Near-infrared (NIR) spectroscopy is a useful tool in determining glucose in biological matrices. Because α-anomers and β-anomers of glucose are in equilibrium in the solution, the observed NIR bands may come from the overlapping of vibrational modes of the anomers. We have conducted NIR and mid-infrared (MIR) absorption spectra measurements to determine the nature of the observed NIR features by using two-dimensional (2D) NIR and MIR correlation spectroscopy. The 2D NIR correlation spectra and 2D synchronous NIR and MIR correlation heterospectra are constructed based on the spectral changes of individual anomers upon mutarotation. We have identified a new NIR feature at 4350 cm<sup>-1</sup> for the α-anomers and two new NIR features at 4200 and 4250 cm<sup>-1</sup> for the β-anomers. The 4350-cm<sup>-1</sup> band of the α-anomers could be assigned to the combination band of C-H stretch at 2945 cm<sup>-1</sup> and a mixed mode of C-H bending and O-H bending at 1415 cm<sup>-1</sup>. The 4200/4250-cm<sup>-1</sup> bands of the β-anomers might be tentatively assigned to the combination band of C-H stretching at 2870 cm<sup>-1</sup> and a mixed mode of C-H bending and O-H bending at 1315/1370 cm<sup>-1</sup>. This finding provides the spectral information needed for implementation of a highly selective coherent two-dimensional vibrational spectroscopic method, the DOVE-Raman four wave mixing for selective identification of glucose anomers from aqueous solutions.
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