Hplc

A large number of chiral amino acids and peptides have been imprinted. Several MIPs selective for pharmaceuticals have also been described. The most widely used method has been bulk polymerization followed by grinding, sieving and packing into HPLC columns. Alternatively, the polymers can be prepared by any of the methods discussed in 'Affinity Separation: Imprint Polymers' in this encyclopedia. Some examples of MIP CSPs are shown in Table 1.

The selectivities are in many cases comparable to those of commercially available CSPs. For example, a separation factor (a) of 17.8 was found for the separation of the two enantiomers of a dipeptide on poly(methacrylic acid-co-EDMA) (EDMA = ethy-

lene glycol dimethacrylate) imprinted with one of the enantiomers (Figure 2).

The specificity and selectivity of MIPs can be fine-tuned by careful choice of monomers and solvent, and by optimizing the molar ratios of the components in the polymerization mixture. The recognition relies on multiple interaction points. The more fun-tionalized the print molecule is, the more interactions are possible. An example of a highly selective polymer is po/y(4-vinylpyridine-co-EDMA) imprinted with Z-l-aspartic acid (1). The chromatogram in Figure 3A shows the separation of racemic Z-aspartic acid on this CSP. Aspartic acid and glutamic acid differ by only one methylene unit, but despite this small difference, the Z-aspartic acid-imprinted CSP was not able to resolve racemic Z-glutamic acid (2) (Figure 3B). The side chain of Z-l-glutamic acid cannot be accommodated into the recognition site in a way that allows specific interaction between the carboxy functionality and the positioned pyridine group of the polymer. The same type of polymer, imprinted with Z-l-glutamic acid, was able to resolve racemic Z-glutamic acid, but not racemic Z-aspartic acid. Similar observations have been done on po/y(methacrylic acid-co-EDMA) imprinted with these print molecules.

oh
2

Polymers imprinted with Z-l-phenylalanine (3a) were able to separate racemic Z-phenylalanine efficiently (Table 2). Racemic Z-alanine (3b) could also be separated on these CSPs, even if lower separation factors were observed. When the amino-group of the racemate was protected with tert-butyloxycarbonyl (Boc) (3c) or 9-fluorenylmethyloxycarbonyl (Fmoc) (3d), the separations were poorer than with the ra-cemate of the print molecule, which was protected with benzyloxycarbonyl (Z). In contrast to the CSPs described above, which were unable to separate the racemate of a molecule which only differed from the

Table 1 A selection of molecularly imprinted CSPs for HPLCa

Print molecule

Polymer

ac

RSd

f/ge

Amino acids

H-L-Phe-OHf

poly(CuVBIDA-co-EDMA)

1.45

n.d.

n.d.

H-L-Phe-NHPhg

poly(MAA-co-EDMA)

13

n.d.

n.d.

H-D-p-NH2Phe-NHPhh

poly(MAA-co-EDMA)

15

n.d.

n.d.

Ac-D-Trp-OMei

poly(MAA-co-EDMA)

3.92

2.2

1.0

Ac-L-Trp-OH'

poly (AA-co-EDMA)

3.24

2.02

n.d.

Boc-L-Trp-OHi

poly(MAA-co-2VPy-co-EDMA)

4.35

1.9

1.0

Fmoc-L-Phe-OHk

poly(MAA-co-EDMA)

1.36

n.d.

n.d.

Z-l-Asp-OH'

poly(4VPy-co-EDMA)

2.81

1.22

0.81

Z-L-Phe-OHm

poly(MAA-co-TRIM)

2.29

3.14

1.00

Z-L-Tyr-OHm

poly(MAA-co-PETRA)

2.86

5.47

1.00

Dansyl-L-Phe-OHi

poly(MAA-co-2VPy-co-EDMA)

3.15

1.6

0.96

Small peptides

H-L-Phe-Gly-NHPhn

poly(MAA-co-EDMA)

5.1

n.d.

n.d.

Boc-L-Phe-Gly-OEtm

poly(MAA-co-TRIM)

3.04

3.44

1.00

Z-L-Ala-L-Ala-OMem

poly(MAA-co-TRIM)

3.19

4.50

1.00

Ac-L-Phe-L-Trp-OMeo

poly(MAA-co-EDMA)

17.8

n.d.

1.00

Z-L-Ala-Gly-L-Phe-OMem

poly(MAA-co-TRIM)

3.60

4.15

1.00

Pharmaceuticals

(S)-Timololp

poly(MAA-co-EDMA)

2.9

2.0

n.d.

