Anal. Chem. 1985, 57. 237-242
fering stabilities. Since traditional normal- and reversed-phase packings separate solutes by entirely different mechanisms (i.e., adsorption or partition), they cannot be expected to achieve selectivities analogous to those of cyclodextrin-bonded packings. An evaluation of the quantitative effects of solvent composition and temperature on k' and efficiency for this system will be discussed in a subsequent publication. Registry No. 0-Cyclodextrin, 7585-39-9; benzo[a]pyrene, 50-32-8; benzo[e]pyrene, 192-97-2; 1,2:3,4-dibenzanthracene, 215-58-7; 1,25,6-dibenzanthracene,53-70-3; phenanthrene, 85-01-8; anthracene, 120-12-7;prostaglandin Al, 14152-28-4;prostaglandin B1, 13345-51-2;prostaglandin A2, 13345-50-1;prostaglandin B,, 13367-85-6;vitamin D,, 50-14-6;lumisterol, 474-69-1;previtamin D2,21307-05-1;a-naphthol, 90-15-3; @-naphthol,135-19-3;a-naphthoflavone, 604-59-1; 0-naphthoflavone, 6051-87-2; a-ethylphenethyl alcohol, 701-70-2; @-ethylphenethylalcohol, 2035-94-1; 1,2-naphthoquinone, 524-42-5; 1,4-naphthoquinone, 130-15-4; quinoline, 91-22-5; isoquinoline, 119-65-3; o-xylene, 95-47-6; m-xylene, 108-38-3;p-xylene, 106-42-3;0-cresol, 95-48-7; m-cresol, 108-39-4;p-cresol, 106-44-5;o-nitrophenol, 88-75-5;m-nitrophenol, 554-84-7; p-nitrophenol, 100-02-7; o-nitroaniline, 88-74-4; mnitroaniline, 99-09-2;p-nitroaniline,100-01-6;o-bromobenzoicacid, 88-65-3; m-bromobenzoic acid, 585-76-2; p-bromobenzoic acid, 586-76-5; o-aminobenzoic acid, 118-92-3; m-aminobenzoic acid, 99-05-8; p-aminobenzoic acid, 150-13-0;1-methylindole,603-76-9; 2-methylindole, 95-20-5; 3-methylindole, 83-34-1;5-methylindole, 614-96-0; 7-methylindole, 933-67-5; cis-clomiphene, 15690-55-8; trans-clomiphene, 15690-57-0; cis-stilbene,645-49-8; trans-stilbene, 60657-25-2; 103-30-0; cis-7,8-dihydrobenzo[a]pyrene-7,8-diol, trans-7,8-dihydrobenzo[a]pyrene-7,8-diol, 57404-88-3;cis-3-hexsyn-azobenzene, en-l-ol,928-96-1;trans-3-hexen-l-ol,928-97-2; 1080-16-6;anti-azobenzene, 17082-12-1;estriol, 50-27-1; 16-epiestriol, 547-81-9;17-epiestriol,1228-72-4;16,17-epiestriol,793-89-5; testosterone, 58-22-0; 17-epitestosterone,481-30-1; 17a-estradiol, 57-91-0; 17@-estradiol,50-28-2;Ha-hydroxyprogesterone, 80-75-1; ll@-hydroxyprogesterone,600-57-7;20a-hydroxy-4-pregnen-3-one, 145-14-2;20@-hydroxy-4-pregnen-3-one, 145-15-3;5a-androstan3,17-dione, 846-46-8; 5@-androstan-3,17-dione,1229-12-5; 50-
237
androstan-3@-01-17-one,571-31-3; 5@-androstan-3a-ol-17-one, 53-42-9; &aldosterone, 52-39-1; 17-isoaldosterone, 13479-36-2; 5@-androstan-3@-01-17-one,571-31-3;5a-androstan-3@-01-17-one, 481-29-8; 5a-androstan-3a-ol-17-one, 53-41-8; 5O-androstan-3aol-17-one, 53-42-9; 2,2'-biphenol, 1806-29-7;4,4'-biphenol, 92-88-6.
