Optimization of Polymer Sieving Matrix Composition for DNA Fragment Separation in a Laboratory-Built Multicapillary Electrophoresis System

Capillary electrophoresis (CE) is widely used for DNA fragment analysis, but its separation performance depends strongly on the composition of the polymer sieving matrix, especially in multicapillary systems where matrix operability and reproducibility are critical. Herein, a laboratory-built multicapillary electrophoresis system was used to optimize the sieving matrix for high-throughput DNA fragment separation. The system integrated twelve fused-silica capillaries, high-voltage electrokinetic injection, fluorescence detection, and digital electropherogram acquisition. Using a 100 bp DNA ladder as the model sample, the effects of poly(ethylene oxide) (PEO), Tween 20, and glycerol on electropherogram quality were systematically investigated. PEO concentration was the dominant factor controlling the dynamic sieving network: 0.1% PEO provided insufficient separation, whereas 0.8–1.0% PEO produced clearly resolved DNA peaks. Tween 20 improved peak regularity and electropherogram quality under the tested conditions, with 0.05% providing sufficient improvement without prolonging migration time. Glycerol affected peak distribution by increasing apparent migration resistance in the polymer matrix; however, excessive glycerol slowed DNA migration and markedly extended the separation window. Considering separation quality, matrix operability, and analytical efficiency, 1.0% PEO, 0.05% Tween 20, 2.5% glycerol, 1× SYBR Gold, and 0.5× TBE were selected as the optimized sieving matrix. Under the optimized matrix, inter-capillary migration-time alignment improved the consistency of parallel capillary outputs, with corrected migration-time RSD values generally below 0.5%. The optimized formulation provides a practical basis for high-throughput CE-based DNA fragment analysis.