sa2
Anal. Chem. 1986, 58,582-584
naphthalenetrisulfonate, 5182-30-9.
LITERATURE CITED Figure I. Simplified model showing a “membrane” or “bilayer-like” area when surfactant adsorbs on LC bonded media. Adsorbed surfactant can significantly alter the thickness and microviscosity of the “bonded-phase’’ as well as provide a hydrophilic boundary layer.
can only take place from one side, the ”bilayer” is asymmetric, etc., in the chromatographic case), it is apparent that there is a hydrophobic layer bounded by a very polar layer of surfactant head groups, ions, and associated waters of hydration. A hydrophobic solute would have to traverse the polar boundary to gain access to or leave the stationary phase. An amphiphilic solute would tend to fit into the stationary phase “layer” so that both its hydrophilic and hydrophobic requirements are fulfilled. The thickness of the “active layer” of the stationary phase can also be increased by the adsorbed surfactant as can the viscosity. All of these factors can contribute to poor stationary phase mass transfer. One must consider this as the simplest-case-model. Both the bonded phase and surfactant type can significantly affect the properties and behavior of the stationary phase. It may even be possible for a liquid-crystal-like environment to exist when appreciable concentrations of surfactant are concentrated at the stationary phase surface. Mobile phase mass transfer effects might also be observed if a system is devised that minimizes these stationary phase effects. Registry No. SDS, 151-21-3; SMS, 512-42-5; @-naphthol, 135-19-3;p-nitroaniline, 100-01-6;p-nitrophenol, 100-02-7;disodium 2,6-naphthalenedisulfonate,1655-45-4;disodium 4,5-dihydroxynaphthalene-2,7-disulfonate,129-96-4;trisodium 1,3,6-
(1) Armstrong, D. W.; Fendler, J. H. Biochim. Siophys. Acta 1977, 4 8 , 75-80. (2) Armstrong, D. W.; Terrill, R. Q. Anal. Chem. 1979, 57,2160-2163. (3) Armstrong, D. W.; Henry, S . J. J. Ll9. Chromatogr. 1980, 3 , 657-662. (4) Armstrong, D. W.; Nome, F. Anal. Cbem. 1981, 5 3 , 1662-1666. (5) Kirkman, C. M. Ph.D. Dissertation, University of Massachusetts, Amherst, MA, 1985. (6) Kirkman, C. M.; Charvat, S.B.; Elliot, W. G.; Stengle, T. R.; Uden, P. C. 1985 Pittsburgh Conference, New Orleans, LA, Abs. No. 1008. (7) Klrkman, C. M.; Zu-Ben, C.; Uden, P. C.; Stratton, W. J.; Henderson, D. E. J. Chromatogr. 1984, 317, 569-578. 1 Armstrong, D. W.; Hinze, W. L.; Bui, K. H.; Singh, H. N. Anal. Lett. 1981, 74, 1659-1667. Weinberger, R.; Yarmchuk, P.; Cline Love, L. J. Anal. Chem. 1982, 54. 1552-1558. Landy, J. S.;Dorsey, J. G. J. Chromatogr. Sci. 1984, 22, 68-70. Dorsey, J. G.; Khaledi, M. G.; Landy, J. S.; Lin, J. L. J. Chromatogr. 1984. 376. 183-191. Khaledi, M: G.; Dorsey, J. G. Anal. Chem. 1985, 5 7 , 2190-2196. Pelizzetti, E.; Pramauro, E. Anal. Cbim. Acta 1985, 769, 1-29. Armstrong, D. W. S e p . Furif. Methods 1985, 74, 213-304. Dorsey, J. G.; DeEchegary, M. T.; Landy, J. S. Anal. Chem. 1983, 55, 924-928. Yarmchuk, P.; Weinberger, R.; Hirsch, R. F.; Cline Love, L. J. J . Chromatogr. 1984, 2 8 3 , 47-60. Borgerding, M. F.; Hinze, W. L. Anal. Chem. 1985, 5 7 , 2183-2190. Taylor, G. I. Proc. R . SOC.London, A 1953, 279, 186-203. Taylor, G. 1. Proc. R . SOC.London, A 1954, 223, 446-460. Arb, R. Proc. R . SOC.London, A 1958, 235, 67. Aris, R. Proc. R . SOC.London, A 1954, 252, 538-551. Evans, D. F.; Chen, C.; Lamartlne, B. C. J. Am. Chem. SOC. 1977, 99, 6492-6496. Tljssen, R. Anal. Chim. Acta 1980, 774, 71-89. Longworth, L. G. J. Am. Chem. SOC. 1953, 74, 5705.