LITERATURE CITED (1) Davankov, V. A. Adv. Chromatogr. 1080, 18, 139-195. (2) Davankov, V. A.; Kurganov, A. A; Bochkov, A. S. Adv. Chromafogr. 1080, 2 2 , 71-116. (3) Armstrong, D. W. J . Llq. Chromatogr. 1084, 7 , Suppl. 2, 353-367. (4) Houx, N. W. H.; Voerman, S. J . Chromafogr. 1078, 129, 456-459. (5) Heath, R. R.; Tumlinson, J. H.; Doolittle, R. E.; Duncan, J. H. J . Chromafogr. Scl. 1077, 15, 10-13. (6) Lam, S.; Grushka, E. J . Chromatogr. Scl. 1077, 15, 234-238. (7) Heath, R. R.; Sonnet, P. E. J . Llq. Chromatogr. 1880, 3 , 1129-1135. (8) Siouffi, A. M.; Traynard, J.-C.; Guiochon, G. J . Chromatogr. Scl. 1077, 15, 469-474. (9) Tlyoshi, T.; Kodama, M; Ito, M.; Kawamota, M.; Takahashi, K. J . Nuh. Scl. Vlfamlnol. 1077, 2 3 , 263-204. (10) Paanakker, J. E.; Groenendijk, G. W. T. J . Chromafogr. 1070, 168, 125-132. (11) E. Merck Technical Bulietln. No. 74-11, Darmstadt, Germany. (12) Redel, J. J.; Capilllon. J. "Steroid Analysis by HPLC", Kautsky, M. N., Ed.; Marcel Dekker: New York, 1981; Vol. 16, pp 343-358. (13) Lln, J.-T.; Heftmann, E. J . Chromatogr. 1081, 212, 239-244. (14) Lin, J.-T.; Heftmann. E. J . Chromatogr. 1082, 237, 215-224. (15) Lin, J.-T. Llq. Chromafogr. Mag. 1084, 2 , 135-138. (18) Ogan, K.; Katz, E. J . Chromatogr. 1080, 188, 115-127. (17) May, W. E.; Wise, S. A. Anal. Chem. 1084, 5 6 , 225-232. (18) Sander, L. C.; Wise, S. A. Anal. Chem. 1984, 56, 504-510. (19) Bender, M. L.; Komlyama, M. "Cyclodextrin Chemistry"; Springer-Verlag: Berlin, 1978. (20) Hlnze, W. L. Sep. furif. Methods 1981, 10, 159-237. (2 1) Szejtli, J. "Cyclodextrins and Their Inclusion Complexes"; Akademlai Kiado; Budapest, 1982. (22) Hinze, W. L.; Armstrong, D. W. Anal. Left. 1080, 73, 1093-1104. (23) Burkert. W. G.: Owensbv, C. N.; Hlnze, W. L. J . Lla. Chromatow. 1981, 4 , 1085-1085. (24) Fujimura, K.; Veda, T.; Ando, T. Anal. Chem. 1083, 5 5 , 446-450. (25) Kawaguchl, Y.; Tanaka, M.; Nakae, M.; Funazo, K.; Sheno, T. Anal. Chem. 1083, 55, 1852-1857. (26) Armstrong, D. W.; DeMond, W. J . Chromafogr. Sci. 1084, 2 2 , 411-415.
RECEIVED for review July 30,1984. Accepted October 9,1984.
Liquid Chromatographic Separation of Enantiomers Using a Chiral ,8-Cyclodextrin-Bonded Stationary Phase and Conventional Aqueous-Organic Mobile Phases Willie L. Hinze* and Terrence E. Riehl Department of Chemistry, Wake Forest University, Box 7486, Winston-Salem, North Carolina 27109 Daniel W. Armstrong,* Wade DeMond, Ala Alak, and Tim Ward Department of Chemistry, Texas Tech University, Lubbock, Texas 79409 A chlral statlonary phase composed of chemically bonded @-cyclodextrln (@-CD)molecules was used to separate enantiomers of dansylsulfonamlde, pnaphthamlde, or @-naphthyl ester derlvatlves of amlno aclds, barbiturates, substituted phenylacetic aclds, and dloxolanes. The separatlons are reasonably ratlonallred In terms of the lncluslon process between the enantlomers and @-cyclodextrlnand consideration of a three-polnt attachment model. The effects of mobllephase composltlon, temperature, and flow rate upon the observed enantiomeric selectivity and resolution were crltlcally assessed. Lastly, a brlef prospectus on the usefulness of cyclodextrln chlral stationary phases In high-performance liquid chromatographlc enantiomeric separations Is presented.
