Biosensors and Bioelectronics 20 (2004) 10981105

A direct comparison of the performance of ground, beaded

and silica-grafted MIPs in HPLC and Turbulent Flow
Chromatography applications
Robert E. Fairhurst
b,
, Christophe Chassaing
a
, Richard F. Venn
a
, Andrew G. Mayes
b
a
Pzer Global R&D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
b
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
Received 1 December 2003; received in revised form 27 January 2004; accepted 27 January 2004
Available online 11 March 2004
Abstract
Spherical molecularlyimprintedpolymers (MIPs) specic tothe -blocker propranolol have beensynthesisedusingtwodifferent approaches
and compared to traditional ground monolithic MIPs in HPLC and TFC applications. TFC is a LC technique used for rapid extraction of
compounds directly from complex matrices. It can be easily coupled to HPLC and MS for automation of an extraction/analysis procedure.
Spherical MIP beads were produced using a suspension polymerisation technique and silica/MIP composite beads by grafting MIP to spherical
silica particles using a surface-bound initiator species. Synthesis of both beaded and silica-grafted MIPs was more practical than using the
traditional grinding method and yields of spherical particles of the required size between 80 and 100% were routinely achieved. Under HPLC
conditions, beaded and ground MIP materials showed a degree of chiral separation for all of the nine -blockers tested. The beaded MIP,
however, showed much better ow properties and peak shape than the ground material. Silica-grafted MIP showed some separation in ve of
the drugs and a large improvement in peak shape and analysis times compared with both ground and beaded MIPs. The materials prepared
were also used in extraction columns for Turbulent Flow Chromatography (TFC). Although no imprinting effect was observed under typical
TFC conditions, beaded polymer materials showed promise for use as TFC extraction columns due to the good ow properties and clean
extracts obtained.
2004 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting; Turbulent Flow Chromatography; HPLC; Propranolol; Spherical polymers; Silica grafting
1. Introduction
Molecularly imprinted polymers (MIPs) are highly
crosslinked polymers with recognition towards a tar-
get molecule or class of molecules. This is achieved by
imprinting a molecule within the polymer during synthesis
by covalent or, more commonly, non-covalent interactions
between the imprint molecule and polymer (Sellergren,
2001a). The expanding interest in MIPs has led to use in
a number of application areas such as catalysis (Wulff,
2002), separations (Sellergren, 2001b; Martin et al., 2003),
slow-release devices for drugs (Allender et al., 2000) and
sensor technology (Haupt and Mosbach, 2000), where their
durability and ease of preparation makes them an attrac-

Corresponding author.
E-mail address: robert.fairhurst@uea.ac.uk (R.E. Fairhurst).
tive alternative to biomolecules such as proteins. The rigid
and insoluble nature of monolithic MIPs, however, often
means long preparation times and can adversely affect the
properties of the materials. Amongst the techniques used to
address this problem have been suspension polymerisation
(Mayes and Mosbach, 1996), multi-step swelling (Hosoya
and Frechet, 1993) and grafting directly to a suitable sup-
port (Rckert et al., 2002; Schweitz, 2002; Nakayama et al.,
2002). In this study, three types of MIPs have been prepared
and compared for their ability to retain and enantiomerically
separate a number of drugs in the -blocker class under
HPLC and Turbulent Flow Chromatography (TFC) condi-
tions. TFC is a relatively new technique used for rapid ex-
traction and analysis of drugs from biological uids (Ayrton
et al., 1997; Chaissang et al., 2001). The solvent front
prole observed with TFC is of a plug nature rather than
parabolic (Pretorius and Smuts, 1966). The formation of
eddies promoted cross channel mass transfer and increases
0956-5663/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.01.020
R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105 1099
the diffusion of small drug molecules into pores containing
reverse-phase material. Separation of plasma proteins from
bound drug is achieved by size-exclusion (60 pore size)
and slow diffusion of proteins into pores. As a result, TFC
allows the rapid passage of large biomolecules through the
column with simultaneous retention of small analytes.
2. Experimental
2.1. Chemicals
Ethylene glycol dimethacrylate (EDMA), methacrylic
acid (MAA), 2,2

