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Raman and infrared (IR) are two complementary vibrational spectroscopy techniques that enable label-free, noninvasive, and nondestructive structural characterization of analyzed specimens. IR spectroscopy is broadly utilized in various research areas ranging from food chemistry and agriculture to geology and medicine. Recently, Raman spectroscopy (RS) has attracted the interest of researchers from these fields because of its minimal interference from water and significant reduction of equipment costs. In this study, we evaluated the complementarity of RS and IR for structural characterization of epicuticular waxes, sophisticated chemical mixtures of long fatty acids, and their derivatives excreted by plants. We show that IR can sense R-O-H vibrations, which are characteristic for alcohols and sugars, as well as carbonyl groups, which can be assigned to aldehydes, ketones, acids, and esters in the chloroform-extracted waxes of Sorghum bicolor. At the same time, RS can detect only C-C, C-H, and CH2 vibrations of these molecules in the same wax extracts. Using theoretical calculations, we were able to elucidate the origin of this phenomenon. It was found that an increase in the aliphatic chain length in carboxylic acids resulted in a quadratic and linear increase in the intensity of aliphatic vibrations in the corresponding Raman and IR spectra, respectively. Thus, the complementary of RS and IR that holds for small molecules may not be always observed for large biological molecules.

Complementarity of Raman and Infrared Spectroscopy for Structural Characterization of Plant Epicuticular Waxes