Isotopic Separation of [14N]- and [15N]Aniline by Capillary

W. Hampton Henley, Richard T. Wilburn, Andrew M. Crouch, and James W. Jorgenson. Analytical Chemistry 2005 77 (21), 7024-7031. Abstract | Full Text HT...
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Anal. Chem. 1998, 70, 3286-3290

Isotopic Separation of [14N]- and [15N]Aniline by Capillary Electrophoresis Using SurfactantControlled 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 [14N]- and [15N]aniline was achieved using capillary electrophoresis based on the isotopic effect on pKa. 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 cm2/(V‚s)). The resolution of [14N]- and [15N]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 18O for 16O in the carboxyl group of benzoic acid causes a slight shift in acid dissociation constant of benzoic acid. A suppressed cathodic EOF counterbalances 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 (35Cl- and 37Cl-) by mobility counterbalance.4 The cathodic EOF was varied using buffer pH. Precise manipulation 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 surfactants, 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 optimization of the separation of a cationic isotopically labeled compound, [14N]- and [15N]aniline. BACKGROUND Isotopic Effect of Ionization. The isotope composition within an ionizable functional group affects the dissociation equilibrium of the solute. The dissociation constant (Ka) for the solute is larger with the lighter isotope than for the same solute with the heavier isotope.8,9 For example, Ka(14N)/Ka(15N) ) 1.019 for aniline and Ka(16O)/Ka(18O) ) 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 [16O]- and [18O]benzoic acid, [16O]- and [18O]-p-nitrophenol, [14N]- and [15N]aniline, and [14N]- and [15N]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: [email protected]. (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.

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occurred when the difference in ionization degree was maximized by adjusting the pH. In addition, Bushey et al. performed a similar separation of dansylated methylamine and dansylated methylamine-d3 using micellar electrokinetic capillary chromatography.14 In CE, separation is based on a difference in the electrophoretic mobility of the solutes. The isotopic effect on the dissociation constants (Ka) causes a slight shift in the ionization of an isotopically labeled compound. Since only the ionized form has a mobility, a difference in mobilities results. However, such mobility differences are small and so must be accentuated to yield a separation. Resolution. An excellent theoretical treatment of ultrahighresolution separations in CE was given previously by Terabe et al.3 In brief, the resolution (R) of two solutes in electrophoresis is described by

R)

xN ∆µa 4 µ ja

(1)

where N is the efficiency of the separation, ∆µa is the difference in the mobilities of the solutes, and µj a is the average apparent mobility. The apparent mobility is defined as the sum of the analytes’ average electrophoretic mobility (µep) and the electroosmotic mobility (µeo).

µa ) µep + µeo

(2)

(3)

where V is the voltage applied, and D is the diffusion coefficient of the analytes. Lt and Ld are the capillary’s total length and length to detector. Combining eqs 1-3 gives the following expression:

R)

x

V Ld ∆µep 1 32D Lt µ j ep1/2 (1 + µeo/µ j ep)1/2

(4)

According to eq 4, the resolution is dependent on three main terms: (a) instrumental factors such as the applied voltage and the capillary lengths; (b) the ratio of the intrinsic difference in mobility between the two components and their average electrophoretic mobility; and (c) the relative magnitude of the electoosmotic mobility and the analytes’ average electrophoretic mobility. The first factor is usually determined by the experimental apparatus and cannot be altered to a great extent. The other two factors can be easily altered and so are most important in maximizing the resolution. First, the difference in mobilities for ionizable compounds can be altered by the pH as described above. Terabe et al.3 derived an expression to determine the optimum pH for the electrophoretic separation of [16O]- and [18O]benzoic acid. It is obtained by relating the mobility (µep) to the fraction of the solute in the ionized form (R). The electrophoretic mobility (14) Bushey M. M.; Jorgenson, J. W. Anal. Chem., 1989, 61, 491.