RECEIVED for review July 15, 1985. Accepted October 7, 1985. Support of this work by the Department of Energy, Office of Basic Energy Research (DE-AS084ER 13159), is gratefully acknowledged.
Thin-Layer Chromatographic Separation of Optical, Geometrical, and Structural Isomers Ala Alak and Daniel W. Armstrong* Department of Chemistry, Texas Tech University, Lubbock, Texas 79409
A number of dlfferent slllca gels and blnders were evaluated in the development of a P-cyclodextrin bonded TLC phase wlth the capabliity to separate enantiomeric compounds. Planar chromatographic conditions were optimlred for the separation of nine racemlc mixtures, three diastereomerlc mlxtures, and SIXstructural Isomers. Mobile phases and eiutlon behavlor of all solutes were analogous to the related LC technlque lndlcatlng the two methods are sufficiently cornpatable to be used In conjunction wlth one another. Thls should provide one with much greater fiexlbillty in the field of isomerlc separations.
The chromatographic separation of enantiomers is an important and rapidly expanding field. In addition to the gas chromatographic separations that have been reported, there has been much recent work on the liquid chromatographic (LC) separation of optical isomers. Despite the fact that a
variety of LC approaches to the separation of enantiomers have been utilized successfully (I-3), there have been few reports on the thin-layer chromatographic (TLC) separation of enantiomers. Yuasa first reported the TLC separation of D,L-tryptophan on a crystalline cellulose coated plate (4). Wainer et al. ( 5 ) separated racemic 2,2,2-trifluoro-1-(9anthry1)ethanol on TLC media to which (R)-N-(3,4-dinitrobenzoy1)phenylglycine was bonded. Weinstein (6),Grinberg and Weinstein (7), and Gunther et al. (8) reported the enantiomeric separation of several dansyl amino acids and underivatized amino acids on reversed-phase plates impregnated with copper(I1) complexes of chiral alkyl a-amino acid derivatives. In the present work, @-cyclodextrin(@-CD)bonded TLC plates were developed and used to separate several enantiomers, diastereomers, and structural isomers. Furthermore, these plates are compatible with the chiral LC columns recently developed in this laboratory (9-12) and can be used to evaluate potential solvent systems and other chromatographic conditions. The advantages of TLC (e.g., low cost,
0003-2700/86/0358-0582$01.50/00 1986 American Chemlcai Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
E
A
simultaneous sample development) are now becoming available to those interested in enantiomeric and general isomeric separations.
