Anal. Chem. 1996, 68, 1948-1953
Antibody-Mimicking Polymers as Chiral Stationary Phases in HPLC Maria Kempe*
Department of Pure and Applied Biochemistry, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden
Antibody-mimicking synthetic polymers, selective for various optically active amino acid derivatives and peptides, were prepared by noncovalent molecular imprinting. A novel approach, in which the branched, trifunctional cross-linkers pentaerythritol triacrylate and 2,2-bis(hydroxymethyl)butanol trimethacrylate were copolymerized with methacrylic acid, is described. The polymers were subsequently applied as chiral stationary phases in highperformance liquid chromatography. They were superior to previously reported noncovalent molecularly imprinted polymers used for chiral separations in that they showed considerably higher load capacity, increased selectivity, and better resolving capability. There are a myriad of processes in nature that rely on highly selective molecular recognition events, e.g., the interactions between antigen-antibody, substrate-enzyme, protein-DNA, and hormone-receptor. It is a challenging task to mimic these natural recognition systems in the design of artificial receptors. The aim is to construct systems possessing steric and electronic features complementary to those of the ligand. The efforts in the field have resulted in artificial receptors such as crown ethers, cyclodextrins, cyclophanes, and various molecular clefts and cavities.1-4 Molecular imprinting, sometimes referred to as template polymerization,5-10 presents a more direct approach to the design of artificial receptor-like binding sites. The sites are tailor-made in situ by copolymerization of functional monomers and crosslinkers around the template molecules, also referred to as the print molecules. The print molecules are subsequently extracted from the polymer, leaving complementary recognition sites in the polymer network. The highly cross-linked polymers prepared this way can selectively recognize and rebind the print molecules. Noncovalent molecular imprinting relies on noncovalent interactions between the print molecules and the functional mono-
mers. This approach has been used for the preparation of synthetic polymers selective for amino acid derivatives and peptides,11-21 carbohydrates,22,23 and drugs,24,25 by copolymerizing the bifunctional cross-linker ethylene glycol dimethacrylate (EDMA) with various functional monomers, e.g., methacrylic acid (MAA),11-14,16-24,26-29 itaconic acid,24 4-vinylpyridine,15,25 and 2-vinylpyridine.16 The polymers were selective for their respective print molecules and proved to be practically useful as (i) stationary phases in HPLC,11-16,18,20-25 TLC,17 and CE,19 (ii) artificial antibody binding mimics in ligand-binding assays,26,27 and (iii) recognizing elements in sensors.28-30 Molecular imprinting has also been employed for the preparation of both polymers selective for proteins21,31 and polymers exhibiting catalytic activity.32-34 The present paper details a recent development in the field of noncovalent molecular imprinting.35 Cross-linkers used for the preparation of tailor-made polymers by noncovalent molecular imprinting have, until recently,35 been restricted to monomers with two vinyl groups. Previous efforts to develop novel polymer systems have focused mainly on varying the functional monomers and testing their capability of producing polymers with selective recognition sites. In general, the polymers were prepared with EDMA as the cross-linking agent. Alternative approaches, in
* Current address: Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455. E-mail:
[email protected] or
[email protected]. (1) Cram, D. J. Angew. Chem., Int. Ed. Engl. 1986, 25, 1039-1057. (2) Lehn, J.-M. Angew. Chem., Int. Ed. Engl. 1988, 27, 89-112. (3) Rebek, J., Jr. Angew. Chem., Int. Ed. Engl. 1990, 29, 245-255. (4) Cram, D. J. Nature 1992, 356, 29-36. (5) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9-14. (6) Shea, K. Trends Polym. Sci. 1994, 2, 166-173. (7) Steinke, J.; Sherrington, D. C.; Dunkin, I. R. Adv. Polym. Sci. 1995, 123, 81-125. (8) Kempe, M.; Mosbach, K. J. Chromatogr. A 1995, 694, 3-13. (9) Nicholls, I.; Andersson, L. I.; Mosbach, K.; Ekberg, B. Trends Biotechnol. 1995, 13, 47-51. (10) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (11) Sellergren, B.; Lepisto ¨, M.; Mosbach, K. J. Am. Chem. Soc. 1988, 110, 58535860. (12) O’Shannessy, D. J.; Ekberg, B.; Andersson, L. I.; Mosbach, K. J. Chromatogr. 1989, 470, 391-399.
