Improved Chiral Separations Using a Polymerized ... - ACS Publications

Crystal W. Harrell, Matthew E. McCarroll, Kevin F. Morris, Eugene J. Billiot, and .... Robin S. Hickok , Seamus A. Wedge , Alexandar L. Hansen , Kevin...
0 downloads 0 Views 168KB Size
Download PDF
Anal. Chem. 1997, 69, 2980-2987

Improved Chiral Separations Using a Polymerized Dipeptide Anionic Chiral Surfactant in Electrokinetic Chromatography: Separations of Basic, Acidic, and Neutral Racemates Shahab A. Shamsi, Javier Macossay, and I. M. Warner*

Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803

Two polymeric chiral anionic surfactants [poly(sodium N-undecylenyl-L-valine) (poly-L-SUV) and poly(sodium N-undecylenyl-L-valine-valine) (poly-L-SUVV)] are compared as pseudostationary phases for chiral separations of basic, acidic, and neutral enantiomers. Parameters such as pH, concentration and type of background electrolyte, concentration of polymerized chiral surfactants, and injection size were studied to investigate the migration behavior and optimize the chiral resolution of several racemic analytes. At equivalent monomer concentrations, the migration factors for cationic enantiomers were larger with poly-L-SUV than with poly-L-SUVV. In contrast, the reverse was true for anionic enantiomers. However, in both cases, chiral recognition was significantly enhanced with poly-L-SUVV as compared to that with poly-L-SUV. It is interesting to note that the separation selectivity and resolution of a neutral racemate were slightly better with the latter, but only at the expense of longer analysis time and lower efficiencies.

and saponin23). Recent reports on two other types of surfactants (6-aminopenicillanic acid24 and L-R-palmitoyllysophosphatidylcholine25) have also been published. A number of studies have demonstrated that electrokinetic chromatography (EKC) using polymerized surfactants has a high potential as an alternative to MEKC for the separation of achiral and chiral compounds.26-34 The separation mechanism in this technique is similar to that in MEKC, except that micelles are replaced with covalently polymerized surfactants. Since these pseudostationary phases are very stable and have greater tolerance to organic solvents, these phases have been synthesized and utilized for achiral separations.26-28 Enantiomeric separations employing polymerized surfactants were pioneered by us.29 Using poly(sodium N-undecylenyl-L-valine) (poly-L-SUV), we have reported the optical resolution of (()-1,1′-bi-2-naphthol and DLlaudonosine. Later, an almost identical approach was employed by Dobashi et al. for the enantiomeric separation of 3,5-dinitrobenzoyl amino acid esters.30 Recently, we have extended the utility of poly-L-SUV and its antipode, poly-D-SUV, for the chiral separa-

Micellar electrokinetic chromatography (MEKC) using chiral micelles has attracted considerable attention in past years for the separation of both neutral and charged enantiomers. Three major classes of monomeric chiral surfactants have been used in MEKC to date: (1) amino acid head type surfactants (acyl amino acids1-8 and alkoxyacyl amino acids9-13), (2) sugar head type surfactants (glucopyranoside-based nonionic14,15 and ionic surfactants16), and (3) surfactants with steroidal backbone (bile salts,17-22 digitonin,3,6 (1) Dobashi, A.; Ono, T.; Hara, S.; Yamaguchi, J. Anal. Chem. 1989, 61, 19841986. (2) Dobashi, A.; Ono, T.; Hara, S.; Yamaguchi, J. J. Chromatogr. 1989, 480, 413-420. (3) Otsuka, K.; Terabe, S. J. Chromatogr. 1990, 515, 221-226. (4) Otsuka, K.; Terabe, S. Electrophoresis 1990, 11, 982-984. (5) Otsuka, K.; Terabe, S. J. Chromatogr. 1991, 559, 209-214. (6) Otsuka, K.; Kashihara, M.; Kawaguchi, Y.; Koike, R.; Itisamitsu, T.; Terabe, S. J. Chromatogr. 1993, 652, 253-257. (7) Otsuka, K.; Karuhaka, K.; Higashimori, M.; Terabe, S. J. Chromatogr. 1994, 680, 317-320. (8) Otsuka, K.; Kawakami, H.; Tamaki, W.; Terabe, S. J. Chromatogr. 1995, 716, 319-322. (9) Mazzeo, J. R.; Grover, E. G.; Swartz, M. E.; Peterson, J. S. J. Chromatogr. 1994, 680, 125-135. (10) Mazzeo, J. R.; Grover, E. G.; Swartz, M. E. Anal. Chem. 1995, 67, 29662973. (11) Hove, E. V.; Sandra, P. J. Liq. Chromatogr. 1995, 18, 3675-3683. (12) Swartz, M. E.; Mazzeo, J. R.; Grover, E. G.; Brown, P. R. J. Chromatogr. 1996, 735, 303-310. (13) Peterson, A. G.; Ahuja, E. S.; Foley, J. P. J. Chromatogr. B 1996, 683, 1528.

