Effects of Pushing Agents on the Separation and Detection of Debranched Amylopectin by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

Abstract

The effects of acetate and nitrate as pushing agents on the high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC-PAD) for the separation and detection of debranched amylopectin were evaluated. Our experimental data indicated that nitrate could differentiate branched from linear isomers, whereas acetate could not. Therefore, the nitrate system had better separating power than the acetate system provided that the chromatograms under both gradient systems were completed within 100 min. Nitrate was more compatible with the detector than acetate; thus, a higher detector response resulted. Under our experimental conditions, with a signal-to-noise ratio of two, the number of DP (degree of polymerization) at various concentrations that was resolved by the nitrate system (62 at 0.5 mg/mL, 65 at 1.0 mg/mL, and 66 at 1.5 mg/mL) was larger than those resolved by the acetate system (56 at 0.5 mg/mL, 58 at 1.0 mg/mL, and 58 at 1.5 mg/mL). The chromatograms obtained from the nitrate gradient system were more reproducible than those from the acetate gradient system in terms of the consistencies of retention time and the peak area of the chromatographic peak. To various concentrations of the component with the same DP, the detector responses were more consistent with nitrate as a pushing agent compared with acetate. Neither pushing agent, however, resulted in a quantitative response for homologous debranched amylopectin.