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Optimizing Quantitative Reproducibility in High-Performance Capillary Electrophoresis (HPCE) Separations of Cereal Proteins¹

¹Cooperative investigations, United States Department of Agriculture, Agricultural Research Service (USDA-ARS) and the Department of Grain Science and Industry, Kansas State University. Contribution 01-153-J, Department of Grain Science and Industry, Kansas State Agricultural Experiment Station, Manhattan KS 66506. ²Dept. Grain Science and Industry, Kansas State University, Manhattan, KS 66506. ³USDA-ARS, Grain Marketing and Production Research Center, and Kansas State University, Manhattan, KS 66502. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.

High-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.