Separation techniques using open tubes, such as Capillary Chromatography and Capillary Electrophoresis (CE) have become universal and important tools for separating inorganic anions, peptides and proteins. This is because of the high separation efficiency, the good resolution, the faster separation, and the lower consumption of samples and solutions that can generally be achieved compared to liquid chromatographic techniques.
Although CE has been known for several decades, new developments and applications continue to be forthcoming with approximately 3,000 papers presently published annually in the field. (Hu et al, Anal. Chem. 2002, 74, 2833-2850.) One area of research within the field of CE is the study of wall chemistry. This research focuses on ways to prevent wall adsorption and suppress EOF, which in turn improve the separation efficiency and resolution of different analytes. For example, inorganic anions are very difficult to separate with native silica capillaries unless very high salt concentrations and very low pH are used to suppress the EOF, because the EOF direction is opposite to the electrophoretic movement of anions. For anions with high electrophoretic mobility, the separation time becomes much longer, whereas anions having low electrophoretic mobility may in extreme cases be transported out of the capillary by the EOF at the injection end of the capillary. The negative charge that the native silica capillary surface expresses over a wide pH range is also problematic in the separation of positive charge molecules such as basic proteins because of electrostatic interaction. Therefore, the EOF must be suppressed or even reversed by shielding or altering the surface charge, in order to obtain fast separations and improve separation efficiency. Many approaches, including adjusting the concentration or pH of the buffer, adding small cationic-type solutes or various detergents or polymers, and attaching a static covalently-bonded polymer layer on the lumen surface have been developed to fulfill this task. (Rodrigueza et al, Anal. Chem. Acta 1999, 383, 1-26; Righetti et al, Electrophoresis 2001, 22, 603-611; Liu, Electrophoresis 2001, 22, 612-628; Horvath et al, Electrophoresis 2001, 22, 644-655; Chen et al, Anal. Chem. 1993, 65, 2770-2775; Pietryk et al, J. Chromatogr., A 1997, 775, 327-338; Minnoor et al, J. Chromatogr., A 2000, 884, 297-309.) All these modifications have shown varying degrees of improvement on the separation of different samples.
Although zwitterionic separation materials, prepared either by dynamically coating or covalently bonding zwitterionic functionalities, have been used for long time in liquid chromatography (Hu et al, P.R. Trends Anal. Chem. 1998, 17, 73-79; Nesterenko et al, Anal. Sci. 2000, 16, 565-574; Jiang et al, Anal. Chem. 1999, 71, 333-344; Anal. Chem. 2001, 73, 1993-2003; Anal. Chem. 2002, 74, 4682-4687; Viklund et al, Macromolecules 2000, 33, 2539-2544; Viklund et al, Anal. Chem. 2001, 73, 444-452.), there are only limited studies involving modified capillaries in CE with zwitterionic functionality. Most of these studies are focused on suppressing the EOF and preventing wall adsorption by zwitterionic surfactants. The different approaches have demonstrated a potential for improving the separation efficiency of inorganic and organic anions or cations, peptides, and proteins. (U.S. Pat. No. 5,415,747; Swedberg, J. Chromatogr. 1990, 503, 449-452; Strege et al, J. Liq. Chromatogr. 1993, 16, 51-68; Greve et al, J. Chromatogr., A 1994, 680, 15-24; Gong et al, Electrophoresis 1997, 18, 732-735; Yeung et al, Anal. Chem. 1997, 69, 3435-3441; Baryla et al, Anal. Chem. 2000, 72, 2280-2284; Woodland et al, Analyst 2001, 126, 28-32; Castelletti et al, J. Chromatogr., A 2000, 894, 281-289; Yokoyama et al, Anal. Chem. 2001, 371, 502-506; Mori et al, Anal. Bioanal. Chem., 2002, 372, 181-186.) Nonetheless, the dynamic coating procedure requires that the zwitterionic surfactant be added to the separation solution in order to get a reproducible surface, a prerequisite for good reproducibility in the separation.
The addition of zwitterionic surfactant results a more complicated separation matrix; their presence causes interference in certain detection schemes such as mass spectrometry (Baryla et al, J. Chromatogr., A 2002, 956, 271-277; Cunliffe et al, Anal. Chem. 2002, 74, 776-783.) and also adds to the complexity of samples that are fraction collected for subsequent use or analysis. Although Cunliffe et al. have tried to use the zwitterionic surfactant with double carbon chains to obtain semi-permanent layers on the capillary surface, the stability of the layer is still an important issue. This is because the mixture containing the zwitterionic surfactant and other chemicals has to be used to flush the capillary between runs of separating proteins, which results longer experiment time. (Cunliffe et al, Anal. Chem. 2002, 74, 776-783.) Accordingly, there is a need in the art for synthesis of capillaries with covalently-bonded zwitterionic functionalities both in order to investigate basic separation properties and for comparison with capillaries dynamically coated with zwitterionic surfactants. U.S. Pat. No. 5,415,747 suggests that a zwitterionic species may be bonded to capillaries though a direct silicon-carbon linkage. However, no results are provided regarding a capillary which comprises covalently bonded zwitterionic groups. Consequently, there is a need for further improvements in the field of capillary electrophoresis and capillary chromatography.