(14) Nimura, H.; Mitsuno, C.; Itoh, H.; Kinoshita, T.; Hanai, T. Proceedings of 12th Symposium on Capillary Electrophoresis, Himeji, Japan, 1992; pp 4546. (15) Smith, J. T.; Nashabeh, W.; El Rassi, Z. E. Anal. Chem. 1994, 66, 11191133. (16) Tickel, D. C.; Okafo, G. N.; Camilleri, P.; Jones, R. F. D.; Kirby, A. J. Anal. Chem. 1994, 66, 4121-4126. (17) Sepaniak, M. J.; Cole, R. O.; Hinze, W. L. J. High Resolut. Chromatogr. 1990, 13, 579-582. (18) Terabe, S.; Shiata, M.; Myashita, Y. J. Chromatogr. 1989, 480, 403-411. (19) Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S. J. Chromatogr. 1990, 515, 233-243. (20) Nishi, H.; Fukuyama, T., Matsuo, M.; Terabe, S. Anal. Chim. Acta 1990, 236, 281. (21) Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S. J. Microcolumn Sep. 1989, 1, 234. (22) Mechref, Y.; El Rassi, S. J. Chromatogr. 1996, 724, 285-296. (23) Ishihama, T.; Terabe, S. J. Liq. Chromatogr. 1993, 16, 933-944. (24) Bouzige, M.; Okafo, G.; Dhanak, D.; Camilleri, P. J. Chem. Soc., Chem. Commun. 1996, 671-672. (25) Nimura, H.; Mitsuno, C.; Itoh, H.; Kinoshita, T. Proceedings of Separation Sciences ‘94, Tokyo, Japan, 1994; pp 283-285. (26) Palmer, C. P.; Terabe, S. J. Microcolumn Sep. 1996, 8 (2), 115-121. (27) Palmer, C. P.; Khaled, M. Y.; McNair, H. M. J. High Resolut. Chromatogr. 1992, 15, 756-762. (28) Shamsi, S. A.; Mathison, S. M.; Dewees, S.; Wang, J.; Warner, I. M. Pittsburgh Conference 96, Chicago, IL, March 3-8, 1996; Poster 84. (29) Wang, J.; Warner, I. M. Anal. Chem. 1994, 66, 3773-3776. (30) Dobashi, A.; Hamada, M.; Dobashi, Y. Anal. Chem. 1995, 67, 3011-3017. (31) Wang, J.; Warner, I. M. J. Chromatogr. 1995, 711, 297-304. (32) Agnew-Heard, K. A.; Sanchez-Pena, M.; Shamsi, S. A.; Warner, I. M. Anal. Chem. 1997, 69, 958-964. (33) Ozaki, H.; Ichihara, A.; Terabe, S. J. Chromatogr. A 1995, 709, 3-10. (34) Terabe, S.; Ozaki, H.; Tanaka, Y. J. Chin. Chem. Soc. 1994, 41, 251-257.

2980 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

S0003-2700(97)00037-1 CCC: $14.00

© 1997 American Chemical Society

Figure 1. Chemical structures of the chiral polymeric surfactants.

tions of a variety of acidic, basic, and neutral compounds.31,32 The successful outcome of the results obtained with use of poly-L- and -D-SUV has encouraged us to embark on a program aimed at the design, synthesis, and applications of many different kinds of polymerized alkyl amino acid surfactants. One approach that we have recently found useful is the introduction of multiple chiral centers onto the polar head group of a polymerized surfactant. Thus, in this first series of papers, our results suggest that increasing the number of stereogenic centers on the ionic head group of the polymerized surfactant, i.e., increasing chiral density, enhances chiral recognition in EKC. In this article, we present data in which the chiral recognition ability of a dipeptide surfactant is compared with that of a single amino acid surfactant. Figure 1 depicts the structure of both polymeric chiral surfactants. As shown, the structural differences between poly-L-SUV and poly(sodium N-undecylenyl-L-valinevaline) (poly-L-SUVV) is primarily the number of valine amino acid groups attached to the hydrocarbon chain. Both chains contain a polymerizable double bond at the end of the hydrocarbon tail. Poly-L-SUV contains one chiral center and the amino acid valine. In contrast, poly-L-SUVV is a dipeptide surfactant in which the R-carboxyl group of one amino acid is attached to the R-amino group of another amino acid by a peptide bond. This results in two amido groups and two chiral centers of the same optical configuration. Both polymeric surfactants exist predominantly as monoanions at pH g 6 due to one ionizable carboxyl group. Several parameters, such as type and concentration of polymerized surfactant, pH, and injection size, as well as type and concentration of background electrolyte (BGE), were found to influence the migration, resolution, efficiency, and sensitivity. For this reason, we have chosen to compare the chiral separation behavior of two cationic racemates [(()-propanolol (PROP) and (()-alprenolol (ALP)], one anionic racemate [(()-1,1′-binaphthyl2,2′-diyl hydrogen phosphate (BNP)], and one nonionic racemate [(()-trifluoro-1-(9-anthryl)ethanol (TFAE)]. EXPERIMENTAL SECTION Materials. The analytes (()-1,1′-binaphthyl-2,2′-diyl hydrogen phosphate (BNP, 99%), propranolol (PROP, 99%), and trifluoro-1(9-anthryl)ethanol (TFAE, 98+%) were purchased from Aldrich Chemical Co. (Milwaukee, WI). The racemic mixture of (()alprenolol (ALP, 99%) was obtained from Sigma (St. Louis, MO). All analytes were used as received. Buffers (Na2HPO4, Na2B4O7)

