离子色谱分离保留因子计算定性分析低聚壳寡糖的研究

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×250 mm）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R²≥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，利用离子色谱分离，保留因子计算来定性壳寡糖聚合度的方法是可行的。

Objective: To developed a method for qualitative analysis of chitosan oligosaccharides using an anion exchange chromatography and retention factor calculation.Methods: Five kinds of chitosan oligosaccharides[degree of polymerization(DP)=2-6] were separated by high performance anion exchange chromatography with integrated pulsed ampere detection(HPAEC-IPAD).A DIONEX CarboPac^® PA10 guard column(4.0 mm×50 mm)and a DIONEX CarboPac^® PA10(4.0 mm×250 mm)separation column were used for the chitosan oligosaccharides separation.The mobile phase was sodium hydroxide.An integrated pulsed ampere detection was utilized for detection.Then,the chitooligosaccharide samples were separated and characterized by the established separation method.The retention factors of sample were calculated in accordance with their retention time and linear correlation between the DP of chitosan oligosaccharides and retention factors.Finally,unknown chitosan oligosaccharides were qualitatively determined according to the above relationship.Results: There was a good linear correlation(R²≥0.998 7)between logk'(retention factor)value of chitosan oligosaccharide and corresponding log DP value.The DP of the unknown chitosan oligosaccharides in the sample were calculated and 4 chitosan oligosaccharides(chitoheptaose,chitooctaose,chitononaose,chitodecaose) were qualitatively determined by established linear equation.The error was below 5.66%.Result of mass spectrometry test also showed 8 chitosan oligosaccharides(DP=2-8)existed in the real sample.Conclusion: The established method can be used as a qualitative method for chitosan oligosaccharide homogeneous when there is a lack of reference substance.

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