EXPERIMENTAL SECTION Materials. p-Cyclodextrin was obtained from Advanced Separation Technologies, Inc. (ASTEC). Seven types of silica gel were utilized in this study. They were (1)Thorn Smith silica gel (TLC-7GF),(2) ASTEC-40 pm diameter, 300--4pore size silica gel, (3) ASTEC-5-40 ym diameter 60-A pore size silica gel, (4) ASTEC-10 ym diameter, 60-A pore size silica gel, ( 5 ) ASTEC-3 ym diameter, 60-Apore size silica gel, (6) Whatman K5, 150-A pore size silica gel, and (7) Macherey Nagel 5-20 ym, 60-A pore size silica gel. The three types of binders used in this study were (1)ASTEC acrylate binder, (2) poly(ethy1ene glycol) (Aldrich), and (3) ASTEC "All Solvent Binder". HPLC grade methanol and water were obtained from Fischer Co. All dansyl amino acids and P-naphthylamide amino acids were obtained from Sigma. Nitroaniline, nitrophenol, and stilbene isomers were obtained from Aldrich. cis- and trans-Benzo[a]pyrene-7,8diol were the generous gift of H. J. Isaaq (Frederick Cancer Research Facility). Ferrocene enantiomers were produced as previously reported (12),as were the hydrolytically stable 0-cyclodextrin bonded phases (9). Methods. TLC plates (5 X 20 cm) were prepared by mixing 1.5 g of p-CD bonded silica gel in 15 mL of 50% methanol (aq) with 0.002 g of binder. The slurry was spread to a thickness of 3 mm on a clean glass plate and left to air-dry. The plate was then heated in an oven to 75 "C for 15 min before use. The stationary phase thickness was (1 mm on the finished plate. All developments were done at room temperature (20 "C) in an ll3I4 in. long, 4 in. wide, and lO3I4in. high Chromaflex developing chamber. Additional separation parameters are given in Tables I and 11. Spot visualization was done by use of a fixed-wavelength (254-nm) lamp. RESULTS AND DISCUSSION In order to develop a p-CD bonded phase TLC technique capable of separating enantiomers, three parameters had to be optimized. The first was the silica gel support, which affected both the efficiency of the separation and the coverage of p-CD. The second parameter was the binder, which not only gave the plates the proper physical and mechanical properties but also affected efficiency, development time, and selectivity. Lastly, the proper mobile phases, which could enhance isomeric separations, were evaluated. The relative amount of (3-CD bonded to silica gel (via a 7-10 atom spacer) could be estimated by using the separation behavior of 0- and p-nitroaniline (9, IO). For example, pnitroaniline always had a higher R, value than o-nitroaniline on bonded phases containing no p-CD (when using methanol/water mobile phases). Conversely, p-nitroaniline was more strongly retained than o-nitroaniline if an appreciable amount of bonded p-CD was present. At high 0-CD coverages (where p-nitroaniline remains near the origin) one begins to see enantioselectivity. Equal amounts of P-CD could not be attached to all silica gels. Figure 1A shows solute elution behavior on the bonded phase containing the least p-CD, while Figure 1C illustrates the related elution behavior on a high-coverage media. Note the different retention behaviors of D,L-leucine as well as 0- and p-nitroaniline. Figure 1B shows typical results of an intermediate-quality media. As expected, the physical properties of the silica gel also had a significant affect on efficiency (spot size). The Macherey Nagel silica gel appeared to combine the best coverage of P-CD with high efficiencies (Figure 1). It is well-known that a TLC binder is necessary to assure that the stationary phase adheres to the support and has sufficient mechanical strength to withstand spotting, development, and general handling procedures. However, the binder can also alter the efficiency, selectivity, development time, and detection unless properly utilized. In the case of
583
r L
Figure 1. Three planar chromatograms Illustrating the effect of sllica gel type on the P C D coverage and on spot size. Chromatogram A used Thorn Smith silica gel, B used Whatman, and C used Macherey Nagel silica gel (see Experimental Section for further details). L represents dansyl-L-leucine; D,L represents dansyl-o,L-leucine, 0,m, and p represent 0-,m-, and p-nitroaniline, respectively. The mobile phase was 50/50methanol/l Yo aqueous triethylammonium acetate (pH 4.1).
A
7.:. ;,
, ., ,', ,,
..
. ,.,. .:.. .
,
: ,.,.'.
.:.:, :..... :. '. ,
',:., ;.,,..,, , . ,.. .,;
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.:.... .. ,. '
.' , ; . 1
.;
.:...
::..;:.;:; ., .;': ...... .. . ., ..... .:. . . ' ,
.;:. ,.
..... .."' . , ..
D,L
D,L
Val
Leu
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Flgure 2. Three planar chromatograms showing the effect of binder on spot size. ASTEC 10 pm silica gel was used in all cases. Chromatogram A had no binder; chromatogram B contains an acrylate polymer; and chromatogram C contains ASTEC "All Solvent Binder (see Experimental Section for further details). The mobile phase was 50/50 methanol/ 1YO aqueous triethylammonium acetate (pH 4.1).