The liquid chromatographic separation of enantiomers is
an important and challenging task. The enantioselectivity of biological systems is well-known to most scientists and is often of paramount importance to many pharmacologists and biochemists. The determination of enantiomeric purity and the routine separation of these isomers are also of great use to many synthetic organic chemists, kineticists, and researchers interested in geochronology, etc. Traditionally, the resolution of enantiomers was a time-consuming often inefficient process that involved the use of naturally occurring optically active compounds to cocrystallize with the desired isomer. In some cases, enzymatic systems were useful as separation tools as a result of their stereospecific influence on reactivity. These more traditional techniques (despite their successes) are often considered tedious and/or not generally applicable. As a result there has been a considerable impetus toward the development of chromatography as a tool for en-
0003-2700/85/0357-0237$01.50/0 0 1984 American Chemlcal Soclety
238
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
antiomeric separation (1-4). An added benefit of an efficient chromatographic technique would be the ability to experimentally evaluate certain thermodynamic and theoretical parameters for enantiomers as was previously done for nonchiral compounds (5). There have been several different approaches to the LC separation of enantiomers. One of the first involved the use of chiral mobile-phase additives ( 2 , 3 ,6). This method was shown to be effective for certain compounds. Chemically binding chiral molecules to the stationary phase is a common starting point for several investigators (1-3, 7,8). Some have used the bonded chiral molecules as ligands in conjunction with an appropriate transition-metal additive (9). When enantiomeric compounds, which also serve as ligands, are injected into such a system, separation can sometimes be achieved. In a different approach, entire proteins have been attached to the stationary phase in order to effect optical resolution. Perhaps the most generally useful technique involves binding of relatively simple chiral molecules to the stationary phase which can interact with a variety of enantiomers via charge-transfer interactions, hydrogen bonding, and/or a or various dipolar interactions (1-3, 7-11). These are generally amino acids or amino acid derivatives. In this work, the use and effectiveness of a somewhat different type of chiral stationary phase (CSP) for the separation of optical isomers is reported. The CSP evaluated consists of @-cyclodextrin(cycloheptaamylose) host molecules covalently bonded to silica gel via a seven-to-nine atom spacer (4, 12). /3-Cyclodextrin (p-CD) is a nonionic, cyclic, chiral carbohydrate composed of seven D(+)-glucopyranose units which are connected to each other via a-1,4 bonds (12-14). 0-CD has the shape of a hollow truncated cone in which the side of the torus with the greater circumference has 14 secondary hydroxyl groups while that of the smaller side has 7 primary hydroxyl groups. The interior of the cavity is relatively hydrophobic compared to the hydrophilic hydroxylcontaining faces. Due primarily to favorable hydrophobic and/or hydrogen-bonding interactions, guest molecules can bind and form inclusion complexes with fl-CD. However, since the molar volume of the p-CD cavity is only approximately 161 cm3/mol (14),only those guest molecules of appropriate size and shape can form strong inclusion complexes with it. Because inclusion complex formation is a spatial interaction, there can be appreciable differences in the strengths for the complexes formed from an enantiomeric pair of guests (12-15). This forms the basis for possible enantiomeric resolution using the p-CD CSP. (3-CD has previously been employed in various optical separation and enrichment schemes (15, 16). For example, enantiomeric enrichments through stereoselectiveprecipitation with 0-CD or enantioselective p-CD catalysis of racemic substrates reactions have been achieved for several compounds (15,17,18). Aqueous solutions of 0-CD have been employed as the mobile phase in the reversed-phase HPLC resolution (partial) of several enantiomers (19,20). Cross-linked polymeric p-CD gels or resins have also been utilized as the stationary phase in the column chromatographic optical resolution of mandelic acid derivatives and indole alkaloids (21,22). Columns containing these polymeric p-CD gel or p-CD impregnated phases are reportedly unlikely candidates as CSPs in HPLC due to their low efficiency (20) and low mechanical strength which necessitates long analytical run times (23,24). Recently, two papers have described the preparation of HPLC columns which contain p-CD chemically bonded to silica gel (23,24). However, no enantiomeric separations using these phases were reported by the authors. As with many of the currently popular CSP, the fi-CD was bonded to the silica via hydrolytically unstable amine or amide linkages. This
necessitates strict limitations on the polarity of the mobile phase since highly polar media can leach the chiral molecules from the support and/or degrade the column rapidly (4,12). In this work, we demonstrate the successful HPLC enantiomeric resolution of a series of amino acid, barbiturate, dioxolane, and phenylacetic acid derivatives on a p-CD CSP. The pertinent chromatographic parameters are summarized and where possible are compared to those obtained for the separation of these enantiomers on other types of CSP. It will be shown that this CSP is compatible with traditional mixed aqueous-organic mobile phases. The effects of variation of the mobile-phase composition, flow rate, and temperature upon the enantiomeric resolution obtained with the p-CD column are briefly discussed.
