A Water-Soluble Tetraethylsulfonate Derivative of 2

Anal. Chem. 1998, 70, 907-912

A Water-Soluble Tetraethylsulfonate Derivative of 2-Methylresorcinarene as an Additive for Capillary Electrophoresis Philip Britz-Mckibbin and David D. Y. Chen*

Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR), an aromatic-based, bowlshaped macrocycle, was used as an additive in capillary electrophoresis. Several phenol derivatives are used as analytes to demonstrate the effect of this highly charged additive. TESMR is observed to interact differently with a mixture of positional isomers and other types of phenol derivatives. A comparative study of separations with two charged additives, TESMR and sulfobutyl ether-β-cyclodextrin (SBE-β-CD), provided insight into the selectivity exhibited by these additives. The influence of buffer pH, ionic strength, and organic modifier content on separation and peak shape is investigated. Peak asymmetry caused by the use of highly charged additives at lower pH is minimized by the addition of small amounts of a polar aprotic organic solvent to the run buffer. The effects of mixing a charged additive with a neutral additive are also discussed. Resorcinarenes1,2 are cyclic tetramers of resorcinol having a bowl-shaped polyhydroxy aromatic cavity. They are a class of synthetic macrocyclic molecules which have been the subject of research for many years.3 Various names, such as calix[4]resorcinarenes, resorcinol-derived calix[4]arenes, Ho¨gberg compound, and octols, have been used in the literature, but the name resorcinarenes seems to be the choice in many recent studies2 and, therefore, is used in this paper. Similar to cyclodextrins (CDs), resorcinarenes are capable of forming stable inclusion complexes with a wide variety of small molecules.2,4-6 However, due to the smaller size and the presence of π-electrons in the aromatic groups inside the cavity, resorcinarenes can be expected to exhibit different affinities than the sugar-based CD additives. Most types of resorcinarenes synthesized thus far are relatively hydrophobic, and their complexation characteristics have been * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: (604) 822-0878. Fax: (604) 822-2847. (1) Schneider, U.; Schneider, H. J. Chem. Ber. 1994, 127, 2455. (2) Timmerman, P.; Berboom, W.; Reinhoudt, D. N. Tetrahedron 1996, 52, 2663-2704. (3) Bohmer, V. Angew. Chem., Int. Ed. Engl. (Reviews) 1995, 34, 713-745. (4) Ba¨chmann, K.; Bazzanella, A.; Haag, I.; Han, K. Anal. Chem. 1995, 67, 1722-1726. (5) Sun, S. X.; Sepaniak, M. J.; Wang, J. S.; Gutsche, C. D. Anal. Chem. 1997, 69, 344-348. (6) Dickert, F. L.; Ba¨umler, U. P. A.; Stathopulos, H. Anal. Chem. 1997, 69, 1000-1005. S0003-2700(97)01059-7 CCC: $15.00 Published on Web 01/27/1998

© 1998 American Chemical Society

Figure 1. Structure of the tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR).

studied mainly in apolar organic solvents. Recently, Kobayashi et al.7 synthesized a highly water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR), as shown in Figure 1. The ability of TESMR to form complexes with of a variety of alcohols, saccharides, nucleosides, nucleotides, and amino acids in aqueous solution was studied.7,8 (7) Kobayashi, K.; Asakawa, Y.; Kato, Y.; Aoyama, Y. J. Am. Chem. Soc. 1992, 114, 10307-10313.

