Disentangling starch polymer topology: A multimethod approach integrating SEC-DRI-MALS, FACE, and HPAEC

This study employed an integrated multimethod approach to investigate how size-exclusion chromatography (SEC) operational parameters influence the starch hydrodynamic and topological characterization. The effects of two column configuration (GRAM 10000/100 vs. 3000/100) and sample concentration on starches with varying amylose contents were systematically evaluated using SEC coupled with differential refractive index and multi-angle light scattering (SEC-DRI-MALS). Results demonstrated that column pore size critically affected the resolution of large amylopectin molecules (hydrodynamic radius, R h  ≥ 100 nm), while amylose-rich fractions ( R h < 100 nm) were less sensitive. Increasing concentration from 1 mg/mL to 4 mg/mL induced amylopectin aggregation, elevating apparent density ( ρ ). A strong positive ρ - R h correlation in amylopectin topology indicated that large amylopectin molecules achieve their size through numerous short branches rather than fewer long chains. This topological model was unequivocally supported by chain length distribution (CLD) analysis. A comparative evaluation of fluorophore-assisted carbohydrate electrophoresis (FACE) and high-performance anion-exchange chromatography (HPAEC) revealed that FACE provides superior resolution for short and long branches, while HPAEC was limited by co-elution, peak broadening, and a restricted dynamic range (DP ∼ 70). Our findings establish an optimized analytical framework for starch topology analysis, highlight critical SEC methodological considerations for reliable structure-function correlations.