American Journal of Anal yt ical Chemistry, 2011, 2, 909-918
doi:10.4236/ajac.2011.28105 Published Online December 2011 (
Copyright © 2011 SciRes. AJAC
Selective Recognition and Detection of L-Aspartic Acid by
Molecularly Imprinted Polymer in Aqueous Solution
Nazia Tarannum, Meenakshi Singh*
Department of C hemi st ry , Mahila Mahav idyalaya, Banaras Hindu University, Varanasi, In di a
E-mail: *
Received August 26, 2011; revised October 1, 2011; accepted October 8, 2011
Molecularly imprinted polymers selective for L-aspartic acid (LAA) have been prepared using the carboxy-
betaine polymer bearing zwitterionic centres along the backbone. LAA is well known to promote good me-
tabolism, treat fatigue and depression along with its significance in accurate age estimation in the field of
forensic science and is an important constituent of ‘aspartame’, the low calorie sweetener. In order to study
the intermolecular interactions in the prepolymerization mixture between the monomer and the template
(LAA)/non-template (DAA), a computational approach was developed. It was based on the binding energy
of the complex between the template and functional monomer. The results demonstrate that electrostatic in-
teractions primarily guide the imprinting protocol. The MIP was able to selectively and specifically take up
LAA from aqueous solution, human blood serum and certain pharmaceutical samples quantitatively. Hence,
a facile, specific and selective technique to detect the amino acid, LAA in the presence of various interfer-
rants, in different kinds of matrices is presented.
Keywords: Amino Acid, L-Aspartic Acid, Molecularly Imprinted Polymer (MIP), Computational Modeling,
Carboxybetaine Polymer, Selective Recognition
1. Introduction
L-Aspartic acid promotes robust metabolism and is oc-
casionally used to treat fatigue and depression. The citric
acid cycle, in which other amino acids and biochemicals
(for example aspargine, arginine, lysine, methionine,
threonine and isoleucine) are synthesized, requires aspar-
tic acid as an important intermediary [1]. Moreover, as-
partic acid treats chronic fatigue by playing a significant
role in generating cellular energy. It moves the coenzyme
NADH molecules from the main body of the cell to its
mitochondria to facilitate ATP synthesis. It removes ex-
cess toxins from the cells, particularly ammonia, which
is detrimental to the brain, nervous system and liver also.
It also assists transportation of minerals to form healthy
RNA and DNA to the cells, and support the immune
system by promoting increased production of immu-
noglobulins and antibodies (immune system proteins). It
keeps our mind sharp by increasing NADH level in the
brain, which boosts the production of neurotransmitters
and chemicals needed for healthy mental state. Aspartic
acid is found in dairy, beef, poultry, sugarcane and mo-
lasses. People with diets low in protein or with eating
disorders or malnutrition may develop a deficiency, not
only in aspartic acid but in other amino acids as well, and
experience fatigue or depression. Athletes may need to
take aspartic acid which can be found in protein supple-
ments such as amino acid tablets and whey protein
drinks/bars, and are often marketed as energy boosters.
Furthermore, the world market for this amino acid is ever
increasing since its direct application as a raw material
for “Aspartame”, a low calorie sweetener. Additionally,
the analysis of aspartic acid racemization of dentine pro-
tein is used for accurate estimation of age in forensic
science [2]. Such vital estimations with their importance
in neurotransmission and other significant physiological
activities and related dysfunctions warrant the develop-
ment of an efficient, accurate, precise and selective tool
for the isolation, identification and determination of as-
partic acid from biological fluids as well as from other
pharmaceutical samples.
The polymerization of monomers in the presence of a
target molecule that imprints structural information into
resulting network polymers, called molecularly im-
printed polymer (MIP) is a scientific field that is rapidly
gaining significance for a wide range of applications in
chemistry, biotechnology and pharmaceutical research.
