Separation and zeta-potential determination of proteins and their oligomers using electrical asymmetrical flow field-flow fractionation (EAF4).

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.