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The migration behavior of analytes in CE can be modified by using additives in the buffer solution, as demonstrated by Terabe et al.9,10 and many other researchers. There have been only a few reported applications of resorcinarenes (or related calixarene derivatives) as potential additives in CE. Ba¨chman et al.4 used alkyl-substituted resorcinarene tetraanions in the separation of a mixture of polycyclic aromatic hydrocarbons (PAHs). Poor water solubility and possible aggregate formation limited the use of these additives. Recently, Sun et al. studied the effects of a charged p-(carboxyethyl)calix[7]arene on the separation of a group of substituted aromatic hydrocarbons.5 The influences of pH, organic solvent, and field strength on the separation were studied. Exploring new types of additives is vital for the continuing development of CE. The aim of this study is to explore the usefulness of TESMR (Figure 1) as an additive in CE. This resorcinarene derivative was selected as a potential additive for several reasons: (1) it has high solubility in aqueous solutions (up to 0.4 M); (2) it is too hydrophilic to form micelles or aggregates;7 (3) it is highly charged, inducing a large difference between the mobilities of the free analyte and the complex (producing a large elution range); (4) it has different cavity properties compared with CD-type additives (size and affinity); (5) it is a single-component additive, in contrast to CD derivatives, which are often multicomponent additives; (6) a variety of functional analogues have been synthesized and well characterized; and (7) it requires only inexpensive starting materials that are readily available. Positional isomers of nitrophenol and a group of para-substituted phenols were used as test analytes to study the influence of steric effect and substituent polarity on the complexation with the resorcinarene additive. Also, the influences of additive concentration, buffer ionic strength, pH, and organic modifier content are investigated to determine optimum separation conditions. A comparative study between the resorcinarene and a charged CD of the complexation affinity with the analytes provided insights regarding the analyteadditive interactions. The benefits of a mixed-additive CE separation system, consisting of R-CD and the charged resorcinarene, are also discussed. EXPERIMENTAL SECTION Chemicals. Sodium phosphate monobasic, p-, m-, and onitrophenols, HPLC grade acetonitrile (ACN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and methanol (MeOH) were purchased from Fisher Scientific (Pittsburgh, PA). Methylresorcinol, p-bromo-, p-ethyl-, p-hydroxy-, and p-phenylphenols, and biphenol were from Aldrich (Milwaukee, WI); R- and β-cyclodextrins were from Sigma (St. Louis, MO). Anhydrous sodium sulfite and sodium hydroxide were purchased from BDH Chemicals (Toronto, Canada). Sulfobutyl ether β-cyclodextrin (SBE-β-CD (IV), also known as Advasep), with an average degree of substitution of 4, was kindly provided by Cydex, L.C. (Overland Park, KS). Syntheses and Characterization. The tetraethylsulfonated 2-methylresorcinarene was synthesized via an acid-catalyzed (8) Kobayashi, K.; Tominaga, M.; Asakawa, Y.; Aoyama, Y. Tetrahedron Lett. 1993, 34 (32), 5121-5124. (9) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113. (10) Terabe, S.; Ozaki, H.; Otsuka, K.; Ando, T. J. Chromatogr. 1985, 332, 211217.

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Figure 2. Absorption spectrum of 2 × 10-5 M TESMR in 30 mM phosphate buffer at pH 4.5.

