Two-dimensional (2D) correlation analysis was applied to characterize the attenuated total reflection (ATR) spectral intensity fluctuations of native cotton fibers with various water contents. Prior to 2D analysis, the spectra were leveled to zero at the peak intensity of 1800 cm<sup>–1</sup> and then were normalized at the peak intensity of 660 cm<sup>–1</sup> to subjectively correct the changes resulting from water diffusion in fibers and resultant density dilution. Next, a new spectral set was subjected to principal component analysis (PCA) and two clusters of hydrated (≥13.3%) and dehydrated (<13.3%) fibers were obtained. Synchronous and asynchronous 2D correlation spectra from individual ATR spectral sets enhanced spectral resolution and provided insights about water-content-dependent intensity variations not readily accessible from one-dimensional ATR spectra. The 2D results revealed remarkable differences corresponding to water loss between the hydrated and dehydrated fibers. Of interest were that: (1) the intensity of the 1640 cm<sup>–1</sup> water band remains in a steady state for hydrated fibers but decreases for dehydrated fibers; (2) during the desorption process of adsorbed water, small and water-soluble carbonyl species (i.e., esters, acids, carboxylates, and proteins) begin to accumulate on the cotton surface, resulting in possible changes in the coloration and surface chemistry of native cotton fibers that were rained on prior to harvesting; (3) intensities of bands in the 1200 to 950 cm<sup>–1</sup> region exhibit a more apparent intensity increase than those in the 1500 to 1200 cm<sup>–1</sup> region, indicating the sensitivity of the 1200 to 950 cm<sup>–1</sup> infrared (IR) region to intra- and inter-molecular hydrogen bonding in fiber celluloses; and (4) the 750 cm<sup>–1</sup> band, ascribed to the unstable I<sub>α</sub> phase in amorphous regions, might originate from the cellulose–water complex through hydrogen bonding.

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