Recovery, overloading, and protein interactions in asymmetrical flow field-flow fractionation

Analytical and Bioanalytical Chemistry
Maria Marioli, Wim Th Kok

Abstract

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 p...Continue Reading

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Citations

Apr 7, 2020·Small·Irina AlbergRudolf Zentel
Nov 25, 2020·Journal of Chromatography. a·Iro K VentouriPeter J Schoenmakers
Feb 6, 2021·Macromolecular Bioscience·Irina AlbergRudolf Zentel
Feb 2, 2021·Drug Delivery and Translational Research·Federico QuattriniMaría José Alonso
Mar 1, 2021·Journal of Pharmaceutical Sciences·Per-Olof WahlundChristian Rischel
Oct 5, 2020·Journal of Pharmaceutical and Biomedical Analysis·Yuchen FanKelly Zhang
Dec 24, 2020·International Journal of Molecular Sciences·Adrian GołębiowskiBogusław Buszewski

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size exclusion chromatography

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