How can the pattern of acetylation be determined?
To date, there is only one established method to test the pattern of acetylation for randomness, namely diad-analysis by nuclear magnetic resonance (NMR) spectroscopy. This method allows to quantify the frequency with which a N-acetylglucosamine (GlcNAc) or a glucosamine (GlcN) residue is located next to a GlcNAc or next to a GlcN residue. The frequencies of these four so-called diads (GlcNAc-GlcNAc, GlcNAc-GlcN, GlcN-GlcNAc, GlcN-GlcN) expected for a random distribution (i.e., a random pattern of acetylation) can easily be calculated from the degree of acetylation of the chitosan investigated. There is a formula which calculates a PA value from the diad frequencies which gives a value of PA = 1 for a random distribution, a PA = 0 for a perfectly blockwise distribution, and a PA of up to 2 (the value will depend on the degree of acetylation, it can reach 2 for a DA of 50% only) for a perfectly regular distribution.