Combined Chiral Crown Ether and [3-Cyclodextrin for the Separation of o-, m-, and p-fluoro-d,l-phenylalanine by Capillary Gel Eiectrophoresis

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1 Combined Chiral Crown Ether and [3-Cyclodextrin for the Separation of o-, m-, and p-fluoro-d,l-phenylalanine by Capillary Gel Eiectrophoresis J.-M. Lin* / T. Nakagama / T. obo Department of ndustrial Chemistry, Faculty of Engineering, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, achioji-shi, Tokyo , Japan Key Words Capillary gel electrophoresis (CGE) Chiral separation Fluorophenylalanine 18-Crown-6-tetracarboxylic acid ~-Cyclodextrin Summary A combination of (13-CD) with a chiral crown ether, 18- crown-6-tetracarboxylic acid (18C6TA), has been used for the chiral capillary gel electrophoretic (CGE) separation of the six enantiomers and positional isomers in a mixture of o-, m-, and p-fluoro-d,l-phenylalanine; the isomers were successfully resolved in 0.3 % agarose -2 x 10-2 mol L -1 18C6TA -2 x 10-2 mol L CD. The method has also been successfully extended to the separation of the enantiomers of 13 other amino acid or primary amine substrates. The role of the chiral crown ether was studied. The effects of the concentrations of 18C6TA, 13-CD and agarose, and of p and applied field strength on the migration time and separation factors of the analytes are reported. ntroduction XVith the increasing demand for optically pure amino COmpounds in many fields, the need for rapid resolution of enantiomers with high enantiomeric purity has prompted development of new resolution methods. The separation of enantiomers has been investigated by liquid chromatography [1, 2], gas chromatography [3, 4] and other methods. n the past few years the use of capillary electrophoresis (CE) has grown rapidly, and many new applications have been introduced in the field of analytical separations [5]. Some reviews on the application of CE for the separation of chiral compounds have also been published [6-10]. Most of this research has been based on the use of cyclodextrins or their derivatives; until now it is also very difficult to separate compounds with both positional and optically active isomers. The crown ethers, macrocyclic molecules were discovered by Pederson in 1967 [11, 12], selectively incorporate cations into their cavities [11-13]. They bind with high selectivity and affinity, not only with alkaline cations [14] but also with protonated amines [15-21]. As shown in Figure 1 A for complexes with R-N3 the substrate is anchored in the center and top of the ring and the R group extends above the macrocycle. Theoretical [22, 23] and experimental results [24, 25] indicate that the positive charge is mainly distributed over the hydrogen atoms in N4 + and R-N3 + groups. The binding scheme may be considered to involve an array of three linear N-a+...-O bonds completed by a set of six electrostatic interactions between the partial charges on the atoms with the neighboring O atoms, which may also be taken as six weaker, bent bonds [26, 27]. n such complexes the interactions between ammonium and crown ether acid can be schematically distinguished as: (1) central recognition at the anchoring site, and (2) lateral recognition by the interaction between the lateral branches -COO of 18C6TA and R of RN3 + (see the example in Figure 1B). Furthermore, when the substrate and receptor are both chiral, enantiomeric differentiation may be observed upon complexation. The crown ether used in this research displays only one type of recognition. Ammonium or primary amines held inside the cavity are bound by three hydrogen bonds in a tripod arrangement. Because these hostguest complexes do.not show chiral recognition, however, secondary lateral interactions between the sub- Original /96/ $ 03.00/0 Chromatographia Vo. 42, No. 9/10, May Friedr. Vieweg & Sohn Verlagsgesellschaft mb 559