EXPERIMENTAL SECTION Apparatus. Two liquid chromatographic systems were used in this investigation. The first, which will be referred to as system I , consisted of two Waters Model 510 pumps, a Waters Model 680 automated gradient controller,a Waters Model U6K injector with a 20-pL loop, and a variable-wavelength UV-vis detector, Waters Lambda-Max Model 481, equipped with a 14-pL cell (path length = 1 cm). The chromatograms were obtained by using a strip chart recorder, Heath Model EU-20B. The second chromatograpic system, system ZZ, was a Shimadzu LC-4A liquid chromatograph equipped with a 200-pL injection loop and a variable-wavelength detector (cell volume = 13 pL). Unless otherwise noted, system I was operated at a temperature of 22 "C while that of system I1 was 20 "C. Ultraviolet-visible absorption spectra of samples were obtained with a Cary 219 recording spectrophotometer. Circular dichroism (CD) spectra were measured in a 1cm path length cell by using a Jasco 500 A spectropolarimeter. Optical rotations were measured with a polarimeter in a water-jacketed cell monitored at 25 "C. Chromatography. The cyclobond columns, 10 cm or 25 cm x 4.6 mm id., were obtained from Advanced Separations Technology, Inc. (Whippany, NJ). The cyclobond I column has chiral p-cyclodextrin molecules chemically bonded to a spherical silica gel support through a non-nitrogen-containing spacer arm. The preparation and characterization of this column has been more fully described elsewhere (4, 12,25). The cyclobond I column gave about 8000 theoretical plates per 25 cm (3600 plates per 10-cm columns) [at a linear velocity, p, length of column (centimeters)/dead volume (seconds) = 0.151 for dansylthreonine using 50% aqueous methanol as the mobile phase at a flow rate of 1.0 mL/min. When not in use, the column was stored with 100% methanol. The column performance appeared to be unchanged for at least a period of 3 months. The mobile phases used were mixtures of methanol-water (10:90-100:0, v/v), ethanol-water (5:95-60:40, v/v), and acetonitrile-water (5:95-55:45, v/v). The flow rate was 1.0 mL/min unless otherwise specified, and the column back pressures at this flow rate ranged from 1650 to 2100 psi and 650 to 1150 psi for the 25- and 10-cm Cyclobond I columns, respectively. The mobile phase was generally filtered through a 2-pm frit before use. The enantiomeric compounds were dissolved in methanol to give concentrations of 0.01-0.1 mM. Typically, 5 p L of these standard reference substances were injected. The wavelength utilized for detection was generally the optimum for the enantiomeric compounds being studied. The elution order was established from consecutive injections of the individual pure enantiomer as well as samples containing the racemic form unspiked and spiked with one enantiomer. Precautions were taken to ensure that the chromatographic peaks attributed to the solute enantiomers actually were those of the enantiomers. Namely, the absence of peak overlap, caused by possible contaminants, was demonstrated by the constant peak-height ratios obtained on detection at different wavelengths. Also, in some instances, the optical rotation and circular dichroism of eluting fractions were measured in order to confirm the separations. Chemicals. HPLC grade acetonitrile, methanol, n-hexane, and water were obtained from Fisher Scientific Co. (Raleigh, NC). Absolute ethanol (USP grade) was obtained from AAPER Alcohol and Chemical Co. (Louisville, KY). The ethanol was filtered through a 0.45-pm Gelman filter prior to use as a mobile-phase
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
component. D,L- and L-Amino acids, D,L- and L-dansylaminoacids, N-t-BOC-amino acids (N-tert-butoxycarbonyl derivatives of alanine, leucine, and methionine), DNPyr-amino acids (N-3,5dinitro-2-pyridylderivatives of D-and L-alanine and methionine), N-CBZ-amino acids (N-carbobenzoxy derivatives of D,Lmethionine), hexobarbital (5-cyclohexenyl-3,5-dimethylbarbituric acid), mephobarbital (5-phenyl-5-ethyl-3-methylbarbituric acid), D,L-methionine p-naphthylamide, and dansyl chloride were used as received from Sigma Chemical Co. (St. Louis, MO). The enantiomers of 4,5-bis(diphenylphosphinomethyl)-2,2-dimethyl-l,3-dioxolane and a-methoxy-a-(trifluoromethyljphenylacetic acid were obtained from Fluka Chemical Co. (Hauppauge, NY). Thionyl chloride, ~,~-alanine-2-naphthylamide, d- and l-mandelic acid, (+)- and (-)-trans-a-( 1-naphthy1)ethylamine, cyclohexylphenylacetic acid, carbobenzyloxy-D,L-amino acids of proline and alanine, enantiomers of 2,2-dimethyl-1,3dioxolane-4-methanol, 2-naphthol, and L-(-)- and &(+)-amethylbenzylamine were obtained from Aldrich Chemical Co. (Milwaukee, WI). The purity of these standard reference enantiomers was confirmed by microanalysis, polarimetry, and melting-point determinations. The remaining chemicals and solvents used were of the best reagent grade materials available. Procedures. Some dansylaminoacids were prepared via reaction of the appropriate amino acid with dansyl chloride following an established procedure (26). The b-naphthol ester derivatives of amino acids were prepared in the following manner: First, the amino acid was placed in 1-2 mL of thionyl chloride in a test tube and warmed slightly until it dissolved. Next, b-naphthol was added. After 15-20 min, this reaction mixture was added dropwise to a mixture containing NaHC03 and ice-water. The neutralized solution was then extracted 3 times with diethyl ether. After removal of the ether, the residue was redissolved in methanol prior to injection and HPLC analysis. Since almost all ions and molecules, including the typical column void volume indicators, such as C1-, Br-, I-, SCN-, NO3-, MeOH, and H,O, partially bind to b-CD (27,28), they cannot be directly employed to determine column void volumes or times. Consequently the following procedure