(13) Andersson, L. I.; Mosbach, K. J. Chromatogr. 1990, 516, 313-322. (14) Kempe, M.; Mosbach, K. Anal. Lett. 1991, 24, 1137-1145. (15) Kempe, M.; Fischer, L.; Mosbach, K. J. Mol. Recognit. 1993, 6, 25-29. (16) Ramstro¨m, O.; Andersson, L. I.; Mosbach, K. J. Org. Chem. 1993, 58, 75627564. (17) Kriz, D.; Berggren Kriz, C.; Andersson, L. I.; Mosbach, K. Anal. Chem. 1994, 66, 2636-2639. (18) Ramstro ¨m, O.; Nicholls, I. A.; Mosbach, K. Tetrahedron: Asymmetry 1994, 5, 649-656. (19) Nilsson, K.; Lindell, J.; Norrlo ¨w, O.; Sellergren, B. J. Chromatogr. A 1994, 680, 57-61. (20) Kempe, M.; Mosbach, K. Int. J. Pept. Protein Res. 1994, 44, 603-606. (21) Kempe, M.; Mosbach, K. J. Chromatogr. A 1995, 691, 317-323. (22) Nilsson, K. G. I.; Sakaguchi, K.; Gemeiner, P.; Mosbach, K. J. Chromatogr. A 1995, 707, 199-203. (23) Mayes, A. G.; Andersson, L. I.; Mosbach, K. Anal. Biochem. 1994, 222, 483-488. (24) Fischer, L.; Mu ¨ ller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360. (25) Kempe, M.; Mosbach, K. J. Chromatogr. A 1994, 664, 276-279. (26) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (27) Andersson, L. I.; Mu ¨ ller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4788-4792. (28) Hedborg, E.; Winquist, F.; Lundstro ¨m, I.; Andersson. L. I.; Mosbach, K. Sens. Actuators A 1993, 37-38, 796-799. (29) Kriz, D.; Ramstro ¨m, O.; Svensson, A.; Mosbach, K. Anal. Chem. 1995, 67, 2142-2144. (30) Kriz, D.; Mosbach, K. Anal. Chim. Acta 1995, 300, 71-75. (31) Kempe, M.; Glad, M.; Mosbach, K. J. Mol. Recognit. 1995, 8, 35-39 . (32) Leonhardt, A.; Mosbach, K. React. Polym. 1987, 6, 285-290. (33) Robinson, D. K.; Mosbach, K. J. Chem. Soc., Chem. Commun. 1989, 4, 969970. (34) Sellergren, B.; Shea, K. J. Tetrahedron: Asymmetry 1994, 5, 1403-1406. (35) Kempe, M.; Mosbach, K. Tetrahedron Lett. 1995, 36, 3563-3566.
1948 Analytical Chemistry, Vol. 68, No. 11, June 1, 1996
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© 1996 American Chemical Society
Table 1. Preparation of Molecularly Imprinted Polymers
Figure 1. Structures of cross-linkers: pentaerythritol triacrylate (PETRA, 1), 2,2-bis(hydroxymethyl)butanol trimethacrylate (TRIM, 2), and pentaerythritol tetraacrylate (PETEA, 3).
