Capillary coatings for protein separations in capillary electrophoresis.

摘要

Capillary electrophoresis (CE) is a separation technique based on the differential migration of ions in an electric field. In CE, control of the electroosmotic flow (EOF) and wall chemistry are critical for many separations. The separation of proteins is particularly challenging due to their strong adsorption to the capillary wall. Herein, the use of surfactant-based capillary wall coatings are investigated for improved protein separations as well as EOF control.; The single-chained zwitterionic surfactant coco (amidopropyl) ammoniumdimethylsulfobetaine (CAS U) formed a wall coating that prevented protein adsorption while maintaining a strong EOF so that both cationic and anionic proteins could be simultaneously separated. Efficiencies up to 1.5 million plates/m and recoveries greater than 91% were observed. However, single-chained surfactants must be present in the buffer to maintain the coating. Alternatively, the cationic double-chained surfactant didodecyldimethylammonium (DDAB) forms a highly stable coating at the capillary wall such that excess surfactant can be removed from the capillary.; Organization of surfactant aggregates on the silica surface was probed using atomic force microscopy (AFM). Single-chained surfactants such as cetyltrimethylammonium (CTAB) and CAS U form spherical aggregates while double-chained surfactants such as DDAB form flat bilayers on silica. The increased coverage afforded by the bilayer minimizes protein adsorption.; To achieve a stable coating and the ability to separate both cationic and anionic proteins, a double-chained zwitterionic surfactant 1,2-dilauroyl- sn-phosphafdylcholine (DLPC) was investigated. DLPC forms a semi-permanent coating at the capillary wall, allowing excess surfactant to be removed from the capillary prior to separation. A DLPC-coated capillary yielded high efficiencies (>1 million plates/m) and good protein recovery (up to 93%) for both cationic and anionic proteins.; The ability to vary the EOF within the capillary using a CAS U coating was used for achieving pH independent large-volume sample stacking of positive or negative analytes. This online sample concentration technique improved detection limits up to 85 fold, while retaining the ability to use buffer pH to optimize the separation.; Finally, coatings comprised of mixtures of DDAB and DLPC were used to control the EOF. Tuning the EOF in this manner allowed optimization of the separation of inorganic anions and proteins.