2 X, ~ O. ~,X f ', +N'R, ' T X r ~'0.ri,,,]-. 9 0~ $~X (A) wavelength was either 254 nm or 235 nm, depending on the absorbance spectrum of the analyte. The separation temperature was maintained at approximately 20 ~ Column cooling was achieved using a laboratory fan. Most experiments were performed using 0.01 tool L -1 Tris, adjusted to p 2.5 with citric acid, containing 0.02 mol L crown-6-tetracarboxylic acid and 0.02 mol L cyclodextrin. o O..o-<o [ o~o (B) Figure 1 Schematic representations of (A) the +3NR complex and (B) the complex of 18C6TA with o-fiuoro-l-phenylalanine. stituents of the crown ether ring and the ligands are necessary for chiral discrimination. Chiral recognition might occur either by a steric barrier mechanism or by hydrogen bonding between the guest molecule and the carboxylic acid groups on the crown ether. The use of a crown ether for the capillary electrophoretic separation of racemic amino acids was first proposed by Kuhn et al. [28-30]; they reported the separation of more than 22 amines, including many amino acids, by capillary zone electrophoresis (CZE) with 18-crown-6-tetracarboxylic acid at p 2.5. oehne et al. [31] reported the resolution of the enantiomers of amino alcohols by CZE using the same crown ether at a lower p. n this work, agarose gel incorporating 18C6TA and 15- cyclodextrin was used for capillary electrophoresis. t was very interesting to find that the method can be used for the separation of the optical isomers and positional isomers. The separation and detection of some other amino acid enantiomers was also demonstrated. Experimental CE nstrumentation The instrumentation comprised a UV-8011 detector (Tosoh, Japan), a regulated high-voltage power supply (ER-30R-LCDW, Matsusada Precision Devices. nc., Japan) and a C-R5A Chromatopac (Shimadzu, Japan). Electrophoresis was performed in 30 cm x 50/.tm i.d. fused silica capillary tubing (GL Sciences, Tokyo, Japan). Sixteen cm from the end, approximately 0.5 cm of polyimide coating was carefully burned off to expose the fused silica for on-column detection. The detection Chemicals Chemicals were reagent grade, unless otherwise stated. Agarose LE (low electroendosmosis), o-, m-, and p- fluoro-d,l-phenylalanine, D,L-tryptophan, ~- cyclodextrin, D,L-1fhenylalanine, and 18-crown-6- tetracarboxylic acid (18C6TA) were obtained from Kanto Chemical Co. nc. (Tokyo, Japan). D,Lnoradrenaline and D,L-3-(3,4-dihydroxyphenyl)alanine were purchased from Nakarai Chemicals (Kyoto, Japan). D,L-tyrosine, D,L-a-phenylglycine, R,S-(+)-- (1-naphthyl)ethylamine, citric acid, and sorbitol were obtained from Tokyo Kasei (Japan). All other enantiomers were obtained from Sigma (USA). Water was purified from tap water using an Aquarius Automatic Water Distillation apparatus (Advantec Toyo, Japan). Buffer solution was filtered through a 0.1-~tm pore-size filter (Advantec Toyo) and vacuum degassed before use. Vacuum degassing of the agarose solution was followed by a helium-purge step in order to minimize the amount of dissolved oxygen. Capillary Electrophoresis Before filling with agarose gel, the capillaries were washed with 0.1 mol L -1 NaO solution, water, and electrophoretic buffer solution, each for 10 min. The agarose gel was prepared by dissolving agarose (0.30 g) in Tris-citric acid (0.01 mol L-l; 100 ml) buffer