Semi-permanent cationic coating for protein separations

Abstract

Capillary electrophoresis has been used extensively for protein separations, but interactions of proteins with the negative charge on the surface of fused silica capillary create band broadening and diminish the separation efficiency. Coatings developed to mask the negative charge of the capillary affect the electroosmotic flow. The method presented in this work addresses these concerns through the use of a two-layer coating of a semi-permanent phospholipid substrate and cetyltrimethylammonium bromide (CTAB). When used alone, phospholipid coating suppresses the electroosmotic flow but cannot be used to simultaneously separate anionic and cationic proteins. When used alone, CTAB creates a dynamic coating that facilitates the separation of cationic proteins with good efficiency, but reduces the separation efficiency of anionic proteins. The use of a hybrid phospholipid-CTAB surface coating alleviates protein adsorption, as demonstrated through a comparison of protein separations obtained with a bare fused silica capillary. The hybrid phospholipid-CTAB surface enables high efficiency separations of cationic and anionic proteins simultaneously. This work verifies the role of the hydrophobic tail of CTAB in developing a stable coating with an electroosmotic flow of 3.14 × 10^-4 cm²V^-1s^-1 (n = 10) from the cathode to the anode at a pH of 7. The coating yields a stable electroosmotic flow even after 2 h of flushing with background electrolyte devoid of CTAB (n = 3) and six consecutive protein injections with no flush sequence between runs. The coating can be used with background electrolytes with pH values ranging from 4 to 8 while maintaining 1% RSD (n = 10) in the electroosmotic flow for each background electrolyte. Six model proteins, lysozyme, ribonuclease A, α-chymotrypsinogen A, enolase, transferrin, and α-1-antitrypsin, with pI values ranging from 4.4 to 11 were used to demonstrate the stability of the phospholipid-CTAB coating, the lack of protein interaction with the wall, and the utility of the coating for the separation of proteins of similar isoelectric points and of protein isoforms.

Keywords: Capillary electrophoresis; Electroomostic flow; Protein adsorption; Semi-permanent coating; Surface modification; pH-stability.