6-CD bonded phase plates one must be careful that the binder does not form a strong inclusion complex with 0-CD thereby rendering it ineffective for further separations. To prevent complex formation, it was decided to use polymeric binders that were too large to fit into the cyclodextrin cavity. Figure 2 shows the effect of various polymeric binders on analogous TLC chromatograms. Binders B and C resulted in plates of excellent stability and mechanical properties. However, binder B became unstable in the presence of >50% water, whereas binder C was stable in any solvent matrix. Furthermore, binder C gave chromatograms with smaller tighter spots without affecting enantioselectivity (Figure 2). Increasing the concentration of binder C (see Experimental Section) increased development time, however. Poly(ethy1ene glycol) was totally ineffective as a binder in that plates produced with it were analogous to those with no binder. Given previous LC experience, it was thought that methanollwater and acetonitrilelwater mobile phases would be best suited for p-CD TLC. Indeed this was the case. It was also found that use of buffers such as 0.1-1% triethylammonium acetate (TEAA), p H 4.1, instead of pure water,
584
ANALYTICAL CHEMISTRY, VOL.
58,NO. 3, MARCH 1986
Table I. Separation Data for Enantiomeric Compounds Generated by Using 5 X 20 cm (3-CD Bonded Phase Plates' compd
Rf
dansyl-D-leucine dansyl-L-leucine dansyl-D-methionine dansyl-L-methionine dansyl-D-alanine dansyl-L-alanine dansyh-valine dansyl-L-valine
0.49 0.66 0.28 0.43 0.25 0.33 0.31 0.42 D-alanine-(3-naphthylamide 0.16 L-alanine-(3-naphthylamide 0.25 D-methionine-P-naphthylamide 0.16 L-methionine-6-naphthylamide 0.24 (-)-I-ferrocenyl-1-methoxyethane' 0.31 (+)-1-ferrocenyl-1-methoxyethane' 0.42 (-)-1-ferrocenyl-2-methylpr~panol~ 0.33 (+)-l-ferrocenyl-2-methyl0.39 propanol' (-)-S-( 1-ferrocenylethyl)thio0.37 glycolic acid' (+)-S-(1-ferrocenylethy1)thio0.44 glycolic acidc
mobile phaseb
detection method
40160
fluorescence
25/75
fluorescence
25/75
fluorescence
25/75
fluorescence
30170
ninhydrin
30170
ninhydrin
90/10
visible
90/10
visible
90/10
visible
'TLC stationary phase consisted of (3-CD bonded through a spacer to Macherey Nagel silica gel plus binder no. 3 (see Experimental Section). bNumbers represent the volume ratio of methanol to 1% triethylammonium acetate (pH 4.1). 'In these compounds, the (+) or (-) refers to the Cotton effect at 250 nm and not to the oDtical rotation at the sodium D line. Table 11. Separation Data for Diastereomers and Structural Isomers Generated by Using 5 X 20 cm P-CD Bonded Phase Plates' compd
R,
0.38 0.46 0.38 0.48 benzo[a]pyrene-trans-7,8-diol 0.46 benzo[a]pyrene-cis-7,8-diol 0.52 o-nitroaniline 0.49 rn-nitroaniline 0.56 p-nitroaniline 0.22 o-nitrophenol 0.56 rn-nitrophenol 0.60 p-nitrophenol 0.46 quinine quinidine trans-stilbene cis-stilbene
