Department of Food Technology, Engineering and Nutrition, Lund University, 22100 Lund, Sweden.
Swedish Orphan Biovitrum AB (publ.), 11276 Stockholm, Sweden.
J Chromatogr A. 2020 Dec 6;1633:461625. doi: 10.1016/j.chroma.2020.461625. Epub 2020 Oct 14.
Electrical asymmetrical flow field-flow fractionation (EAF4) is an interesting new analytical technique that separates proteins based on size or molecular weight and simultaneously determines the electrical characteristics of each population. However, until now, the research using EAF4 has not been published except for the proof-of-concept in the original publication by Johann et. al. in 2015 [1]. Hence the methods capabilities and optimized conditions need to be further investigated, such as composition of the carrier liquid, pH stability and effect of the electric field strength. The pH instability was observed in the initial method of EAF4 due to the electrolysis products when applied electric field. Therefore, we have investigated and provided a modified method for rapid pH stabilization through additional focusing step with the electric field. Then, the electrical properties such as the zeta-potential and effective net charge of the monomer and oligomers of three different proteins (GA-Z, BSA, and Ferritin) were determined based on their electrophoretic mobility from EAF4. The results showed that there were limitations to the applicability of separation by EAF4 to proteins. Nevertheless, this study shows that EAF4 is an interesting new technique that can examine the zeta-potential of individual proteins in mixtures (or monomers and oligomers) not accessible by other techniques.
电非对称流场流分离（EAF4）是一种有趣的新型分析技术，可根据大小或分子量分离蛋白质，并同时确定每个群体的电学特性。然而，到目前为止，除了 Johann 等人在 2015 年的原始出版物中提出的概念验证外，尚未发表使用 EAF4 的研究结果[1]。因此，需要进一步研究其方法的能力和优化条件，例如载液的组成、pH 值稳定性和电场强度的影响。由于施加电场时会产生电解产物，因此在 EAF4 的初始方法中观察到 pH 值不稳定。因此，我们已经研究并提供了一种通过外加电场的附加聚焦步骤进行快速 pH 值稳定化的改进方法。然后，根据三种不同蛋白质（GA-Z、BSA 和铁蛋白）的单体和低聚物的电泳迁移率，确定其电特性，如zeta-电位和有效净电荷。结果表明，EAF4 对蛋白质的分离适用性存在局限性。尽管如此，这项研究表明，EAF4 是一种有趣的新技术，可以检查混合物（或单体和低聚物）中单个蛋白质的 zeta-电位，这是其他技术无法实现的。