MIPs are stable to physical and chemical treatment, in-
cluding high temperature, pressure, extreme pH, organic
solvents, acids and bases [3]. In addition, MIPs can be
reused many times by repeating extraction with suitable
solvents and without remarkable decrease in the adsorp-
tion capacity for template molecules [4]. These proper-
ties have made them extremely attractive for solving
problems in the fields of preparative chemical separa-
tions, chemical sensing or selective catalysis. Due to
their analytically useful properties, such as selectivity,
shelf stability, robustness and reusability, MIP offers
potential for the synthesis of artificial recognition mate-
rial and they are being proposed for the development of
novel biorecognition techniques for human health and
bioterrorism protection technologies.
MIP for aspartic acid was synthesized by the Mosbach
group as a part of novel utilization of MIPs in the enzy-
matic synthesis of “aspartame” and Syritski et al. [5,6]
have also successfully imprinted aspartic acid on the
electrochemically synthesized film of polypyrrole but
many more aspects affecting the imprinting protocol
need to be developed and endorsed. Herein we attempted
the imprinting of LAA with a different approach, keep-
ing in mind their native nature of zwitterionic composi-
tion, the polymer format chosen for imprinting is also a
novel zwitterionic polymer synthesized in our laboratory
[7] to enable the electrostatic interactions to direct in the
template- monomer interactions responsible for selective
and specific imprinting. In conventional molecular im-
printing, a high level of cross-linking is used to ensure
robustness. Template binding specificity and the slow
rebinding kinetics arising from the inner diffusion of
target molecules toward the recognition sites which are
totally embedded in the polymer matrices hinder and
slow down the overall uptake of the template. To prevail
over this limitation, here in this novel polymer format
external crosslinker is not needed as in this class of car-
boxybetaine polymers or zwitterionic polymers in gen-
eral, the electrostatic interactions between the two charge
centres keep them in collapsed state, which can be con-
sidered as self crosslinking forces. Low mol wt electro-
lytes or other stimuli are needed to break these coulom-
bic interactions. The biocompatible and biomimetic na-
ture of the carboxybetaines has recently been applied for
wide range of biomedical applications [8-11].
A typical imprinting protocol is tedious, time con-
suming and labour-intensive. An attempt was made to
simulate the adduct or complex formed between the
monomer/template (LAA) and the monomer/non-tem-
plate (DAA), the enantiomer of LAA. For assessment of
the template and monomer affinities, binding energy (E)
of the template and monomer can be used as a measure
of their interaction [12-16]. Additionally Mulliken
charges on atoms and dipole moment has also been ana-
lysed to see the effect on imprinting.
2. Materials and Methods
2.1. Reagents
p-Phenylene diamine, glutaraldehyde, dimethylforma-
mide (DMF),
-butyro lactone, L-aspartic acid (LAA)
and D-aspartic acid (DAA) were purchased from Loba
Chemie (Mumbai, India). Aspartame was obtained from
Sigma Aldrich (Steinheim, Germany). The interferrants
studied like urea, oxalic acid, itaconic acid, malonic acid,
L-glutamic acid and 4-aminobutyric acid were purchased
from Spectrochem Pvt. Ltd. (Mumbai, India). Butanol,
acetic acid and ninhydrin used for paper chromatography
were procured from Loba Chemie (Mumbai, India). All
the chemicals were of AR grade and used as received.
Water used was triple distilled deionozed water (0.05 -
0.08) × 10–6 S/cm. Human blood serum was collected
from a local pathology centre and used as received. The
pharmaceutical samples analyzed were Sugar free gold
sachet (artificial sweetener) from Acme diet care Pvt. Ltd.
(Chhatral, India). Sugar free gold was pretreated before
2.2. Equipments
A Cary 50 Bio (Varian Instrument Inc., Melbourne, Aus-
tralia) was used for all spectrophotometric measurements.
The infrared spectra of the materials were recorded using
Jasco FTIR 5300 from 400 - 4000 cm–1. pH of the solu-
tions were determined by Systronics pH system 361.
