Analytical Chemistry Group, van't Hoff Institute for Molecular Sciences, University of Amsterdam, Postbus 94157, 1090 GD, Amsterdam, The Netherlands.
Anal Bioanal Chem. 2019 Apr;411(11):2327-2338. doi: 10.1007/s00216-019-01673-w. Epub 2019 Feb 21.
In asymmetrical flow field-flow fractionation (AF4), similar to other separation techniques, mass recovery and overloading require special attention in order to obtain quantitative results. We conducted a systematic study with five globular proteins of different molecular weight (36.7-669 kDa) and isoelectric point (4.0-6.5), and ultrafiltration membranes that are commonly used in aqueous AF4, regenerated cellulose (RC) and polyethersulfone (PES). Phosphate-buffered saline (PBS) with ionic strength 0.15 M and pH 7.2 was used as the carrier liquid in this study. The actual molecular weight cutoff (MWCO) was found to be higher than the nominal value and varied between membranes of different chemistry but the same nominal MWCO. Adsorption on the membrane was found to be dependent on the membrane chemistry (RC had lower adsorption compared to PES), and independent of the protein standard for the examined proteins. On the other hand, the mass overloading effects (i.e., higher retention times, peak broadening, and fronting peaks) were significantly more pronounced for γ-globulin than for the other proteins. The overloading effects could be rationalized with the increase of the local viscosity close to the membrane, depending on the properties of the proteins, and we derived theoretical equations that related the dependency of the migration velocity on the protein concentration through this non-ideal viscosity effect.
在不对称流场流分离（AF4）中，与其他分离技术类似，质量回收率和过载需要特别注意，才能获得定量结果。我们使用具有不同分子量（36.7-669 kDa）和等电点（4.0-6.5）的五种球形蛋白质以及在水相 AF4 中常用的超滤膜，即再生纤维素（RC）和聚醚砜（PES）进行了系统研究。在这项研究中，磷酸盐缓冲盐水（PBS）的离子强度为 0.15 M，pH 值为 7.2，用作载液。实际的分子量截止值（MWCO）高于标称值，并且在不同化学性质但相同标称 MWCO 的膜之间变化。发现膜吸附取决于膜化学性质（RC 的吸附比 PES 低），而与所研究蛋白质的标准无关。另一方面，与其他蛋白质相比，γ-球蛋白的质量过载效应（即，更高的保留时间，峰展宽和前沿峰）更为明显。过载效应可以根据蛋白质的性质，用靠近膜的局部粘度增加来合理化，并且我们通过这种非理想粘度效应导出了与蛋白质浓度相关的迁移速度依赖性的理论方程。