condensation of sodium 2-formylethane-1-sulfonate with 2-methylresorcinol according to the method reported by Kobayashi et al.7 1H NMR spectra of the product were taken in D2O using a 200-MHz Bru¨ker AC-200E spectrometer to confirm its purity. The spectra were identical with that previously reported for TESMR.7 Singlet aromatic H and methyl H signals were observed in the spectra, which was consistent with the symmetrical bowl-shaped conformation of the tetramer.7 The molecular weight of TESMR is 1064, which was verified by running a liquid secondary ion mass spectrum (LSIMS) in a thioglycerol/water matrix. The spectrum showed two main peaks at m/z 1065 and 1087 for the proton adduct and the sodium adduct, respectively. The orange TESMR exhibited two main absorption bands in the UV region in phosphate buffer at pH 4.5, a stronger band in deep UV, and a much weaker band centered at 280 nm. The absorption spectrum at this pH is shown in Figure 2, and the absorption coefficients at 200, 280, and 305 nm are 9.4 × 104, 7.0 × 103, and 40 M-1 cm-1, respectively. The purity of TESMR was also assessed by CE with UV absorption detection at 220 nm at pH 4.5, which showed trace impurities, with the peak area being under 4% of the main component peak. Apparatus and Procedure. All experiments were performed on a Beckman P/ACE 5500 automated CE system with a diode array detector at 25 °C. Unless otherwise specified, the run buffer contained 40 mM phosphate at pH 4.5, with different concentrations of various additives and organic modifiers. Fused silica capillaries (Polymicro Technologies, Phoenix, AZ) had an i.d. of 50 µm, an o.d. of 375 µm, and, unless otherwise specified, a length of 27 cm (20 cm to detector). New capillaries were rinsed first with 0.1 M NaOH for 3 min, followed by a rinse with deionized water for 5 min and phosphate buffer for 10 min, which was then allowed to equilibrate overnight. Stock solutions of the analytes (1 × 10-2 M) were prepared in aqueous solutions that contained 50% methanol. The concentrations of the analytes in the injected sample mixtures were 7 × 10-4 M for the nitrophenols and 3 × 10-3 M for bromo-, ethyl-, hydroxy-, and phenylphenols and biphenol. Samples were introduced in the capillary by using a low-pressure (0.5 psi) injection for 3 s. Analyte peaks were identified by spiking the sample solution with the appropriate stock solution of each analyte.

RESULTS AND DISCUSSION Effects of TESMR on the Migration Behavior of Nitrophenols. Three fundamental parameters11,12 influence the apparent electrophoretic mobility of an analyte when using additives in CE: the electrophoretic mobilities of the free analyte (µep, A) and of the analyte-additive complex (µep, AC) and the capacity factor of the interaction, k′AC (which is the product of the equilibrium constant and the additive concentration, K[C], for 1:1 interactions). The apparent electrophoretic mobility of the analyte is given by11,13

νµAep )

k′AC 1 µ + µ 1 + k′AC ep,A 1 + k′AC ep,AC

(1)

where ν is the correction factor that converts the net analyte mobility to an ideal-state mobility where the additive concentration approaches zero. The correction factor ensures that the change in the net analyte mobility is solely the result of the shifts in equilibria. For neutral analytes, because µep,A ) 0, the net analyte mobility is

νµAep )

k′AC µ 1 + k′AC ep,AC

(2)

Equations 1 and 2 illustrate an important advantage in using charged additives in CE. As demonstrated by the large number of applications of charged micelles, charged additives can effectively change the mobility of both charged and neutral analytes through differential interaction reflected by differences in k′ and µep,AC. Neutral additives do not affect the mobility of neutral analytes because both µep,A and µep,AC are 0. According to eq 2, a highly charged additive can increase the mobility of neutral or minimally charged analytes significantly since the net analyte mobility is proportional to the complex mobility. It is for these reasons that charged cyclodextrins are increasingly being used, especially for chiral separations in CE.14-18 TESMR was observed to have a large negative electrophoretic mobility of -3.91 × 10-4 cm2 V-1 s-1 at pH 4.5, compared to a relatively low electroosmotic flow of 1.51 × 10-4 cm2 V-1 s-1. This suggests that TESMR is highly charged, even under the acidic conditions used throughout the investigation. The analyteadditive complex migrates toward the cathode against the EOF, resulting in increased analyte migration times. The net mobility of the analyte is determined by the fractions of the free and complexed analyte and is a weighted average of the free and complex mobilities, as shown in eq 1. The fractions are determined by the capacity factor, which is determined by both the (11) Peng, X.; Bowser, M. T.; Britz-Mckibbin, P.; Bebault, G. M.; Morris, J. M.; Chen, D. D. Y. Electrophoresis 1997, 18, 706-716. (12) Britz-Mckibbin, P.; Chen, D. D. Y. J. Chromatogr. A 1997, 781, 23-34. (13) Peng, X.; Bebault, G. M.; Sacks, S. L.; Chen, D. D. Y. Can. J. Chem. 1997, 75, 507-517. (14) Tait, R. J.; Thompson, D. O.; Stella, V. J.; Stobaugh, J. F. Anal. Chem. 1994, 66, 4013-4018. (15) Chankvetadze, B.; Endresz, G.; Blaschke, G. J. Chromatogr. A 1995, 700, 43-49. (16) Desiderio, C.; Fanali, S. J. Chromatogr. 1995, 716, 183-196. (17) Janini, G. M.; Muschik, G. M.; Issaq, H. J. Electrophoresis 1996, 17, 15751583. (18) Lurie, I. S.; Klein, R. F.; Dal Cason, T. A.; LeBelle, M. J.; Brenneisen, R.; Weinberger, R. E. Anal. Chem. 1994, 66, 4019-26.