solution containing 18C6TA (0.02 mol L -1) and 15-CD (0.02 mol L -1) at 85 ~ n order to improve the stability of the gel, a small amount of sorbitol (0.01%) was added to the reaction mixture. After filling with the solution of agarose and the chiral selectors, the capillaries were cooled at ambient temperature for approximately 2 h before use. The samples were typically electrokinetically injected for 5 s under a field of 250 V cm -1. The applied field strength for electrophoresis is indicated in the respective figure legends. Samples were dissolved in the separation buffer or deionized water at a concentration of 5 x x 10-4 mol L -1. The resolution, Rs, was defined as: R s = 2x [(t 2 - tl)/(w + w2) ] 0) and the chiral separation factor, a, as: a = t2/tl (2) where t is the migration time and w the peak base width. 560 Chromatographia Vol. 42, No. 9/10, May 1996 Original

3 lz 1.2 Figure 2 Dependence of separation factor (ct) on 18C6TA concentration in the gel and buffer solution. Test mixture: (1) D,L-tryptophan, (2) ~ (3) m-fluoro-d,l- phenylalanine. _Buffer: 0.01 m'ol L -q Tris-citric acid (p = 2.5); gel: 0.3 % agarose, 0.02 rnol L -1 ~-CD, 0.01 mol L -1 Tris - citric acid (p = 2.5); Capillary: 160 mm x 0.05 mm i.d., 300 V cm-1; Electrokinetic injection: 250 V cm -1, 5 s; Detection wavelength: 254 nm. 1o o (1) S0 Concentration of 18C6TA (xl0 "3 mol! "1) 9 ~ 1 m i i i ~ s i / Migration time (min) i~igure 3 Effect of 13-cyclodextrin concentration on the separation of o-, m-, and p-fluoro-d,l-phenylalanine. 1. o-fluoro-l-phenylalanine; 2, o-fluoro-d-phenylalanine; 3, m-fluoro-l-phenylalanine; 4, m- ~Uoro-D-phenylalanine; 5, p-fluoro-llphenylalanine; 6. p-fluoro- _~ Buffer: 0.01 mol L- Tris-citric acid containing tj.02 rnol L -1 18C6TA (p = 2.5); gel: 0.3 % agarose in 0.01 mol L' Tris - citric acid containing 0.02 mol L -1 18C6TA (p = 2.5);.Capillary: 160 mm 0.05 mm i.d., 300 V cm-1; Electrokinetic in- JeCtion: 250 V cm -1, 5 s; Detection wavelength: 254 rim. d t 3 d~ 6 Results and Discussion Effect of the Concentration of 18C6TA n our initial attempt to separate the mixture of o-, m-, and p-fluoro-d,l-phenylalanine by capillary electrophoresis with only 13-cyclodextrin incorporated in the agarose gel, only four peaks were observed, although the enantiomers of the individual positional isomers were readily separated. As shown in Figure 2, it was of interest to find that the separation factors started to increase on addition of 18C6TA to the gel and buffer solution. Similar behavior is also apparent from Figure 3 if the migration times of the enantiomers are measured and plotted as a function of concentration of 13-cyclodextrin in the electrophoretic buffer and gel. The combination of 2 x 10-2 mol L -1 18C6TA with 0.01 mol L -1 ~-cyclodextrin led to almost complete separation of all six o-, rn-, and p-positional isomers of fluorophenylalanine. From Figure 2 it is apparent that the separation factors (ct) tend to plateau at a high 18C6TA concentrations. The migration times also increase with increasing concentrations of 18C6TA and the peak shapes became poorer. The higher the concentration of 18C6TA, the higher the electrophoretic current, possibly because of an increase in the internal temperature of the capillary; this resulted in reduced lifetime of the agarose gel capillary. An 18C6TA concentration of 0.02 mol L -1 was chosen for this work. Otherwise mixed samples of o-, m-, and p-fluoro-d,lphenylalanine could not be completely separated using only 18C6TA as chiral selector, although the optical isomers of the individual positional isomers of fluorophenylalanine were