Scanning electron microscopy was performed by FE-
SEM Quanta200F.
2.3. Computational Method
All computer simulations were undertaken by the soft-
ware Gaussian software [17]. The 3D chemical structures
were generated using Gaussian View [18]. Further, ge-
ometry optimization and energy calculations were per-
formed using DFT method. The DFT calculation is
commonly applied to MIP studies as it has the advan-
tages of high accuracy level of information, reliability
and reasonable computational costs in comparison with
other computational methods (e.g. ab initio) [19]. There-
fore, DFT method was selected to set up the calculations.
The geometry optimizations were performed at B3LYP/
6-31G level of theory. Since the molecules under con-
sideration are large in size and charged molecules, a lar-
ger basis set was adopted and the molecules were opti-
Copyright © 2011 SciRes. AJAC
mized again with the method [B3LYP/6-311G (d, p)]. To
obtain better interaction energies, additional single point
calculations were performed at the optimized geometries
using the method [B3LYP/6-311G (d, p)].
2.4. Preparation of Molecularly Imprinted
Moleculary imprinted polymer and corresponding non-
imprinted polymer poly (N-phenylene N’imino pentyl)
imminium butane carboxylate were prepared and char-
acterized as reported earlier [7]. In short, equimolar solu-
tions of p-phenylene diamine were added to a solution of
glutaraldehyde in DMF and the mixture was heated at 45
°C for three hours. The reaction mixture was subse-
quently treated with
-butyrolactone. Before the gelation
starts, 3.5 ml of aqueous solution of LAA [1.082 g/L, pH
= 2.80] was added. Gelation started after 20 minutes and
imprinted adduct was obtained. It is to mention here that
the polymer is not a hydrogel but an organogel. The cor-
responding structures of MIP and adduct are shown in
Scheme 1.
The concentrations of LAA in the solutions were de-
termined using a spectrophotometer at 200 nm. Standard
calibration plot was constructed at 200 nm and various
parameters (e.g. extracting solvent, time, pH) were opti-
mized at 200 nm. Deionized water at 40˚C was used to
extract LAA template from adduct (volume 10 mL, tem-
perature 40˚C, n = 4, time 20min). A weighed amount of
imprinted polymer was dipped in the template solution
under optimized conditions and subsequently washed
with deionized water (40˚C). Further, the concentrations
of solutions were calculated using standard calibration
plot of LAA. All the measurements were made in tripli-
cate (RSD 1.2%).
Scheme 1. Schematic route showing binding/rebinding of
print molecule (LAA) in the polymer matrix.
3. Results and Discussion
Aspartic acid bears a net negative charge at neutral pH
and no charge at pI (2.8) as the zwitterionic character of
the amino acids cancel out the positive charge of –3
and negative charge of –COO moiety at their respective
pI. Thus we chose a zwitterionic polymer to imprint as-
partic acid, which could provide electrostatic interactions
with respective counter charges along with additional
H-bonding and other hydrophobic interactions to gener-
ate specific recognition sites in the imprinting network.
The biocompatibility of this polymer format chosen for
imprinting LAA would be an added advantage for its
utilization in developing the biorecognition techniques
and applying the tool to biological fluids and other such
pharmaceutical samples without any kind of pre-treat-
ment with their application in forensic sciences also.
3.1. Calculation of Interaction Energy
The recognition ability of MIP is generally evaluated by
affinity for template. The factors affecting affinity of the
template for MIP are strength and quantity of interac-
tions between monomers in polymer network and the
template. The optimized geometries of non-imprinted
polymer, imprinted polymer with template L-aspartic
acid and complex with non-template D-aspartic acid are
shown in Figure 1. In designing MIP via computational
approach, monomers giving highest E with the template
have been assumed to offer corresponding MIP with
highest affinity for the chosen template. The interaction
energies of the template with complex were calculated
from Equation (1):
complex templatemonomer
EE EE  (1)
where Ecomplex is the energy of the complex, Etemplate is the
energy of the template and Emonomer is energy of the
monomer unit.
