高效阴离子交换色谱法分析杜氏盐藻多糖的单糖组成

选用CarboPacPA20分析柱(3mmi.d.×150mm),以H2O-250mmol/LNaOH-1mol/LNaAc为流动相,建立了三元梯度洗脱分离、积分脉冲安培检测多糖中常见的8种中性单糖和2种糖醛酸的高效阴离子交换色谱分析方法;利用离子交换色谱柱(DEAESepharoseCL6Bfastflow,3.5cm×30cm)和凝胶过滤色谱柱(SepharoseCL6B,2.6cm×100cm)从提取出β-胡萝卜素的杜氏盐藻残渣中分离出PD1、PD2、PD3、PD4a和PD4b共5个多糖级分,选用适当的条件水解后,利用高效阴离子交换色谱法测定了各多糖级分的单糖组成。其中具有显著生物活性的多糖级分PD4a中主要含有半乳糖、阿拉伯糖、木糖、葡萄糖、盐藻糖及鼠李糖6种中性糖,还含有少量糖醛酸;PD4b中主要含有核糖、葡萄糖、半乳糖、阿拉伯糖及木糖5种中性糖,不含糖醛酸。该结果与PMP柱前衍生化反相高效液相色谱法测定的单糖摩尔比基本一致。10种单糖的离子色谱法的检出限为0.76～2.92nmol/L(以3倍信噪比计),峰面积相对标淮偏差RSD为0.72%～3.25%。

Analysis of Monosaccharide Compositions in Polysaccharides from D. Salina by High-performance Anion-exchange Chromatography

A simple and sensitive method for simultaneous determination of eight neutral and two acidic monosaccharides has been described by high-performance anion-exchange chromatography(HPAEC)with pulsed amperometric detection using gradient elution procedure of H2O-250 mmol/L NaOH-1 mol/L NaAc as mobile phase on CarboPac PA20 column(3 mmi.d.×150mm).Five polysaccharides fractions(PD1,PD2,PD3,PD4a and PD4b)were isolated using ion-exchange chromatography column(DEAE Sepharose CL6B fast flow,3.5cm×30cm)and gel filtration chromatography column(Sepharose CL6B,2.6cm×100cm)from the residue produced in extraction of β-carotene using Duanaliella salina,and monosaccharide compositions of their hydrolysates prepared using optimum acid hydrolysis condition were directly analyzed by HPAEC.Bioactivity polysaccharides fractions PD4a contains galactose,arabinose,xylose,glucose,fucose and rhamnose mostly,and a little of galacturonic acid and glucuronic acid.Another bioactivity fractions PD4b contains ribose,glucose,galactose,arabinose and xylose mostly.The results were agreement basically with values obtained by pre-column derivatization high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone as reagent.The detection limits of ten monosaccharides for HPAEC were 0.76～2.92nmol/L at the signal-to-noise ratio of 3,and RSDs of peak area were 0.72～3.25%.