Starch Amylose and Amylopectin Molar Mass and Size Distributions by FFF-MALS

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The Application Notebook

The Application Notebook, The Application Notebook-02-02-2021, Volume 39, Issue 2
Pages: 102

Starch contains large homopolymers of linear amylose (AMY) and branched amylopectin (AMP). Its functional properties are influenced by the ratio and molar masses of its macromolecular constituents, which vary with source, crop year, and climate.

With average radii in the hundreds of nanometers, and molecular weights ranging into the hundreds of millions, starch polymeric components cannot be separated by GPC. However, field-flow fractionation coupled to multi-angle light scattering (FFF-MALS) is suitable for separation and characterization of polymers and nanoparticles from 1 to 1000 nm, and hence is applicable to starch analysis. FFF performs non-shearing separation by hydrodynamic size, and MALS analyzes absolute molar mass and size regardless of conformation or retention properties.

This note demonstrates the application of FFF-MALS to separating AMY and AMP in order to calculate their mass ratio, determine molar mass distributions, the average molecular weights of AMY and AMP, and the z-average root-mean-square radius Rg,z and polydispersity Mw/Mn of the AMP component.

Materials and Methods

Separation was performed by an Eclipse® FFF system with short channel, a 350 μm spacer, and a 10 kDa cutoff regenerated cellulose membrane. Detection was accomplished with DAWN® 18-angle MALS and Optilab® refractive index (RI) detectors. 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 switched to 0.0 mL/min.

Results and Discussion

Figure 1 shows that FFF-RI fractograms separate into distinct peaks for AMY and AMP. The molar mass distributions determined for these samples ranged from 10 kDa to 1 GDa, though there is evidence for smaller starch components as well.

Table I summarizes the results of the analyses. Integration of the respective peak areas enabled calculation of the AMY:AMP ratios, in excellent agreement with the nominal values. The values for Mw and Rg,z fall within the generally accepted limits found in the literature. Polymer conformation is assessed by comparing Rg with molar mass. Conformational plots for the AMP component (not shown) indicate a log-log slope of 0.39–0.41 for all starches measured, verifying their branched nature. Species-specific differences in the overall density of the amylopectins are apparent in the vertical offsets of the conformation plots.

Conclusions

Starch characterization by size-exclusion chromatography can be limited due to shear degradation or column adsorption of the higher mass fractions of AMP, and SEC columns cannot cover the entire size range. Due to branching, the usual linear calibration standards would not have been applicable. The open-channel separation of FFF eliminates these limitations and enables recovery of intact AMP. The molar masses and mass ratios of AMY:AMP were accurately determined over five orders of magnitude. Only FFF-MALS fully addresses the multiple analytical challenges presented by starch.

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