The results are shown in Ta bles 1- 3. Tables 1-2 show
the Mulliken atomic charges in complexes L and D. For
the sake of brevity only those atoms involved in interac-
tions responsible for imprinting such as electrostatic in-
teractions, H-bonding and other weak forces are shown
in the tables. Figure 2 shows the electrostatic interac-
tions and H-bonding interactions between template and
monomer. As the dipole moments of complex L as well
as D are much larger, the electrostatic interactions seem
to play a primary role in the interaction of template with
MIP cavities.
A comparison of Mulliken charges before imprinting
and after imprinting on the atoms involved either in elec-
trostatic interaction or in H-bonding suggests a more
stable structure [20]. Hence, imprinting the polymer with
Copyright © 2011 SciRes. AJAC
Figure 1. Optimized structures of (a) NIP, (b) Complex L
and (c) Complex D.
Table 1. Mulliken charges on the atoms of Complex L.
Atom Atom Number Mulliken atomic charge
C 1 0.121
H 2 0.137
C 15 –0.058
H 16 0.225
H 17 0.122
O 25 –0.538
O 26 –0.680
C 27 0.290
N 37 –0.460
N 43 –0.481
H 44 0.320
H 45 0.327
H 46 0.368
O 49 –0.624
H 56 0.275
Table 2. Mulliken charges on the atoms of Complex D.
Atom Atom Number Mulliken atomic charge
C 1 0.136
H 2 0.176
C 15 –0.086
H 16 0.283
H 17 0.180
O 25 –0.481
O 26 –0.612
C 27 0.299
N 37 –0.586
N 44 –0.696
H 47 0.362
H 48 0.387
H 56 0.468
the template LAA could be proceeded experimentally.
As shown in Table 3, dipole moments of the nonim-
printed polymer, L-aspartic acid imprinted polymer
(complex L) and complex D with non-template possess
Copyright © 2011 SciRes. AJAC
Electrostatic Interactions
Hydrogen Bonding
Electrostatic Interactions
Hydrogen Bonding
Figure 2. Interactions between monomer and template in (a)
complex L and non-template in (b) complex D.
quite high dipole moment. Dipole moment is a measure
of polarity of the molecule in general and polarity of the
bond in particular. When molecules (here template and
monomers) approach each other, the initial contact arises
from long-range electrostatic forces and when they ap-
proach each other, these electrostatic forces are supple-
mented by the weak forces (H-bonding, Vander Waal
forces, hydrophobic interactions and π-π interactions)
between complementary functional groups located on
template molecule and on the monomers thus fitting the
template into thus created specific MIP cavities. The ΔE
value of complex L as shown in the Table 3 is higher
than that of complex D. Thus the LAA imprinted in the
carboxybetaine polymeric network is more stable. Hence,
the imprinting of LAA was conceded further.
3.2. Infrared Spectrum
In MIP technology, FTIR analysis is useful for prelimi-
nary determination of template-MIP interaction which
proves to be vital for prediction of stability and interac-
tion capacity of recognition cavities towards template
molecule and detection of the efficacy extraction and
rebinding procedures. The FTIR spectra of adduct and
MIP has been shown in Figure 3.
In the non-imprinted polymer (NIP), the –NH2 group
from phenylene diamine end groups and –OH stretch
(moisture) conferred an overlapped absorption band at
–3430. The C-H asymm st at 2925 cm–1 and C-H symm
st at 2859 cm–1 characterize the –CH2 groups of the
skeleton. The carboxylate group (COO) is well evinced
by characteristic absorption bands at 1167 cm–1 (C-CO-C)
with shoulders at 1093 and 1035 cm–1. In the adduct, the
characteristic –3
st of LAA is observed at 3370
cm–1 and 3399 cm–1 as a part of overlapped broad ab-
sorption band. Since the –3 groups are shielded by
electrostatic binding with the COO group of carboxybe-
taine group of polymer, the characteristic COO absorp-
tions at 1173, 1120 and 1083 are subdued in intensity.
