Isotopic Separation of [ 14 N]- and [ 15 N]Aniline by Capillary Electrophoresis Using Surfactant- Controlled Reversed Electroosmotic Flow Ken K.-C. Yeung and Charles A. Lucy* Department of Chemistry, The University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4 Separation of isotopically labeled [ 14 N]- and [ 15 N]aniline was achieved using capillary electrophoresis based on the isotopic effect on pK a . The effects of the buffer co-ion, pH, and electroosmotic mobility on the resolution are investigated in this paper. Electroosmotic flow (EOF) was controlled using the zwitterionic surfactant Rewoteric AM CAS U as buffer additive. The resultant EOF was anodic (reversed) and low in magnitude (0 .6 × 10 -4 cm 2 / (V‚s)). The resolution of [ 14 N]- and [ 15 N]aniline was 1.22. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the zwitterionic surfactant increased the magnitude of the anodic EOF. This EOF improved the resolution to 1.33 based on mobility counterbalance. Electroosmotic flow (EOF) plays a very important role in capillary electrophoresis (CE) separations. Rapid separations are achieved when the EOF flows in the same direction as the analyte mobility. 1,2 Alternatively, differences in analyte mobility are accentuated when the EOF flows against the analyte mobility. If the magnitude of the counter EOF is comparable to that of the analyte, ultrahigh-resolution separations are achieved. This was first demonstrated by Terabe et al. in the separation of oxygen isotopic benzoic acid. 3 Substitution of 18 O for 16 O in the carboxyl group of benzoic acid causes a slight shift in acid dissociation constant of benzoic acid. A suppressed cathodic EOF counterbal- ances the mobility of benzoic acid and accentuates the isotopic effect. The EOF suppression was achieved using hydroxypropyl cellulose as a buffer modifier. Previously, our group separated the chloride isotopes ( 35 Cl - and 37 Cl - ) by mobility counterbalance. 4 The cathodic EOF was varied using buffer pH. Precise manipula- tion of the EOF is critical to achieving mobility counterbalance and then performing ultrahigh-resolution CE separation. Such control was lacking for anodic (reversed) EOF, making isotopic separations of cationic species impossible. Recently, we demonstrated that the zwitterionic surfactant, CAS U, could be used as a buffer additive for EOF modification. 5 Addition of low concentrations of CAS U suppresses the EOF to near zero. This EOF suppression results from the formation of a dynamic hemimicellar coating at the capillary wall. The modified electroosmotic mobility was virtually independent of the buffer pH and the CAS U concentration. 5 Furthermore, cationic surfac- tants, such as cetyltrimethylammonium bromide (CTAB) or tetradecyltrimethylammonium bromide (TTAB), incorporate into the CAS U hemimicelle, allowing alteration of the EOF. The ratio of the cationic to zwitterionic surfactants determines the charge of the hemimicelle coating and thus the magnitude of the resultant EOF. In such a mixed surfactant system, varying the ratio of cationic surfactant to CAS U allows monotonic alteration of the EOF from fully reversed (cationic surfactant alone) 6 to near zero (CAS U alone). 7 Thus, control of EOF in the anodic (reversed) direction is obtained. This paper investigates the use of such mixed surfactant systems to modify the anodic EOF for optimiza- tion of the separation of a cationic isotopically labeled compound, [ 14 N]- and [ 15 N] aniline. BACKGROUND Isotopic Effect of Ionization. The isotope composition within an ionizable functional group affects the dissociation equilibrium of the solute. The dissociation constant ( K a ) for the solute is larger with the lighter isotope than for the same solute with the heavier isotope. 8,9 For example, K a ( 14 N)/ K a ( 15 N) ) 1.019 for aniline and K a ( 16 O)/ K a ( 18 O) ) 1.020 for benzoic acid. 10 Consequently, there is a difference in the degree of ionization between the isotopic species. Using this isotopic effect of dissociation, Tanaka et al. demonstrated a series of isotopic separations of [ 16 O]- and [ 18 O]- benzoic acid, [ 16 O]- and [ 18 O]- p-nitrophenol, [ 14 N] - and [ 15 N] aniline, and [ 14 N]- and [ 15 N]dimethylaniline using reversed-phase liquid chromatography (RPLC). 10-13 In RPLC, the neutral form of a solute is retained much longer than the ionized form. Isotopic effects on ionization yield differences in the fraction of ionization; thus, chromatographic separation results. Optimum separation * To whom correspondence should be addressed. Facsimile: 403-289-9488. Electronic mail: Lucy@ chem.ucalgary.ca. (1) Zemann, A.; Volgger, D. Anal. Chem. 1997 , 69, 3243. (2) Dabek-Zlotozynska, E.; Dlouhy, J. F. J. Chromatogr. 1994 , 671, 389. (3) Terabe, S.; Yashima, T.; Tanaka, N.; Araki, M. Anal. Chem. 1988 , 60, 1673. (4) Lucy, C. A.; McDonald, T. L. Anal. Chem. 1995 , 67, 1074. (5) Yeung, K. K.-C.; Lucy, C. A. Anal. Chem. 1997 , 69, 3435. (6) Lucy, C. A.; Underhill, R. S. Anal. Chem. 1996 , 68, 300. (7) Yeung, K. K.-C.; Lucy, C. A. J. Chromatogr. A 1998 , 804, 319. (8) Ellison, S. L. R.; Robinson, M. J. T. J. Chem. Soc., Chem. Commun. 1983 , 745. (9) Thornton, E. R. J. Am. Chem. Soc. 1962 , 84, 2474. (10) Tanaka, N.; Hosoya, K.; Nomura, K.; Yoshimura, T.; Ohki, T.; Yamaoka, R.; Kimata, K.; Araki, M. Nature. 1989 , 341, 727. (11) Tanaka, N.; Araki, M. J. Am. Chem. Soc. 1985 , 107, 7780. (12) Tanaka, N.; Yamaguchi, A.; Araki, M. J. Am. Chem. Soc. 1985 , 107, 7781. (13) Tanaka, N.; Yamaguchi, A.; Hashizume, K.; Araki, M.; Wada, A.; Kimata, K. J. High Resolut. Chromatogr. Chromatogr. Commun. 1986 , 9, 683. Anal. Chem. 1998, 70, 3286-3290 3286 Analytical Chemistry, Vol. 70, No. 15, August 1, 1998 S0003-2700(98)00156-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/03/1998