基于非对称场流分离技术分离表征天麻多糖:空间位阻转变效应  

Separation and characterization of Gastrodia elata polysaccharides based on asymmetrical flow field-flow fractionation:steric transition phenomenon

作　　者：王沐 张西瑞 窦雨薇 叶红[3] 窦海洋 WANG Mu;ZHANG Xirui;DOU Yuwei;YE Hong;DOU Haiyang(College of Basic Medical Sciences,Hebei University,Baoding 071000,China;College of Public Health,Hebei University,Baoding 071000,China;Health Science Center,Hebei University,Baoding 071000,China)

机构地区：[1]河北大学基础医学院,河北保定071000 [2]河北大学公共卫生学院,河北保定071000 [3]河北大学医学部,河北保定071000

出　　处：《色谱》2023年第8期714-721,共8页Chinese Journal of Chromatography

基　　金：河北省自然科学基金(H2020201295);河北省重点研究计划(22372505D)。

摘　　要：采用非对称场流分离(AF4)在线联用多角度光散射(MALS)检测器和示差折光(dRI)检测器(AF4-MALS-dRI)对天麻多糖(GEPs)的相对分子质量(M)、回转半径(Rg)和构象进行了表征。GEPs粒径分布较宽,在AF4分离过程中存在空间位阻转变效应,导致粒径大小不同的GEPs分子被同时洗脱,无法准确表征其M、Rg分布和构象。本文研究了恒定交叉流流速、指数衰减交叉流流速、样品质量浓度和垫片高度对空间位阻转变效应的影响。结果表明,较高的样品质量浓度会导致空间位阻转变现象的发生;垫片高度会影响样品的分离度和保留时间,改变空间位阻转变点(di);对于多分散GEPs样品,指数衰减交叉流流速不仅能调整di,而且能改善样品的分离度,缩短分析时间。在样品质量浓度为0.50 mg/mL、进样体积为50μL、检测器流速为1.0 mL/min、垫片高度为350μm、初始交叉流流速由0.2 mL/min呈指数衰减至0.05 mL/min、半衰期为2 min的条件下,解决了AF4分离GEPs时存在的空间位阻转变效应问题。此外,在最佳洗脱条件下研究了不同产地及不同超声时间对GEPs的M、Rg和构象的影响。结果表明,随着超声时间的增加,云南和四川产地GEPs的Rg和M均减小,分布变窄;当超声时间为15 min时,云南产地GEPs为松散的高度支化构象,四川产地GEPs为球状构象;当超声时间增加到30 min或60 min时,两产地GEPs的构象主要为高度支化结构,表明GEPs发生了降解。实验结果证明,在最佳洗脱条件下,AF4-MALS-dRI对GEPs的表征具有良好的重复性,GEPs的Rg和M相对标准偏差分别为0.5%和1.7%。Asymmetrical flow field-flow fractionation(AF4),a gentle tool for the separation and characterization of particles and macromolecules,has attracted increased interest in recent years owing to its broad dynamic size range and utilization of“open channel”voids in the packing or stationary phase.A steric transition phenomenon in which the sample elution mode change from the normal mode to the steric/hyperlayer mode occurs.Accurate characterization by AF4 requires the absence of steric transition,particularly when the sample has a broad size distribution,because the effect of the combination of different modes is difficult to interpret.In this study,the relative molecular mass(M),radius of gyration(R g),and conformation of Gastrodia elata polysaccharides(GEPs)were characterized using AF4 coupled with online multi-angle light scattering(MALS)and differential refractive index(dRI)detection(AF4-MALS-dRI).Steric transition was observed during GEP separation by AF4 owing to the broad size distribution of the molecules.This phenomenon would result in the inaccurate characterization of the GEPs in terms of M and R g because two GEP groups of different sizes may elute together.In this study,the effects of constant and exponentially decaying cross-flow rates,sample mass concentration,and spacer thickness on steric transition were systematically investigated.The results indicated that a high GEP mass concentration(i.e.,0.75 mg/mL)can lead to steric transition.The spacer thickness affected the resolution and retention time of the GEPs and changed the steric transition point(d i).An exponentially decaying cross-flow rate not only adjusted the d i of the polydisperse GEP samples but also improved the GEP resolution and shortened the analysis time.The influence of steric transition was solved under the following operating conditions:injected GEP mass concentration=0.5 mg/mL;injection volume=50μL;spacer thickness=350μm;detector flow rate=1.0 mL/min;and cross-flow rate exponentially decayed from 0.2 to 0.05 mL/min with a half-

关 键 词：非对称场流分离 天麻多糖 空间位阻转变 粒径表征 

分 类 号：O658[理学—分析化学]