which the imprinted EDMA-based polymers were grafted onto preformed poly(2,2-bis(hydroxymethyl)butanol trimethacrylate) beads, were presented recently.36,37 The aim of the study reported here was to change the network of the imprinted polymer itself in a more profound way. This was done by using tri- and tetrafunctional monomers as cross-linkers (Figure 1, 1-3), with the hope that the resulting recognition sites in the polymers would be more well-defined and of better quality, which would be reflected in higher selectivities and load capacities when the polymers are used as chiral stationary phases in HPLC. The preparation and applications of imprinted poly(pentaerythritol triacrylate-co-methacrylic acid) and poly(2,2-bis(hydroxymethyl)butanol trimethacrylate-co-methacrylic acid) chiral stationary phases are described. EXPERIMENTAL SECTION Nomenclature. The abbreviations used for amino acids and the designation of peptides follow the rules of the IUPAC-IUB Commission on Biochemical Nomenclature (J. Biol. Chem. 1972, 247, 977-983). The following additional abbreviations are used: AIBN, 2,2′-azobis(2-methylpropionitrile); Boc, tert-butyloxycarbonyl; CE, capillary electrophoresis; CSP, chiral stationary phase; DCC, N,N′-dicyclohexylcarbodiimide; EDMA, ethylene glycol dimethacrylate; HOBt, 1-hydroxybenzotriazole; HPLC, highperformance liquid chromatography; MAA, methacrylic acid; OEt, ethyl ester; OMe, methyl ester; PETEA, pentaerythritol tetraacry(36) Glad, M.; Reinholdsson, P.; Mosbach, K. React. Polym. 1995, 25, 47-54. (37) Dhal, P. K.; Vidyasankar, S.; Arnold, F. H. Chem. Mater. 1995, 7, 154162.
polymer
print molecule
1a 1b 2a 2b 3 4 5 6 7a 7b
Z-L-Tyr-OH Z-L-Tyr-OH Z-L-Phe-OH Z-DL-Phe-OH Z-L-Glu-OH Boc-L-Phe-Gly-OEt Z-L-Ala-L-Ala-OMe Z-L-Ala-Gly-L-Phe-OMe Z-L-Phe-OH Z-L-Phe-OH
amount of amount amount of print molecule of MAA cross- cross-linker (mmol) (mmol) linker (mmol) 7.5 5 7.5 7.5 3.75 5 5 3.75 7.5 7.5
30 20 30 30 30 30 30 30 30 30
1 1 2 2 2 2 2 2 3 3
30 40 30 30 30 30 30 30 30 20
late; PETRA, pentaerythritol triacrylate; SEM, scanning electron microscopy; TLC, thin-layer chromatography; TRIM, 2,2-bis(hydroxymethyl)butanol trimethacrylate; Z, benzyloxycarbonyl. Materials. Boc-Phe-Gly-OEt, Boc-D-Phe-Gly-OEt, Z-Ala-GlyPhe-OMe, Z-D-Ala-Gly-D-Phe-OMe, Z-L-Ala-D-Ala-OMe, and Z-D-AlaL-Ala-OMe were synthesized in solution according to the method of Ko¨nig and Geiger using DCC and HOBt as reagents.38 All other peptides and amino acid derivatives were purchased from Sigma Chemicals (St. Louis, MO), Bachem AG (Bubendorf, Switzerland), or Nova Biochem (La¨ufelfingen, Switzerland). DCC and HOBt were from Nova Biochem. MAA and AIBN were obtained from Janssen Chimica (Geel, Belgium). PETRA was from Aldrich Chemical Co. (Milwaukee, WI), TRIM was from Aldrich Chemie (Steinheim, Germany), and PETEA was from Monomer-Polymer Laboratories (Windham, NH). All organic solvents were of analytical or HPLC grade. Preparation of Polymers. Print molecule, MAA, and one of the cross-linkers PETRA (1, Figure 1), TRIM (2), or PETEA (3) were dissolved, according to the amounts indicated in Table 1, in 20 mL of chloroform. AIBN (0.7 mmol) was added, and the solutions were sonicated and deoxygenated with a stream of nitrogen gas for 10 min and then irradiated for 48 h at 366 nm in 4 °C. The resulting bulk polymers were ground in an end runner mill (Model RM O, Retsch, Haan, Germany) and wet-sieved by hand with water and ethanol through a 25 µm sieve (Retsch). The particles that passed the sieve were collected and dried with methanol on a sintered glass filter funnel. The fine particles were removed by repetitive sedimentations and decantations in acetone. Porosimetry Analysis. Nitrogen adsorption/desorption isotherms39 were recorded on a Micromeritics ASAP 2400 (Chemical Engineering, University of Lund, Sweden) using a 72-point pressure table and 10 s equilibration times after particles of polymer 5 were degassed at 150 °C for 3 h. The average pore diameter was 282 Å, and the average pore volume was 0.022 mL/g (calculations based on the BJH model on the adsorption isotherm, pore diameter ) 4 × pore volume/pore area). Specific Surface Area. Single-point surface area measurements were performed on a Micromeritics FlowSorb II 2300 after particles were degassed at 150 °C for 4 h. The specific surface areas were determined to be (in m2/g) 21.4 (polymer 1a), 3.8 (polymer 2a), 15.9 (polymer 3), 5.0 (polymer 4), and 4.2 (polymer 5). Scanning Electron Microscopy. SEM was performed on Polymer 5 particles using an ISI-100 A instrument. (38) Ko ¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788-798. (39) Emig, G.; Hofmann, H. J. Catal. 1967, 8, 303-306.
