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 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 in order to calculate a mass ratio, to determine the molar mass distributions, the average molecular weights (Mw), and the mean-square radius (Rz) of the AMP component. We applied the technique to starches with AMY:AMP ratios covering a wide range.
Figure 1: AF4-MALS-RI results for five native starches of varying AMY:AMP ratio: AF4-RI fractograms with molar mass distributions overlayed. (Cross-flow (Vx) = 1.0 to 0.1 mL/min in 10 min, then Vx = 0.0 mL/min).
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 RI detectors (both instruments 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 2: Conformation plot (log Rz versus log Mw) for the amylopectin component of five starches (Slopes 0.39â0.41 indicative of branching).
Integration of RI peak areas enabled calculation of the AMY:AMP ratios, in excellent agreement with the nominal values. The values for Mw and Rz fall within the generally accepted limits found in the literature. Conformational plots for the AMP component verify its branched nature.
This note graciously submitted by Rick White and Eija Chiaramonte, Global Analytical Sciences—Personal Health, The Procter & Gamble Company, Mason, OH.
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SEC-MALS of Antibody Therapeutics—A Robust Method for In-Depth Sample Characterization
June 1st 2022Monoclonal antibodies (mAbs) are effective therapeutics for cancers, auto-immune diseases, viral infections, and other diseases. Recent developments in antibody therapeutics aim to add more specific binding regions (bi- and multi-specificity) to increase their effectiveness and/or to downsize the molecule to the specific binding regions (for example, scFv or Fab fragment) to achieve better penetration of the tissue. As the molecule gets more complex, the possible high and low molecular weight (H/LMW) impurities become more complex, too. In order to accurately analyze the various species, more advanced detection than ultraviolet (UV) is required to characterize a mAb sample.