Figure 3. Investigation of the effect of TESMR on the separation of three neutral geometric isomers using (A) 0 and (B) 1.0 mM TESMR. The length of the capillary was 37 cm, the separation voltage was 28 kV, and UV absorption was monitored at 305 nm. Peaks: 1, p-nitrophenol; 2, m-nitrophenol; and 3, o-nitrophenol.

equilibrium constant and the concentration of the additive. The direction of the net mobility of the analyte can be reversed if the TESMR concentration is sufficiently high. Figure 3 shows the effect of TESMR on the separation of nitrophenol isomers. p-Nitrophenol was partially separated from the other two in the absence of TESMR because of its relatively stronger acidity, but the other two coelute with the EOF marker, as shown in electropherogram A. As little as 1 mM TESMR is required to fully resolve all three isomers, as shown in electropherogram B in Figure 3. The relative elution order of the nitrophenol isomers indicates that the affinity of TESMR is the strongest for o-nitrophenol and the weakest for p-nitrophenol because of the spatial constraints dictated by the size of the cavity. Analyte Peak Asymmetry. As shown in Figure 3, significant peak tailing was observed for the analytes when TESMR was added to the buffer. Both the magnitude of the tailing and the migration times increased with increasing resorcinarene concentrations. Peak tailing is a serious problem when using these potent novel additives. This phenomenon has been studied by a number of groups when highly charged additives such as SBEβ-CD14-18 are used. Peak asymmetry in CE has been generally attributed to two main sources:16,18-22 electrokinetic dispersion and surface adsorption. Electrokinetic dispersion occurs when there is an unstable moving boundary in the sample plug, caused by perturbations in the electric field distributed along the capillary due to conductivity or pH differences between sample and run buffer zones. Peak tailing is prominent when the conductivity of the sample zone is different from the conductivity of the buffer zone. Peak asymmetry in this case can be minimized, when using charged additives, by reducing analyte concentration and/or minimizing additive concentration. Because the concentration of TESMR (19) Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979, 169, 1-10. (20) Hjerten, S. Electrophoresis 1990, 11, 665-690. (21) Roberts, G. O.; Rhodes, P. H.; Snyder, R. S. J. Chromatogr. A 1989, 480, 35-67. (22) Xu, X.; Kok, W. T.; Poppe, H. J. Chromatogr. A 1996, 742, 211-227.

