AFFF–MALS–RI for Determining the Mass and Size Distributions of Amylose and Amylopectins in Starch

Starch is used for a variety of industrial and nutritional purposes. Its functional properties are influenced by the ratio and molar masses of its macromolecular constituents, which vary with source, crop year, and climate. Starch contains large homopolymers of amylose (AMY) and amylopectin (AMP).

Linear AMY consists of long chains of (1⇨4)-α-D-glucose linkages, while the higher molar mass AMP is a branched structure containing a mixture of (1⇨4)-α- and (1⇨6)-α-D-glucose linked residues. The goal of this work was to apply AF4–MALS–RI to separate AMY and AMP to calculate a mass ratio, to determine the molar mass distributions, the average molecular weights (M_w), and the mean-square radius (R_z) of the AMP component. We applied the technique to starches with AMY:AMP ratios covering a wide range.

An Eclipse AF4 system (Wyatt Technology) was equipped with a short (18 cm) channel, a 350 μm spacer, and a regenerated cellulose (10 kDa cutoff) membrane. Detection was accomplished with DAWN multi-angle light scattering (MALS) and Optilab refractive index (RI) detectors (both Wyatt Technology). The channel flow was maintained at 1.0 mL/min and the cross-flow was varied linearly from 1.0 to 0.1 mL/min for 10 min, then abruptly switched to 0.0 mL/min.

Figure 1: AF4–MALS–RI results for five native starches of varying AMY:AMP ratio: AF4–RI fractograms with molar mass distributions overlaid. (Cross-flow [Vx] = 1.0 to 0.1 mL/min in 10 min, then Vx = 0.0 mL/min).

Integration of RI peak areas enabled calculation of the AMY:AMP ratios, in excellent agreement with the nominal values. The values for M_w and R_z fall within the generally accepted limits found in the literature. Conformational plots for the AMP component verify its branched nature.

Figure 2: Conformation plot (log Rz versus log Mw) for the amylopectin component of five starches (Slopes 0.39–0.41 indicative of branching).

Starch characterization by size-exclusion methods can be limited because of shear degradation and/or column adsorption of the higher mass fractions of AMP. The open-channel separation of AF4 obviates these limitations and enables recovery of intact AMP. Resolution of AMY and AMP was achieved by varying the cross-flow. Coupled with MALS and RI detection, the mass ratios of AMY:AMP were accurately determined and the molar masses of these macromolecules were estimated without the need for calibration standards.

This note was graciously submitted by Rick White and Eija Chiaramonte, Global Analytical Sciences—Personal Health, The Procter & Gamble Company, Mason, Ohio, USA.

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