was used to estimate column void times and volumes. The retention times (or volumes) of several low molecular weight alcohols (C1-C3) were determined. Than a plot of the retention times of the alcohols vs. their formation binding constant for b-CD complexation (28)was made. The extrapolated retention time (volume) for a binding constant equal to zero was taken as the column void volume. For the 25-cm Cyclobond I column and chromatographic system I, the void volume was determined to be 2.92 mL (to = 175 s) for flow rate = 1.00 mL/min. This volume agreed well with that determined by obseving the base-line disturbance (“glitch”)caused by some unknown impurity upon injection. In most cases, methanol was also used as an internal, relative retention marker. RESULTS AND DISCUSSION Basis for Chiral Recognition by Cyclodextrins. The factors reportedly responsible for the enantioselectivity previously observed in the binding and reactivity of racemates when included by cyclodextrins (15,17,18,2%31) are the same ones which provide the rationale for their use in chromatographic enantiomeric separations (12,20). The chiral recognition was rationalized in terms of several possible interactions between the p-CD host and guest racemates. (i) The p-CD molecule contains 35 chiral centers, and guest solutes can interact via van der Waals-London dispersion forces with its hydrophobic cavity. For this interaction to be effective, the guest must be of a suitable size so that it can tightly bind the p-CD cavity, whose diameters and depth are 6-8 and 7 A, respectively. In most cases, the cylindrical binding cavity of P-CD is too symmetrical itself to induce large enantioselectivities. Consequently, there must be other points of interaction in order to achieve chiral recognition. (ii) p-CD has a C7 symmetry axis and 14 hydroxyl groups situated about the mouth of the cavity. The seven which are attached to the Cz carbon of the glucose are pointed in a clockwise direction while those attached to the C3 carbon point counterclockwise. The opposite rim of the cavity has seven
239
Table I. Optical Resolution of the Enantiomers of Dansylamino Acidsn capacity factor ( k 9 b separation resolution mobile amino acid solutes” L. D factor (a)‘ (RJd phase‘
a-amino-n-butyric 2.69 acid arginine 20.7 methionine 3.18 norleucine 1.90 norvaline 2.81 phenylalanine 3.10 serine 2.67 17Ah tryptophan threonine 1.70 2.20 valine
3.00
1.12
0.60
5056
24.2 3.66
1.17 1.15 1.26 1.13 1.23 1.12 1.00 1.24 1.18
0.60 0.70 2.30 0.83 1.10 0.43 h 2.00 2.10
70:36 50:56 50:56 50:5d 5545f 505d 50:5W 50:5d 50:5d
2.40 3.18 3.80 3.00 17.8h 2.10 2.60
Solutes are dansyl derivatives of the D,L-amino acids (Le. l-N,-
N-(dimethylamino)naphthalene-5-sulfonamides).* Capacity fac-
tor, k’ = (retention volume of enantiomer-void volume)/void volume. CSelectivityfactor, a = k$/k’,. dResolution factor, R, = 2(distance between peaks)/(sum of the bandwidths of the two peaks). eNumber~represent the volume percent of methanol to water. The flow rate was 0.50 mL/min and the temperature 20 “C. f Data obtained using a 10-cm p-cyclodextrin Cyclobond I column on chromatographic system 11. #Data obtained using a 25-cm @cyclodextrin Cyclobond I column, flow rate 1.00 mL/min on chromatographic system I. Not resolved.
primary hydroxyl groups. Thus, the possibility exists for a number of potential interactions between these hydroxyl groups and substituent(s) present in the guest enantiomer molecule. For instance, if the enantiomer has suitable polar substituent(s), one or more favorable hydrogen bonds can form. Additionally, repulsive steric interaction(s) could also occur between any group of the guest and the CD hydroxyl groups (29). The observation of enantioselectivity appears to require three points of interaction between the enantiomer and p-CD (17,20, 29). At least one substituent of the guest must be tightly interacting with the CD cavity. In addition, the arrangement of appropriate substituents bonded to an asymmetric atom must be in close proximity to the CD rim hydroxyl groups so that two other points of interaction can occur (13,31). These findings are in harmony with the popular three-point attachment concept initially introduced by Dalgliesh in order to explain optical resolution by chromatography (2-4). Consequently, for appropriate enantiomers, the possiblity of chiral recognition and subsequent chromatographic optical resolution using p-CD exists. Enantiomeric Separations. It was found that the p-CD column exhibited excellent enantioselectivity for certain amino acid derivatives using aqueous-methanol mobile phases. Table I summarizes some of the optical separation data for a variety of representative dansylamino acids. As can be seen, excellent resolutions have been achieved for various D,L pairs with R, values greater than 2.00 in several instances. Note that most of the data in Table I was generated on a 10-cm column and that the resulting separation factors and resolutions obtained often exceed those previously reported for longer CSP columns and/or more complex chiral mobile-phase systems (2,6). A typical chromatogram is shown in Figure 1which i l l w a t e s the separation of a mixture of dansyl-D,L-threonine and phenylalanine as well as that of (f)-trans-a,a-(2,2-dimethyl-l,3-dioxlane-4,5-diyl)bis(diphenylmethanol).This chromatogram was obtained under isocratic conditions although as will be discussed later both solvent gradients and/or flow programming can be used to further optimize the separations. While underivatized amino acids can be partially separated from each other with the p-CD column, all racemic amino acids
240
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 Dns Thr
35% MeOH
"w
tom
r
DDDD
0
2 5 % MeOH
In N
E
W z V
0
Lo
m a
N
K
h
m a
0 c
0 v)
a m
~ o o o l ~
K v) 0
m
1
a
-
R, = 0.7
R, = 1.0 Time --$
-
d
5
IO
20
30
40
60
T i m e , min.
