Anal. Chem. 2000, 72, 2280-2284
Simultaneous Separation of Cationic and Anionic Proteins Using Zwitterionic Surfactants in Capillary Electrophoresis Nicole E. Baryla and Charles A. Lucy*
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
The zwitterionic surfactant Rewoteric AM CAS U forms a dynamic wall coating that prevents the adsorption of cationic proteins as well as suppresses the electroosmotic flow (EOF). Addition of polarizable anions to buffers containing this zwitterionic surfactant increases the once suppressed EOF to values nearing +3 × 10-4 cm2/(V s). The retention of the EOF allows for the separation of analytes of widely different mobilities and is demonstrated by the simultaneous separation of cationic and anionic proteins. Using a buffer containing optimal amounts of the polarizable anion perchlorate and surfactant CAS U, the proteins lysozyme, ribonuclease A, r-chymotrypsinogen A, and myoglobin are separated in less than 15 min. Efficiencies as high as 1.5 million plates/m and recoveries greater than 91% are observed for proteins injected in distilled water. Migration time reproducibility is ∼1% RSD within 1 day and ∼3% RSD from day to day. The anionic and cationic proteins can be separated over a pH range of 5.5-9, all yielding good efficiencies. In capillary electrophoresis, control of the electroosmotic flow (EOF) and wall chemistry is critical for many separations. The separation of proteins is particularly challenging because of their strong adsorption onto the capillary wall.1,2 This adsorption results in a loss of efficiency, low protein recovery, and poor reproducibility in migration times. There have been a number of approaches developed to suppress the EOF and/or prevent wall adsorption so as to obtain high-efficiency protein separations.3 Mazzeo and Krull identified four characteristics that an ideal coating should exhibit:4 (1) separation efficiency (in theory, this should approach 1-2 million plates/m), (2) protein recovery (this should approach 100%), (3) reproducibility of migration time from run to run and day to day, and (4) retention of the EOF so that cationic and anionic proteins can be separated in the same run. To these we would add that the coating procedure should also ideally be (5) easy to apply, (6) inexpensive, and (7) applicable over a wide range of buffer conditions. * To whom correspondence should be addressed. Fax: (780) 492-6742. E-mail:
[email protected]. (1) Towns, J. K.; Regnier, F. E. Anal. Chem. 1992, 64, 2473. (2) Bushey, M. M.; Jorgenson, J. W. J. Chromatogr. 1989, 480, 301. (3) Rodriguez, I.; Li, S. F. Y. Anal. Chim. Acta 1999, 383, 1. (4) Mazzeo, J. R.; Krull, I. S. In Handbook of Capillary Electrophoresis; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1994; Chapter 18.
2280 Analytical Chemistry, Vol. 72, No. 10, May 15, 2000
Many approaches to wall coatings involve permanent chemical modification of the inner capillary wall5-11. Some of these coatings have achieved excellent efficiencies10,11 up to 1.8 million plates/ m, and the poly(vinyl alcohol) coating of Giles et al.11 achieved outstanding reproducibility (1.3-2.1% RSD). However these coatings are often laborious and time consuming to apply. For example, Srinivasan et al.10 applied a cross-linked polymer that yielded excellent protein efficiencies (1 million plates/m) but required an 18 h drying step in the preparation of the coating. As an alternative, dynamic coatings are desirable because of their low cost and simplicity of application. Many dynamic coatings have been investigated. One recently reported involved several layers dynamically coated onto the capillary. Graul and Schlenoff12 applied 6.5 layers of alternating positively and negatively charged polyelectrolyte polymer onto the capillary wall. They achieved a maximum of 700 000 plates/m for their protein separations, but over 1 h was required to apply the coating. Other approaches to dynamic coatings required even longer rinse times. For instance, Wang and Dubin’s13 noncovalent polycation coating took 16 h to apply. Furthermore, while convenient, many dynamic coatings do not yield high efficiencies. Gong and Ho14 achieved only 50 000 plates/m for lysozyme with their zwitterionic surfactant coating. In 1997, Yeung and Lucy15 demonstrated that low concentrations of the zwitterionic surfactant Rewoteric AM CAS U yields extremely high efficiencies for cationic proteins (>750 000 plates/ m) such as lysozyme and R-chymotrypsinogen A. A strongly suppressed cathodic EOF (