Analytical Chemistry, Vol. 68, No. 11, June 1, 1996
1949
Table 2. Separation of 1 mg of the Indicated Racemates on Molecularly Imprinted CSPs polymer
separated species
eluent (v/v)
nD
nL
k′D
k′L
R
f/g
1a 1b 4 5
Z-DL-Tyr-OH Z-DL-Tyr-OH Boc-DL-Phe-Gly-OEt Z-D-Ala-D-Ala-OMe, Z-L-Ala-L-Ala-OMe Z-D-Ala-Gly-D-Phe-OMe, Z-L-Ala-Gly-L-Phe-OMe
chloroform-HOAc (99:1) chloroform-HOAc (99:1) chloroform chloroform-HOAc (99.75:0.25)
176 260 298 289
29 61 85 113
4.79 3.24 0.84 0.93
9.89 5.44 1.37 1.79
2.06 1.68 1.63 1.92
0.93 0.86 0.67 0.93
chloroform-HOAc (99.5:0.5)
216
59
0.88
1.82
2.07
0.80
6
Table 3. Separation of 100 µg of the Indicated Racemate on Molecularly Imprinted CSPsa polymer
separated species
1a 2a 3 4 5
Z-DL-Tyr-OH Z-DL-Phe-OH Z-DL-Glu-OH Boc-DL-Phe-OEt Z-D-Ala-D-Ala-OMe, Z-L-Ala-L-Ala-OMe Z-L-Ala-Gly-L-Phe-OMe, Z-D-Ala-Gly-D-Phe-OMe Z-DL-Phe-OH Z-DL-Phe-OH
6 7a 7b
elution conditions
eluent A (v/v)
eluent B (v/v)
gradient I gradient II gradient III gradient IV gradient V
chloroform-HOAc (96:4) chloroform-HOAc (99.9:0.1) chloroform-HOAc (96:4) chloroform-HOAc (99.9:0.1) chloroform-HOAc (99.75:0.25)
chloroform-HOAc (8:2) chloroform-HOAc (98:2) chloroform-HOAc (9:1) chloroform-HOAc (98.2) chloroform-HOAc (8:2)
244 963 2.32 6.63 2.86 5.47 166 947 5.07 11.59 2.29 3.14 204 861 1.77 4.34 2.45 3.10 175 1539 1.50 4.56 3.04 3.44 286 780 1.34 4.28 3.19 4.50
1.0 1.0 1.0 1.0 1.0
gradient VI
chloroform-HOAc (99:1)
chloroform-HOAc (9:1)
228 2172 0.86
3.10 3.60
4.15
1.0
isocratic isocratic
chloroform chloroform
340 491
3.72 2.14 2.07 1.74
nd nd
0.93 0.92
nD
nL
k′D
52 1.74 121 1.19
k′L
Rs
R
f/g
a 20 µg was injected on polymer 2a. The gradients are given in the legends of Figures 3-6. n, number of theoretical plates; k′, capacity factor; R, separation factor; Rs, resolution factor according to ref 40 (nd, not determined); f/g, resolution factor according to ref 41.
Table 4. Separation of 20 µg of the Indicated Racemates on Z-L-Phe-OH-Imprinted Poly(TRIM-co-MAA) CSP (Polymer 2a) Using Isocratic Elution with Chloroform-HOAc (99.9:0.1 v/v) at 1 mL/min
Figure 2. Separation of 100 µg of racemic Z-Tyr-OH on Z-L-TyrOH-imprinted poly(PETRA-co-MAA) CSP (polymer 1a). Gradient elution at 1 mL/min with chloroform-HOAc (96:4 v/v) and chloroformHOAc (8:2 v/v) (B): 0-23 min, 0% B; 23-24 min, 0-100% B; 2443 min, 100% B; 43-47 min, 100% B (gradient I).