R, = 1.2
Figure 2. Enantiomeric separation and resolution of hexobarbital on the @ C D chiral stationary phase as a function of the percentage of methanol In the mobile phase. Condltions: 10-cm @-CD column, chromatographic system 11, flow rate 1.0 mL/min, 22 O C , mobile phase was methanol-water at the indicated percent composition.
Flgure 1. Chromatographic separation of the enantiomers of a mixture of racemic (f)-trans-cr,cr-(2,2dimethyl-1,3dioxolane-4,5-diyl)bls(diphenylmethanol) [DDDD], dansyl-D,L-threonine [Dns Thr], and dansyl-o,L-phenylalanine [Dns Phe] on the @ C D CSP. Conditions: chromatographic system I, 25-cm column, flow rate 1.00 mL/min, mobile phase 5050 methanoVwater, 25 O C , the first peak Is methanol, added as a retention marker.
examined eluted as a single peak. This result was not surprising since it had been previously reported that no enantioselectivity was observed in binding studies of various amino acids and simple dipeptides with @-CD(30, 32). The unsubstituted amino acids and dipeptides are much too small to tightly bind to the CD cavity. Likewise, the enantiomers of N-t-BOC-amino acids, CBZamino acids, and DNF'ry-amino acids were also not resolvable upon the @-CDCSP. Apparently the phenyl and pyridyl groups are not quite large enough to provide the tight binding situation required for chiral recognition. Molecules possessing a substituent of approximately the size of naphthalene or a little larger, however, can form strong inclusion complexes with @-CD(1&15,31,33). Consequently, chiral recognition is possible for the dansylamino acids as was already illustrated. In terms of the three-point attachment model, one would expect that the (dimethy1amino)naphthyl group of the dansylamino acid could penetrate and tightly bind the @-CDcavity. This would put the chiral carbon atom, along with its substituents, in close proximity to the hydroxyl groups on the CD rim where its carboxylate and amine moieties could form hydrogen bonds. Additionally, it may be possible for the sulfonyl oxygens to also participate in hydrogen bond formation (17). Good base-line separations were also observed for the enantiomeric @-naphthylamideand @-naphthylester derivatives of amino acids on the /3-CD columns (refer to representative entries 1-3, Table 11). In these derivatives, the naphthyl ring tightly binds the CD cavity and allows the carbonyl oxygen and primary amine functionality to interact with the CD's hydroxyl groups. It is apparent that one can obtain chiral recognition on the @-CDCyclobond I column by either derivatizing the amino or the carboxylate groups of the amino acid. For some of the dansyl, @-naphthylamide, and @naphthyl ester derivatives of amino acids, it is possible to detect as little as 0.50% of one isomer in the presence of the other (12). Judging from the literature, this appears to be one
V L ', 200
250
h ,nm
300
200
250
300
h ,nm
Flgure 3. CD spectra of the eluting enantiomers of hexobarbital resolved by the Cyclobond I column as shown in Figure 2. Fractions of the first (1) as well as second (2) eluting isomers were collected from
two successive injections each of which contained the racemic hexobarbital. of the more sensitive means by which to detect the enantiomeric purity of amino qcids (2, 3). In addition to the amino acids and some of their derivatives, the resolving power of the @-cyclodextrincolumn for a series of racemic barbiturates, 1,3-dioxolanes,and phenylacetic acids was also examined (Table 11). It was found that the P-CD CSP successfully resolved the enantiomers of the barbiturates, Evipan and Prominal (Table 11, entries 4 and 5). Figure 2 shows the chromatograms for the separation of (f)-hexobarbital using different methanol-water mobile phase compositions. The resolution obtained for the separation of the optical isomers of hexobarbital using the 15% methanol mobile phase was comparable to that reported for its separation on microcrystalline cellulose triacetate (3). The separation was confirmed by collecting fractions from the first and second eluting isomers and recording the respective CD spectra. As can be seen from the CD spectra shown in Figure 3, the two enantiomers were indeed resolved. The enantiomers of the substituted 1,3-dioxolanes,DIOP and DDDD, both of which possess four phenyl groups, were also partially separated on the 0-CD stationary phase. By contrast, 2,2-dimethyl-l,3-dioxolane-4-methannol (DDM) could not be resolved at all. Presumably, the DDM is too small to adequately bind to the CD cavity. The same thing can be said
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 _ I _ _ _ _ I
-.