well separated. A synergistic effect [6] was observed when an appropriate amount of 13-cyclodextrin was added to the electrolytic buffer and gel containing 18C6TA. As shown in Figure 3, with no cyclodextrin in the buffer and gel, the influence of the crown ether on the separation of the structural isomer was small. This result is in accord with the suggestion of Kuhn et al. [6]. t is obvious that when the 13-cyclodextrin concentration is increased, the resolution improves. The peak width was also affected by the concentration of 13-cyclodextrin; the peaks became broad when its concentration was more than 0.03 mol L -1. Migration times increased with increasing concentrations of ]-cyclodextrin. A [3- cyclodextrin concentration of 0.02 mol L -1 was, therefore, selected for the following experiment. Effect of p Many papers have reported the effect of p on the separation of enantiomers by CE. Almost all the results indicated that p was one of the most important experimental parameters in c hiral recognition. Rawjee and coworkers [32] proposed an equilibrium model to describe the relationship between p and the concentration of 13-cyclodextrin in the electrolyte buffer solution in the enantioseparation of chiral weak acids. n Original Chromatographia Vol. 42, No. 9/10, May

4 p=2,0 p=2.5 p=3.0 s ! 2 p= S 3 S A Mil~ration time (min) Figure 4 Effect of electrolyte buffer p on the separation of fluorophenylalanine using 18C6TA and ~--cyclodextrin as chiral selectors. CE parameters: 300 V cm -1 electrode voltage. 20 ~ capillary temperature; electrolyte buffer was 0.01 mol L-'Tris-citric acid containing 0.02 mol L-' 18C6TA and 0.02 mol L -' 13-cyclodextrin (p = 2.5). Other conditions as for Figure 2. 1, o-fluoro-l-phenylalanine; 2, o-fluoro-d-phenylalanine, 3, m-fluoro-l-phenylalanine; 4, m-fluoro-d-phenylalanine; 5, p-fluoro-l-phenylalanine; 6, p-fluoro-d-phenylalanine. this work, although the 13-cyclodextrin was also employed as chiral selector for the separation of amino acid enantiomers, another selector, 18C6TA was combined with 13-cyclodextrin in agarose gel and buffer solution to achieve full separation of o-, m-, and p-fluoro- D,L-phenylalanine. This made our system more complex because both 18C6TA and the samples are weak acids and their form in solution thus depends on p. The dissociation constants of 18C6TA [33] are 4.88, 4.29, 2.84 and 2.13, and the m-amino acids have pka values for the acid moiety of approximately These data indicate that higher p does not benefit chiral recognition. At low p, however, conductivity increases rapidly, which is also unsuitable for separation. We examined the effect on the resolution of the samples of changing the operating buffer p value over the range 2.0 to 4.0. As shown in Figure 4, when the p exceeds 3.0, the mixture of fluorophenylalanine isomers cannot be well separated because at high p the carboxylic acid groups of 18C6TA lose some of their hydrogen atoms, resulting in weakening of the interaction of 18C6TA with the analytes. The signal peak shapes also became broad and asymmetric. Taking into consideration the other samples in our experiment, the p was controlled between 2.2 and 2.5 which should be the most acceptable for all analytes. Effect of Agarose Concentration on the Separation of the Fiuorophenylalanine somers Although polyacrylamide gels have been the most commonly used gel in capillary get electrophoresis, the preparation of polyacrylamide gel results in a gel containing ammonium peroxydisulfate and acrylamide, which will complex with 18C6TA. n order to enhance selectivity in the separation of fluorophenylalanine, an