Identification of chitin allomorphs in poorly crystalline samples based on the complexation with ethylenediamine

Chitin is a key component of hard parts in many organisms, but the biosynthesis of the two distinctive chitin allomorphs, α- and β-chitin, is not well-understood. The accurate determination of chitin allomorphs in natural biomaterials is vital. Many chitin-secreting living organisms, however, produce poorly crystalline chitin which leads to spectrums with only broad lines and imprecise peak positions under conventional analytical methods such as X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and solid state nuclear magnetic resonance spectroscopy (NMR), resulting in inconclusive identification of chitin allomorphs. Here, we developed a novel method for discerning chitin allomorphs based on their different complexation capacity and guest selectivity, using ethylenediamine (EDA) as a complexing agent. From the peak shift observed in XRD profiles of the chitin/EDA complex, the chitin allomorphs can be clearly discerned. By testing this method on a series of samples with different chitin allomorphs and crystallinity, we show that the sensitivity is sufficiently high to detect the chitin allomorphs even in near-amorphous, very poorly crystalline samples. This is a powerful tool for determining the chitin allomorphs in phylogenetically important chitin-producing organisms and will pave the way to clarify the evolution and mechanism of chitin biosynthesis.