High-Performance Liquid Chromatography. The sieved polymer particles were slurried in chloroform-acetone (17:3 v/v) and packed with acetone into stainless-steel HPLC columns (polymers 2a and 2b, 100 × 4.6 mm; polymers 1a, 1b, 3-6, 7a, and 7b, 250 × 4.6 mm) at 300 bar using an air-driven fluid pump (Haskel Engineering Supply Co., Burbank, CA). The print molecules were extracted from the polymer network by elution with methanol-acetic acid (7:3 v/v) at 1 mL/min until a stable baseline was obtained. The HPLC analyses were performed using a Kontron HPLC system comprising a Model 420 HPLC pump, a Model 425 gradient former, and a Model 432 variable absorbance detector. The pump was controlled by a Toshiba T 1000 personal computer. The elutions were performed at room temperature and were monitored spectrophotometrically at 260 nm. The flow rate was 1.0 mL/min. The retention times were determined by injection of 0.02-1 mg of the racemates (dissolved in 20 µL of the eluent). The capacity factors (k′L and k′D) were calculated as k′ ) (t - to)/to, t being the retention time of the analyte and to (the void volume) the retention time of toluene. The separation factors (R) were calculated as R ) k′L/k′D. The number of theoretical plates (n) 1950 Analytical Chemistry, Vol. 68, No. 11, June 1, 1996
separated species
nD
nL
k′D
k′L
R
Rs
Z-DL-Phe-OH Boc-DL-Phe-OH Z-DL-Ala-OH Fmoc-DL-Phe-OH
174 204 223 178
50 104 130 80
5.07 6.11 8.41 3.37
12.63 10.85 13.37 5.59
2.49 1.78 1.59 1.66
2.43 1.78 1.60 1.39
was calculated as n ) 5.54(t/wh)2, t being the retention time of the analyte and wh the peak width at half of the peak height. The resolution factors were calculated according to Wulff et al. (Rs)40 or Meyer (f/g).41 Frontal Chromatography. The polymer particles were packed into HPLC columns (50 mm × 4.6 mm). Dissociation constants and numbers of binding sites of polymers 4 and 5 were detemined in the HPLC mode by elution with chloroform at 0.5 mL/min, as previously described in ref 14 and references therein. RESULTS AND DISCUSSION Two block copolymers were prepared by radical solution polymerization of PETRA (1) and MAA in the presence of the print molecule Z-L-Tyr-OH: one with a molar ratio PETRA-MAA of 1:1 (polymer 1a, Table 1), and the other with a ratio of 2:1 (polymer 1b). Both polymers were able to resolve Z-DL-Tyr-OH when applied as stationary phases in HPLC. The main interactions responsible for the recognition of Z-L-Tyr-OH are most likely hydrogen bonds between carboxy groups in the polymer and the carboxy- and carbamate functionalities of the print molecule. Furthermore, the hydroxyl groups originating from PETRA can (40) Wulff, G.; Poll, H.-G.; Mina´rik, M. J. Liq. Chromatogr. 1986, 9, 385-405. (41) Meyer, V. R. Chromatographia 1987, 24, 639-645.
a
b a
c
b
d c
Figure 3. Chromatographic resolution of various racemic NRprotected amino acids on Z-L-Phe-OH-imprinted poly(TRIM-co-MAA) CSP (polymer 2a). (a) Gradient elution of 20 µg of racemic Z-PheOH with chloroform-HOAc (99.9:0.1 v/v) and chloroform-HOAc (98:2 v/v) (B) at 1 mL/min: 0-12 min, 0% B; 12-13 min, 0-25% B; 1324 min, 25% B; 24-26 min, 25-0% B (gradient II). (b) Isocratic elution of 20 µg of racemic Boc-Phe-OH with chloroform-HOAc (99.9:0.1 v/v) at 1 mL/min. (c) Isocratic elution of 20 µg of racemic Fmoc-PheOH with chloroform-HOAc (99.9:0.1 v/v) at 1 mL/min. (d) Isocratic elution of 20 µg of racemic Z-Ala-OH with chloroform-HOAc (99.9: 0.1 v/v) at 1 mL/min.
