Cationic surfactant based coatings for protein separations and control of electroosmotic flow in capillary electrophoresis

• Author / Creator

  Bahnasy, Mahmoud FM

• Capillary electrophoresis (CE) is a fast and high efficiency separation technique based on the differential migration of charged species in an electric field. CE is useful for the separation of a wide range of analytes from small ions to large biomolecules. However, CE separations of proteins are challenging due to the adsorption of protein onto the capillary silica surface. Capillary coatings are the most common way to minimize this adsorption. This thesis focuses on the use of two-tailed cationic surfactant based coatings as means of preventing protein adsorption.

      Factors affecting the stability of two-tailed cationic surfactant coatings have been investigated. The impact of small i.d. capillaries (5-25) µm on enhanced stability of surfactant bilayer cationic coatings and on the efficiency of separation of basic proteins was studied. Using a dioctadecyldimethylammonium bromide (DODAB) coated 5 µm i.d. capillary, exceptional short term stability (210 consecutive runs) and long term stability (300 runs over a 30 day period) were achieved. The average separation efficiency of four basic model proteins was 1.4-2 millions plates/m. DODAB coatings were stable over a pH range of 3-8 as demonstrated by strong anodic magnitude of electroosmotic flow (EOF) and good EOF reproducibility. Surprisingly, at pH ≥ 9, EOF became less anodic and even became suppressed cathodic. The reason is unclear. Chemical degradation of DODAB at high pH was excluded. Increased vesicle size at high pH and/or accelerated desorption may be involved.  
  
     A surfactant bilayer/diblock copolymer coating was developed to tune the EOF and prevent protein adsorption. The coating consisted of a DODAB bilayer which served as a strong anchor to the capillary wall and polyoxyethylene (POE) stearate to suppress the EOF. The coating has been applied successfully to the capillary zone electrophoretic separation of basic, acidic and histone proteins, and to capillary isoelectric focusing. The ability to tune the EOF enabled both single-step capillary isoelectric focusing (cIEF) and two-step cIEF to be performed. A strongly suppressed EOF coating provided a linear pH gradient and allowed for the separation of two hemoglobin variants HbA and HbS. Factors affecting the stability and EOF of the developed surfactant bilayer/diblock copolymer coating were studied. The magnitude of the anodic EOF can be tuned by varying the hydrophilic block POE chain length. The hydrophobic block of the diblock copolymer accounts for stability of the coating, with a longer (stearate) block giving the best stability. The sequential coating provided a stable and suppressed EOF over a broad range of pH 3.0-11.5. The EOF was suppressed and anodic at low pH. As the pH increases, the EOF was still suppressed but became cathodic. This reversal in EOF of the sequential coating is consistent with the reported applications of the sequential coating, and the behavior of the underlying DODAB bilayer. The sequential coating shows a good stability in buffers containing up to 20% v/v acetonitrile.
  

• Subjects / Keywords

  • Capillary electrophoresis
  • Capillary coatings
  • Surfactants
  • Capillary isoelectric focusing
  • Protein adsorption
  • Electroosmotic flow

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R38S4K08P

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.