Ka µep ) µionR ) µion + [H ] + Ka

(5)

The predicted optimum pH for maximum mobility difference (∆µa) is ∼0.3 pH unit (log 2) below the pKa of the analytes.

optimum pH (anionic) ) pKa - log 2

(6)

At this pH, the intrinsic difference in the mobility of a [16O]- and [18O]carboxylic acid is maximized, i.e., the ∆µep/µj ep1/2 term in eq 4. Terabe et al. experimentally verified this optimum condition for the separation of [16O]- and [18O]benzoic acid.3 In addition, they showed that, as the pH deviates from the optimum pH in either direction (higher or lower), the resolution drops in a Gaussian distribution fashion.3 An expression similar to eq 5 can be obtained for cationic species using the fractional composition expression (R) for cationic ionizable compounds. The predicted optimum pH for separation of an isotopically labeled cationic species is 0.3 pH unit above the pKa:

optimum pH (cationic) ) pKa + log 2

Assuming longitudinal diffusion is the major source of bandbroadening, the efficiency is expressed as

V Ld N ) µa 2D Lt

of an ionizable species at a given pH is calculated from the mobility of its fully ionized form (µion) and the fraction of this species in the ionized form.15,16

(7)

Therefore, the intrinsic difference in mobility of [14N]- and [15N]aniline should show a maximum at the pH predicted by eq 7. This will be investigated below. Finally, according to the third term in eq 4, the resolution can be improved by adjusting the electroosmotic mobility. Equation 4 states that resolution is maximized when the mean mobility is zero; i.e., a counterbalance is maintained between the analyte’s intrinsic mobility and the EOF (µj ep ) -µeo). The penalty of this approach is increased analysis time. The effect of EOF on resolution will be investigated by adjusting the electroosmotic mobility using the mixed surfactants described above. EXPERIMENTAL SECTION Apparatus. A Beckman P/ACE 2100 (Fullerton, CA) equipped with an ultraviolet detector was used for all experiments. Untreated silica capillaries (Polymicro Technologies, Phoenix, AZ) with an inner diameter of 50 µm, an outer diameter of 365 µm, and a total length of 47 cm (40 cm to the detector) were used. The capillary was thermostated at 25 °C in all experiments. Direct UV detection at 214 nm was used for the isotopic separations and at 254 nm for the EOF measurements. A detector rise time of 1.0 s and a data collection rate of 5 Hz were used. Data acquisition using System Gold software (Beckman, version 8.10) was performed on a 386-based microcomputer. Chemicals. Tetraethylammonium acetate buffers were prepared from reagent grade glacial acetic acid (BDH, Darmstadt, West Germany) and tetraethylammonium hydroxide (35 wt % (15) Morris, D. J. O. R.; Morris, P. Separation Methods in Biochemistry; Interscience Publishers: New York, 1963; Chapter 18. (16) Smith, S. C.; Khaledi, M. G. Anal. Chem. 1993, 65, 193.

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Figure 1. Chemical structure of Rewoteric AM CAS U: cocoamidopropylhydroxyldimethylsulfobetaine (CAS U). R ) C8-C18.