used as BGEs were of analytical reagent grade and were also obtained from Sigma. The migration order for each enantiomer was established by spiking the racemic mixture with the corresponding (+)- or (-)-enantiomer obtained from either Sigma or Aldrich. The monosodium salt of naphthalenemonosulfonate (NMS, 99.5% purity) was purchased from American Tokyo Kasei (Portland, OR). This compound was used as a light-absorbing electrolyte to detect monomeric surfactants. Synthesis of Poly(sodium N-undecylenyl-L-valine) and Poly(sodium N-undecylenyl-L-valine-valine). The monomeric amino acid surfactant, N-undecylenyl-L-valine (L-UV), was synthesized according to the procedure of Wang and Warner after some modifications. A similar procedure was used for the synthesis of the dipeptide surfactant, N-undecylenyl-L-valine-valine (L-UVV). The modified synthetic strategies for L-UV and L-UVV developed in our research laboratory are reported in detail elsewhere.35 The acid forms of both L-UV and L-UVV were then converted to their corresponding sodium salt forms by adding an equimolar solution of sodium bicarbonate in the presence of THF. This was followed by solvent evaporation and freeze-drying to obtain the desired L-SUV and L-SUVV surfactants. Polymerization of both surfactants was achieved by 60Co γ-irradiation (70 krad/h), for about 36-48 h (total dose, 3-4 Mrad), of 100 mM each of the above-mentioned surfactant solutions. After irradiation, both poly-L-SUV and polyL-SUVV solutions were dialyzed against bulk H2O using a regenerated cellulose membrane with a 2000 molecular weight cutoff. Finally, the dialyzed products were lyophilized to obtain the dry products of poly-L-SUV and poly-L-SUVV. Proton NMR spectroscopy was used to follow the polymerization process. For both poly-L-SUV and poly-L-SUVV surfactants, NMR indicated the disappearance of the double bond proton signals at 5.8 and 5.0 ppm. Polymerization also produced broadening of the remaining peaks. The polymers were found to be 99% pure, as calculated from elemental analysis. Further characterization, such as molecular weight, partial specific volume, and size of these polymers, is under study in our laboratory. Electrokinetic Chromatography Instrumentation. A Beckman (Fullerton, CA) P/ACE Model 5510 capillary electrophoresis (CE) instrument was employed in electrokinetic chromatography (EKC) for the separation of (()-BNP. This CE instrument was equipped with (1) 0-30 kV high-voltage built-in power supply, (2) 200, 214, 254, and 280 nm selectable wavelength filters for UV detection, and (3) System Gold software for system control and data handling. However, the enantiomeric separations of (()PROP, (()-ALP, and (()-TFAE were performed on a HewlettPackard (Palo Alto, CA) 3D-CE instrument. Data processing for the HP instrument was performed by use of an HP Vectra personal computer (5/90) with HPCE Chemstation software. Separations were performed in both the Beckman and the HP instruments using uncoated fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) of 50 µm i.d. with total lengths of 47 and 64 cm [40 and 60 cm to detector window (Ld)], respectively. The capillary in the Beckman instrument was thermostated by use of a fluoroorganic fluid, whereas the HP instrument was equipped with a Peltier element for forced air cooling and temperature control of the capillary. The study for reverse migration order of (()BNP was performed with a poly(vinyl alcohol) (PVA)-coated capillary that was 50 µm × 64.5 cm (56 cm effective length, Ld), purchased from HP. (35) Macossay, J.; Shamsi, S. A.; Warner, I. M. Manuscript in preparation.

Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

2981

Capillary Electrophoresis Procedures. All new capillaries were prepared by use of a standard wash cycle of 1 N NaOH for 1 h before use. A daily routine procedure also involved flushing the capillary with 1 N NaOH (15 min), triply deionized water (2 min), and the running EKC buffer (10 min). For the separations performed at pH e 7.0, the capillary was flushed only with the running EKC buffer for 3 min between injections. However, when a basic pH between 8 and 10 was employed, the capillary under such pH conditions was flushed for 3 min each with 0.1 N NaOH and the EKC buffer. This procedure resulted in improved peak shapes and a good migration time reproducibility range of 1.01.7% RSD, n ) 5. Preparation of EKC Buffers and Standard Solutions. For all EKC experiments, the BGE consisted of either dibasic sodium phosphate or borate buffer. Before the pH of these buffers was adjusted, an appropriate amount (% w/v) of poly-L-SUV or polyL-SUVV surfactant was added to the BGE. The desired pH values were achieved by adding either HCl or NaOH to the BGE containing the polymeric surfactant. After the pH was adjusted, the final EKC running buffers were filtered through a 0.45 µm nylon syringe filter (Nalgene, Rochester, NY) by creating a vacuum inside the syringe. This was followed by ultrasonication for about 10 min to ensure appropriately degassed EKC running buffers. All stock standard solutions were prepared in 50% (v/v) methanol/water at concentrations of about 1-2 mg/mL each. Appropriate dilutions of stock solutions were obtained with a 50% (v/v) methanol/water mixture. Calculations. The migration factors (k′), resolution factor (Rs), and N were calculated using the following equations:36

tr-t0 t0

(1)

2(tr2-tr1) w1 + w2

(2)

( )

(3)

k′ )

Rs )

N ) 5.54

tr w1/2

2

where to and tr are the respective migration times of the unretained species and the enantiomer; w is the peak width at the baseline of each enantiomer, designated as “1” and “2”, respectively, and w1/2 stands for the peak width at half-height. RESULTS AND DISCUSSION Physicochemical Properties of Monomeric Chiral Surfactants. Table 1 provides a comparison of the structural features and physicochemical properties of the sodium N-undecyl-L-valine (L-SUV) and sodium N-undecyl-L-valine-valine (L-SUVV) surfactants. The cmc for both chiral surfactants was measured by following a linear decrease of the surface tension with an increase in concentration of L-SUV or L-SUVV surfactants up to a point. After this point, no appreciable change in surface tension was observed. The point of intersection of the two lines (one linear and the other relatively flat) was taken as the cmc of the surfactants. The greater hydrophobicity of L-SUVV than that of L-SUV resulted in a relatively lower cmc for the former surfactant. (36) Terable, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113.