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used in this study is rather small, electrokinetic dispersion is unlikely to be the cause of the observed peak asymmetry. Analyte adsorption on the inner capillary walls is generally attributed to electrostatic and/or hydrophobic forces. Since the resorcinarene and the capillary wall are both negatively charged, it is unlikely that the resorcinarene is adsorbed electrostatically. When no TESMR is added to the run buffer, the analytes are close to neutral at pH 4.5, and they all coelute with the EOF marker. There is no peak tailing observed. When the mobility of TESMR is measured by injecting a plug into the capillary, no peak tailing was observed, either. These observations suggest that neither the nitrophenols nor TESMR alone is adsorbed by the capillary wall. However, when the nitrophenols are injected onto the capillary in the presence of TESMR in the run buffer under acidic conditions, severe tailing is observed. This suggests that, with the presence of highly charged additives such as TESMR, the hydrophobic interactions between the analytes and the silanol groups on the capillary wall23 were enhanced, resulting in severely skewed peaks. The presence of highly charged ions in the solution induces stronger solvent-solvent interactions, and thus weaker solvent-solute interactions and stronger solute-solute interactions or hydrophobic interactions. This argument is further supported by the fact that increasing the analyte concentration led to increased peak asymmetry. Experiments were conducted to investigate the influences of buffer ionic strength, pH, and organic modifiers on peak tailing. Various concentrations of phosphate buffer (40, 80, 120, and 160 mM) at pH 4.5 were used with 2 mM TESMR, and the peak shapes of the nitrophenol isomers were examined. No significant improvement in peak shape was observed with increased ionic strength. Increased resolution due to the slower EOF was observed at the cost of greater peak tailing, higher current, and more Joule heating. The pH of the run buffer was observed to have a dramatic effect on peak shape. The use of an increasingly basic pH buffer resulted in much sharper peaks over a wide range of TESMR concentrations. When the pH of the 40 mM phosphate buffer was changed incrementally from 4.5 to 9.0, significant improvement in peak shape was found to occur at pH > 7.6. These observations suggest that the silanol groups on the capillary wall are sufficiently deprotonated at higher pH and that the hydrophobic interaction of the analyte with the capillary wall is significantly reduced. Peak tailing was also observed by others when charged cyclodextrins were used in acidic buffer conditions.14-18 The drawbacks of using basic buffers with resorcinarene additives include the increase in background noise due to the strong absorption of the TESMR throughout the UV region at higher pH. In cases where acidic conditions have to be used for optimum separation, organic modifiers can be used to increase the solvent strength, and, therefore, minimize the hydrophobic interactions between the analytes and the capillary wall, and to improve the peak shape. Four types of organic modifiers are used in this study: MeOH, ACN, DMF, and DMSO. As shown in Figure 4, each of the organic modifiers has a different effect on the separation and peak asymmetry. The addition of the polar protic solvent, i.e., methanol, to the run buffer has a minimal effect on peak shape but results in a small reduction in resolution. (23) Weinberger, R.; Sapp, E.; Moring, S. J. Chromatogr. 1990, 516, 271-285.

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Figure 4. Influence of organic modifiers on the resolution and analyte peak asymmetry using (A) 5% MeOH, (B) 5% ACN, (C) 5% DMF, and (D) 5% DMSO. CE conditions are the same as in Figure 3, and the concentration of TESMR was 1.5 mM. Analyte peaks are numbered the same as in Figure 3.

Figure 5. Comparison of the migration orders of nitrophenol isomers using two different negatively charged additives: (A) 6 mM TESMR (2.5% DMSO) and (B) 2 mM SBE-β-CD (30% DMSO). The analyte peaks are numbered the same as in Figure 3. The separation voltage was 20 kV for electropherogram A and 25 kV for electropherogram B.

However, the polar aprotic solvents were observed to have a significant improvement on peak shape, albeit with a much larger reduction in resolution. When DMSO is used, even though the resolution is not as good when compared to those of DMF and ACN, it gives the largest improvement in peak shape when higher concentrations of highly charged additives are used. Therefore, DMSO was chosen as the most effective organic modifier. It is apparent that the reduced analyte-wall interaction is achieved at the cost of reduced analyte-additive interaction because of the increased solvent strength (stronger solvent-solute interaction). Peak tailing was minimized to a great extent when using only a small percentage of DMSO (2-5%) in the run buffer. Excellent resolution and peak symmetry of the nitrophenol isomers were achieved with 2.5% DMSO in the run buffer using 6 mM TESMR, as shown in Figure 5A. One of the other advantages of using organic modifiers is that the reduced conductivity of the run buffer allows the application of higher voltages for more efficient separations. The main disadvantage of using an organic modifier

