杜氏盐藻多糖的高效体积排阻色谱的保留特性及其分析方法的研究

Study on the Retention Behaviors and Analytical Methods of Polysaccharides from Duanaliella salina by High Performance Size-Exclusion Chromatography

通过对从杜氏盐藻中提取出的不同多糖级分在高效体积排阻色谱柱(Waters Ultrahydragel Linear,7.8 mmi.d.×300 mm,2根串联)上的保留特性的考察及其分离分析条件的优化,建立了高效体积排阻色谱分析盐藻多糖平均相对分子质量及其分布的方法。结果表明:流动相中盐的种类及其浓度、pH值对3种酸性多糖级分(特别是硫酸化多糖级分PD4a)的保留行为有显著影响;在柱温为45℃,流速为0.9 mL/min条件下,使用0.1 mol/L的NaAc水溶液作流动相基本上能消除非特异性吸附作用及分子间缔合等因素的干扰,使各多糖级分基本以非缔合状态按立体排除机制保留和分离。在优化的色谱条件下,测得的盐藻多糖5个级分的重均相对分子质量(Mw)分别为1 548 000,33 000,67 000,424 000,10 000;测得的硫酸化多糖级分PD4a的Mw和峰面积的相对标准偏差分别为1.7%和0.88%(n=5)。

There are many polysaccharides with biological activities including anti-virus and anti-tumor in the residue produced by the extraction of β-carotene from Duanaliella salina. In this paper, a method of high performance size-exclusion chromatography coupled with refractive index detection has been developed for the relative molecular mass analysis of the polysaccharides isolated from the residue. The effects of salt and pH values of mobile phase on retention behaviors of five polysaccharides fractions were investigated on two HPSEC columns （ Waters Ultrahydragel Linear, 7.8 mm i. d. × 300 mm） connected in series. The results showed that 0. 1 mol/L NaAc buffer solution may be utilized as mobile phase during HPSEC under the conditions of column temperature of 45 ℃ and flow rate of 0.9 mL/min to minimize nonspecific interaction of sulfated polysaccharides fraction （PD4a）, complex carbohydrate containing oligonucleic acid （PD4b） and acidic polysaccharide fraction （PD1） with the column matrix. In ad- dition, under the conditions the association effect of the polysaccharide molecules was eliminated. Thereby, the polysaccharide molecules were eluted and separated following equilibrium exclusion mechanism mainly. The weight mean molecular masses （Mw） of five polysaccharide fractions from D. salina determined under optimum chromatographic conditions were 1 548 000 for PD1, 33 000 for PD2, 67 000 for PD3, 424 000 for PD4a and l0 000 for PD4b. For sulfated polysaccharide fraction PD4a, the relative standard deviations were 1.7% and 0.88% for Mw and peak area, respectively.