(S)-Naproxenq

poly(4VPy-co-EDMA)

1.65

n.d.

n.d.

(S,R)-Ephedriner

poly(MAA-co-TRIM)

3.42

1.6

n.d.

(S,S)-Pseudoephedriner

poly(MAA-co-TRIM)

3.19

1.8

n.d.

aThe print molecules and their optical antipodes were separated.

bAA, Acrylamide; CuVBIDA, Cu(II)[W-(4-vinylbenzyl)]iminodiacetate; EDMA, ethylene glycol dimethacrylate; Ita, itaconic acid; MAA, methacrylic acid; PETRA, pentaerythritol triacrylate; TRIM, trimethylolpropane trimethacrylate; 2VPy, 2-vinlypyridine; 4VPy, 4-vin-lypyridine.

cThe separation factor were calculated as a = kprint molecule/koptical antipode; k' = (t— t0)/t0; t is the retention time of the analyte and t0 is the retention time of unretained compound (the void).

dThe resolution factors (RS) were calculated according to Wulff G, Poll HG and Minarik M (1986) Enzyme-analogue built polymers. XIX. Racemic resolution on polymers containing chiral cavities. Journal ofLiquid Chromatography 9: 385-405.

eThe resolution factors (f/g) were calculated according to Meyer VR (1987) Some aspects of the preparative separation of enantiomers on chiral stationary phases. Chromatographia 24: 639-645.

fData from Vidyasankar S, Ru M and Arnold FH (1997) Molecularly imprinted ligand-exchange adsorbents for the chiral separation of underivatized amino acids. Journal of ChromatographyA 775: 51-63.

gData from Sellergren B and Shea KJ (1993) Chiral ion-exchange chromatography. Correlation between solute retention and a theoretical ion-exchange model using imprinted polymers. Journal ofChromatographyA 654: 17-28.

hData from Sellergren B and Nilsson KGI (1989) Molecular imprinting by multiple noncovalent host-guest interactions: Synthetic polymers with induced specificity. Methods in Molecular and Cellular Biology 1: 59-62.

iData from Ramstrom O, Andersson LI and Mosbach K (1993) Recognition sites incorporating both pyridinyl and carboxy functionalities prepared by molecular imprinting. Journal of Organic Chemistry 58: 7562-7564.

'Data from Yu C and Mosbach K (1997) Molecular imprinting utilizing an amide functional group for hydrogen bonding leading to highly efficient polymers. Journal ofOrganic Chemistry 62: 4057-4064.

kData from Kempe M and Mosbach K (1994) Chiral recognition of ^-protected amino acids and derivatives in non-covalently molecularly imprinted polymers. InternationalJournalofPeptide and Protein Research 44: 603-606.

'Data from Kempe M, Fischer L and Mosbach K(1993) Chiral separation using molecularly imprinted heteroaromatic polymers. Journal ofMolecularRecognition 6: 25-29.

mData from Kempe M (1996) Antibody-mimicking polymers as chiral stationary phases in HPLC. Analytical Chemistry 68: 1948-1953. "Data from Andersson LI, O'Shannessy DJ and Mosbach K (1990) Molecular recognition in synthetic polymers: preparation of chiral stationary phases by molecular imprinting of amino acid amides. JournalofChromatography 513: 167-179. "Data from Ramstrom O, Nicholls IA and Mosbach K (1994) Synthetic peptide receptor mimics: Highly stereoselective recognition in non-covalent molecularly imprinted polymers. Tetrahedron: Asymmetry5: 649-656.

pData from Fischer L, Muller R, Ekberg B and Mosbach K (1991) Direct enantioseparation of ^-adrenergic blockers using a chiral stationary phase prepared by molecular imprinting. Journal ofthe American Chemistry Society 113: 9358-9360. qData from Kempe M and Mosbach K (1994) Direct resolution of naproxen on a non-covalently molecularly imprinted chiral stationary phase. Journal ofChromatographyA 664: 276-279.

rData from Ramstrom O, Yu C and Mosbach K (1996) Chiral recognition in adrenergic receptor binding mimics prepared by molecular imprinting. Journal ofMolecularRecognition 9: 691-696.

