Anal. Chem. 1997, 69, 3239-3242
Enantiomeric Separations by Use of Calixarene Electrokinetic Chromatography Montserrat Sa´nchez Pen˜a, Yuling Zhang, and Isiah M. Warner*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
Chiral acylcalix[4]arene amino acid derivatives have been synthesized and used as pseudostationary phases in capillary electrophoresis (CE). Use of these calix[4]arene (CX4) derivatives with electrokinetic capillary chromatography (4-EKC) resulted in enantioseparation of three binaphthyl derivatives at pH values above 7. The structures of the CX4 derivatives, i.e., (N-L-alaninoacyl)calix[4]arene (CX4-L-Ala) and (N-L-valinoacyl)calix[4]arene (CX4-L-Val), and the buffer pH were found to be important factors in chiral separation. When CX4-L-Ala was used as a pseudostationary phase in the CE buffer, baseline resolution was achieved for 1,1′-bi-2-naphthol (BINOL) and 1,1′-binaphthyl-2,2′-diamine (BNA). The addition of sodium dodecyl sulfate (SDS) to the CX4-L-Ala solution resulted in baseline resolution of the enantiomers of 1,1′binaphthyldiyl hydrogen phosphate (BNHP). In contrast, chiral separations of BNHP, BINOL, and BNA were successfully achieved in a single run with use of CX4-LVal as a chiral selector in the absence of SDS. The utility of these new chiral selectors for chiral separation in CE is compared and discussed in detail. Capillary zone electrophoresis (CZE) is a powerful separation technique for many compounds. Separation in CZE is based on a difference in electrophoretic mobilities of ions and is, therefore, proportional to charge-to-mass ratio. Different separation modes have been developed for separation in CZE, e.g., capillary gel electrophoresis (CGE) and micellar electrokinetic chromatography (MEKC). A variation of CZE, termed electrokinetic chromatography (EKC), can separate both neutral and ionic molecules, based on the chromatographic principle of partitioning the solute between a pseudostationary phase (in this case, calix[4]arene) and the mobile phases. In addition, the separation is based on the differential migration of the two phases.1 To achieve chiral separation in CZE, some modifications are required. Chiral separations in CZE require the addition of chiral selectors as mobile phase additives. As examples, chiral separations have been accomplished by use of cyclodextrins,2,3 chiral surfactants,4,5 chiral micelle polymers,6,7 and crown ethers.8,9 We have also recently reported initial results in chiral separations (1) Terabe, S. Trends Anal. Chem. 1996, 8, 129-134. (2) Fanali, S. J. J. Chromatogr. 1989, 474, 441-446. (3) Wren, S. A. C.; Roove, R. C. J. Chromatogr. 1992, 603, 235-241. (4) Dobashi, A.; Ono, T.; Hara, S.; Yamaguchi, J. Anal. Chem. 1989, 61, 19841986. (5) Otsuka, K.; Terabe, S. J. Chromatogr. A 1990, 515, 221-226. (6) Wang, J.; Warner, I. Anal. Chem. 1994, 66, 3773-3776. (7) Agnew-Heard, K. A.; Sa´nchez Pen ˜a, M.; Shamsi, S. A.; Warner, I. M. Anal. Chem. 1997, 69, 958-964. (8) Kuhn, R.; Erni, F.; Bereuter, T.; Hausler, J. Anal. Chem. 1992, 64, 28152820. S0003-2700(96)01319-4 CCC: $14.00
© 1997 American Chemical Society
using a chiral calix[4]arene (CX4) derivative as a mobile phase additive in capillary electrophoresis (CE).10 Other separations using achiral CX4 can also be cited.11,12 Interest in calix[4]arenes as additives in CE is largely due to their complexation properties. Calixarenes are a class of cyclic phenols linked by methylene groups with defined cavities, having host-guest properties similar to those of cyclodextrins.13,14 One of the advantages of CX4s over naturally occurring host molecules, such as cyclodextrins, is that the size of the internal cavity of the macrocycle is more flexible. The most common forms of CX4s are slightly soluble in several organic solvents but are practically insoluble in water and do not have chiral recognition properties. To enhance the water solubility of CX4 