Table I gives the separation data and optimized conditions for nine enantiomeric compounds. All enantiomers were completely resolved. The separation of D,L-methionine was particularly impressive, although this mixture may have been in the sulfoxide form. Note that the D enantiomer of all amino acid derivatives and the (-) enantiomer of all metallocenes elute first. Table I1 lists representative data for the separation of diastereomers and structural isomers. This indicates that p-CD TLC is applicable t o a wide variety of separations including enantiomers, diastereomers, structural isomers, and nonisomeric compounds. Registry NO.P-CD, 7585-39-9; D-leUCine, 328-38-1;L-leucine, 61-90-5; DL-leUCine, 328-39-2; D-methionine, 348-67-4; Lmethionine, 63-68-3; DL-methionine, 59-51-8; D-alanine, 338-69-2; L-danine, 56-41-7;DL-danine, 302-72-7;D-valine, 640-68-6; L-valine, 72-18-4; DL-valine, 516-06-3; dansyl-D-alanine, 56176-32-0; dansyl-L-alanine, 35021-10-4; dansyl-D-methionine, 77481-10-8; dansyl-L-methionine, 17039-58-6; D-alanine-p-naphthylamide, 20723-89-1; L-alanine-p-naphthylamide, 720-82-1;D-methionineP-naphthyhmide, 85827-87-8; L-methionine-@-naphthylamide, 7424-16-0; ferrocenyl-1-methoxyethane,12512-90-2;ferrocenyl2-methylpropanol, 51177-17-4;S-(1-ferrocenylethy1)thioglycolic acid, 56483-18-2; quinine, 130-95-0; quinidine, 56-54-2; transstilbene, 103-30-0; cis-stilbene, 645-49-8; benzo[a]pyrene-trans7,8-diol, 57404-88-3; benzo[a]pyrene-cis-7,8-diol,60657-25-2; onitroaniline, 88-74-4; m-nitroaniline, 99-09-2; p-nitroaniline, 100-01-6; o-nitrophenol, 88-75-5; m-nitrophenol, 554-84-7; pnitrophenol, 100-02-7; DL-methionine-P-naphthylamide, 9857579-2; dansyl-DL-alanine, 42808-04-8; dansyl-DL-methionine, 48208-47-5; poly(ethy1ene glycol), 25322-68-3; Astec Acrylate Binder, 99746-91-5; Astec All Solvent Binder, 99746-92-6.
LITERATURE CITED
mobile phaseb
detection method
25/75
fluorescence
(1) Davankov, V. A. "Advances In Chromatography"; Giddings, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1980; Vol. 18, p 139 and references therein. (2) Davankov, V. A.; Kurganov, A. A.; Bockkov, A. S. "Advances in Chromatography";Giddings, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1983; Vol. 22, p 71 and refer-
80/20
0.1% KMnO,
(3)
80120
0.1% KMn0,
(4) (5)
ences therein. Armstrong, D. W. J . Liq. Chromatogr. Suppl. 2 1984, 7,353, and references therein. Yuhsa, S.; Shimado, A.; Kameyama, K.; Yasui, M.; Adzuma, K. J . Chromatogr. Sci. 1980, 18, 311. Wainer. I. W.; Brunner, C. A,; Doyle, T. A. J . Chromatogr. 1983, 264, 154.
40160
UV (254 nm)
(6) (7) (8)
40160
UV (254 nm)
"TLC stationary phase consisted of p-CD bonded through a spacer to Macherey Nagel silica gel plus binder no. 3 (see Experimental Section). bNumbers represent the volume ratio of methanol to 1% triethylammonium acetate (pH 4.1). tended to increase the resolution and efficiency of most ionizable solutes. Other buffers compatible with cyclodextrin bonded phases (such as ammonium acetate and phosphate buffers) could also be used.
Weinstein, S. Tetrahedron Lett. 1984, 25,985. Grinberg, N.; Weinstein, S. J . Chromatogr. 1984, 303, 251. Gunther, K.; Martens, J.; Schickedanz, M. Angew. Chem., I n t . Ed.
Engl. 1984, 23, 506. (9) Armstrong, D. W.; DeMond, W. J . Chromatogr. Sci. 1984, 22, 411. (10) Armstrong, D. W. U S . Patent 4539399, Sept 3, 1985. (1 1) Armstrong, D. W.; DeMond, W.; Alak, A,; Hinze, W. L.; Riehl, T. E.; Bul, K. H. Anal. Chem. 1985, 57,234. (12) Armstrong, D. W.; DeMond, W.; Czech, B. P. Anal. Chem. 1985, 57, 481.
RECEIVED for review August 19, 1985. Accepted October 15, 1985. The authors gratefully acknowledge the support of the
U.S. Department of Energy, Office of Basic Energy Science (DE-AS0584ER13159)