Figure 2 Separation of 10 ^g of a mixture of Ac-L-Phe-L-Trp-OMe and Ac-D-Phe-D-Trp-OMe on a po/y(methacrylic acid-co-EDMA) CSP (4.6x200 mm column) imprinted with Ac-L-Phe-L-Trp-OMe. Isocratic elution at 1 mLmin~1 with CHCl3-HOAc (99 : 1). Attentu-ation was increased 10-fold at 10min. (Adapted from Ramstrom O, Nicholls IA and Mosbach K (1994) Tetrahedron: Asymmetry 5: 649-656, © 1994, with permission from Elsevier Science, UK.)

Figure 2 Separation of 10 ^g of a mixture of Ac-L-Phe-L-Trp-OMe and Ac-D-Phe-D-Trp-OMe on a po/y(methacrylic acid-co-EDMA) CSP (4.6x200 mm column) imprinted with Ac-L-Phe-L-Trp-OMe. Isocratic elution at 1 mLmin~1 with CHCl3-HOAc (99 : 1). Attentu-ation was increased 10-fold at 10min. (Adapted from Ramstrom O, Nicholls IA and Mosbach K (1994) Tetrahedron: Asymmetry 5: 649-656, © 1994, with permission from Elsevier Science, UK.)

print molecule by one methylene unit, these polymers were able to separate all of the tested structurally related racemates, even if the separations were not as good as with the print molecule and its optical antipode. This shows that the polymer recognizes both

Figure 3 Po/y(4-vinylpyridine-co-EDMA) CSP (4.6x200 mm column) imprinted with Z-L-aspartic acid. (A) 20 ^g of racemic Z-aspartic acid was applied. Isocratic elution at 0.5 mL min~1 with tetrahydrofuran-HOAc (24 : 1) and detection at 260 nm. (B) 20 ^g of racemic Z-glumatic acid was applied. Isocratic elution at 0.5 mL min with tetrahydrofuran- HOAc (199: 1) and detection at 260 nm. (Adapted from Kempe M, Fischer L and Mosbach K (1993) Journal of/Molecular Recognition 6: 25-29, © 1993, with permission from John Wiley & Sons, UK.)

the ammo-protecting group and the side chain, but that an exact fit is not necessary for enantioseparation in these cases.

Figure 3 Po/y(4-vinylpyridine-co-EDMA) CSP (4.6x200 mm column) imprinted with Z-L-aspartic acid. (A) 20 ^g of racemic Z-aspartic acid was applied. Isocratic elution at 0.5 mL min~1 with tetrahydrofuran-HOAc (24 : 1) and detection at 260 nm. (B) 20 ^g of racemic Z-glumatic acid was applied. Isocratic elution at 0.5 mL min with tetrahydrofuran- HOAc (199: 1) and detection at 260 nm. (Adapted from Kempe M, Fischer L and Mosbach K (1993) Journal of/Molecular Recognition 6: 25-29, © 1993, with permission from John Wiley & Sons, UK.)

In a study on ^-adrenergic blockers, (S)-timolol was imprinted in EDMA-based polymers. When the functional monomer was methacrylic acid, the

Table 2 Separation of racemic amino acid derivatives on Z-L-Phe-OH-imprinted CSPs

Racemate poly (MAA-co-EDMA)abc poly (MAA-co-TRIM)abd a a

Boc-Phe-OH 1.31 1.78

Fmoc-Phe-OH 1.21 1.66

aEDMA, Ethylene glycol dimethacrylate; MAA, methacrylicacid; TRIM, trimethylolpropane trimethacrylate.

bThe separation factors were calculated as a = kL/kD; k= (t— i0)/t0; tis the retention time of the analyte and t0 is the retention time of unretained compound (the void). cData from Kempe M and Mosbach K (1994) Chiral recognition of NT-protected amino acids and derivatives in non-covalently molecularly imprinted polymers. International Journal ofPeptide and Protein Research 44: 603-606.

dData from Kempe M (1996) Antibody-mimicking polymers as chiral stationary phases in HPLC. AnalyticalChemistry 68: 1948-1953.

polymer was able to resolve not only racemic timolol (4), but also racemic propanolol (5). In contrast to this, a timolol-imprinted polymer prepared with itaconic acid as the functional monomer instead of methacrylic acid could only resolve racemic timolol out of a number of racemates of structurally related ft-blockers (Figure 4). This clearly demonstrates that the selectivity of MIPs can be highly dependent on the functional monomer used.

