Critical parameters for achieving high efficiency and reproducible double-chained cationic surfactant coatings for protein separation by capillary electrophoresis

Abstract

Analyzing intact proteins by capillary electrophoresis (CE) is challenging but highly valuable, as it holds potential for future clinical applications via the separation and quantification of protein biomarkers. To achieve this, the development of CE methods requires improvements in separation efficiency, reproducibility, and reduction of sample adsorption on the capillary wall. Double-chained surfactant coatings were described as potential candidate leading to high separation efficiency. In this work, a capillary coating based on dioctadecyldimethylammonium bromide (DODAB), a double-chained cationic surfactant, was investigated by assessing the critical parameters affecting DODAB-vesicle preparation and coating performances. These parameters include temperature & sonication of the coating solution, nature of the coating buffer, and capillary inner diameter. The plate height (H) of proteins as a function of their linear migration velocity (u) was systematically plotted to evaluate the separation performances under different kinetic conditions by applying different separation voltages. DODAB coating showed high surface homogeneity leading to low slope (p) of the H vs u curve (2.23 ± 0.99 ms in average on 3 different capillaries) on 5 model basic proteins (lysozyme, carbonic anhydrase, ribonuclease A, β-lactoglobulin A and myoglobin) using 2 M acetic acid, pH 2.2, as background electrolyte in a 40 cm total length and 25 μm inner diameter capillary. Excellent separation performances were achieved reaching up to 606,000 ± 27,000 plates/m at −12 kV for lysozyme, with excellent repeatability (RSD on migration times = 0.21 %).