Since neither of the carboxyl groups are free (from poly-
carboxybetaine or from LAA, the intensity of the peaks
are diminished to a larger extent. But on extracting the
LAA from adduct, in the resulting MIP (Figure 3), the
characteristic carboxylate absorption bands regained
their intensity at 1175 cm–1, 1100 cm-1and 1061 cm-1
attesting the removal of LAA which shrouded the
charged centres of polycarboxybetaine. Since imine cen-
tres are devoid of any covalently bonded H, similar
analogy on the charged centres of imine could not be
applied in absence of N-H bond. The other characteristic
absorption band of the polymeric chain shifts from 2859
(C-H sym st) to 2890 cm–1 in adduct and again to 2835
cm–1 in MIP. This shift might be attributed to a more
ordered orientation of functional groups with the struc-
tural backbone also. Whereas in NIP, the structural
backbone was free to orient in absence of any template
only according to its own functional groups but in MIP
and adduct, the presence of template dictates the spatial
orientation of groups, hence a little change is observed in
C-H st absorptions. The (C-CO-C) absorption of car-
boxyl have lost its sharpness in adduct evincing interac-
tion of template LAA with MIP via electrostatic interac-
tion of carboxyl groups of carboxybetaine polymer. The
absorption bands at 1167, 1093, 1035 cm–1, with 1767
cm–1, the typical absorptions of carboxylate entity, has
lost their sharpness as they are involved in electrostatic
nteractions with template as shown in Scheme 1.
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
Table 3. The interaction energies and dipole moments of monomer constituents and complexes between monomer and tem-
plate (LAA)/non-template (DAA).
S. No. Compounds Energy (Hartree) in gas phase DFT B3LYP/6311G(d,p)Interaction Energy (E) in KJ/mol Dipole moment
1. Glutaraldehyde
(monomer) –345.85 - 2.957
2. p-Phenylene diamine
(monomer) –342.04 - 0.001
-butyrolactone –306.58 - 4.548
4. Nonimprinted polymer –918.99 - 16.313
5. Complex L –1431.16 11468 24.571
6. Complex D –1431.45 11425 23.210
7. LAA(template) –512.47 - 3.126
8. DAA (non-template) –512.46 - 1.093
Figure 3. Comparative FTIR of adduct and molecularly
imprinted polymer (MIP).
Electrostatic interaction has been known to play a pri-
mary guiding role in inducing the other kinds of
non-covalent weak interactions to specifically and selec-
tively create the imprinted cavities for the amino acid as
the template has been reported by earlier workers also [21].
The adduct shows NH2 wagging at 797 cm–1. The other
absorption bands were characteristic of polymer network
as reported earlier [7]. The fingerprint region clearly dis-
tinguishes MIP and adduct as shown in Figure 3.
3.3. Scanning Electron Microscopy
The scanning electron microscopy images of the NIP,
adduct and MIP are shown in Figure 4. The images of
adduct and the MIP are shown in Figures 4(b) and (c).
Figure 4(b) shows some compactness which after tem-
plate removal turned microporous and appeared to be
consisted of cavities reflecting a highly porous material
with microvoids. While the nonimprinted polymer sur-
face appeared to have smooth surface and few pore
structures as shown in Figure 4(a). Here the polymeriza-
tion was carried out in the absence of template. The
morphological changes are well-consistent with the im-
printing results as discussed in subsequent section.