derivatives and to catalyze enantioselective reactions, chiral centers have been attached to the rims of CX4s.10,15-17 For example, Shinkai et al. reported the synthesis of a chiral calixarene bearing aliphatic chains15 and amino acid moieties16 on the lower and upper rim, respectively. In addition, Bayard et al. modified the upper rim of calixarene by the introduction of two chiral units of 2-(methoxymethyl)pyrrolidine.17 There are several advantages to using CX4s as chiral selectors in CE. First, as indicated above, the size of the cavity is more flexible than that of cyclodextrins. Second, CX4s can be easily synthesized and derivatized. Finally, since CX4s are not UV-transparent at certain wavelengths, they can be employed for indirect detection. As indicated earlier, we have recently synthesized a new chiral calixarene. Our modified calix[4]arene possesses four amino acid residues on the lower rim.10 These CX4s provide not only a hydrophobic cavity (benzene rings) but also a hydrophilic and a chiral rim (amino acid residues). Under acidic conditions, the carboxylic groups of the amino acids are protonated and the CX4 is neutral. Thus, this CX4 would move with the electroosmotic flow (EOF) in the CE system. At neutral and basic pH values, the CX4 is negatively charged, and therefore mobility is opposite to that of the EOF. Because of this property, the CX4 is a strong (9) Hohne, E.; Gubitz, G.; Krauss, G.-J. J. High Resolut. Chromatogr. 1992, 15, 698-700. (10) Sa´nchez Pen ˜a, M.; Zhang, Y.; Thibodeaux, S.; McLaughlin, M. L.; Mun ˜oz de la Pen ˜a, A.; Warner, I. M. Tetrahedron Lett. 1996, 37, 5841-5844. (11) Shohat, D.; Grushka, E. Anal. Chem. 1994, 66, 747. (12) Sun, S.; Sepaniak, M. J.; Wang, J.-S.; Gutsche, C. D. Anal. Chem. 1997, 69, 344. (13) Gutsche, C. D. In Calixarenes, Monographs in Supramolecular Chemistry; Stoddar, J. F., Ed.; The Royal Society of Chemistry: Cambridge, 1989; Vol. 1. (14) Gutsche, C. D.; Muthukrishnan, R. J. Org. Chem. 1978, 43, 4905. (15) Shinkai, S.; Arimura, T.; Satoh, H.; Manabe, O. J. Chem. Soc., Chem. Commun. 1987, 1495. (16) Nagasaki, T.; Tajiri, Y.; Shinkai, S. Recl. Trav. Chim. Pays-Bas 1993, 112, 407-411. (17) Bayard, F.; Fenet, B.; Lamartine, R.; Petit-Ramel, M.; Royer, J. J. Chim. Phys. 1995, 92, 13-21.
Analytical Chemistry, Vol. 69, No. 16, August 15, 1997 3239
chiral selector and is able to resolve charged and uncharged enantiomers. It should also be noted that the negative charges on the calixarene may produce additional interactions between the calixarene and the analyte. In this paper, we describe the first chiral separations of three binaphthyl derivatives using calix[4]arene derivatives in CX4-EKC. The two chiral calix[4]arenes used in this study were synthesized in our laboratory and are designated (N-L-alaninoacyl)calix[4]arene (CX4-L-Ala) and (N-L-valinoacyl)calix[4]arene (CX4-L-Val). The structures of these CX4 derivatives and the influences of the CX4 concentration added to BGE, the buffer pH, and the surfactant concentration on the enantioseparation have been examined. EXPERIMENTAL SECTION Reagents. The calix[4]arenes used in this study were synthesized in our laboratory. The procedures for the synthesis and the characterization of the calix[4]arene L-alanine derivative have been recently reported.10 The calix[4]arene L-valine derivative was prepared according to the same procedure (1H NMR (200 MHz, CDCl3) δ 0.88 (t, 24H, CH(CH3)2, 1.00 (s, 36H, ArCMe3), 1.44 (s, 36H, CO2CMe3), 2.1 (m, 4H, CH(CH3)2), 3.15 (d, 4H, ArCH2Ar), 4.47-4.79 (m, 16H, CHCO2, CH2CON, and ArCH2Ar), 6.73 (s, 8H, ArH), 7.35 (d, 4H, NH). R (c ) 1 in methanol ) -21.73. Found: C, 70.33; H, 8.89; N, 3.80. Anal. Calcd for C80H116N4O16: C, 70.35; H, 8.86; N, 3.73. (()-1,1′-Bi-2-naphthol (BINOL, 99%), (R)-(+)-1,1′-bi-2-naphthol (99%), (S)-(-)-1,1′-bi-2-naphthol (99%), (R)-(+)-1,1′-binaphthyl-2,2′diamine (BNA, 99%), (S)-(-)-1,1′-binaphthyl-2,2′-diamine (99%), (()-1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNHP, 99%), (R)-(-)-1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (99%), and (S)-(+)-1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (99%) were purchased from Aldrich (Milwaukee, WI). These items were used as received. Water was distilled and doubly deionized with a Pure Lab UV/UF water system (U.S. Filter Corp., Rockford, IL). CE Procedure. Calixarene electrokinetic capillary chromatography experiments were conducted using an automated capillary electrophoresis apparatus (Hewlett Packard, Palo Alto, CA). A fused-silica capillary (64.5 cm × 50 µm i.d.) was purchased from Hewlett Packard. The length of the capillary between the injection and detection points was 56 cm. Detection was performed at 300 or 254 nm. Data were processed using HP software. All analyses were conducted at 25 kV and 25 °C. The solution was buffered at different pHs using 40 mM Na2HPO4. The calix[4]arenes were added directly to the buffer system. Before use, the buffer solutions were filtered through a 0.20 µm filter. All samples were prepared in methanol/water (50: 50) containing 0.33 mg/mL of analyte and were introduced from the anodic end of the capillary. RESULTS AND DISCUSSION Calix[4]arene was modified at the lower rim by attaching four amino acid residues (Figure 1). The carboxylic groups of the calix[4]arene derivatives are negatively charged at the pHs studied here, and thus the CX4s are soluble in the aqueous buffer. Three binaphthyl derivatives were used for the study of the chiral recognition properties of our CX4 derivatives. The structures of these compounds are shown in Figure 1. BINOL, BNHP, and BNA are atropisomeric compounds, whose chirality is due to an asymmetrical plane, instead of an asymmetrical carbon center. It is useful to note that BINOL is useful as a chiral shift reagent in 3240 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
Figure 1. Structures of chiral calixarenes and the chiral analytes.
Figure 2. Electropherograms of the separation of a racemic mixture of BNHP (1), BINOL (2), and BNA (3) using 10 mM CX4-L-Ala. Conditions: 40 mM phosphate buffer; applied voltage, 25 kV; detection at 300 nm; analyte concentration, 0.33 mg/mL. 1H
NMR spectroscopy for estimating the enantiomeric purity of a variety of organic compounds.18 Under the conditions studied, BINOL and BNHP were negatively charged, while BNA was electrically neutral. Thus, the negatively charged calix[4]arene as well as the analytes BINOL and BNHP would tend to move toward the anode. In contrast, BNA would tend to move toward the detector, i.e., with the electroosmotic flow (EOF). However, the analytes and the calix[4]arene will ultimately move toward the detector under the strong EOF conditions present in our CE system. The calix[4]arene (negatively charged), which forms complexes with the analytes, causes an increase in the migration times of all three analytes. Effect of pH. The effect of pH on the enantioseparation of these three binaphthyl derivatives has been studied using 40 mM phosphate buffer containing 10 mM CX4-L-Ala. The pH range used in our studies was pH 7-11. Figure 2 shows the separation of the three racemic mixtures in buffers of different pHs. In all cases, the elution order was BNHP > BINOL > BNA. The resolution as a function of pH studied is plotted in Figure 3. The migration times of all compounds increased with an increase in pH. Although the results confirm a strong complex between BNA and CX4, which is strongly affected by the pH, chiral (18) Toda, F.; Mori, K.; Okada, J.; Node, M.; Itoh, A.; Oomine, K.; Fuji, K. Chem. Lett. 1988, 131-134.
Table 1. Effect of CX4-L-Ala Concentration on Migration Time (tR), Separation Factor (r), and Resolution (Rs) for the Separation of Binaphthyl Derivativesa CX4-L-Ala concn (mM) analyte
parameters
0.25
0.5
1
5
10
15
BNHP
t0 t1 t2 R Rs t1 t2 R Rs t1 t2 R Rs
3.23 5.00 5.00
