色谱

色谱

• 研究论文 • 上一篇    下一篇

氨基苯甲酸衍生化高效液相色谱法
分析多糖中的单糖及糖醛酸组成

郝桂堂1,陈尚卫2,朱松2,尹鸿萍3,戴军2,曹玉华1

  

  1. 1.School of Chemistry and Material Engineering of Southern Yangtze University, Wuxi 214036, China;
    2.Testing & Analysis Center of Southern Yangtze University, Wuxi 214036, China; 3.School of
    Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
  • 收稿日期:2006-05-16 修回日期:2006-09-28 出版日期:2007-01-30 发布日期:1986-03-25
  • 通讯作者: 戴军

Analysis of Monosaccharides and Uronic Acids in Polysaccharides
by Pre-column Derivatization with p-Aminobenzoic Acid
and High Performance Liquid Chromatography

HAO Guitang1, CHEN Shangwei2, ZHU Song2, YIN Hongping3, DAI Jun2, CAO Yuhua1

  

  1. 1.School of Chemistry and Material Engineering of Southern Yangtze University, Wuxi 214036, China;
    2.Testing & Analysis Center of Southern Yangtze University, Wuxi 214036, China; 3.School of
    Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
  • Received:2006-05-16 Revised:2006-09-28 Online:2007-01-30 Published:1986-03-25
  • Contact: DAI Jun

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摘要：

衍生反相离子对色谱法同时分离检测多糖中单糖及糖醛酸组成的方法,筛选出适合于p-AMBA糖衍生物分离的色谱柱,考察了流动相组成对9种单糖和两种糖醛酸的p-AMBA衍生化产物的保留值及分离的影响,优化了反应温度和反应时间等衍生化条件,并应用优化的分析方法测定了螺旋藻中的单糖和糖醛酸的组成。采用紫外检测时,方法的检出限为 (2.55～13.4)×107mol/L;采用荧光检测时,方法的检出限为(3.38～176)×108 mol/L。

关键词: 单糖, 对氨基苯甲酸, 多糖 , 高效液相色谱法, 糖醛酸, 柱前衍生化

Abstract:

An ion-pair reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of carbohydrate and uronic acids was developed. p-Aminobenzoic acid (p-AMBA) was used for pre-column derivatization of the analytes, enabling fluorescence (λex=313 nm, λem=358 nm) or ultraviolet (UV at 303 nm) detection. Reaction conditions such as reaction temperature and reaction time were optimized. Atlantis dC18 column with hydrophilic end capping was selected for the separation of derivatives. Effects of mobile phase compositions such as ion pairs and their concentrations and pH on the retention behaviors and separation results of 9 monosaccharides and 2 uronic acids were investigated. Derivatives of fructose, galactose, glucose, mannose, xylose, arabinose, ribose, galacturonic acid, fucose, glucuronic acid and rhamnose were separated within 42 min, applying tetrabutyl ammonium hydrogen bisulfate (TBAHSO4) as the ion pair reagent. The detection limits were between 3.38×108 mol/L and 176×108 mol/L for fluorescence detection and between 2.55×107 mol/L and 13.4×107mol/L for UV detection. Good linearities were obtained with correlation coefficients (r2) above 0.99. The relative standard deviations (RSDs) of the peak area of the derivatives in 1251 h after derivatization were from 2.5% to 3.9%. This method has been applied for the determination of mono-/disaccharides and uronic acids in spirulina polysaccharide after dissolved in trifluoroacetic acid solution (2 mol/L). The results showed this method is suitable for the analysis of monosaccharide compositions in polysaccharides.

Key words: high performance liquid chromatography (HPLC), monosaccharide, p-aminobenzoic acid, polysaccharides , uronic acid, pre-column derivatization