Dynamic capillary coatings with zwitterionic surfactants for capillary isoelectric focusing

Abstract

The use of surfactants as additives was demonstrated for the first time in capillary isoelectric focusing (CIEF) to dynamically modify the surfaces of bare fused silica capillaries. These surfactants were zwitterionic sulfobetaines: dodecyldimethyl (3-sulfopropyl) ammonium hydroxide (C₁₂N₃SO₃), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide (C₁₆N₃SO₃) and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U). They were added directly to the protein–ampholyte mixture, and remained in the capillary during isoelectric focusing and mobilization. The C₁₆N₃SO₃ and CAS U coatings were shown effective in CIEF. Separation of seven IEF protein standards was obtained, with significantly improved resolution compared to that from an uncoated silica capillary. The effect of these surfactants on the electroosmotic flow (EOF) in CIEF was determined. CAS U was effective in suppressing the EOF at neutral and alkaline pH conditions, C₁₆N₃SO₃ was effective in suppressing EOF at acidic and neutral pH conditions. C₁₂N₃SO₃ however had little effect on the EOF. The pH gradients formed inside these surfactant coated capillaries were recta-linear at pH 6 to 9 (R² ∼ 0.99). Reproducibility of migration time and peak area was determined. For all three coatings, the migration time standard deviations were less than 1.6 min, and the relative standard deviations of area were below 10%. The protein recovery in the CAS U-modified capillary was quantitative or near-quantitative for five of the seven proteins studied.