2982 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

Table 1. Comparison of the Physicochemical Properties of Undecylenyl-L-valine (L-UVal) and Undecylenyl-L-valine-valine (L-UVal-Val) Monomeric Surfactants characteristic molecular weight no. of stereogenic centers hydrophobic tail no. of amido groups no. of carboxylic groups critical micelle concentrationa (cmc) [mM] optical rotation [R]25D effective electrophoretic mobilityb (µep) [cm-2 V-1 s-1]

L-UVal

L-UVal-Val

305 1 1 1 11 13

404 2 1 2 1 10

-5.29° -1.5 × 10-4

-26.14° -1.2 × 10-4

a Determined in pure water using surface tension measurement. Determined using capillary zone electrophoresis/indirect photometric detection at 280 nm, using NMS as light-absorbing electrolyte in 100 mM boric acid and 5 mM Na2B4O7, pH 8.0. Methanol was used as the EOF marker. b

In addition, an increase in the molecular weight decreases the mobility of L-SUVV toward the injector end. Thus, L-SUVV elutes faster than L-SUV as it is carried more rapidly by the bulk electroosmotic flow (EOF) toward the detection window. Note that optical resolution is substantially enhanced by use of L-SUVV as compared to L-SUV surfactant. Selection of Buffer pH for EKC Separations. Buffer pH is an important factor in EKC separations of basic, acidic, and neutral optical isomers using polymeric ionic surfactants. This is because alterations in pH can affect the charge on the analyte and the chiral pseudophase. In addition, the polymer conformation and the EOF may also vary with changes in pH, thus influencing chiral resolution. In an attempt to optimize the pH for the separation of basic, acidic, and neutral enantiomers, detailed studies were conducted in which 0.5% (w/v) poly-L-SUV or poly-L-SUVV was added to phosphate or borate buffer in the pH range of 5.5-11.0 (data not shown). However, pH values below 5.5 were not applicable because the surfactants tended to precipitate out of solution, probably due to a decrease in the ionization of the carboxylate functionality of both poly-L-SUV and poly-L-SUVV surfactants. Based on our pH optimization study, enantioseparation of the cationic enantiomers PROP and ALP was found to be optimal at pH 9.2. In contrast, the anionic (BNP) and neutral (TFAE) enantiomers were best resolved at respective pH values of 7.0 and 10.2. Under these optimum pH conditions for the resolution of each of these enantiomers, further optimization experiments were conducted by varying the concentration and type of polymeric chiral pseudophase and the BGE concentration. The details of these optimization procedures are described in the following sections. Separation of Basic Enantiomers. PROP and ALP are two examples of basic drugs that are commonly called β-adrenergic blockers (β-blockers). These cationic drugs have been used for the treatment of hypertension.37 Typically, the (S)-(-)-enantiomer of these drugs is more potent than the corresponding (R)-(+)form, and the latter may also be toxic.38 PROP and ALP possess similar structural features: an alkanolamine side chain terminating (37) Israili, Z. H. In Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography; Wong, S. H. Y., Ed.; Chromatographic Science Series 32; Marcel Dekker, Inc.: New York, NY, 1985; Chapter 13, pp 367-374, (38) Nelson, W. L.; Burkes, T. R. J. Org. Chem. 1978, 43, 3641-3645.

Figure 2. Comparison of polymerized anionic surfactants for the separation of basic enantiomers. EKC conditions: 0.57% and 0.50% (w/v) of poly-L-SUV or poly-L-SUVV, respectively, in 50 mM Na2B4O7 buffered at pH 9.2. Peak identification: 0.2 mg/mL each of one (S)-(-)-ALP, (1′) (R)-(+)-ALP; 0.1 mg/mL each of (2) (S)-(-)-PROP, (2′) (R)-(+)-PROP. Pressure injection for 2 s; +20 kV applied for separation; current, 85 µA for poly-L-SUV and 56 µA for poly-L-SUVV. UV detection was at 214 nm.

in a secondary amino group and an aromatic group (for their structures see Figure 2 inset). The pKa value of the ionizable nitrogen is in the region of 9.2-9.6.39,40 Thus, it is not too surprising that the best pH for enantioseparation can be seen near the pKa of these analytes. As indicated in the previous section, a pH value of 9.2 was, indeed, the optimum for such separations. The electrokinetic data for separation of PROP enantiomers under analogous pH and BGE concentrations for various concentrations of poly-L-SUV and poly-L-SUVV are provided in Table 2. When chiral resolution is obtained, the k′ and N values given in Table 2 are those of the first enantiomers of PROP. As expected for any electrokinetic separation, increasing the concentration of the polymeric chiral anionic pseudophase (poly-L-SUV or poly-LSUVV) increases the k′ for PROP. This is directly related to an increase in hydrophobic and electrostatic interactions of this positive charged racemate with the polymeric anionic surfactant. Although poly-L-SUVV is more hydrophobic than poly-L-SUV, the use of the former polymer almost always produced separations of PROP enantiomers with shorter k′ values and much improved resolutions under equivalent monomer concentrations. Similar trends for k′ were also seen for ALP enantiomers (data not shown). Baseline resolutions (Rs >1.5) with the highest possible N values were obtained for both PROP enantiomers at 0.5% (w/v) poly-L-SUVV. In contrast, no Rs for PROP was possible at any concentration of the poly-L-SUV surfactant. Higher concentrations (>1% (w/v)) of either polymer did not improve the Rs but led to markedly longer k′ values. Figure 2 compares the electrokinetic (39) Marko, V., DeZeeiew, R. A., Eds. Determination of Beta Blockers in Biological Material; Elsevier: Amsterdam, 1989; p 77. (40) Laxer, M.; Capomacchia, A. C.; Hardee, G. E. Talanta 1981, 28, 973.

