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Anal. Chem. 1991, 63,2979-2981
CORRESPONDENCE ChiraI Separation of Naphthalene-2,3-dicar boxaldehyde- Labeled Amino Acid Enantiomers by Cyclodextrin-Modified Micellar Electrokinetic Chromatography with Laser-Induced Fluorescence Detection Sir: The enantiomeric separation of racemic mixtures of amino acids and various drugs is an important subject of interest to the pharmaceutical and biotechnology fields. The achievement of chiral separation of amino acids by cyclodextrin-modified micellar electrokinetic chromatography at attomole levels with laser-induced fluorescence (LIF)detection is the subject of this report. It is of interest to note that recent advances in high-performance liquid chromatography (HPLC) offer several new approaches to the separation of enantiomeric mixtures, for example, through the diastereoisomericinteraction of sample molecules with chiral reagents (1). Such approaches include the use of chiral reagents as (a) part of the stationary phase, (b) an addition to the mobile phase for direct separation, or (c) a precolumn derivatizing agent with separation of the resulting diastereoisomers. However, the design of chiral stationary phases is rather complex and such phases are usually applied to only a limited number of racemates. The use of a chiral mobile phase is limited due to the consumption of large amounts of the chiral reagent. Recently, high-performance capillary electrophoresis (HPCE) has made remarkable progress as a relatively fast separation method providing high resolution. This progress is due to the development of several different separation modes, such as capillary zone electrophoresis (2),electrokinetic chromatography (31, capillary gel electrophoresis (41, and capillary isoelectric focusing (5). The versatility of these methods is now being expanded to include chiral separations of racemic compounds. The reported separations are based on (a) a different metal-chelate complexation (6, 7), (b) a distribution of the solute between a chiral micelle and an aqueous phase (€0,and (c) the use of cyclodextrins (CDs) as complexing agents (3, 9, 10). CDs and their derivatives have been applied to the separation of racemic mixtures of dansylated amino acids and catecholamines by HPCE under minimized electroosmotic flow. CDs incorporated into a polyacrylamide gel capillary column, where electroosmotic flow velocity was negligibly small, behaved as a pseudostationary phase for chiral separation of dansylated amino acids (9). The effect of the CD shape on the effective mobility of several catecholamineswas investigated by Fanali (10) to achieve the enantiomeric separation of the solutes. Complete resolution of the racemates was obtained by adding heptakis(2,6-di-O-methyl-fl-CD) to a low-pH buffer solution with a coated capillary column so that the electroosmotic flow was minimized. Theaeseparations allowed only charged molecules to migrate toward the detector because of this flow minimization. Terabe et al. (3) prepared the compound 6-[(3-aminoethyl)amino]-6-deoxy-/3-cyclodextrin (CDen), which acted as a charged carrier to facilitate the chiral separation of dansylated amino acids. Since the electroosmotic flow was suppressed by the addition of hydroxypropyl cellulose at pH 3, a neutral solute, which is not partitioned into the cavity of CDen. will not elute at all. 0003-2700/91/0363-2979$02.50/0
Micellar electrokinetic chromatography (MEKC) is somewhat similar to reversed-phase chromatography,where solutes in a sample are separated due to the differences in hydrophobic interaction of the solutes with the stationary phase bonded to the surface of the packing materials (11). Separation in MEKC results from the distribution of solutes between the carrier micelle and the aqueous phase in the presence of electroosmoticflow. Usually, electroosmoticflow velocity is faster than the electrophoretic mobility of the micelles, and all the components migrate toward the detector at a velocity that is primarily dependent on the hydrophobicity of the solute molecule. When CD is added to the electrophoretic buffer solution in MEKC, the selectivity of sample solutes may change, since CD can introduce an additional distribution mechanism. CD itself will migrate at a velocity identical to the electroosmotic flow. Consequently, when a solute tends to interact with CD through the formation of an inclusion complex, the migration time for that solute becomes less than that obtained in MEKC. The difference in migration time for a solute, with and without CD, is undoubtedly very dependent on the degree of complexation between that solute and CD. Thus, the selectivity may change considerably with the addition of CD in MEKC. Recently, Terabe et al. (12)used CDs for separating highly hydrophobic compounds whose separations are generally difficult by MEKC or other related electrophoretic techniques. They named this technique CD-modified MEKC (CDMEKC). In this communication, this technique is used to separate the DL forms of several amino acids, which have been derivatized with naphthalene-2,3-dicarboxaldehyde(NDA) in the presence of cyanide ion to form 1-cyano-2-substituted-benzv]isoindoles (CBIs). As will be demonstrated herein, it is not necessary to use derivatized CDs to elicit the enantiomeric separation with CBI derivatives.
