Abstract
A simple, robust and reproducible capillary isoelectric focusing method using dynamically coated fused silica capillaries was developed. Simultaneous focusing and mobilization was successfully carried out using the hydrophilic polymer polyethylene oxide (PEO), which was added during sample preparation. The effects of preconditioning and anolyte and catholyte concentrations on the resolution and reproducibility of the method were optimized. Statistical screening of the significance of the effects of pressure, voltage, PEO concentration, and molecular weight on the efficiency of dynamic coating was carried out. Full-factorial design and analysis of variance (ANOVA) were used in order to determine the significance of each variable on the performance of the dynamic coat. Three response variables were selected to evaluate the method performance: migration time of a basic protein marker, migration time of an acidic protein marker, and difference in migration time between both markers. An empirically determined second order polynomial was employed to describe the relationship between the migration time and the isoelectric point under the experimental conditions studied in order to validate the method performance. The method was used under non-denaturing conditions to resolve a mixture of four standard proteins having isoelectric points covering a wide pH range (pH 3–11).
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