Optimizing Quantitative Reproducibility in High‐Performance Capillary Electrophoresis (HPCE) Separations of Cereal Proteins

Abstract

ABSTRACTHigh‐performance capillary electrophoresis (HPCE) is capable of producing high‐resolution, rapid separations of cereal proteins. Furthermore, HPCE is highly reproducible in terms of migration time. However, little work has focused on the quantitative reproducibility of cereal protein separations. Several factors such as sample matrix, sample evaporation, voltage ramp‐up time, sample injection time, and capillary end‐cut were evaluated for involvement in quantitative reproducibility. These experiments showed that preventing sample evaporation, using optimum injection times, and ensuring a clean, square cut on the capillary all improved the reproducibility of peak areas. Combining these factors into an optimized procedure produced reproducibility with peak areas varying by 1.76% relative standard deviation (RSD). Migration time was also excellent under these conditions, varying by only 0.45% RSD. Other variables such as peak area percent, peak height, and peak height percent also showed good reproducibility with RSD < 4%. Increasing the voltage ramp‐up time from 0.17 to 0.68 increased peak efficiency by ≈150%. This factor had no effect on quantitative reproducibility, however. The gradual buildup of contaminants on the capillary walls occurred over time and decreased both separation efficiency and reproducibility. Rinsing capillaries periodically with appropriate solvents delayed this effect. Peak efficiency was a good marker for capillary performance and lifetime.