In this study, a new method, a three-stage infrared spectroscopy (Fourier transform infrared spectroscopy (FT-IR) integrated with second derivative infrared spectroscopy and two-dimensional correlation infrared spectroscopy (2D-IR)) was developed to analyze the organic and inorganic compositions of three different horns (Cornu Antelopis, Cornu Bubali and Pulvis Cornus Bubali Concentratus). In IR spectra, all the three horns had their own macroscopic fingerprints especially for those compositions containing amide groups, CH groups and Ca(3)(PO(4))(2). Their second derivative spectra amplified the differences and revealed the potentially characteristic IR absorption bands 1350-400 cm(-1) to be investigated in 2D-IR. Subsequently, many covered characteristic fingerprints were disclosed in 2D-IR spectra in the range of 1350-400 cm(-1) and the three horns were therefore effectively discriminated. Meanwhile, the analysis results of inorganic constituents were verified by atomic spectroscopy. Furthermore, thirty different horn samples including ten of each horn were also successfully classified by soft independent modeling of class analogy (SIMCA). It was demonstrated that the above three-stage infrared spectroscopy could be applicable for quick, non-destructive and effective analysis and identification of very complicated and similar mixture systems (e.g. traditional Chinese medicines).
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