This reports the first detection of chemical heterogeneity in octenyl succinic anhydride modified single starch granules using a Fourier transform infrared (FT-IR) microspectroscopical technique that combines diffraction-limited infrared microspectroscopy with a step size that is less than the mask projected spot size focused on the plane of the sample. The high spatial resolution was achieved with the combination of the application of a synchrotron infrared source and the confocal image plane masking system of the double-pass single-mask Continuμm<sup>®</sup> infrared microscope. Starch from grains such as corn and wheat exists in granules. The size of the granules depends on the plant producing the starch. Granules used in this study typically had a median size of 15 μm. In the production of modified starch, an acid anhydride typically is reacted with OH groups of the starch polymer. The resulting esterification adds the ester carbonyl (1723 cm<sup>−1</sup>) organic functional group to the polymer and the hydrocarbon chain of the ester contributes to the CH<sub>2</sub> stretching vibration to enhance the intensity of the 2927 cm<sup>−1</sup> band. Detection of the relative modifying population on a single granule was accomplished by ratioing the baseline adjusted peak area of the carbonyl functional group to that of a carbohydrate band. By stepping a confocally defined infrared beam as small as 5 μm × 5 μm across a starch granule 1 μm at a time in both the <i>x</i> and <i>y</i> directions, the heterogeneity is detected with the highest possible spatial resolution.

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