K. Hrobo nˇ ová, A. Lomenova
of optical isomers is still increasing [1]. The determination
of the individual enantiomeric forms is essential for quality
control and stability studies of drug preparations mainly in
pharmaceutical chemistry. The frequently used analytical
methods of separation of enantiomers are chromatographic
methods [GC and HPLC with chiral stationary phase
summarized in Table 1. Molecularly imprinted polymers
were prepared by utilizing a block or suspension poly-
merization methods using phenylalanine enantiomer (L- or
D-) as the template. Underivatized forms or derivatives
(anilides, tert-butyloxycarbonyl-, dansyl-) are used [5].
In the present study, a resolution of amino acid racemate
was done using MIP CSP prepared by a modified poly-
merization method with L-phenylalanine as template
molecule. The analytical performance of MIP CSP was
evaluated. The effects of various operating parameters,
such as the selection of a suitable mobile phase composi-
tion, flow rate, column length, and column temperature,
were investigated for the purpose of achieving efficient
separation of phenylalanine enantiomers. The enthalpic and
entropic contributions to chromatographic retention of the
phenylalanine enantiomers were calculated.
(
CSP), chiral additive in mobile phase or with chiral
derivatization] and electromigration methods (CE with
chiral additives in electrolyte or with chiral derivatization,
CEC with chiral stationary phase). At present, attention is
oriented to a chiral polymeric sorbents prepared by
molecular imprinting technology [2]. On the base of
presence of specific recognition sites, molecularly imprin-
ted polymers (MIP) have ability to recognize and bind
target molecule, template. Beside selectivity toward tem-
plate or structural analogues, they are characterized by high
mechanical and chemical stabilities, good adsorption
capacity, and reusability [3]. Components of polymeriza-
tion mixture for MIP synthesis include template, functional
monomer, crosslinking monomer, initiator, and porogenic
solvent. For the preparation of MIP sorbent with high
selectivity, sufficient capacity, and suitable morphology,
the optimization of composition of polymerization mixture
is needed. When MIP has been utilized as HPLC stationary
phase for separation of enantiomers, the imprinting process
has been done using pure enantiomeric form as template.
The advantage of this approach to chiral separation, being
that the order of elution is predictable, is that the imprinted
enantiomer will bind to CSP strongly (higher value of
elution time) than the non-imprinted enantiomer (lower
value of elution time). The disadvantages of MIP CSPs
include unsuitability for some types of templates, low
sample capacity, low chromatographic peak efficiency
arising from slow mass-transfer kinetics. The applications
of molecularly imprinted polymer as separation media in
HPLC, and also in SFC and CEC, for chiral separation
have been extensively reviewed [4–6].
Results and discussion
Synthesis of MIP
MIPs were synthesized by bulk polymerization with non-
covalent approach. The properties of resulting sorbent
mostly depend on the polymerization mixture composition
(type and amount of template, functional monomer, poro-
genic solvent, crosslinking monomer), synthesis condi-
tions, and polymerization method used. In MIP-L-
phenylalanine synthesis, EGDMA was used as crosslinking
monomer and initiation was performed thermally at 60 °C
with using of AIBN. In the first step of preparation, we
selected type of functional monomer and type of porogenic
solvent. In the case of MAA as functional monomer, the
monomer–template prepolymerization complex was
formed through hydrogen bonding between carboxyl group
of monomer and amino group of template, and also
monomer hydrophilically interacts with the C=O bond in
template [16]. Hydrogen bonding interaction takes place
between the carbonyl and amino groups of AA and tem-
plate functional groups. Since the interactions between
functional monomers and target molecule are done mainly
through hydrogen bonds, thus a less polar solvent with
lower dielectric constant, acetonitrile, was chosen as an
appropriate porogenic medium. This solvent optimized the
formation of monomer–template prepolymerization com-
plex, while in the more polar porogenic solvents the for-
mation of monomer–porogen complexes is preferred.
Besides, we optimize the mole ratio of functional mono-
mer, template, and crosslinking monomer for synthesis. As
documented in Table 2, in the case of MIP I–III, resulting
polymers were soft and not suitable for application as
HPLC stationary phases. The formation of blocks of
polymers was not obtained with an increasing of
Phenylalanine is an essential amino acid that exists in
two isomeric forms. Due to the different biological natures
and pharmacological activities of both enantiomers, it is
important to develop methods for the effective separation
of them. In our previous study [7], HPLC method by uti-
lization of macrocyclic antibiotics, b-cyclodextrin, and
cyclofructan 6-based CSPs in reversed-phase, normal-
phase, and polar-organic phase separation modes was used
for separation of D,L-phenylalanine. The best enantiosepa-
ration was obtained on teicoplanin-based CSP in reversed-
phase mode. The limited lifetime and cost of commercial
CSPs provide the motivation to prepare MIP-based chiral
sorbent for separation of phenylalanine enantiomers. The
examples of phenylalanine enantiomers imprinting for
applicability of resulting polymers such as HPLC CSP,
sensors, and electrode for voltammetry [8–15] are
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Hroboňová, Katarína