_---
-___-.
241
-
Table 11. Retention, Selectivity, and Resolution Parameters for the Separation of Other Enantiomers upon the Chiral Cyclobond I Stationary Phase
capocityfactors no. 1 2 3 4 5 6 7
8 9 10 11 12
compounds
k'l
k'2
alanine-b-naphthyl ester alanine-P-naphthylamide methionine-P-naphthylamide hexobarbital (Evipan)b mephobarbital (Prominal)d DIOP DDDDf DDMh MTPA] cyclohexylphenylacetic acid mandelic acid a-(1-naphthy1)ethylamine
1.0 5.1 2.7 9.38 6.64 10.56 0.60 0.58 7.49 1.60 0.67 2.46
1.8 6.1 3.6 10.70 7.43 11.84 0.74 0.58 9.48 1.68 0.67 2.50
configuration of CD sign of first enantiomer
separation factor (a)
resolution
1.80 1.20 1.33 1.14 1.16 1.12 1.23 1.00 1.27 1.05 1.00 1.02
2.6 2.0 2.4 1.51 1.60 1.20 0.83
L L L
(-) at 245 nm (-) at 237 nm
-
+
i k i S-(-)
(R,)
mobile phase" 50:5OC 505OC 505OC 1585" 20:809 48:52c 50:509 50:509 50:509 50509 50:509 40609
2
0.58 2
i i
" Numbers represent the volume percent of methanol to water. The flow rate was 0.50 mL/min and the temperature 22 OC. 5-Cyclohexenyl-3,5-dimethylbarbituricacid. Data obtained using a IO-cm Cyclobond I column on system 11. d5-Phenyl-5-ethyl-3-methylbarbituric acid. e4,5-Bis(diphenylphosphinomethyl)-2,2-dimethyl-1,3-dioxolane. fTrans-a,ol-(2,2-Dimethyl-1,3-dioxolane-4.5-diyl)bis(diphenylmethanol). g Data obtained using a 25-cm Cyclobond I column on system I, flow rate 1.00 mL/min. 2,2-Dimethyl-l,3-dioxolane-4-methanol. 'Enantiomers not resolved. a-Methoxy-a-(trifluoromethy1)phenylacetic acid. Not determined. of the phenylacetic acid derivatives studied (Table 11). Only for the enantiomers of a-methoxy-a-(trifluoromethy1)phenylacetic acid (MTPA) was any resolution noted. Whereas the enantiomers of a-methylbenzylamine could not be resolved, some separation was observed for a - ( l naphthy1)ethylamine. This again demonstrates the strict size requirements for tight binding to the CD cavity. The behavior of a-(1-naphthy1)ethylaminewas unusual in that individual injections of the pure enantiomers indicated that a good separation was possible while injection of the mixture gave poor resolution. However, the trapped halves of the eluate fractions resulted in negative and positive optical rotations which indicate that partial separation had occurred. Based upon the optical separations provided by the chiral p-CD stationary phase for the limited number of racemic compounds examined in this study, it appears that the Cyclobond I column will prove to be useful in numerous other optical resolutions. The examination of molecular models in context with the required three-point attachment model should prove valuable in predicting which enantiomers should be separable due to chiral recognition upon inclusion with the 0-CD. Effect of Environmental Variations on Enantiomeric Separations. The effects of environmental changes on the resolution process were studied by varying the mobile-phase composition (type and percentage of organic solvent in water), flow rate, and temperature. First, the capacity factor and resolution of several enantiomeric compounds were measured by changing the methanol-water ratio in the mobile phase from 75:25 to 15:85. In all instances, there was observed an increase of the capacity factor of enantiomers as the percentage of methanol decreased. A plot of k'vs. percent MeOH for the D and L enantiomers of dansylthreonine which illustrates this general behavior is shown in Figure 4. More importantly, there was usually a marked improvement in the separation factor and resolution obtained on the p-CD column as the methanol content of the mobile phase was reduced. For example, in mobile phases containing 262% MeOH, the (&) enantiomers of DDDD could not be resolved. However, on going from 50% to 35% MeOH, the resolution improved from R , 0.85 to 1.18. Figure 2 shows a similar improvement in resolution of (&)-hexobarbital. Enantiomeric separations were also possble by using ethanol-water or acetonitrile-water mobile phases and the P-CD column. Some of the data obtained is summarized in Table 111. In general, lower percent compositions of ethanol or
I
I 25 0
550
45 0
35 0
65 0
75 0
% MeOH
Flgure 4. Influence of percentage methanol in the aqueous mobile phase on the capacity factors of D,L-dansylthreonine. Conditions: 25-cm Cyclobond I column, chromatographic system I, flow rate 1.00 mL/min, 24 OC.
