Batch and Size-Exclusion Chromatographic Characterization of Ultra-high Molar Mass Sodium Hyaluronate Containing Low Amounts of Strongly Scattering Impurities by Dual Low Angle Light Scattering/Refractometric Detection
Batch and Size-Exclusion Chromatographic Characterization of Ultra-high Molar Mass Sodium Hyaluronate Containing Low Amounts of Strongly Scattering Impurities by Dual Low Angle Light Scattering/Refractometric Detection
复制标题
DOI：
10.1080/10826070802479984
复制
发表时间：
2008-10
影响因子：
1.3
通讯作者：
B. Porsch;R. Laga;Č. Koňák
中科院分区：
文献类型：
--
作者：
B. Porsch;R. Laga;Č. Koňák
摘要研究表明，纯透明质酸钠（HA）中含有少量的强散射杂质，这些杂质在质量尺度上用折射检测法检测不到，但在静态光散射实验中用小角光散射检测法可以清楚地检测到。其溶液的尺寸过滤不会去除这些杂质，仅根据过滤器孔隙率减少其量。通过疏水性滤膜上的疏水性吸附，可完全去除这些颗粒杂质，与过滤器孔隙率无关。使用0.1 M NaCl作为移动的相，尺寸排阻色谱（SEC）柱也通过疏水吸附去除杂质;因此，当在本体光散射实验中使用疏水过滤器时，从两种技术获得的摩尔质量一致。不同的流体动力学流动阻滞效应，包括障碍色谱行为显示，大大偏置的超高摩尔质量（UHM）HA获得的摩尔质量分布，除非在SEC分析中的流速降低到0.1 mL/min以下。过高的注射浓度（C注射）被证明是引入HA柱降解的发病。UHM HA的正确的多分散性指数和摩尔质量分布从SEC以0.09 mL/min的流速和优化的c inj .
Abstract It is shown that very pure sodium hyaluronate (HA) contains small amounts of strongly scattering impurities not detectable on mass scale by refractometric detection, but clearly detectable using low angle light scattering detection during a static bulk light scattering experiment. Size filtration of its solutions does not remove these impurities, only reduces their amount depending on filter porosity. Complete removal of these particle impurities, independent of filter porosity, is achieved by hydrophobic adsorption on hydrophobic filter membranes. Using 0.1 M NaCl as a mobile phase, size exclusion chromatography (SEC) column removes the impurities by hydrophobic adsorption as well; molar masses obtained from both techniques thus agree when hydrophobic filters are used in bulk light scattering experiments. Diverse hydrodynamic flow retardation effects including slalom chromatography behavior are shown to substantially bias molar mass distributions obtained for ultra-high molar mass (UHM) HA, unless the flow rate in SEC analysis is reduced below 0.1 mL/min. Too high injected concentration (c inj ) is shown to introduce the onset of HA on column degradation. Correct polydispersity indices and molar mass distributions of UHM HA are obtained from SEC at a flow rate of 0.09 mL/min and optimized c inj .