interact with hydrogen bond acceptors of the print molecule. The separation was better on polymer 1a than on polymer 1b when 1 mg of the racemate was eluted on analytically sized columns (Table 2). These poly(PETRA-co-MAA) CSPs showed considerably higher load capacities than the conventional poly(EDMA-coMAA) CSPs; the latter has been reported to separate only 10 µg of racemic NR-protected amino acids with separation factors in the range 1.67-2.45 under similar conditions.13 Since the separation was better on polymer 1a than on the more cross-linked polymer 1b, the molar ratio used for polymer 1a was chosen for subsequent polymers. Figure 2 shows the separation of 100 µg of racemic Z-Tyr-OH on polymer 1a. Gradient elution was used to enhance the elution rate of the enantiomer used as print molecule, which was, as expected, more retarded than its optical antipode (Table 3). The resolution factor was remarkably high (Rs ) 5.47) for a molecularly imprinted polymer. The previously discussed poly(EDMA-co-MAA) CSPs imprinted with NR-protected amino acids gave resolution factors in the range of 0.1-1.4.13 TRIM (2) was copolymerized with MAA, and the resulting polymers, imprinted with various amino acid derivatives and peptides (polymers 2a, 3-6, Table 1), were demonstrated to
d
Figure 4. Chromatographic resolution of racemates on molecularly imprinted poly(TRIM-co-MAA) CSPs. (a) Gradient elution of 100 µg of racemic Z-Glu-OH on Z-L-Glu-OH-imprinted poly(TRIM-co-MAA) CSP (polymer 3) with chloroform-HOAc (96:4 v/v) and chloroformHOAc (9:1 v/v) (B) at 1 mL/min: 0-13 min, 0% B; 13-14 min, 0-100% B; 14-25% min, 100% B; 25-30 min, 100-0% B (gradient III). (b) Gradient elution of 100 µg of racemic Boc-Phe-Gly-OEt on Boc-L-Phe-Gly-OEt-imprinted poly(TRIM-co-MAA) CSP (polymer 4) with chloroform-HOAc (99.9:0.1 v/v) and chloroform-HOAc (98:2 v/v) (B) at 1 mL/min: 0-10.5 min, 0% B; 10.5-15 min, 0-50% B; 15-30 min, 50% B; 30-35 min, 50-0% B (gradient IV). (c) Gradient elution of 100 µg of a mixture of Z-L-Ala-L-Ala-OMe and Z-D-Ala-DAla-OMe on Z-L-Ala-L-Ala-OMe-imprinted poly(TRIM-co-MAA) CSP (polymer 5) with chloroform-HOAc (99.75:0.25 v/v) and chloroformHOAc (8:2 v/v) (B) at 1 mL/min: 0-10 min, 0% B; 10-18 min, 0-5% B; 18-22 min, 5% B; 22-24 min, 5-0% B (gradient V). (d) Gradient elution of 100 µg of a mixture of Z-L-Ala-Gly-L-Phe-OMe and Z-DAla-Gly-D-Phe-OMe on Z-L-Ala-Gly-L-Phe-OMe-imprinted poly(TRIMco-MAA) CSP (polymer 6) with chloroform-HOAc (99:1 v/v) and chloroform-HOAc (9:1 v/v) (B) at 1 mL/min: 0-7 min, 0% B; 7-9 min, 0-100% B; 9-17 min, 100% B; 17-22 min, 100-0% B (gradient VI).
