Separation and Characterization of Barley Starch Polymers by a Flow Field‐Flow Fractionation Technique in Combination with Multiangle Light Scattering and Differential Refractive Index Detection

Flow field‐flow fractionation (flow FFF) with frit inlet and frit outlet mode (FIFO) was coupled online to multiangle light scattering (MALS) and refractive index (RI) detectors to investigate the molecular characteristics of normal and zero amylose barley starch polymers. Application of two different cross‐flows, 0.35 mL/min followed by 0.1 mL/min, and constant channel and frit flows of 0.1 and 1.0 mL/min, respectively, permitted a complete separation of amylose and amylopectin. The improved signals from the detectors due to application of the FIFO mode enabled the proper characterization of the small molecular weight species, as well as significantly enhanced the reproducibility of the measurements. The weight‐average molecular weight (M_w) and zaverage root‐mean‐square (RMS) radii of gyration (R_g) values for amylose and amylopectin in the normal starch samples were 2.3 × 10⁶ and 280 × 10⁶, and 107 and 260 nm, respectively. The M_w and R_g of amylopectin in the zero amylose starch samples were 360 × 10⁶ and 267 nm, respectively. The slopes (α) obtained by plotting log M_w versus log R_g for amylose and amylopectin were 0.6 and 0.3, respectively. These results are in good agreement with the theoretical prediction of the molecular conformation of amylose and amylopectin.