Optimizing Salt Concentration for Reliable Aqueous Size-Exclusion Chromatography of Water-Soluble Polymers

Size-exclusion chromatography (SEC) or gel-permeation chromatography (GPC) is an essential tool for determining the molecular weight and polydispersity of water-soluble polymers, including biopolymers used in hydrogels, sealants, bioinks, and other biomedical materials. However, aqueous SEC of polyelectrolytes, i.e., charged polymers, is often complicated by non-size interactions among polymer chains, porous column beads, pore surfaces, frits, tubing, and mobile phase. Salt addition to eluent is commonly used to screen these interactions, but the minimum salt concentration required to restore reliable SEC behavior remains poorly defined, and excessive salt may introduce tailing, refractive-index artifacts, deposits, or instrument concerns. In this study, aqueous SEC with refractive index (RI) and right-angle light scattering (RALS) detection was used to evaluate the effect of salt (Na2SO4) concentration on poly(ethylene oxide) (PEO), a nominally neutral reference standard polymer, and sodium alginate as a model anionic biopolymer. PEO retained a single bell-shaped peak across the tested salt range, but its elution volume and SEC/RALS-derived molecular weights varied slightly with salt concentration, showing that even a nominally neutral reference polymer is affected by mobile-phase conditions. Alginate showed much stronger salt dependence: eluent at very low salt concentration produced broad, noisy, and convoluted chromatograms, whereas increasing salt concentration progressively narrowed the main peak. The first condition that produced a clear, approximately symmetric RI/RALS main peak was 6.25×10−3 M Na2SO4, identifying it as the minimum effective salt concentration for this alginate/column/instrument system. To rigorously validate these observations, we propose a set of both qualitative and quantitative peak analyses that objectively confirm the optimal mobile-phase conditions. Ultimately, these results provide a practical workflow for identifying the minimum effective salt concentration required for reliable SEC analysis of water-soluble polymers.