solution, Aldrich). Tetraethylammonium is chosen as the buffer counterion to minimize electrodispersion by mobility matching with aniline in this study.17,18 The zwitterionic surfactant Rewoteric AM CAS U (Witco, Dublin, OH) and the cationic surfactant cetyltrimethylammonium bromide (CTAB, Aldrich) were used as received. The structure of CAS U is given in Figure 1. The Rgroup represents an alkyl chain with variable chain length from C8 to C18. The molecular weight of CAS U was estimated to be ∼450 g/mol based on gas chromatographic analysis of the acid hydrolysis products.19 Isotopic samples of aniline were prepared from [15N]aniline (98%, Cambridge Isotope Laboratory, Andover, MA) and natural abundance aniline (99.6% 14N, BDH). A 1 mM mesityl oxide (Aldrich) solution was used as the EOF marker. All solutions were prepared in Nanopure ultrapure water (Barnstead, Dubuque, IA). Isotopic Separations. Isotopic [14N]- and [15N]aniline were separated in a 50 mM tetraethylammonium acetate buffer containing 0.5 mM CAS U. Isotopic aniline sample solution (0.03 mM [15N]aniline and 0.07 mM [14N]aniline) was introduced onto the capillary using low-pressure (0.5 psi) hydrodynamic injection for 5.0 s (∼5 nL). New capillaries were conditioned with 0.1 M tetraethylammonium hydroxide solution. Between runs, the capillary was rinsed at high pressure (20 psi) with 0.1 M tetraethylammonium hydroxide for 2 min, distilled water for 2 min, and buffer for 4 min. EOF Measurements. The magnitude of the EOF is greatly suppressed when using CAS U as a buffer additive. Hence, the sequential injection method introduced by Williams and Vigh20 was used to measure the EOF. In this method, mesityl oxide was injected onto the capillary using low pressure. Constant voltage (15 kV) was applied to induce the EOF for a fixed period of time (e.g., 5 min). A low pressure was then applied to hydrodynamically push the mesityl band off the capillary. The magnitude of the EOF is calculated by the displacement of the mesityl oxide band during the time that voltage was applied. The precise procedure is described in our previous paper.5 RESULTS AND DISCUSSION EOF Modification with Zwitterionic Surfactant. As discussed above, the mobility difference between the [14N]- and [15N]aniline can be accentuated by mobility counterbalance with the EOF. The unmodified normal EOF is cathodic. Thus, it comigrates with the ionized aniline (µep ) 1.2 × 10-4 cm2/(V‚s) at pH 4.9). One way to reverse the direction of the EOF is by addition of cationic surfactants at concentrations above their critical micelle concentration.6 The resultant reversed EOF is, however, much larger (µeo ) -6 × 10-4 cm2/(V‚s)) than the aniline mobility. As (17) Hjerte´n, S. Electrophoresis 1990, 11, 665. (18) Grossman, P. D.; Colburn, J. C. Capillary Electrophoresis Theory & Practice; Academic Press: San Diego, CA, 1992. (19) Mannhardt, K.; Novosad, J. J. J. Pet. Sci. Eng. 1991, 5, 89. (20) Williams, B. A.; Vigh, Gy. Anal. Chem. 1996, 68, 1174.

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Figure 2. Resolution of [14N]- and [15N]aniline at various buffer pH. Experimental conditions: buffer, 0.5 mM CAS U and 50 mM tetraethylammonium acetate; voltage, +15 kV; capillary length, 47 cm (40 cm to detector); pH adjusted with tetraethylammonium hydroxide.

Figure 3. Average intrinic mobility of [14N]- and [15N]aniline (2), average apparent mobility (9), and electroosmotic mobility (O) at various buffer pH. Conditions are as in Figure 2.

a result, the aniline is simply swept to the anode with little change in resolution. To obtain the low-magnitude reversed EOF required, CAS U and CTAB surfactant systems will be explored herein.5,7 Previously, a suppressed cathodic EOF (∼+0.2 × 10-4 cm2/ (V‚s)) was observed when 0.5 mM CAS U was added to 10 mM sodium phosphate buffer.5 In this experiment, acetate is used to buffer the pH in the range of interest (pH 3-5). Surprisingly, under these conditions a suppressed EOF in the reversed (anodic) direction (∼-0.6 × 10-4 cm2/(V‚s) at pH 4.9) was observed. This weak anodic EOF is ideal for mobility counterbalance with aniline. However, the cause of the reverse direction of the suppressed EOF is not clearly understood at this point and is currently under investigation. Effect of pH on Resolution. Isotopic separation of [14N]- and 15 [ N]aniline is based on the isotope effect on the dissociation of

Figure 4. Separation of [14N]- and [15N]aniline under modified EOF. (A) No CTAB; (B) 1.0 µM CTAB; (C) 1.25 µM CTAB; (D) 1.35 µM CTAB. Resolution: 1.22, 1.27, 1.32, and 1.33, respectively. Experimental conditions are as in Figure 2 except the following: voltage, +20 kV; buffer, 0.5 mM CAS U plus indicated CTAB in 50 mM tetraethylammonium acetate (pH 4.90).