EXPERIMENTAL SECTION Apparatus. CD-MEKC was performed in a fused-silica capillary (Scientific Glass Engineering, Ringwood, Victoria, Australia),50-pm i.d., 29O-Mm o.d., 70 cm in length. A Model EH30R.3 high-voltage power supply (Glassman High Voltage Inc., Whitehouse Station, NJ) was used to generate the electric field across the capillary. Each end of the capillary was immersed in a separate glass bottle fiied with micellar solution for each electrophoretic run. Platinum-wire electrodes were inserted into the buffer solutions for electrical connection. The high-voltage end of the capillary was enclosed in a Plexiglas box with a safety interlock. For temperature control, the capillary was inserted into a 40-cm length of Teflon tubing (0.8-mm i.d., 1.6-mm o.d.), which was connected to two PEEK T-joints so that the water could be circulated outside the capillary. Laser-induced fluorescence detection was performed with a Model 4207NB helium-admium (He-Cd) laser (Liconix, Santa Clara, CA), operating at an output power of ca. 7 mW at 442 nm as a light source. For on-column detection, a 2-mm length of the polyimide coating was removed at a distance 50 cm from the inlet side of the capillary. The laser beam was divided by a beam splitter; one beam was monitored by a photodiode to provide a 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
reference channel to compensatefor the noise and drift. The other fluorescent excitation beam was focused on the capillary with a 1OX microscope objective lens. The fluorescent emission from the capillary was collected at right angles to the excitation beam with a 20X microscope objective lens and passed through an iris diaphragm, polarizing filter, and a band-pass filter with a center wavelength of 490 nm and a half-width of 20 nm (Omega Optical Inc., Brattleboro, VT). The filtered fluorescence was detected with a R1527 photomultiplier tube (Hamamatsu Corp., Bridgewater, NJ) operating at -900 V. The current-voltage amplifiers converted the current from both the photodiode and photomultiplier to voltages; the output voltage from the photomultiplier tube was divided by the reference voltage with an analog ratio circuit for compensation purposes. The resulting voltage was displayed on a Model CR501 data processor (Shimadzu Scientific Instruments, Columbia, MD). Reagents. NDA was obtained from Tokyo Kasei (Tokyo, Japan) and dissolved in HPLC grade acetonitrile. Sodium hydroxide of semiconductor grade, boric acid, sodium dodecyl sulfate (SDS), and CDs were purchased from Aldrich (Milwaukee,WI), and sodium cyanide was obtained from Fluka Chemical (Ronkonkoma, NY).Amino acids were obtained from Sigma (St. Louis, MO) and dissolved in 0.1 N hydrochloric acid. DL-aminO acid solutions at 2 mM concentration were prepared as stock solutions and serially diluted with 0.1 N hydrochloric acid to obtain the desired fiial concentration. Nanopure water (SybronBarnstead, Boston, MA), filtered through a membrane filter of 0.2-pm pore size, was used throughout. Derivatization Procedure. NDA was used to derivatize the amino acids for high-sensitivityfluorescence detection. The amino acids were derivatized with NDA by the following procedure. To 700 pL of borate buffer (100 mM, pH 9.5) in a 1.5 mL vial were added 100 p L of sodium cyanide solution (10 mM) and 100 p L of amino acid solution (4 p M for each pair of DL-amino acids), and the solution was mixed. Next, 100 pL of NDA solution (1 mM) was added to the vial. After gentle shaking, the reaction was allowed to proceed for 30 min. Sample solutions were each introduced into the capillary by a hydrodynamicinjection method with the injection volume being estimated by the method described by Rose and Jorgenson (13). Peak identification was performed by spiking the sample mixture with a known amino acid.
