低场核磁法测定凝胶过滤介质的孔径分布

Determination of pore size distribution of gel filtration media by low-field NMR

凝胶过滤介质在蛋白质、多糖等生物大分子的分离纯化过程中具有非常重要的作用,其中介质的孔径分布是决定分离纯化效果的关键因素。由于绝大部分凝胶过滤介质是软凝胶,因此很难用常规的方法如压汞法、低温氮吸附法等测定其孔径分布。本文探索了利用低场核磁共振测定凝胶过滤介质的孔径分布的方法。首先通过抽滤、自制琼脂糖凝胶块等实验确定了峰的归属,明确了介质孔内水、介质间隙水和自由水在核磁共振横向弛豫时间(T2)图谱上的分布范围;随后与逆体积排阻层析法(ISEC)测定的结果相对比,得出层析介质孔径和介质孔内水弛豫时间的关系;最后通过高斯正态拟合得到了介质的孔径分布。实验结果证实了低场核磁共振法测定凝胶过滤介质孔径分布的可行性。该法操作简单、测定迅速,并可以为其他层析介质孔径分布的测定提供参考。

Gel filtration media play a very important role in separation and purification of bio-macromolecules such as proteins,polysaccharides.Their pore structure directly influences protein transport and adsorption in chromatographic separation column.The pore size distribution（PSD）plays a major role beyond simply the mean pore size.Various methods have been employed to determine the PSD of porous materials,including gas adsorption-desorption（BET）,mercury intrusion porosimetry（MIP）.Since most of gel filtration media is soft gel,so it is difficult to measure their PSD by conventional method.In this paper,a new method,low-field NMR,was explored to determine the pore size distribution of gel filtration media.First,the distribution range of internal water,external water and free water in transverse relaxation time（T2）of low-field NMR were setting-out; then comparing the results with that from inverse size-exclusion chromatography（ISEC）and finding relationship between the pore size and relaxation time in internal water; finally,the pore size distribution of media was fitted by the Gaussian normal distribution.The results demonstrated that low-field NMR is an effective method for PSD determination of porous chromatographic media.The method is easy to operate,simple and quick,and can provide a reference for other determination of chromatography media pore size distribution.