Table 2. Comparison of Migration Factors, Resolution, and Efficiency for Propranolol Enantiomers, Obtained Using Various Concentrations of Poly-L-SUV and Poly-L-SUVV Surfactantsa concentration [% (w/v)] 0.075 poly-L-SUV 0.10 poly-L-SUVV 0.19 poly-L-SUV 0.25 poly-L-SUVV 0.38 poly-L-SUV 0.50 poly-L-SUVV 0.57 poly-L-SUV 0.75 poly-L-SUVV 0.76 poly-L-SUV 1.00 poly-L-SUVV

equivalent monomer concn [mM]

} } } } }

2.5 6.2 12.4 18.6 24.8

k′

Rs

N

0.63 {0.64 1.69 {1.36 3.20 {2.03 4.80 {2.53 7.02 {2.99

0.0 1.1 0.0 1.5 0.0 2.3 0.0 1.7 0.1 1.4

107 500 104 060 113 500 186 500 114 200 290 000 116 600 248 600 127 400 231 700

a Using 50 mM Na B O buffered at pH 9.2. Pressure injection for 2 4 7 2 s (0.1 mg/mL) for propranolol. Separation voltage, +20 kV; current, 50-87 µA. Detection was at 214 nm.

chromatograms for the simultaneous separations of the ALP and PROP enantiomers using optimized concentrations of poly-L-SUV and poly-L-SUVV surfactants. Using either of these surfactants, (()-ALP and (()-PROP eluted in order of increasing hydrophobicity. However, the (S)-(-)-enantiomer of each racemate always eluted faster than the corresponding (R)-(+) form. Thus, the migration times and order are a direct consequence of the analyte/ surfactant binding. Furthermore, the electrokinetic chromatogram (Figure 2a) clearly shows that an increase in the migration time for both (()-ALP and (()-PROP enantiomers using poly-LSUV does not lead to enhanced chiral separations. Our results Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

2983

Table 3. Comparison of Migration Factors, Resolution, and Efficiency for Alprenolol and Propranolol Enantiomers, Obtained Using Various Concentrations of Borate Buffera concn [mM]

k′

Rs

N

5.0 12.5 25.0 50.0 75.0 100.0

Alprenolol 1.06 1.12 1.19 1.39 1.44 1.57

0.2 0.5 0.7 1.5 1.2 0.9

319 600 329 900 155 540 136 000 75 300 65 800

5.0 12.5 25.0 50.0 75.0 100.0

Propranolol 1.14 1.30 1.53 2.03 2.44 3.01

0.3 0.5 0.9 2.3 2.5 2.6

550 600 650 200 312 700 290 000 194 800 104 600

a Using the optimized 0.5% (w/v) poly-L-SUVV containing variable concentration of Na2B4O7 buffered at pH 9.2. Pressure injection for 2 s, 0.2 mg/mL for alprenolol and 0.1 mg/mL for propranolol. Separation voltage, +20 kV; current, 10-102 µA. Detection was at 214 nm.

are consistent with the equilbrium model studies described by Allenmark41 and Krstulovic,42 as well as Wren and Rowe.43,44 All of these researchers suggest that the success of chiral recognition depends on the strength of association of enantiomers with the selector, the differntial association between the enantiomers, and the concentration of the selector used. The much improved chiral resolution with decreased migration time of cationic racemates using poly-L-SUVV suggests that the retention mechanism and the chiral recognition for such analytes are controlled by steric factors rather than by the hydrophobicity of the chiral pseudophase. Probably, a relatively milder electrostatic attraction between the anionic poly-L-SUVV surfactants and the cationic racemates results in a lower k′ value and better chiral discrimination than those obtained with poly-L-SUV surfactants. In addition, it is reasonable to conclude that an increase in the number of stereogenic centers and hydrogen bonding sites on the ionic head group in poly-L-SUVV may have also contributed to this superior chiral discrimination over poly-L-SUV surfactant. The k′, Rs, and N values of both ALP and PROP enantiomers were further optimized by varying the concentration of borate buffer serving as the BGE. As shown in Table 3, the borate concentration ranging from 5 to 100 mM has some effect on these parameters. Initially, as the borate concentration was increased from 5 to 50 mM, the k′ and Rs values for the two cationic racemates increased at the expense of some decrease in N. Further increases in the concentration of borate in the 75-100 mM range decreased both Rs and N for ALP enantiomers. In contrast, a slight improvement in Rs for PROP enantiomers was noted. This reduction in Rs and N can be explained on the basis of the observed increase in the peak width. Consequently, this band broadening effect is caused by a reduction in EOF and an increase in the viscosity of the electrolyte. Joule heating at high ionic strength is another factor that contributes to a decrease in (41) Allenmark, S. Chromatographic Enantioseparation; Ellis Horwood: New York, 1991; Chapter 5, pp 82-83. (42) Krstulovic, A. M. Chiral Separations by HPLC; Ellis Horwood: New York, 1989; Chapter 16, pp 493-497. (43) Wren, S. A. C.; Rowe, R. C. J. Chromatogr. 1992, 603, 235-241. (44) Wren, S. A. C.; Rowe, R. C. J. Chromatogr. 1992, 609, 363-367.