0
5
15
10
20
Time (min)
Flgure 1. Electropherogram of a mixture of five CBI-ol-aminoacids. Electrolyte composition is as follows: 10 mM @-CD,50 mM SDS, and 100 mM borate buffer, pH 9.0. The capillary is 50 pm i.d. (290 pm o.d.1, 70 cm in length (50 cm to the detector). The concentration for each CBI-a-amino acid is 200 nM. The applied voltage is 15 kV, and the current is 35 FA. D. LSei
RESULTS AND DISCUSSION NDA reacts with primary amines in the presence of cyanide to form CBI derivatives, which possess excellent stability and high quantum efficiency, even in aqueous buffer solutions (14). Another advantage of these compounds is that the excitation maxima coincide closely with the 442-nm output wavelength of the He-Cd laser. Thus, LIF detection can be performed with high sensitivity. The electropherogram shown in Figure 1 illustrates the chiral separation of CBI-DL-amino acids obtained by @-CDMEKC. The electrophoretic buffer contains 10 mM @-CD, 50 mM SDS, and 100 mM borate buffer at pH 9.0. The concentration of each CBI-amino acid was 200 nM. The injection volume is estimated to be 2.5 nL so that the amount of each amino acid injected is 500 amol. At a signal-to-noise ratio of 2, the detection limit for CBI-L-phenylalanine is calculated to be 0.9 amol. Even though the difference in migration time for each pair of amino acids was very small, the high efficiency inherent to HPCE produced a chiral separation of these enantiomers by CD-MEKC. The separation of these compounds could not be obtained without the addition of SDS to the electrophoretic buffer solution. The amino acids were negatively charged under the analytical conditions employed so that the electrophoretic properties of the derivatized amino acids would be very similar thereby causing a poor separation in the CZE mode. However, the addition of SDS to the electrophoretic buffer solution produced prolonged migration times for the amino acids due to the equilibrium between the aqueous phase and the micelle.
-
0
1
,
_
L
5
10 Time (min)
15
20
Figure 2. Electropherogram of a mixture of five CBI-ol-aminoacids. Electrolyte composttion is as follows: 10 mM -y-CD,50 mM SDS, and
100 mM borate buffer,pH 9.0. Other conditions are the same as in Figure 1.
Hence, the separation time scale could be expanded. Therefore, CD molecules in a micellar electrolyte solution tend to shorten the migration times of those compounds that are dependent on the degree of inclusion complex formation. For those CBI-amino acids chirally separated by @-CD-MEKC, the elution order of the D and L enantiomers seemed to be dependent on the amino acid structure. As shown in Figure 1, the D form migrates faster than the L form for aliphatic amino acids (Thr, Asp, and Ile) while the L form migrated faster than the D form for aromatic amino acids (Tyr and Phe). Even though all the CBI-amino acids contain a naphthalene ring in their structure, the functional group on each amino acid residue might be important for their chiral recognition, depending on the extent of the formation of inclusion complex. To further investigate the nature of this chiral separation, 7-CD was employed in place of P-CD with the same micellar solution. Figure 2 shows a typical electropherogram of the chiral separation of CBI-DL-amino acids by yCD-MEKC. Compared with p-CD, 7-CD exhibited different selectivities for CBI-DL-amino acids. For example, the separations of CBI-DL-Thr and DL-Phe are improved with y-CD-MEKC
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ACKNOWLEDGMENT We thank Susan Lunte for her helpful suggestion on the derivatization procedure of the amino acids. Registry No. NDA, 7149-49-7;pCD, 7585-39-9;yCD, 17465-86-0;D - T ~632-20-2; , L - T ~72-19-5; , D-As~,1783-96-6; b h p , 56-84-8; Brie, 319-788;Ale, 73-32-5; LPhe, 63-91-2;DPhe, 67346-3;D-LeU, 328-38-1;L-Leu, 61-90-5; L-Vd, 72-18-4;D-Vd, 640-68-6;L-Ser, 56-45-1;D-Ser, 312-84-5; L-Met, 63-68-3;D-Met, 348-67-4;D-hg, 157-06-2;L - h g , 74-79-3; L-TY, 60-18-4;D - m , 556-02-5;DL-Thr, 80-68-2;DL-ASP,617-45-8; D L - m ,556-03-6; DL-Ile, 443-79-8;DL-Phe, 150-30-1;DL-Leu, 328-39-2;D L - V ~ , 516-06-3; DL-Ser, 302-84-1; DL-Met, 59-51-8;DL-hg, 7200-25-1; sodium cyanide, 143-33-9.