A comparison of six different CSPs, all imprinted with the same print molecule (Z-l-phenylalanine), confirms that the choice of monomers is important for the selectivity of the resulting polymers (Table 3). The selectivity of EDMA-based polymers was higher when vinylpyridines were used, either alone or together with methacrylic acid, than when methacrylic acid was used alone. Methacrylic acid interacts with the print molecule through hydrogen bonds. The beneficial effect of vinylpyridine is attributed to strong ionic interactions between the carboxy groups of the print molecule and the pyridinyl groups. The polymer prepared with acrylamide also showed a higher selectivity than the one prepared with meth-acrylic acid. Acrylamide forms strong hydrogen bonds even in a polar solvent such as acetonitrile.

It is noteworthy that the load capacity and the resolving capability increased when the trifunctional cross-linker pentaerythritol triacrylate (PETRA) was used instead of EDMA. The same features have been seen with polymers prepared with trimethylol-propane trimethacrylate (TRIM), another trifunc-tional cross-linker. Poly(methacrylic acid-co-TRIM) imprinted with a dipeptide was able to resolve 1 mg of the racemate with almost baseline separation (analytical column: 4.6 x 250 mm) (Figure 5).

(S)-Naproxen (6), a nonsteroidal antiinflammatory drug, has been imprinted in po/y(4-vinylpyridine-co-EDMA) by two different approaches. Bulk polymerization followed by grinding and sieving resulted in highly irregular particles and a two-step swelling and polymerization method gave uniformly sized beads. Both materials, used in the chromatographic mode, were able to separate naproxen from the related ibup-rofen (7) and ketoprofen (8). The polymers were also able to resolve racemic naproxen, but not the ra-cemates of ibuprofen and ketoprofen (Figure 6). Even

Figure 4 Separation of 20 ^g of racemic timolol on a poly(itaconic acid-co-EDMA) CSP (4.6x200 mm column) imprinted with (S)-timolol. Isocratic elution at 1mLmin~1 with EtOH-tetrahydrofuran-HOAc (5 : 4 : 1). (Adapted from Fischer L, Muller R, Ekberg B and Mosbach K (1991) JournaloftheAmeri-can Chemical Society 113: 9358-9360, © 1991, with permission from the American Chemical Society, USA.)

Figure 4 Separation of 20 ^g of racemic timolol on a poly(itaconic acid-co-EDMA) CSP (4.6x200 mm column) imprinted with (S)-timolol. Isocratic elution at 1mLmin~1 with EtOH-tetrahydrofuran-HOAc (5 : 4 : 1). (Adapted from Fischer L, Muller R, Ekberg B and Mosbach K (1991) JournaloftheAmeri-can Chemical Society 113: 9358-9360, © 1991, with permission from the American Chemical Society, USA.)

Table 3 Chiral separation of racemic Z-Tyr-OH on molecularly imprinted CSPs

Polymera

Separated amount (fig)

ab

Rsc

f/gd

poly (AA-co-EDMA)e

40

3.62

2.52

n.d.

poly(MAA-co-EDMA)f

10

1.82

n.d.

0.50

poly(4VPy-co-EDMA)g

20

4.00

1.53

0.94

poly(2VPy-co-EDMA)f

10

3.81

1.90

0.95

poly(MAA-co-2VPy-co-EDMA)f

10

4.32

1.90

0.97

poly(MAA-co-PETRA)h

100

2.86

5.47

1.00

poly(MAA-co-PETRA)h

1000

2.06

n.d.

0.93

aAA, Acrylamide; EDMA, ethylene glycol dimethacrylate; MAA, methacrylic acid; 2VPy, 2-vinylpyridine; 4VPy, 4-vinylpyridine; PETRA, pentaerythritol triacrylate. bThe separation factors were calculated as a = kL/kD; k = (t — i0)/t'0; t is the retention time of the analyte and t0 is the retention time of unretained compound (the void). cThe resolution factors (RS) were calculated according to Wulff G, Poll HG and Minarik M (1986) Enzyme-analogue built polymers. XIX. Racemic resolution on polymers containing chiral cavities. Journal ofLiquidChromatography 9: 385-405. dThe resolution factors (f/g) were calculated according to Meyer VR (1987) Some aspects of the preparative separation of enantiomers on chiral stationary phases. Chromato-graphia 24: 639-645.

eData from Yu C and Mosbach K (1997) Molecular imprinting utilizing an amide functional group for hydrogen bonding leading to highly efficient polymers. Journal of Organic Chemistry 62: 4057-4064.

fData from Ramstrom O, Andersson LI and Mosbach K (1993) Recognition sites incorporating both pyridinyl and carboxy functionalities prepared by molecular imprinting. Journal of Organic Chemistry 58: 7562-7564.

gData from Kempe M, Fischer L and Mosbach K (1993) Chiral separation using molecularly imprinted heteroaromatic polymers. Journal ofMolecularRecognition 6: 25-29. hData from Kempe M (1996) Antibody-mimicking polymers as chiral stationary phases in HPLC. AnalyticalChemistry 68: 1948-1953.