agarose gel was used in this experiment. t was found that the resolution obtained with agarose was better than that obtained by CZE. This may be because the electroosmotic flow is suppressed by the agarose gel, an hypothesis supported by the observation that when the concentration of agarose is increased the electrophoretic current becomes smaller, mgarose concentrations of 0.3, 0.4, and 0.5 % were evaluated for the separation of the fluorophenylalanine isomers. ncreasing the agarose concentration increases the resolution to some extent, but the preparation of the agarose-gelfilled capillary becomes more difficult because of the viscosity of agarose solution. An agarose concentration of 0.3 % was recommended for this work. Effect of the Applied Field Strength The effect of the applied field strength on the resolution was studied for the separation of D,L-tryptophan, o- fluoro-d,l-phenylalanine and m-fluoro-d,l-phenylalanine. The electrophoretic conditions were the same as for Figure 3 except that the p was 2.5. CGE was performed at 4, 6, 8, 10, 11, and 14 kv. The results showed that a higher applied field strength resulted in a larger 562 Chromatographia Vol.42,No.9/10,May 1996 Original

5 ~}... L. 25..'"" ""m.~, 1 B.~...m"" "'" o so ~ -. 5, i i i ~ Field strength (kv) Figure 5 nfluence of the applied field strength on the migration time (A, O, ~l, <> ) and resolution (~9 ~ ). 1, o-fluoro-l-phenylatanine; 2, ~ 3, L-tryptophan; 4, D-tryptophan; R, o- fluoro-d,l-phenylalanine; R2, D,L-tryptophan. Conditions as for Figure 4 except for buffer p = 2.5. electrophoretic current. According to Nelson et al. [34], the contribution of the radial temperature gradient-- caused by Joule heating to the total dispersion must be negligible in this work because the capillary dimensions Were mm i.d. and mm o.d. As a result of the Use of an air-cooling fan, the power consumed in the capillaries never exceeded 1.0 W m -1 (the currents ranged from 5 pa to 20 ~ta only). Figure 5 shows that the resolution obtained was optimum at 330 V cm -1 and that poor separation resulted from the use of a higher R2 2.5 voltage. This may be because the interaction of the analytes with 18C6TA and cyclodextrin is a diffusioncontrolled process, which requires sufficient separation time, and so migration velocities which were too high were not good for the resolution. Resolution of other Enantiomers The method was also successfully employed for the resolution of some primary amine enantiomers, including D,L-noradrenaline, D,L-l-(1-naphthyl)ethylamine and other protein amino acids. Table shows the separation factors and resolution obtained for the compounds examined. All the samples were well separated under the conditions described in the experimental section. t is supposed that the method can be used not only for the separation of amino acid enantiomers but also chiral recognition of some ammonium substrates. Conclusion This paper demonstrates that incorporation of 18C6TA with 13-cyclodextrin is very useful as the selector for the separation of enantiomers in capillary agarose gel electrophoresis. Separations of fluorophenylalanine and other enantiomers were satisfactory. Under the recommended conditions, 16 primary amine enantiomers were well separated by this method, the separation efficiency has been improved by suppression of the electroosmotic flow as a result of the use of agarose gel. The gel has a significant effect on migration times, particularly those of slow-migrating species. t is interesting to note that Table. Separations of some enantiomers by capillary agarose gel electrophoresis. No. Compound Structure ct Resolution, Rs t OOC--C--N2 (1) o-fluorophenylalanine c~v OOC-~:--N 2 (2) m-fluorophenylalanine c~1~ F OOC--C--~ (3) p-fluorophenylalanine 1~ OOC--C--Nz (4) Phenylalanine ~ F Original Chromatographia Vol. 42, No. 9/10, May