-azobis(isobutyronitrile) (AIBN), sodium

diethyldithiocarbamate (DTC), 2,2-dimethoxy-2-phenylace-
tophenone (DPA), (R, S, R/S)-propranolol, acebutolol,
atenolol, nadolol and pindolol were purchased from Aldrich,
Dorset, UK. Peruoro-1,3-dimethylcyclohexane (PMC) was
obtained from Apollo, Cheshire, UK and toluene was from
Fisher, Leicestershire, UK. 4-Chloromethylphenyltrimeth-
oxysilane (CPTS) was purchased from Lancaster, Lan-
cashire, UK. Alprenolol, carvedilol, metoprolol and ox-
prenolol were all provided by Pzer Global R&D. Nucleosil
spherical silica (5 m, 120 pore size, 200 m
2
g
1
) was
purchased from Phenomenex, Cheshire, UK and Silicycle
spherical silica (4075 m, 120 pore size, 280 m
2
g
1
)
was purchased from Silicycle, Quebec, Canada. Polymeric
surfactant for bead production was synthesised as described
elsewhere (Mayes and Mosbach, 1996).
MAA was distilled under vacuum before use. EDMA was
washed three times with 0.5 M NaOH then passed through
a plug of MgSO
4
. Both were then stored in a refrigerator
over 4 sieves. Toluene was stored over 4 sieves at room
temperature. Before imprinting, the hydrochloride salt of
(S)-propranolol was extracted into dichloromethane (DCM)
from 0.5 M NaOH, evaporated to dryness and stored in the
refrigerator. All other chemicals were used as received.
2.2. Equipment
2.2.1. HPLC
Empty stainless-steel columns (150 mm 4.6 mm i.d.)
were obtained from Supelco, PA, USA. HPLC columns
were packed in methanol at approximately 2000 psi using
an Alltech Model 1666 slurry packer. Columns packed with
beaded and silica-grafted materials were tted with 0.5 m
frits and ground monolithic polymer columns with 2 m
frits all from Supelco, UK. The standard, achiral column
used as a comparison was a 150 mm4.6 mm i.d. HiChrom
S5-CN (HiChrom, Berkshire, UK). HPLC analysis was per-
formed using a Jasco AS-950-10 Intelligent Autosampler t-
ted with a 250 l loop and a Jasco PU-98 Intelligent HPLC
pump. The column was maintained at 40

C using a Jones
Chromatography Model 7990 column temperature regula-
tor and detection was performed using a Shimadzu SPD-6A
single wavelength UV detector. Wavelengths used for detec-
tion of -blockers were as follows: acebutolol 235 nm, al-
prenolol 220 nm, atenolol 226 nm, carvedilol 241 nm, meto-
prolol, nadolol and oxprenolol 223 nm, pindolol 217 nm,
propranolol 290 nm. A mobile phase composed of 70:30
acetonitrile: phosphate buffer (20 mM, pH 5.1) was used
(Haginaka and Sakai, 2000).
2.2.2. TFC
Turbulent ow conditions are achieved using high ow
rates of lowviscosity solvents in micro-bore columns packed
with particles of a large diameter. A guide to the ow char-
acteristics of a mobile phase in a packed column is given by
the Reynolds number, Re = (D
p
)/, where is the lin-
ear velocity of the mobile phase, D
p
the average diameter of
the stationary phase particles and the mobile phase kine-
matic viscosity. Turbulent ow is described by a Reynolds
number greater than 1. Large particles are therefore used to
encourage turbulent ow conditions whilst simultaneously
lowering backpressure on the column, which is particularly
important due to the high ow rates required.
This study uses TFC in dual column mode, which is per-
formed in three stages. The rst is the sample load where the
sample containing the drug of interest is loaded onto the ex-
traction column using an aqueous mobile phase. Lipophilic
drug molecules are retained on the column and polar mate-
rials are eluted to waste. Reverse ow through the extraction
column using a high organic phase then takes the drugs onto
an analytical column and through a detector of choice. Fi-
nally, the extraction column is re-equilibrated with aqueous
phase ready for the next load. Fig. 1 shows a valve diagramof
the TFC system and Table 2 gives the standard protocol used
for all TFC work throughout this study. The TFC system
consisted of HP1100 series binary and isocratic pumps and
a 2300 HTLC valve module both from Cohesive Technolo-
gies, Buckinghamshire, UK. Previously used TFC columns
from Cohesive Technologies (50 mm1 mm i.d.) were emp-
tied and packed with ground, silica-grafted or beaded mate-
rial in methanol using a home-built rig. Material was slurried
Fig. 1. The double-valve TFC system in dual column mode as used for
this study.
1100 R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105
in methanol and transferred to an empty 250 mm 5 mm
i.d. column with a male end adapter. The empty TFC col-
umn was tted directly to the end of this column and the
material was pumped at 9 ml min
1
followed by 150 ml of
methanol. 5 g ml
1
propranolol in plasma was prepared by
spiking 193.84 ml of centrifuged plasma (10 min at 4