3.4. Optimization of Analytical Parameters
To find out the conditions under which binding of LAA
to the LAA-imprinted polymer is best and much higher
than binding of the NIP; the extracting solvent, its vol-
ume, extraction time and pH of the solution were opti-
mized. Among the solvents chosen (water, ethanol,
ethanol-water, chloroform, acetonitrile, etc.) for extract-
ing the template molecule from MIP-template adducts,
deionized water at 40˚C was found to be best suited. The
optimized extraction conditions are 10 ml deionized wa-
ter at 40˚C. Three washes of optimized extracting solvent
with continuous mechanical stirring (600 rpm) for 20
min were able to extract template completely. The pres-
ence of LAA in final wash was checked by paper chro-
matography (CHCl3(1):CH3OH(2)) using ninhydrin as
developing reagent. In rebinding experiments, as shown
in Figure 5, 40 min were optimum for uptake of the
template in MIP cavities.
Figure 6 shows the concentration dependence of LAA
imprinted polymer. The uptake by MIP was linearly de-
pendent on concentration of the template with saturation
of 1666 ppm by 1 mg of MIP, being attained at 0.02 M
of template LAA in aqueous solution.
As shown in Figure 7, pH range 2.7 - 6.0 was found to
Figure 4. Scanning electron microscopy of (a) NIP (b) ad-
duct and (c) MIP at 10000 × magnification.
Figure 5: Effect of the retention time to rebind the template
on molecularly imprinted polymer (MIP).
Figure 6. Effect of the concentration of template solution on
recovery (%) of the template from aqueous solution.
Figure 7. Effect of pH of the template solution on recovery
be most suitable for recovery of template. Scheme 2
shows the pH dependent ionization behaviour of aspartic
acid. Below pKa1, (pH 1.88) the amino acid LAA is
fully protonated with net charge on the template as (+1)
in aqueous solutions. As pH is increased beyond pKa1,
the net charge is 0 at its pI 2.88, when amino acids are in
their zwitterionic form. The native nature of the polymer,
i.e. zwitterionic nature which has a positive and a nega-
tive charge centres by virtue of the strength of their ion-
isable groups is accountable for this behaviour where the
Copyright © 2011 SciRes. AJAC
electrostatic interactions between the charge centres of
imprinted polymer and the corresponding opposite
charge centres of template LAA as shown in Scheme 2,
dictate/dominate the retention mechanism. In the pH
range 1.88 - 2.88, the electrostatic interactions are ac-
countable for tight binding of the amino acid to its MIP
cavity (Scheme 2). In addition, H-bonding facilitates the
imprinting further. As the pH of the solution increases
further, in the range 2.8 - 6.0, aspartic acid is deproto-
nated further with net charge as (–1), electrostatic forces
again keeps the template and hold them in the cavities
found in MIP. As the Scheme 2 shows, the additional
ionic interaction at pH > 6.0, further tightens the binding,
hence making the imprinting more successful. Hence pH
range 2.8 - 6.0 was experimentally found to be suitable
for this electrostatic force driven assembly of tem-
plate-polymer assembly in this MIP generated.
Figure 8 shows the binding capacity in terms of Re-
covery (%) of MIP and NIP for template LAA. The
known weight of MIP and NIP samples were equili-
brated under optimized conditions in aqueous LAA solu-
tions of various concentrations. The MIP and NIP were
washed and extracted with optimized solvents under the
similar experimental conditions. MIP is envisioned as a
selective binding material with functional groups com-
plementary to the template structural features. The tem-
plate fits into the imprinted cavity driven by the electro-
static interaction in this study followed by aligning of the
functional groups on the MIP around the LAA confor-
mational orientation. In NIP, although the functional
groups are also present in the polymer, they are randomly
arranged in such a manner that it is ineffective for correct
binding with the template. Similarly, an interferrant is
1.8 < pH < 2.8
2.8 < pH < 6.0
electrostatic interaction
hydrogen bonding
Scheme 2. Scheme representing the structure of adduct at
varying pH.