2984 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

Rs and N values. This was experimentally confirmed by plotting current as a function of applied voltage at 100 mM borate. The plot indicated significant deviation from linearity around 12 kV. In general, a borate buffer concentration of 50 mM is optimal for the Rs of ALP and PROP enantiomers. This is because such concentrations do not result in an excessive increase in k′ and a significant loss in N values. In an attempt to improve the signal-to-noise ratio (S/N) of ALP and PROP enantiomers, experiments were conducted to determine the injection time that can be conveniently employed without sacrificing Rs. Figure 3 shows that a tradeoff exists between Rs and S/N. Loading less analyte on the capillary substantially improves the Rs between the enantiomers. However, the S/N deteriorates with longer injection time. From the figures, it is apparent that a different optimal injection size is required in order for each racemate to be sufficiently resolved, depending on the molar absorptivity of the racemate. This is because the racemate with the higher molar absorptivity can be injected at lower concentrations and thus improve the Rs. For instance, it is quite clear that the separation of the ALP enantiomers injected at concentrations of 0.2 mg/mL began to degrade rapidly without any improvement in S/N around an injection time of 6 s. In contrast, the separation of PROP enantiomers injected at concentrations of 0.1 mg/mL using the same injection size is still quite reasonable. However, as the injection time of PROP enanatiomers was increased above 12 s, peak fronting was very pronounced, and the Rs degrades significantly (electrokinetic chromatogram not shown). Separation of Acidic Enantiomers. Many molecules are chiral even without an asymmetric carbon center. As an example, atropisomeric binaphthyl compounds such as BNP belong to a class of molecules that are chiral because they possess an adjacent π system that cannot adopt a coplanar configuration due to steric hindrance and rotational restrictions around a central bond (see Figure 4 inset). The (()-BNP has been widely used as a chiral shift reagent45 for determining the enantiomeric purity of many organic compounds.45,46 In addition, its functions as ligands for dissymetric catalyst47 and as building blocks in the synthesis of macrocylic compounds have been reported.48 The enantiomers of (()-BNP predominantly exist in the anionic form at the optimized pH of 7.0 used in this work. In a second series of experiments, chiral separations of negatively charged (()-BNP were compared using poly-L-SUV and poly-L-SUVV surfactants at an optimized pH value of 7.0. Table 4 shows the influence of the type and concentration of polymeric chiral pseudophase on the k′, Rs, and N values of BNP enantiomers. Again, increases in k′ and Rs with increasing concentration of poly-L-SUV or poly-L-SUVV were observed, indicating that the negatively charged (()-BNP has a more or less pronounced tendency to interact with these polymerized anionic surfactants. However, note that, at each equivalent monomer concentration, better Rs values were obtained with the dipeptide than with the use of the single amino acid surfactant. For example, (()-BNP showed baseline Rs even at 0.25% (w/v) poly-L-SUVV, whereas (45) Jacques, J.; Fouquey, C. Tetrahedron Lett. 1971, 48, 4617-4620. (46) Arnold, W.; Daly, J. J.; Imhof, R.; Kyburz, E. Tetrahedron Lett. 1983, 24, 343-346. (47) Shao, L.; Miyata, S.; Muramatsu, H.; Kaqano, H.; Ishii, Y.; Saburi, M.; Uchida, Y. J. Chem. Soc., Perkin Trans. 1990, 1 (5), 1441-1445. (48) Castro, P. P.; Georgiaddis, T. M.; Diederich, F. J. Org. Chem. 1989, 54, 5835-5839.

Figure 3. Effect of analyte injection size on resolution and S/N ratio. EKC conditions are the same as in Figure 2, except only poly-L-SUVV was used and the indicated injection size was varied.

Table 4. Comparison of Migration Factors, Resolution, and Efficiency for Binaphthol Phosphate (BNP) Enantiomers Using Various Concentrations of Poly-L-SUV and Poly-L-SUVV Surfactantsa concentration [% (w/v)] 0.19 poly-L-SUV 0.25 poly-L-SUVV 0.38 poly-L-SUV 0.50 poly-L-SUVV 0.76 poly-L-SUV 1.00 poly-L-SUVV 1.13 poly-L-SUV 1.50 poly-L-SUVV 2.26 poly-L-SUV 3.00 poly-L-SUVV

equivalent monomer concn [mM]

} } } } }

6.2 12.4 24.8 37.2 74.4

k′

Rs

N

1.25 {1.46 1.65 {1.87 2.46 {2.67 3.42 {3.81 10.43 {11.30

0.1 1.5 0.5 2.2 0.7 3.0 1.2 3.2 0.6 3.1

101 000 114 000 135 000 140 000 167 000 172 000 175 000 87 400 69 000 60 000

a Using 50 mM phosphate (NaH PO /Na HPO ) buffered at pH 7.0. 2 4 2 4 Pressure injection for 4 s (0.1 mg/mL) for BNP racemate. Separation voltage, +20 kV; current, 44-78 µA. Detection was at 214 nm.