LITERATURE CITED
I
io
16 Tlme (mln)
20
26
Flgufs 3. Electropherogram of a mixture of five CBI-m-amino acids. Analytical conditions are the same as in Figure 2.
compared to 8-CD-MEKC. Furthermore, CBI-DL-Ser can be separated with y-CD-MEKC while this species is coeluted with 8-CD-MEKC. On the other hand, CBI-DL-AS~could not be resolved with y-CD-MEKC, as seen earlier in Figure 1 with 8-CD-MEKC. (The unresolved CZE peaks for the DL forms of serine and aspartine are not shown in Figures 1 and 2, respectively.) Future study includes the optimization of the enantiomeric separation of CBI-DL-amino acids with CDMEKC. Generally, y-CD-MEKC seemed to be more effective than 8-CD-MEKC in terms of the enantiomeric selectivity for CBI-~baminoacids under these analytical conditions. Figure 3 shows the separation of CBI-DL-amino acids, which were difficult to resolve with /3-CD-MEKC, except for CBI-DL-Phe. Improved separation of the D L - ~ acids O with yCD-MEKC may arise from the difference in the cavity size between the y and 8 forms. At present, the chiral separation of CBI-DLGlu and His has not been achieved by CD-MEKC. Optimization studies with variation of the analytical conditions such as pH, concentration of SDS and CD, buffer species, and addition of organic solvent will be conducted in an attempt to separate these amino acids. Compared to other methodologies for chiral separation, CD-MEKC offers the following advantages. First, both CDs and SDS or other surfactants are readily available. Secondly, CD-MEKC itself is one of the separation modes in HPCE so that the advantages of HPCE can be easily utilized: improved mass sensitivity, small consumption of both electrophoretic buffer and sample solutions, and high resolving power. CDMEKC with LIF detection appears particularly attractive for the chiral separation of minute quantities of enantiomers. Further investigations are being continued to achieve chiral separation of other amino acids and to understand the mechanism of chiral recognition by CD-MEKC.
(1) Davankov, V. A.; Kwganov, A. A.; Bochkov, A. S. In Advances In ~ t o g v e p h yGiddhgs, ; J. C., Grushka, E., Cares, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1983; Vol. 22, p 71. (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1081, 5 3 , 1298. (3) Terabe, S. TrendsAnal. Chem. 1080, 8 , 129. (4) Cohen, A. S.; Karger, B. L. J . chromatogr. 1987. 397, 409. (5) Hjerten, S.; Zhu, M.D. J . Chrometop. 1085, 246, 265. (8) Cohen, A. S.; Paulus, A.; Karger. B. L. Chfomatographia 1087, 24, 15. (7) Gozel, P.; Gassmann, E.; Mlchelsen, H.; Zare, R. N. Anal. Chem. 1087, 5 9 , 44. (8) Terabe, S.; Shlbata, M.; Miyashka, Y. J . chromatogr. 1089, 480,403. (9) @Maman, A.; Paulus, A.; Cohen. A. S.; Grinberg, N.; Karger. B. L. J . Chfomatogr. 1080, 474, 441. (IO) Fanali, S. J. Chrmtogr. 1088, 448. 41. (11) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1085, 5 7 , 834. (12) Terabe, S.; Miyashita, Y.; Shlbata, 0.; Barnhart, E. R.; Alexander, L. R.: Patterson. D. G.; Karger, B. L.; Hosoya, K.; Tanaka, N. J . Ct"top. 1990, 576, 23. (13) Rose, D. J.; Jorgenson, J. W. Anal. Chem. 1088, 60, 642. (14) Matuszewski, B. K.; Wens, R. S.; Srinlvasachar, K.; Carlson, R. 0.; Higuchi, T. Anal. Chem. 1087, 5 9 , 1102.
To whom correspondenceshould be addressed.
Teruhisa Ueda Fumito Kitamura Rosalind Mitchell Timothy Metcalf Shimadzu-Kansas Research Laboratory in the Center for Bioanalytical Research The University of Kansas 2095 Constant AvenueJCampus West Lawrence, Kansas 66047
Theodore Kuwana* Center for Bioanalytical Research and Department of Chemistry The University of Kansas 2095 Constant AvenueJCampus West Lawrence, Kansas 66047
Akira Nakamoto Analytical Instruments Division Shimadzu Corporation 1 Nishinokyo-Kuwabaracho Nakagyo-ku, Kyoto 604,Japan RECEIVED for review June 24,1991.Accepted September 19, 1991. This work was supported by funds provided by the Kansas Technology Enterprise Corp. and Shimadzu Corp.