resolve successfully their respective print molecules from the corresponding optically active antipodes (Table 3). A reference polymer, prepared using racemic Z-Phe-OH as the print species (polymer 2b), was unable to discriminate Z-L-Phe-OH from Z-DPhe-OH. This proves that the chiral recognition is due to the imprinting procedure and is not inherent to the polymer as such. Analytical Chemistry, Vol. 68, No. 11, June 1, 1996
1951
a
b
c
Figure 5. Separation of 1 mg of racemates on imprinted poly(TRIM-co-MAA) CSPs. (a) Isocratic elution of 1 mg of racemic Boc-Phe-Gly-OEt on Boc-L-Phe-Gly-OEt-imprinted poly(TRIM-co-MAA) CSP (Polymer 4) with chloroform at 1 mL/min. (b) Isocratic elution of 1 mg of a mixture of Z-L-Ala-L-Ala-OMe and Z-D-Ala-D-Ala-OMe on Z-L-Ala-L-Ala-OMe-imprinted poly(TRIM-co-MAA) CSP (polymer 5) with chloroform-HOAc (99.75: 0.25 v/v) at 1 mL/min. (c) Isocratic elution of 1 mg of a mixture of Z-L-Ala-Gly-L-Phe-OMe and Z-D-Ala-Gly-D-Phe-OMe on Z-L-Ala-Gly-L-PheOMe-imprinted poly(TRIM-co-MAA) CSP (polymer 6) with chloroform-HOAc (99.5:0.5 v/v) at 1 mL/min.
Polymer 2a, made selective for Z-L-Phe-OH, was capable of separating not only racemic Z-Phe-OH but also racemic Boc-PheOH, Fmoc-Phe-OH, and Z-Ala-OH (Table 4, Figure 3). The resolutions were much better than has previously been achieved on poly(EDMA-co-MAA) CSPs imprinted with Z-L-Phe-OH.20 Polymers 3-6 (Table 1) were capable of separating 100 µg of the enatiomers used as print molecules from their optically active antipodes, resulting in separation factors in the range of 2.453.60 and resolution factors in the range of 3.10-4.50 (Table 3, Figure 4). High load capacities were demonstrated by separating 1 mg on polymers 4-6 with separation factors as high as 2.07 (Figures 5, Table 2). Separation factors, resolution factors, and load capacities were, in general, considerably higher for poly(TRIM-co-MAA) than for poly(EDMA-co-MAA). A direct comparison between Z-L-Ala-L-Ala-OMe-imprinted poly(EDMA-coMAA) and poly(TRIM-co-MAA) CSPs showed the superiority of the poly(TRIM-co-MAA) polymer; separation of 100 µg of the racemate on poly(EDMA-co-MAA) resulted in a separation factor of 2.22 and a resolution factor of 1.50, which should be compared to 3.19 and 4.50 on poly(TRIM-co-MAA). Furthermore, 1 mg was easily separated on poly(TRIM-co-MAA) (R ) 1.92), whereas the corresponding poly(EDMA-co-MAA) polymer could not resolve the same amount (R ) 1.0). In this context, it should be mentioned that a cross-linking derivative of pentaerythritol containing four vinyl groups (PETEA, 3, Figure 1) did not produce polymers having the same high load capacities and excellent resolving characteristics as seen for those prepared using the trifunctional cross-linkers PETRA and TRIM (Table 3, polymers 7a and 7b). A mixture of all four stereoisomers of Z-Ala-Ala-OMe was eluted on Z-L-Ala-L-Ala-OMe-imprinted poly(TRIM-co-MAA) (Figure 6). Z-L-Ala-L-Ala-OMe was, as expected, the most retarded isomer. Z-DAla-D-Ala-OMe and Z-D-Ala-L-Ala-OMe coeluted and were less retarded than Z-L-Ala-D-Ala-OMe. From the elution order of these species, conclusions regarding the recognition mechanism can be drawn: it seems as the amino-terminal part of the molecule (Z-Ala-) is more important in the recognition than the carboxyterminal part (-Ala-OMe). The higher load capacities and more efficient resolutions achieved on the described polymers, as compared to the data 1952 Analytical Chemistry, Vol. 68, No. 11, June 1, 1996
Figure 6. Isocratic elution of 100 µg of Z-DL-Ala-DL-Ala-OMe on Z-LAla-L-Ala-OMe-imprinted poly(TRIM-co-MAA) CSP (polymer 5) with chloroform at 1 mL/min.