no Rs of the same enantiomers was possible at equivalent concentrations of poly-L-SUV. Moreover, Table 4 clearly shows that, for each polymeric surfactant, there is an optimum concentration at which chiral Rs reaches a maximum value. A maximum Rs of 3.2 was obtained for (()-BNP at 1.50% (w/v) poly-L-SUVV. However, the same enantiomers were best resolved (Rs ) 1.2) using 1.13% (w/v) poly-L-SUV. Further, an increase in concentration of poly-L-SUV (i.e., g2.26% w/v) drastically decreased the enantiomeric Rs of (()-BNP. This is in contrast to an increase in concentration (i.e., g3.0% w/v) of poly-L-SUVV surfactants, which shows either very low or no decrease in Rs values. It seems noteworthy that, for enantiomers of (()-BNP, when using poly-L-SUVV, the k′ values (reported in Table 4) are higher than those of poly-L-SUV at equivalent monomer concentrations

of the surfactants. This is consistent with the theory of EKC in which an increase in the hydrophobicity of the pseudostationary phase should increase the k′ values. However, such k′ data for anionic enantiomers are in sharp contrast to the k′ values reported in Table 2 for cationic enantiomers. Typical electrokinetic chromatograms obtained under the optimized Rs values of 1.2 and 3.0 for (()-BNP using poly-L-SUV and poly-L-SUVV surfactants, respectively, are displayed in Figure 4. Again, much better separation with higher Rs and selectivity was obtained with poly-L-SUVV than with poly-L-SUV. The successful enantioseparation of negatively charged (()-BNP obtained with anionic polymerized surfactant confirms that, although electrostatic attractive interactions can contribute in the binding of charged analytes with oppositely charged polymerized surfactants, these interactions do not always seem to be the major force for chiral recognition. The highly hydrophobic naphthyl moiety and the hydrogen-bonding capability of the phosphate group in (()-BNP are probably major factors in chiral discrimination using polymeric anionic pseudostationary phases. The effect of phosphate buffer as a BGE on poly-L-SUVV surfactant was also investigated. Table 5 shows that the Rs values for (()-BNP enantiomers can be improved to as high as 4.5 with an increase in the phosphate buffer concentrations. However, this occurs only at the expense of longer analysis time and a reduction in N. Concentrations of 25-50 mM are the best working range since the Rs is more than sufficient with a reasonable analysis time. However, note that much faster separation with a value of Rs ) 1.8 and an analysis time under 10 min (k′ ) 1.10) for (()BNP is still possible, even when the concentration of the phosphate buffer in the polymer solution was as low as 2.5 mM. The reversal of the elution order of the two enantiomers is also critical, particularly when trace-level enantiomeric impurities Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

2985

Table 6. Comparison of Migration Factors, Resolutions, and Efficiencies for Trifluoroanthryethanol (TFAE) Enantiomers, Obtained Using Various Concentrations of Poly-L-SUV and Poly-L-SUVV Surfactantsa concentration [% (w/v)] 0.075 poly-L-SUV 0.10 poly-L-SUVV 0.19 poly-L-SUV 0.25 poly-L-SUVV 0.38poly-L-SUV 0.50 poly-L-SUVV 0.76 poly-L-SUV 1.00 poly-L-SUVV

equivalent monomer concn [mM]

} } } }

2.5 6.2 12.4 24.8

k′

Rs

N

3.56 {3.66 4.62 {4.83 7.23 {8.02 8.10 {8.79

1.9 2.1 2.1 2.1 2.5 2.1 2.0 1.7

38 450 59 800 46 200 59 380 49 200 61 530 40 600 55 700

a Using 50 mM sodium borate (Na B O ) buffered at pH 10.2. 2 4 7 Pressure injection for 4 s (0.1 mg/mL) for TFAE racemate. Separation voltage, +20 kV; current, 78-86 µA. Detection was at 254 nm.

Figure 4. Comparison of polymerized anionic surfactants for the separation of acidic enantiomers. EKC conditions: 1.13% and 1.00% (w/v) of poly-L-SUV or poly-L-SUVV, respectively, in 50 mM phosphate (Na2HPO4/NaH2PO4) buffered at pH 7.0. Peak identification: 0.1 mg/ mL each of (1) (S)-(-)-BNP, (1′) (R)-(+)-BNP. Pressure injection for 4 s; +20 kV applied for separation; current, 64 µA for poly-L-SUV and 50 µA for poly-L-SUVV. UV detection was at 214 nm. Table 5. Comparison of Migration Factors, Resolution, and Efficiency for Binaphtholphosphate (BNP) Enantiomers, Obtained Using Various Concentrations of Phosphate Buffera concn [mM]

k′

Rs

N

2.5 5.0 10.0 25.0 50.0 100 150

1.10 1.17 1.33 2.29 2.67 5.21 9.75

1.8 1.9 2.2 3.0 3.0 3.7 4.5

200 000 201 000 225 000 190 000 172 000 90 000 47 000

a Using the optimized 1% (w/v) poly-L-SUVV containing equimolar concentration of phosphate (NaH2PO4/Na2HPO4), buffered at pH 7.0. Pressure injection for 4 s (0.1 mg/mL) for BNP. Separation voltage, +20 kV; current, 14-200 µA. Detection was at 214 nm.

are to be detected. In CE, parameters such as pH and use of chiral selectors with opposite configurations (D and L) have been studied for reversal of the migration order.31,49 Another cause of reversal of migration order is the use of coated capillaries with suppressed or zero EOF. The separation of (()-BNP in a zero EOF environment using poly(vinyl alcohol)-coated capillary was studied (electropherogram not shown). Although the Ld of the coated capillary was about 15 cm shorter, the analysis time was (49) Schmitt, T.; Engelhardt, H. J. Chromatogr. A 1995, 697, 561-570.

