Identification and Characterization of Chitin in Organisms

Model compound chitin and invertebrate cuticles were analysed using pyrolysis-gas chromatography-mass spectrometry, (13) C NMR and C-, N-, and O-Xray Absorption Near Edge Structure (XANES) spectral imaging using Scanning Transmission X-ray Microscopy (STXM) to detect spectra that are characteristic of chitin. Acetylpyridones, acetamidofuran, 3-acetamido-5-methylfuran and 3-acetamido-(2 and 4)-pyrones appear to be characteristic pyrolysis products for chitin. Pyrolysis products with ions of m/z 70, 154, 168, 194 likely derive from diketopiperazine structures and provide potential markers for proteins and peptides in which proline, alanine, valine, arginine and glycine are the dominant amino acids. The C-13 NMR spectra of chitin reveals that amidyl methyl group resonates at 23 ppm, amidyl linked glyocosyl carbon resonates at 56 ppm, glucosyl secondary alcohols resonate between 62 and 84 ppm, and glycosidic carbon absorption is evident at similar to 105 ppm. The presence of protein in the arthropod cuticles is evident by resonance intensity associated with sp(2) bonded carbon associated in unsaturated amino acids (e.g. phenyl alanine, tyrosine, and histidine) occurring at 116, 129, and 137 ppm. Additionally, the protein back bone methine carbon atoms are indicated by resonance intensity at 43 ppm. Additional broad resonance intensity in the 20-30 ppm range is derived both from aliphatic aminoacids (e.g. valine and leucine) as well as the fatty acids associated with the waxy cuticulin layer of the cuticle. High energy resolution C-, N-, and O-XANES spectra provide further functional group level characterization of the biomacromolecular assemblages at spatial scales on the order of 100's of nm. Combining the power of Solid state C-13 NMR, pyrolysis with the micro-analytical capabilities of C-, N-, and O-XANES yields a formidable analytical approach towards detecting and quantitating the presence of chitin in complex biomacromolecular assemblages.