Hybrid phospholipid bilayer coatings for separations of cationic proteins in capillary zone electrophoresis

Protein separations in CZE suffer from nonspecific adsorption of analytes to the capillary surface. Semipermanent phospholipid bilayers have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m^-2, respectively, compared to 17 ± 1 mJ m^-2 for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3-1.9 × 10^-4 cm² V^-1s^-1) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10^-4 cm² V^-1s^-1, 4.8 ± 0.4 × 10^-4 cm² V^-1s^-1, and 6.0 ± 0.2 × 10^-4 cm² V^-1s^-1, respectively), with increased stability compared to phospholipid bilayer coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6%, n ≥ 6) with separation efficiencies as high as 200 000 plates/m.