6 (5) Phenylglycine OOC--C~N 2 () (6) Tyrosine '~OC--C--NB 2 () (7) Tryptophan O OOC--~--N (8) ydroxytryptophan Y OOC--C--N= O (9) R,S-(+)-l-(1-naphthyl)- ethylamine (1o) Noradrenaline? --C--N.j o-~ (ii) 3-(3,4-dihydroxyphenyl)alanine OOC--C--N O (12) Leucine N2.,,Ca OOC--C-C2-C.. Ca (13) soleucine N2 OOC--C--C-C2--C 3 C (14) Valine ~ 2 3C--C--C--COO C (15) Threonine \ /N 2 Ooc/C~c~O C (16) Serine ~ z OOC~C~C2O , Chromatographia Vol.42,No. 9/10,May 1996 Original

7 the mixture of o-, rn-, and p-fluoro-d,l-phenylalanine can be separated well on the same column. The concentrations of 18C6TA and 13-cyclodextrin, and the p value have a significant effect on the separation. This method seems very promising, especially for the separation of aromatic isomers, although much research remains to be performed. Acknowledgement Support of this work by the Sasakawa Science Research Foundation of Japan Science Society (Grant No ) is gratefully acknowledged. References [1] A. Ahuja, ed., Chiral Separations by Liquid Chromatography, American Chemical Society, Washington, DC, [2] K. Sato,. Nakano, T, obo, J. Chromatogr. A 666, 463 [3] K. Watabe, E. gil-av, T obo, S. Suzuki, Anal. Chem. 61, 261 (1989). [4] J. M. Finn, M. Zief,, L. J. Crane, eds., Chromatographic Chiral Separations, Marcel Dekker, New York, [5] S. E Y Li, Capillary Electrophoresis, Principles, Practice and Application, J. Chromatogr. Library, Vol. 52, Elsevier, Amsterdam, 1992, p [6] R. Kuhn, S. offstette-kuhn, Chromatographia 34, 505 (1992). [7] M.M. Rogan, K. D. Altria, D. M. goodall, Chirality 6, 25 [8] T.J. Ward, Anal. Chem. 66, 633A [9] S. Terabe, K. Otsuka,. J. Nishi, J. Chromatogr. 666, 295 [10] M. Novotny,. Soini, M. Stefansson, Anal. Chem. 66, 646 [11] C J. Pederson, J. Am. Chem. Soc. 89, 2495 (1967). [12] C.J. Pederson, J. Am. Chem. Soc. 89, 7017 (1967). [13] J. J. Christensen, D. J. Eatough, R. M. zatt, Chem. Rev. 74, 352 (1974). [14] C.J. Pedersen,. K. Frensdorff, Angew. Chem. nt. Ed. Engl. 11, 16 (1972). [15] V. Prelog, Pure Appl. Chem. 50, 893 (1978). [16] J.M. Lehn, Struct. Bonding (Berlin) 16, 1 (1973). [17] J.M. Lehn, Pure Appl. Chem. 50, 87 (1978). [18] J.M. Lehn, Science 227, 849 (1985). [19] D.J. Cram, J. M. Cram, Ace. Chem. Res. 11, 8 (1978). [20] D.J. Cram, Science 219, 1977 (1983). [21] D.J. Cram, Angew. Chem. nt. Ed. Engl. 25, 1039 (1986). [22] A. Pullman, A. M. Armbruster, Chem. Phys, Lett. 36, 558 (1975). [23] P. Kollman, J. Am. Chem. Soc. 99, 4875 (1977). [24] J. E Griffin, P. Coppen, J. Am. Chem. Soc. 97, 3496 (1975). [25] J.P. Behr, J. M. Lehn, P. Vierling, elv. Chim. Acta 65, 1853 (1982). [26] J.M. Lehn, Pure. Appl. Chem. 50, 871 (1978). [27] J.M. Lehn, Pure. Appl. Chem. 51, 979 (1979). [28] R. E Kuhn, E Stoecklin, E Erni, Chromatographia 33, 32 (1992). [29] R. Kuhn, F. Erni, Anal. Chem. 64, 2815 (1992). [30] R. Kuhn, S. Celine, T. Bereuter, P. aas, E Erni, J. Chromatogr. 666, 367 [31] E. oehne, G. J. Krauss, G. Guebitz, J. igh Resol. Chromatogr. 15, 698 (1992). [32] Y. Y Rawjee, D. U. Staerk, G. Vigh, J. Chromatogr. 635, 291 (1993). [33] P. J. Dutton, T. M. Fyles, S. J. Mcdermid, Can. J. Chem. 66, 1097 (1988). [34] R.J. Nelson, A. Paulus, A. S. Cohen, A. Guttman, B. L. Karger, J. Chromatogr. 480, 111 (1989). Received: Dec 21, 1995 Revised manuscript received: Feb 8, 1996 Accepted: Mar 12, 1996 Original Chromatographia Vol. 42, No. 9/10, May