2986 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

10 min faster than that on an uncoated capillary. This is probably due to the use of a negative polarity on the power supply. Under this configuration, both poly-L-SUV and the analyte ((()-BNP) have a natural mobility toward the anodic end (i.e., detector end used in negative polarity CE). In addition, it was also found that the use of the shorter wavelength of 214 nm improves the S/N of (()-BNP by factors of 10 and 8 as compared to those 254 and 280 nm, respectively (data not shown). This is obviously due to the higher molar absorptivity of (()-BNP at 214 nm. However, baseline stability was not as good at this wavelength as for the use of 254 or 280 nm due to some background absorbance generated by the poly-L-SUVV. Separation of Neutral Enantiomers. (()-TFAE (see Figure 5 inset) was selected as an example of a neutral racemate in order to compare the effect of poly-L-SUV and poly-L-SUVV surfactants on chiral separation. The enantiomers of TFAE have been used as chiral NMR solvating agents for the discrimination of enantiomeric purity of optically active compounds.50 Table 6 compares the effects of both chiral polymeric surfactant concentrations (at optimized pH and BGE concentrations) on k′, Rs, and N values for (()-TFAE. In general, both k′ and N for (()-TFAE increase gradually with an increase in concentration of both polymers. Note that, at equivalent monomer concentrations, the values of k′ and N for (()-TFAE are higher with poly-L-SUVV than those with polyL-SUV. However, enantiomeric Rs increased only slightly with increasing concentrations of poly-L-SUV from 0.075 to 0.38% (w/v) and remained unchanged for poly-L-SUVV in the concentration range of 0.10-0.50% (w/v). Further increases in concentration of both polymers decrease the Rs of (()-TFAE. Figure 5 compares the separation of (()-TFAE at optimized Rs values of 2.5 and 2.1 using poly-L-SUV and poly-L-SUVV surfactants, respectively. It is interesting to note that the higher Rs of (()-TFAE obtained using poly-L-SUV, as compared to that obtained using poly-L-SUVV, occurred only at the expense of longer analysis times and lower electrokinetic efficiencies. CONCLUSIONS Enantioselective separations of two cationic analytes (PROP and ALP), one anionic analyte (BNP), and one neutral analyte (TFAE) were compared with those of a single amino acid (poly(50) Francotte, E. J. Chromatogr. A 1994, 666, 565-601.

Figure 5. Comparison of polymerized anionic surfactants for the separation of neutral enantiomers. EKC conditions: 0.38% and 0.10%(w/v) of poly-L-SUV and poly-L-SUVV in 50 mM Na2B4O7 buffered at pH 10.2. Peak identification: 0.1 mg/mL each of (1) (-)-TFAE, (1′) (+)-TFAE. Pressure injection for 4 s; +20 kV applied for separation; current, 82 µA for poly-L-SUV and 78 µA for poly-L-SUVV. UV detection was at 254 nm.

L-SUV)

vs a dipeptide (poly-L-SUVV) surfactant. Faster migration times with enhanced resolution of the cationic enantiomers by poly-L-SUVV as compared to poly-L-SUV suggest that chiral recognition is predominantly controlled by steric factors rather than by the hydrophobicity of the surfactants. In contrast, increasing hydrogen bonding and hydrophobic interactions of poly-L-SUVV over poly-L-SUV seems to be the dominant factor in enhancing the chiral recognition of anionic enantiomers. Surprisingly, for the neutral racemate, poly-L-SUV showed slightly better chiral discrimination. However, this data set is too limited to allow us draw any specific conclusions in this regard. Furthermore, this occurred only at higher concentrations of poly-L-SUV, which resulted in excessively longer analysis times with reduced efficiencies. We are currently extending this approach further to address the following questions. First, does chiral recognition for amino acids vs dipeptide in EKC vary upon changes in the structural features of the racemates or the type of amino acid/dipeptide surfactants? In other words, we would like to know whether the enhancement of chiral recognition that we have observed with dipeptide surfactants for the above-mentioned analytes is fortuitous and whether it is analyte and surfactant dependent. Preliminary data from ongoing experiments suggest that these observations

are not fortuitous. In some of our ongoing experiments, we are currently studying the chiral discrimination behavior of a variety of acidic, basic, and neutral racemates with diverse structural features. A second important question we would like to answer is whether the order of linking of amino acids in dipeptide surfactants is important in terms of chiral recognition. So far, we have noted some interesting success with this approach. Finally, a very important question needs to be answered: Does an increase in the number of chiral centers on the polar head groups of our pseudostationary phases always produce better chiral recognition ability? ACKNOWLEDGMENT Support of this work was made possible through a grant from the National Institutes of Health (GM39844). I.M.W. also acknowledges the Philip W. West endowment for partial support of this research. Received for review January 13, 1997. Accepted May 9, 1997.X AC970037A X

Abstract published in Advance ACS Abstracts, June 15, 1997.

Analytical Chemistry, Vol. 69, No. 15, August 1, 1997

2987