obtained on previously reported molecularly imprinted polymers, are probably due to a combination of several factors. Compared to the amount of the print molecule usually used in imprinted poly(EDMA-co-MAA), higher amounts (1.3-2.5 times more) were used during the polymerizations of the described poly(PETRAco-MAA) and poly(TRIM-co-MAA) copolymers. This could, in theory, result in more recognition sites in the resulting polymers. The recognition sites of molecularly imprined polymers are “polyclonal”; when the most well-defined sites are studied, the corresponding dissociation constants are lower than those when a larger fraction of the sites, including less specific sites, are studied. The number of recognition sites that can practically be determined by frontal chromatography was in the same range as in poly(EDMA-co-MAA) CSPs:14 polymer 4 had 14 µmol sites/g of polymer, with dissociation constants for the L- and the Denantiomers of 2.7 and 6.0 mM, respectively, and polymer 5 had 22 µmol sites/g of polymer, with dissociation constants of 2.5 and
Figure 7. Scanning electron micrographs of Z-L-Ala-L-Ala-OMeimprinted poly(TRIM-co-MAA) (polymer 5) at (a) 100× magnification and (b) 10 000× magnification.
4.8 mM, respectively. However, the method is limited by the sensitivity of the spectrophotometric detector of the HPLC system, and it is likely that differences between poly(EDMA-co-MAA) and poly(TRIM-co-MAA) would be observed if a smaller number of sites (the most specific sites) could be studied. The textures of dry polymer particles were studied by SEM (Figure 7). The average size of the pores shown in Figure 7 was determined by nitrogen adsorption measurements to be 282 Å. Likewise, the average pore volume was detemined to be 0.022 mL/g. These (42) Sellergren, B.; Shea, K. J. Chromatogr. 1993, 635, 31-49. (43) Rosenberg, J.-E.; Flodin, P. Macromolecules 1987, 20, 1522-1526. (44) Reinholdsson, P.; Hargitai, T.; Isaksson, R.; To ¨rnell, B. Angew. Macromol. Chem. 1991, 192, 113-132. (45) Afeyan, N. B.; Gordon, N. F.; Mazsaroff, I.; Varady, L.; Fulton, S. P. J. Chromatogr. 1990, 519, 1-29.
figures are higher than those for imprinted poly(MAA-co-EDMA) polymers.42 TRIM has previously been reported to give rise to macroporous polymers under some conditions.43,44 Continuous pores (“through pores”) in chromatographic packing materials have been shown to facilitate intraparticle convection, which reduces the mass transport resistance and improves the resolution.45 Nothing indicates that the pores in the imprinted polymers described here are of this type, but it cannot be precluded that a slightly more porous structure contributes to the observed improvements in the resolution. One thing that would improve the efficiency of this type of molecularly imprinted stationary phases even more is the use of spherical beads, prepared by, for example, suspension polymerization. The polymers described here were prepared as bulk polymers, which were subsequently ground, sieved, and sedimented. This resulted in highly irregular particles, as can be seen in Figure 7a. Particles used as stationary phases in HPLC should preferably be spherical and of uniform size to provide optimal chromatographic efficiency. Therefore, the chromatographic performance of the polymers is expected, at least in theory, to be improved by the use of such particles. In conclusion, molecularly imprinted CSPs prepared with the trifunctional cross-linkers pentaerythritol triacrylate and 2,2-bis(hydroxymethyl)butanol trimethacrylate were shown to be superior to previously reported noncovalent molecularly imprinted polymers, in that considerably higher load capacities and better resolutions were achieved. This important progress in the field will hopefully make molecularly imprinted polymers a practically useful alternative to conventional CSPs, in analytical as well as preparative applications. ACKNOWLEDGMENT The author thanks Prof. Klaus Mosbach (Pure and Applied Biochemistry, University of Lund) for supporting this project and for fruitful discussions, Ms. Birgitta Svensson for SEM and porosimetry analysis (Chemical Engineering, University of Lund), and the Royal Swedish Academy for Engineering Sciences’ Hans Werthe´n Foundation for financial support. A preliminary account of part of this work was presented at the 18th International Symposium on Column Liquid Chromatography (HPLC ’94), Minneapolis, MN, May 10, 1994 (lecture by M. Kempe), and in in ref 35. Received for review December 18, 1995. Accepted March 13, 1996.X AC9512160 X
Abstract published in Advance ACS Abstracts, May 1, 1996.
Analytical Chemistry, Vol. 68, No. 11, June 1, 1996
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