High-resolution separation of homogeneous chitooligomers series from 2-mers to 7-mers by ion-exchange chromatography

Highly purified chitooligomers with single degree of polymerization are of significance for studying bioactivity of chitooligomers. However, there are few reports on high-resolution preparative separation of chitooligomers, especially for those oligomers with degree of polymerization higher than 4. This study developed a high-resolution chromatography for the preparative separation of a pure fully deacetylated chitooligomer series. A glucosamine oligomer mixture with low degree of polymerization was prepared by acid hydrolysis of a highly deacetylated chitosan. Then, six fractions were separated from the prepared oligomer mixture by ion-exchange chromatography and analyzed by HPLC and ESI/MS, which primarily contained glucosamine dimers, trimers, tetramers, pentamers, hexamers, and heptamers, respectively, with chromatographic purities over 98% for dimers to hexamers and a purity of 93% for heptamers. The yields of a single round of separation were 75, 60, 60, 55, 35, and 20 mg for glucosamine dimers, trimers, tetramers, pentamers, hexamers, and heptamers, respectively. Furthermore, a chromatographic separation model for GlcN homomers was established. The capacity factor (k) of glucosamine oligomers and their degrees of polymerization (DPs) exhibited a good correlation, lnk = 0.786 + 0.846 lnDP, (R-2 = 0.997). Based on this equation, glucosamine octamers are expected to be separated by this system.