欢迎访问《林产化学与工业》,

林产化学与工业 ›› 2020, Vol. 40 ›› Issue (3): 31-38.doi: 10.3969/j.issn.0253-2417.2020.03.004

• 研究报告 • 上一篇    下一篇

柱前衍生反相高效液相色谱法测定田菁种子水解液中的单糖

张倩男,徐方敏,连之娜,李鑫*(),勇强   

  1. 南京林业大学 化学工程学院, 江苏 南京 210037
  • 收稿日期:2019-12-13 出版日期:2020-06-28 发布日期:2020-06-29
  • 通讯作者: 李鑫 E-mail:xli@njfu.edu.cn
  • 作者简介:张倩男(1996-),女,江苏连云港人,硕士生,主要从事植物资源生物加工研究工作
  • 基金资助:
    国家林业公益性行业科研专项(201404615)

Analysis of Monosaccharide in Sesbania cannabina Seed Enzymatic Hydrolysate by Pre-column Derivatization Reversed-phase High Performance Liquid Chromatography

Qiannan ZHANG,Fangmin XU,Zhina LIAN,Xin LI*(),Qiang YONG   

  1. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
  • Received:2019-12-13 Online:2020-06-28 Published:2020-06-29
  • Contact: Xin LI E-mail:xli@njfu.edu.cn

摘要:

建立了一种柱前衍生反相高效液相色谱法,测定田菁种子酶水解液中的甘露糖、葡萄糖、半乳糖和木糖等4种单糖,用以实现酶法制备半乳甘露低聚糖的定量测定。采用1-苯基-3-甲基-5-吡唑啉酮(PMP)对酶水解液中的单糖进行柱前衍生化,考察不同色谱分析条件和衍生化条件对单糖衍生物峰面积的影响,利用反相高效液相色谱法分析酶水解液中的4种单糖。结果表明:当流动相为乙腈/磷酸二氢钾缓冲溶液(0.02 mol/L,pH值6.5),衍生化条件为PMP用量150 μL、反应时间80 min、反应温度60℃和三氯甲烷萃取3次时,各单糖组分的峰面积较大、分离效果较好;方法学考察结果发现:4种单糖组分标准曲线的R2均大于0.999 4,加标回收率为96.9%~104.8%,相对标准偏差(RSD)小于0.56%,重复性、稳定性和精密度实验结果良好,说明该实验条件下建立的方法有效可信。同时,田菁种子酶水解液中组成半乳甘露聚糖的甘露糖、葡萄糖、半乳糖、木糖等4种单糖的物质的量之比为1:0.44:1.92:0.53。柱前衍生化高效液相色谱法为分析田菁种子酶水解液中单糖组成和含量提供了一种操作简单、准确、分离度高、重现性好的定量方法。

关键词: 田菁种子, 半乳甘露聚糖, 甘露低聚糖, 水解, 单糖

Abstract:

To establish a pre-column derivatization reversed-phase high performance liquid chromatography for the determination of mannose, glucose, galactose and xylose in the enzymatic hydrolysate of Sesbania cannabina seed, the pre-column derivatization was carried out by using 1-phenyl-3-methyl-5-pyrazolone (PMP) as derivatization reagent, and the effects of different chromatographic conditions were investigated. The experimental results showed that when the mobile phase was acetonitrile/potassium dihydrogen phosphate buffer solution (0.02 mol/L, pH value 6.5) and the derivatization conditions were PMP dosage 150 μL, reaction time 80 min, reaction temperature 60℃ and chloroform extraction for 3 times, the peak area of each monosaccharide component was larger and the separation effect was better. The methodological study verified that the R2 values of standard curves of four monosaccharide were over 0.999 4, the recovery rates were 96.9%-104.8%, the RSD values were lower than 0.56%, and the results repeatability, stability and precision tests were good, which indicated that the established method under these conditions was effective and convincible. The mass ratio of mannose, glucose, galactose and xylose was 1:0.44:1.92:0.53. In summary, pre-column derivatization high performance liquid chromatography provided a quantitative method with simple operation, accuracy, high resolution and good reproducibility for analyzing monosaccharide composition and content of the enzymatic hydrolysate of S. cannabina seed.

Key words: Sesbania cannabina seed, galactomannan, manno-oligosaccharides, hydrolysis, monosaccharide

中图分类号: