectral resolution by spreading peaks along the second dimension and facilitates the extraction of information that cannot be obtained easily from one-dimensional spectra. With the rapid progress in the field of generalized 2D correlation spectroscopy, hetero-spectral 2D correlation anal- ysis has recently become a subject of keen interest due to its power in dealing with sets of completely different types of spectra obtained for a system under the same perturbation conditions. 15,16 Two types of hetero-spectral 2D correlations are often used. The first one is concerned with the comparison between closely related spectroscopic techniques, such as infrared/near-infrared (IR/NIR) and Raman/NIR spectroscopy; the second one is hetero-correlation between completely different types of spectroscopy or physical techniques such as IR and X-ray scattering. For example, Liu et al. 17 used hetero- spectral 2D correlation based on the combination of visible and NIR spectroscopy to study the storage and treatment-process dependence of chicken meat. Czarnik-Matusewicz et al. 3 used IR/NIR hetero-spectral correlation to analyze the temperature effect on hydrogen bonded assembly of water. McNavage et al. 18 developed a variant form of hetero-spectral correlation called cross-spectra correlation for time-resolved FT-IR emission study of photolysis reactions. Hyde et al. 19 reported hetero-correlation based on IR and gas chromatography. Hybrid 2D correlation spectroscopy was proposed in 2002. 20 It deals with the 2D correlation analysis between two separately obtained data matrices. 20–22 In hetero-spectral 2D correlation, two sets of data matrices are obtained by making two different types of spectroscopic measurements under the same perturbation; in hybrid correlation, on the other hand, a single type of spectroscopic measurement is usually carried out under multiple perturbation variables. Most importantly, hybrid 2D correlation spectroscopy is often applied to both sample– sample and variable–variable 2D correlation spectroscopy. By coupling with sample–sample correlation, hybrid 2D correla- tion furthermore can potentially explore the latent correlation between different perturbation variables. It explores the similarity or difference between two systems or processes by investigating the symmetry along the diagonal line in the sample–sample hybrid 2D cross-product. The technique so far disclosed the presence of two different reaction mechanisms of hydrogenation of nitrobenaene 20 and the reversibility of polymer processes dependent on temperature and pressure. 21 Here we propose a new application of generalized 2D correlation spectroscopy called ‘‘concatenated’’ 2D correlation analysis. By using this method, one can more clearly and directly identify the structural dissimilarity between two spectral data of related origin. In other words, concatenated 2D correlation analysis deals with the correlation between two sets of spectra by concatenating them together into a new data matrix, instead of separately performing 2D calculation on each data set. The technique may be regarded as a further development or an interesting extension of 2D hetero-spectral (with the same perturbation) or hybrid (with different perturbations) correlation spectroscopy. Unlike the usual 2D hetero-spectral or hybrid processes, concatenated 2D correla- tion analysis probes the correlation between two sets of spectra by means of the so-called vertical concatenation strategy. This approach, we believe, is computationally easier and more efficient and thus offers many potential applications, such as Received 3 June 2009; accepted 22 December 2009. * Author to whom correspondence should be sent. E-mail: yqwu@jlu.edu. cn. Volume 64, Number 3, 2010 APPLIED SPECTROSCOPY 343 0003-7028/10/6403-0343$2.00/0 Ó 2010 Society for Applied Spectroscopy