Figure 6. Comparison of the separation of a group of six parasubstituted phenols using two different negatively charged additives: (A) 16 mM TESMR (2.5% DMSO) and (B) 6 mM SBE-β-CD (50% DMSO). The UV absorption was monitored at 285 nm, and the separation voltage was 10 kV. Peaks: 1, p-hydroxyphenol; 2, p-ethylphenol; 3, p-bromophenol; 4, p-nitrophenol, 5, biphenol; and 6, p-phenylphenol.

is that a higher concentration of additive is often required to achieve the same resolution. However, due to the high solubility of the resorcinarene in aqueous solutions, this is not a serious limitation. Resorcinarenes vs Charged Cyclodextrins. The binding affinities between the nitrophenol isomers and the two different negatively charged macrocyclic molecules, TESMR and SBE-βCD, were compared. Similar to TESMR, the use of SBE-β-CD alone in the run buffer resulted in dramatic peak tailing. A relatively large percentage of DMSO (30%) was required in order to minimize peak asymmetry. It is not clear why a higher concentration of DMSO is required in this case. A possible explanation is that lipophilic analytes may be mass transfer resistant due to the high charge density in the vicinity of highly charged macrocyclic molecules. However, separations at higher temperatures (up to 40 °C, which should have higher reaction rates) do not show significant peak shape improvement. Further study is needed to gain more insight into this band broadening process. Figure 5 shows a comparison of the effects of two charged additives for the separation of the nitrophenol isomers. SBE-βCD was observed to have the greatest affinity for p-nitrophenol, an intermediate interaction with m-nitrophenol, and the weakest interaction to o-nitrophenol. This trend is similar to what has been demonstrated with other cyclodextrins.24 On the other hand, TESMR has the opposite selectivity, resulting in a reversed migration order of the nitrophenol isomers. The unique affinities displayed by each additive reflect the specific shape, size, and polarity of their apolar cavity and the resultant stability of the analyte-additive complex with the nitrophenol isomers. Analyte Size and Polarity and Migration Order. Figure 6 shows the electropherograms for the separation of six parasubstituted phenols with TESMR and SBE-β-CD additives. Because all analytes are close to neutral at the pH of the run buffer, (24) Lucy, C. A.; Brown, R.; Yeung, K. K. C. J. Chromatogr. A 1996, 745, 9-15.

the separation is mainly due to the differential interaction of the analytes with each charged additive. Although the use of TESMR and SBE-β-CD results in opposite migration orders for the positional isomers, they demonstrated similar trends in binding affinity for the group of para-substituted phenols. Phenylsubstituted phenol displayed the strongest interaction with both additives, whereas the hydroxy-substituted phenol (1,4-benzenediol) showed the weakest interaction. The addition of a hydroxy functionality on the phenyl residue (biphenol) resulted in a weaker interaction with the additives in comparison to that of phenylphenol. The remaining three substituted phenols, ethyl-, bromo-, and nitrophenols, displayed similar intermediate affinities with both additives. Lower concentrations of SBE-β-CD were used to separate the mixture of para-substituted phenols. However, SBEβ-CD was observed to be unable to discriminate between p-ethyland p-bromophenols under the conditions used in this study. Since both macrocyclic molecules displayed similar trends in binding affinities for the para-substituted phenols, the driving force for complexation should be hydrophobic in nature for both additives. The steric and functional complementarity between the analyte and the additive molecules seems to play an important role in the separation system. Mixture of Charged and Neutral Additives. Although a variety of conditions can be modified in CE to improve the separation (e.g., ionic strength, pH, organic modifier, temperature, etc.), the use of mixed additives can be one of the most predictable ways to rapidly optimize separation conditions. A systematic approach to the optimization of multiple additives systems has been previously described,11 and the effects of a mixture of charged and neutral additives on analyte migration behavior have also been discussed.25 When TESMR (highly charged) and R-CD (neutral) are used simultaneously, the apparent mobility of an analyte, assuming 1:1 interactions with each additive, can be described by

