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目的:建立阴离子交换色谱分离,并用保留因子计算法定性分析低聚壳寡糖。方法:首先利用高效阴离子交换色谱-积分脉冲安培检测法(HPAEC-IPAD)对5种壳寡糖(聚合度DP=2~6)标准样品进行分离;分离条件:采用DIONEX CarboPac·PA10(4.0 mm×50 mm)保护柱和DIONEX CarboPac·PA10(4.0 mm×250mm)分析柱,淋洗液采用氢氧化钠溶液,积分脉冲安培法检测。然后用建立的分离方法对壳寡糖样品进行分离并定性。再根据所得样品色谱峰的保留时间分别计算保留因子,建立壳寡糖聚合度(DP)与其保留因子(k’)的相关关系,根据建立的相关关系对未知的壳寡糖进行定性,并用质谱方法对未知壳寡糖样品进行验证。结果:壳寡糖聚合度的对数值与其保留因子的对数值具有线性关系,回归系数R~2≥0.998 7,利用建立的线性回归方程对样品中的4种未知壳寡糖样品的定性结果为壳七糖、壳八糖、壳九糖和壳十糖,误差≤5.66%。质谱方法对未知壳寡糖样品进行了验证,验证结果为样品中存在壳二糖、壳三糖、壳四糖、壳五糖、壳六糖、壳七糖和壳八糖。结论:对于壳寡糖样品用离子色谱分离,保留因子计算来定性壳寡糖聚合度的方法是可行的。
OBJECTIVE: To establish an anion exchange chromatographic separation and to determine the oligomeric chitooligosaccharides by retention factor. Methods: Five kinds of chitooligosaccharides (DP = 2 ~ 6) were separated by HPAEC-IPAD. The separation conditions were as follows: DIONEX CarboPac PA10 (4.0 mm × 50 mm) guard column and a DIONEX CarboPac · PA10 (4.0 mm × 250 mm) analytical column. The eluent was detected using sodium hydroxide solution and integrated pulse amperometry. The chitooligosaccharides samples were then separated and characterized by the established separation method. The retention factors were calculated according to the retention times of the obtained chromatographic peaks. The relationship between the degree of oligochitosan polymerization (DP) and its retention factor (k ’) was established. The unknown chitooligosaccharides were characterized by mass spectrometry Methods Samples of unknown chitooligosaccharides were validated. Results: The logarithm of the degree of chitooligosaccharides polymerization had a linear relationship with the logarithm of its retention factor, and the regression coefficient was R ~ 2 ≥ 0.998. Using the established linear regression equation, the qualitative results of four unknown chitooligosaccharides samples were Heptacoshane, chitin, chitosan and chitosan, with an error ≤ 5.66%. Chitosan oligosaccharide samples were verified by mass spectrometry. The results showed that chitobiose, chitotriose, chitotetraose, chitotetraose, chitosan, chitin and chitin were present in the sample. Conclusion: It is feasible to determine the degree of polymerization of chitooligosaccharides by chromato-oligosaccharide samples by ion chromatography and retention factor.