高效阴离子交换色谱法分析杜氏盐藻多糖的单糖组成

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

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%。

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, PIMa 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 %.