νµAep )

k′AC 1 µ + µ + 1 + k′AC + k′AD ep,A 1 + k′AC + k′AD ep,AC k′AD µ (3) 1 + k′AC + k′AD ep,AD

where µep,AC, µep,AD, k′AC, and k′AD are the analyte-additive complex mobilities and capacity factors for the analyte-resorcinarene (AC) and analyte-cyclodextrin (AD) complexes, respectively. When neutral analytes and a neutral cyclodextrin (R-CD) additive are used (µep,A and µep,AD are 0), eq 3 can be simplified to

νµAep )

k′AC µ 1 + k′AC + k′AD ep,AC

(4)

In this case, the apparent mobility of an analyte can be effectively changed by the use of two additives through differences in three fundamental physicochemical properties: k′AC, k′AD, and µep,AC. The migration behavior of an analyte can be better controlled by the appropriate selection of additives to maximize the differences in binding affinities and/or complex mobilities. It was in this spirit (25) Kranack, A. R.; Bowser, M. T.; Britz-Mckibbin, P.; Chen, D. D. Y. Electrophoresis, in press.

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Table 1. Effect of TESMR and r-CD on the Separation of Nitrophenol Isomers µep,para µep,meta µep,ortho [TESMR] [R-CD] (× 10-5 cm2 (× 10-5 cm2 (× 10-5 cm2 Rs Rs (mM) (mM) V-1 s-1) V-1 s-1) V-1 s-1) (p,m) (m,o) 0 6 6

0 0 8

-0.11 -1.62 -0.98

0 -2.62 -1.36

0 -3.74 -3.42

0.31 3.41 3.21

0 4.37 7.08

that TESMR and R-CD were selected as a dual-additive system for the separation of nitrophenol isomers. A previous study24 of the complexation of R-CD with nitrophenol isomers showed that R-CD exhibited the highest affinity for the para isomer and the lowest affinity for the ortho isomer (similar to SBE-β-CD), which is in contrast with the binding trends observed with TESMR. In addition, the complex mobilities are drastically different because TESMR is highly negatively charged and R-CD is neutral. Table 1 lists the mobility values of the three nitrophenols measured from the electropherograms when no additives, TESMR alone, and TESMR and R-CD were added to the run buffer, respectively. Both p- and m-nitrophenols show significant binding to the neutral R-CD and elute with a shorter migration time, resulting in decreased resolution. In contrast, because R-CD displays the weakest interaction with the o-nitrophenol and resorcinarene the strongest, increased resolution of the ortho isomer from the other two is achieved. The resolutions between two vicinity peaks are listed in Table 1. Although the analyte mixture was rather simple in this study, one can envision the potential of modifying the mobilities of specific analytes in a complex mixture through the appropriate use of multiple additive systems.

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CONCLUSION The potential of employing a novel type of macrocyclic resorcinarene additive to enhance CE separations was investigated. The highly soluble TESMR can be used to discriminate between various geometric isomers and different types of substituted phenols. Analyte peak tailing was significant under acidic conditions when using highly negatively charged additives. The use of a basic buffer system and the addition of polar aprotic solvents to the run buffer were found to be the most effective remedies to minimize peak asymmetry. TESMR and SBE-β-CD were shown to have opposite selectivity with a group of nitrophenol isomers in terms of steric constraints dictated by the analyte shape and size. However, both additives exhibited similar binding affinities with respect to analyte functionality, as demonstrated with a group of para-substituted phenols. The benefits of utilizing mixed additive systems for the fine-tuning of resolution in CE was explored using an R-CD and charged TESMR additive pair. The application of new types of additives serves to increase the versatility of CE in chemical separations. ACKNOWLEDGMENT The authors thank Naveen Chopra, Robert Chapman, and Dr. John C. Sherman for valuable discussions. This work is supported by the Natural Sciences and Engineering Research Council of Canada. Beckman Instruments (Canada) Inc. kindly loaned us the P/ACE 5500 system. SBE-β-CD was a gift from Cydex, L.C.

Received for review September 24, 1997. December 17, 1997. AC9710590

Accepted