Koshari Stijn H S, Wagner Norman J, Lenhoff Abraham M
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716, USA.
J Chromatogr A. 2015 Jun 19;1399:45-52. doi: 10.1016/j.chroma.2015.04.042. Epub 2015 Apr 28.
Measurements of the nanoscale structure of chromatographic adsorbents and the associated distribution of sorbed protein within the media can facilitate improvements in such media. We demonstrate a new technique for this purpose using small-angle neutron scattering (SANS) to characterize the nano- to microscale structure of the chromatographic media and sorbed protein under conditions relevant for preparative chromatographic separations. The adsorption of lysozyme on cellulosic S HyperCel™ (Pall Corporation), a strong cation exchanger, was investigated by SANS. The scattering spectrum is reduced to three contributions arising from (1) the chromatographic medium, (2) discrete protein molecules, and (3) the distribution of sorbed protein within the medium. These contributions are quantified for a range of protein loadings. The total concentration of protein in the chromatographic media can be quantified from the SANS spectrum and the protein is observed to retain its tertiary structure upon adsorption, within the resolution of the method. Further analysis of the SANS spectra shows that protein adsorption is uniform in the media. These measurement techniques provide new and valuable nanoscale information about protein sorption in chromatographic media.
对色谱吸附剂的纳米级结构以及介质中吸附蛋白质的相关分布进行测量，有助于改进此类介质。我们展示了一种为此目的的新技术，即使用小角中子散射（SANS）来表征在与制备型色谱分离相关的条件下色谱介质和吸附蛋白质的纳米至微米级结构。通过SANS研究了溶菌酶在强阳离子交换剂纤维素S HyperCel™（颇尔公司）上的吸附。散射光谱可归结为来自以下三方面的贡献：（1）色谱介质，（2）离散的蛋白质分子，以及（3）介质中吸附蛋白质的分布。针对一系列蛋白质负载量对这些贡献进行了量化。色谱介质中蛋白质的总浓度可从SANS光谱中量化得出，并且在该方法的分辨率范围内，观察到蛋白质在吸附时保留其三级结构。对SANS光谱的进一步分析表明，蛋白质在介质中的吸附是均匀的。这些测量技术提供了有关色谱介质中蛋白质吸附的新的有价值的纳米级信息。