Table 111. Comparison of Enantiomeric Separations Using Different Mobile-Phase Components with the P-CD Column Using Chromatographic System I compound
k;
k',
a
R,
mobile phase
Dns The Dns The
2.39 1.37 3.89 6.12 0.93 2.94 3.97 0.10 0.35 5.8
2.77 1.58 4.45 6.61 1.39 3.63 4.73 0.10 0.35 6.9
1.16 1.16 1.14 1.08 1.50 1.23 1.19 1.00 1.00 1.19
0.96 0.74 0.68 c 1.00 0.93 0.69 c
30% AN" 40% AN" 30% AN" 30% EtOHb 40% AN" 30% AN" 30% EtOHb 40% AN" 30% AN" 25% EtOHb
Dns Leu DDDD
c
0.51
"Refers to percent (v/v) of acetonitrile in water as mobile phase. Refers to percent (v/v) of ethanol in water as mobile phase. Not resolved.
acetonitrile were required in order to affect separation compared to methanol. In most cases, the methanol-water mobile phase proved to be superior in terms of obtained resolution of enantiomers. As in the case of methanol, the capacity factors and resolution decreased as the percentage composition of the organic component in water was increased. Although other solvents could be used, it is widely known that the
242
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
T a b l e IV. E f f e c t o f F l o w Rate on t h e C h r o m a t o g r a p h i c S e p a r a b i l i t y F a c t o r and R e s o l u t i o n o f the (+) and (-) E n a n t i o m e r s o f DDDDO
flow rate,
mL/min
+
1.00 0.80 0.60 0.40 0.30 0.20
0.58 0.57 0.56 0.55 0.56 0.53
k’ -
(Y
R,
0.71 0.71 0.70 0.69 0.70 0.66
1.22 1.24 1.24 1.25 1.26 1.24
0.85 0.92 0.97 1.12 1.22 1.20
a Conditions: 25-cm p-CD column, chromatographic system I, using a 5050 (v/v) methanol-water mobile ahase.
magnitude of the binding constant decreases as the amount of added alcohol or dipolar aprotic solvent (e.g., acetonitrile and dimethyl sulfoxide) in water is increased (15). Due to the fact that methanol itself interacts less with P-CD compared to ethanol or acetonitrile (28), it should be the cosolvent of choice for optimum resolution of enantiomers on the 0-CD stationary phase. Table IV shows the effect of mobile-phase flow rate on the separation and resolution of DDDD. Its behavior is typical of that also observed for other enantiomers. There was no appreciable change in the selectivity factor as a function of flow rate. However, there was often a dramatic improvement in resolution as the flow rate was reduced. In general, the improvement was greatest on going from 1.00 to approximately 0.40 mL/min, after which further reduction in flow rate caused only a small gain in resolution. It is known from cyclodextrin binding studies that increases in temperature diminish the extent of complexation between guest molecules and p-CD (15). In fact, most CD complexes in water are reported to be completely dissociated a t temperatures around 60-80 “C. Comparison of some separations conducted at ambient room temperatures of 18 and 31 “C showed that the capacity factors were greater at the lower temperature due to enhanced binding to P-CD. Although a reduction in temperature increases the magnitudes of the separation factors, the resolution does not correspondingly improve, most likely due to a loss of efficiency which is the consequence of poorer mass transfer.
CONCLUSIONS The P-CD CSP can provide efficient, selective separation of a variety of optical isomers. In comparison to the other typical CSP’s now popular, the use of 0-CD CSP appears to offer advantages in terms of flexibility and versatility. In particular, the solvent and experimental requirements for optical resolution appear to be somewhat more relaxed compared to those for other CSP’s. Specifically, the p-CD phase can be used with highly polar mobile-phase combinations whereas most other CSP’s are typically not very stable when used with comparable mobile phases. Consequently their use is limited to solvents of relatively low polarity (8, 34). Ad-
ditionally, unlike other CSP’s, the P-CD phase can be employed in the HPLC separation of many other classes of compounds. For instance, it was possible using the p-CD CSP to easily separate a variety of diastereomeric and structural isomers as well as more routine compounds that were poorly resolved on other types of columns (12,331. The future use of other cyclodextrin bonded phases composed of a-or 6-CD holds great promise for the resolution of other enantiomers due to their ability to form inclusion complexes with different size solutes. In order to further evaluate the applicability, advantages, and limitations of CD CSP’s in enantiomeric separations, additional work is required and is under way in these laboratories.
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RECEIVED for review July 19,1984. Accepted October 9,1984.