Z-D-Ala-D-Ala-OMe

Z-D-Ala-D-Ala-OMe

Z-L-Ala-L-Ala-OMe

Z-L-Ala-L-Ala-OMe

1 T

Figure 5 Separation of mixtures of Z-L-Ala-L-Ala-OMe and Z-D-Ala-D-Ala-OMe on a po/y(methacrylic acid-co-TRIM) CSP (4.6 x250 mm column) imprinted with Z-L-Ala-L-Ala-OMe. (A) 100 ^g was applied. Gradient elution at 1 mL min~1 with CHCl3-HOAc (99.75 : 0.25) and CHCl3-HOAc (4: 1) ( = B). Gradient: 0-10 min, 0% B; 10-18 min, 0-5% B; 18-22 min, 5% B; 22-24 min 5-0% B. Detection at 260 nm. (B) 1 mg was applied. Isocratic elution at 1 mLmin~1 with CHCl3-HOAc (99.75:0.25). Detection at 260 nm. (Adapted from Kempe M (1996) Analytical Chemistry 68: 1948-1953, © 1996, with permission from the American Chemical Society, USA.)

Figure 6 Separation of racemic mixtures of naproxen, ibuprofen and ketoprofen on po/y(4-vinylpyridine-co-EDMA) CSPs (4.6x100 mm columns) imprinted with (S)-naproxen. (A) The column was packed with particles prepared by grinding and sieving a bulk polymer. Isocratic elution at 0.1 mLmin~1 with tetrahydrofuran-heptane-HOAc (250:250: 1) and detection at 260 nm. (Adapted from Kempe M and Mosbach K (1994) JournalofChromatographyA 664: 276-279, © 1994, with permission from Elsevier Science, UK.) (B) The column was packed with beads prepared by a two-step swelling and polymerization method. Isocratic elution at 1.0mLmin~1 with CH3CN-phosphate buffer (20mmolL~1, pH 4.0) (1 :1) and detection at 254nm. (Adapted from Haginaka J, Takehira H, Hosoya Kand Tanaka N (1997) Chemistry Letters, 555-556, © 1997, with permission from the Chemical Society of Japan.)

Figure 6 Separation of racemic mixtures of naproxen, ibuprofen and ketoprofen on po/y(4-vinylpyridine-co-EDMA) CSPs (4.6x100 mm columns) imprinted with (S)-naproxen. (A) The column was packed with particles prepared by grinding and sieving a bulk polymer. Isocratic elution at 0.1 mLmin~1 with tetrahydrofuran-heptane-HOAc (250:250: 1) and detection at 260 nm. (Adapted from Kempe M and Mosbach K (1994) JournalofChromatographyA 664: 276-279, © 1994, with permission from Elsevier Science, UK.) (B) The column was packed with beads prepared by a two-step swelling and polymerization method. Isocratic elution at 1.0mLmin~1 with CH3CN-phosphate buffer (20mmolL~1, pH 4.0) (1 :1) and detection at 254nm. (Adapted from Haginaka J, Takehira H, Hosoya Kand Tanaka N (1997) Chemistry Letters, 555-556, © 1997, with permission from the Chemical Society of Japan.)

if comparisons of the two chromatograms cannot be done because of differing flow rates, it is not obvious that the chromatographic efficiency was better with the uniformly sized beads (Figure 6B) than with the irregular particles (Figure 6A). This may be due to impairment on the selectivity by water interfering with the monomer-print molecule complex, since water was used as the suspension medium in the two-step swelling and polymerization method.

7

In general, the polymerizations in noncovalent molecular imprinting have to be done in nonaqueous solutions to prevent water molecules from interfering with the interactions between the monomers and the templates, as previously discussed. Several reports, however, show that the chromatography can be performed efficiently with buffered aqueous eluents.

An approach has been developed which allows both the imprinting and the chiral separation of free amino acids to be carried out in aqueous solutions. The recognition was based on metal coordination-chelation interactions using N-(4-vinylbenzyl)imino-diacetic acid as the functional monomer. The method worked best for aromatic amino acids (Figure 7).

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