the amine group. Hence, the buffer pH is critical to the separation. Equation 7 predicts the optimum pH be 0.3 pH unit above the pKa. The pKa of aniline at an ionic strength of ∼30 mM is estimated by interpolation to be 4.62.21 Thus, theory predicts that the optimum pH for the isotopic separation should be 4.92. To experimentally determine the optimum pH, experiments were conducted in tetraethylammonium acetate buffers from pH 4.2 to 5.2. Mobility counterbalance was maintained by the use of 0.5 mM CAS U. The resolution between [14N]- and [15N]aniline measured at each pH is shown in Figure 2. A maximum resolution of 1.2 was obtained at pH 4.96. This indeed agrees with the prediction obtained from eq 7. As the buffer pH moves away from the optimum to either side, the resolution decreases rapidly, as predicted by Terabe et al.3 The optimum pH predicted by eq 7 is based solely on maximizing the isotopic effect of amine group dissociation. However, it does not take into account the effect of variation in the electroosmotic mobility due to the pH change. In these experiments, the EOF was suppressed by the addition of 0.5 mM CAS U. The µeo and the µep of aniline are shown in Figure 3. A positive mobility refers to a cathodic mobility, and a negative mobility refers to an anodic mobility. It is observed that the µeo indeed varies with the buffer pH, from -0.46 × 10-4 cm2/(V‚s) at pH 5.2 to -1.4 × 10-4 cm2/(V‚s) at pH 4.2 (O in Figure 3). Nonetheless, the µeo/µj ep ratio in eq 4 was found to be effectively constant (-0.55, with standard deviation of 0.05) over the pH range studied. This indicates that the EOF variation due to pH does not significantly alter the resolution. As a result, the optimum pH predicted by eq 7 (pH 4.92) is in good agreement with the experimental optimum pH (pH 4.96), and the predicted pH from eq 7 is a good estimate of the optimum condition. Effect of EOF. While the EOF variation due to pH has minimal effect on the resolution, control of the EOF (independent of pH) is still an important factor in ultrahigh-resolution (21) Smith, R. M.; Martell, A. E. Critical Stability Constants, Vol. 2: Amines; Plenum Press: New York, 1975.

separations in CE. It is evident from eq 4 that resolution is maximized when the electroosmotic mobility is similar in magnitude to the analyte intrinsic mobility. Such mobility counterbalance is used here to improve the resolution of [14N]- and [15N]aniline. Using solely 0.5 mM CAS U as the buffer additive, an anodic EOF of 0.7 × 10-4 cm2/(V‚s) is obtained in the pH 4.9 tetraethylammonium acetate. This EOF counterbalances ∼60% of the intrinsic mobility of aniline (µep ) 1.2 × 10-4 cm2/(V‚s)), and a resolution of 1.22 is obtained (Figure 4A). As expected, the first peak is [15N]aniline, followed by the [14N]aniline peak. To fine-tune the EOF, small amounts (1.00-1.35 µM) of CTAB were added to buffer containing 0.5 mM CAS U to gradually increase the magnitude of the anodic EOF. The pH was maintained at 4.9, so that the intrinsic mobility of aniline remained constant. The electropherograms obtained are shown in Figure 4B-D. As the amount of CTAB increases, the anodic EOF increases, gradually increasing the counterbalance of aniline’s mobility. The resolution of [14N]- and [15N]aniline improves marginally with the addition of CTAB (from 1.22 to 1.33). The µeo/µj ep ratio was again calculated in these four cases (Figure 4AD) and was found to be -0.58, -0.65, -0.69, and -0.75, respectively. The increasing µeo/µj ep ratio agrees generally with the increasing resolution observed. However, the magnitude of resolution improvement (Rs ) 1.22-1.33) was less than that predicted from the changes in µeo/µj ep (Rs ) 1.22-1.58 based on eq 7). As is evident in Figure 4, decreases in efficiency resulted from the addition of CTAB. The cause of this efficiency degradation is currently under investigation. Increasing CTAB concentration also increases the analysis time and decreases the signal-to-noise ratio, as is evident in Figure 4D. These drawbacks result from the diffusional bandbroadening increasing as the apparent mobility of aniline decreases. It is therefore best to select a condition where the counterbalancing EOF is just strong enough in magnitude to achieve the desired resolution while maintaining reasonable analysis times and signalto-noise. For the separation of [14N]- and [15N]aniline, this optimum is achieved using CAS U alone. Analytical Chemistry, Vol. 70, No. 15, August 1, 1998

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ACKNOWLEDGMENT

and Witco Corp. for their generous gift of Rewoteric AM CAS U.

This work was supported by the Natural Sciences and Engineering Research Council of Canada and by The University of Calgary. K.K.-C.Y. thanks the Killam Foundation for their support. Thanks also to Canada Colors and Chemicals Limited

Received for review February 11, 1998. Accepted May 15, 1998.

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