Figure 8. Comparison of selective recognition of analyte by
NIP and MIP at different conce ntrations of template .
different in size and conformation and might lack key
structural features responsible for fitting into the recog-
nition cavities. In MIPs, the cavities created after re-
moval of the template are complementary to the imprint
molecule in size and coordination geometries. This leads
to much greater affinity for the template molecule by
molecular imprinting, in comparison with non-imprinted
one. As the figure shows, MIP shows an imprinting ef-
fect for the template molecule. The non-specific bindings
are responsible for the template adsorption shown by the
NIP as evident in the figure. At lower concentration, the
non-specific bindings are not able to override the specific
bindings but as the crowding begins at high concentra-
tions of template in solution, the adherence of template
to the non-imprinted polymer network could be noted as
the competition for the specific sites in the imprinted
network becomes high.
In order to evaluate the selectivity of the synthesized
MIP, various structural analogues of L-aspartic acid were
considered (Figure 9 structure of interferrants).
The analogues chosen were either having similar func-
tional groups or its isomer D-asapartic acid (DAA). Fig-
ure 10 shows the response of synthesized MIP toward
template and other non-templates. From Figure 10, it is
evident that the MIP is selective to LAA only even in
presence of DAA or other possible interferrants having
structural similarity. Hence, this study confirms the se-
lectivity of MIP towards template.
The technique thus developed was validated by deter-
mining its performance characteristics regarding linearity,
repeatability and precision. To test the UV response
linearity, a series of standard solutions in the concentra-
tion range 0.005 - 0.015 M was analyzed (at least 15
4. Analytical Applications
This MIP technique was also examined in human blood
serum samples and in certain pharmaceutical samples.
The serum collected from local pathology laboratory was
Copyright © 2011 SciRes. AJAC
Figure 9. Structures of analogues of LAA used in the
cross-selectivity study of MIP.
Figure 10. Cross-selectivity study of adsorption of various
interferrants on LAA-MIP (OA-oxalic acid, UA-urea, IA-
itaconic acid, MA- malonic acid, DAA-D-aspartic acid,
LGA-L-glutamic acid, ABA-4 aminobutyric acid).
diluted 50 times to minimize non-specific bindings due
to intricate matrices of blood serum. Human blood serum
is reported to contain 50 ppm of L-aspartic acid. The
MIP was immersed in dilute serum solution under opti-
mized conditions. The extracted solution was measured
to contain 40 ppm LAA. In this way, the MIP thus de-
veloped was able to recover 80% of LAA present in hu-
man blood serum. This recovery can be enhanced further
if such MIP could be hyphenated to other suitable ex-
traction (Solid phase extraction or Solid phase microex-
traction) techniques. Further the serum sample was
scanned spectrophotometrically before and after treat-
ment with MIP. The key peaks responsible for LAA
were found to be vanished after coming in contact with
MIP under optimized conditions.
The pharmaceutical sample (Sugarfree gold) is re-
ported to contain 35 mg of aspartame, a dipeptide com-
prising of L-aspartic acid and methyl ester of L-pheny-
lalanine. Aspartame in the pharmaceutical sample was
hydrolyzed by acid (6 M HCl) to release the constituent
amino acids. The hydrolytic products were verified by
paper chromatography [butanol (25 mL): acetic acid (5
mL): water (20 mL). The total LAA recovered from the
sample was 75%.
5. Conclusions
The described LAA-MIP has shown enantioselective
feature for the trace-level analysis of d- and l-LAA. For
this a shape complementry cavity in the imprinted poly-
meric network was created. The binding mechanism in-
volved principally electrostatic interactions comple-
mented with hydrogen bonding between the analyte and
MIP, which reduced non-specific bindings of structural
analogues and potential interferents. The developed MIP
can be regenerated for next uses maintaining enantiose-
lective separation. Hence, a facile, highly specific and
selective technique to detect the amino acid LAA in the
presence of various interferrants, in different kinds of
matrices and most importantly without any pre-treatment
or other such complicated pathological/clinical proce-
dures is presented here.
6. Acknowledgements
The authors are grateful to Dr. R. K. Singh for his kind
cooperation extended in Gaussian calculations. Financial
grant was provided by Department of Science and Tech-
nology, New Delhi (SR/S2/CMP-65/2007).
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