C)
with 6.16 l of 324.6 g ml
1
stock (S)-propranolol solu-
tion in acetonitrile:water (70:30). Two hundred microliters
of 1 M monochloroacetic acid solution in methanol:water
(10:90) was then added and the solution was centrifuged for
a further 30 min at 4

C. Two hundred microliters of this so-

lution was then injected onto the column. Clean solutions
were also injected onto the system. These were 5 g ml
1
solutions of the -blocker in acetonitrile:water (70:30). The
standard set up was using a C
18
silica or reference polymer
beads extraction column and a HiChrom S5-CN (150 mm
4.6 mm i.d.) analytical column in dual column mode. De-
tection was performed using a HP1100 series UV detector
and a Merck Hitachi Lachrom L-7480 uorescence detector
in series.
2.2.3. Suspension polymerisation
Solutions were homogenised before polymerisation
using an IKA-Werke Ultra-Turrax T8 homogeniser. An
IKA-Labortechnik Eurostar Digital stirrer was used to
maintain the emulsion throughout polymerisation. Jacketed
reaction vessels and stainless-steel stirrer paddles were cus-
tom built. The neck of the ask where the paddle entered
was sealed with a teon stirrer guide. For UV irradiation,
a UVP Blak-Ray B100 series lamp was used. Thirty min-
utes was allowed for the lamp to reach maximum intensity
before being applied.
2.3. Methods
2.3.1. Imprinted polymers
Astandard imprint mixture containing EDMA(1.0 mmol),
MAA (0.2 mmol), (S)-propranolol (0.03 mmol), initiator
(0.01 mmol) in toluene (2.55 ml) was used for each of the
methods. In the case of a bulk polymerisation the initiator
was AIBN, for beaded polymers the initiator used was DPA
and no initiator was added to the silica-grafted mixture.
Reference polymers were synthesised and washed in exactly
the same way as their imprinted counterparts, but contained
no (S)-propranolol. Polymers were washed according to a
method described previously (Andersson, 1996).
2.3.2. Monolithic polymers
A standard imprinting mixture using AIBN as the initia-
tor was held in a reaction vessel maintained at 25

C by
passing heated water through the jacket. The mixture was
then subjected to UV irradiation from a distance of 5 cm
for approximately 24 h. The polymer was then ground by
hand and size-selected using steel sieves. Particles between
38 and 75 m were collected for TFC and SPE analysis.
Particles smaller than 20 m were subjected to three sedi-
mentation cycles (60 min each) in methanol to remove very
small particles and used for HPLC.
2.3.3. Beaded polymers
DPA, EDMA, MAA and (S)-propranolol were all taken
up in toluene. To this was added 20 ml PMC saturated with
toluene and polymeric surfactant. The amount of surfactant
added depended on the nal use of the beads: 25 mg for SPE
and TFC applications, 95 mg for beads designed for HPLC.
The mixture was homogenised until no surfactant precipi-
tate was visible and then added to a jacketed reaction vessel
held at 25

C by a water heater/recirculator. The mixture was

purged with argon for 5 min and then stirred at 2000 rpm
for 5 min. The UV lamp was positioned at a distance of ap-
proximately 5 cm while the mixture was stirred at 500 rpm.
Exposure to UV continued for 15 min with a positive ar-
gon pressure being maintained throughout. After polymeri-
sation the beads were collected by ltration, washed with
copious quantities of acetone and dried under high vacuum.
PMC was collected to recycle. Beads made using 25 mg
surfactant were passed through sieves and the 3875 m
fraction was collected. Beads made using 95 mg surfac-
tant were subjected to three 60 min sedimentation cycles in
methanol.
2.3.4. Silica-grafted polymers
This was synthesised in two parts. Firstly, spherical sil-
ica gel was modied with a DTC-type free-radical initia-
tor species, the MIP was then grafted to the silica via the
initiator.
2.3.5. Modication of silica with DTC-type initiator
Both type of silica particles (5 m and 4075 m) were
treated in the same way during synthesis. Silica (0.70 g) was
rst added to a 5% solution of CPTS in toluene (7 ml). The
mixture was sealed and stirred for approximately 40 h at
55

C. The silica was collected by ltration and washed with

toluene followed by acetone and dried under high vacuum
then transferred to a 2% DTC solution in THF (3.5 ml).
The silica suspension was stirred for 4 h at 40

C then again
ltered and washed with THF, water and nally methanol
before being dried under high vacuum.
2.3.6. Polymer grafting onto silica
Initiator-modied silica (0.65 g) was added to a standard
imprinted polymer mixture with a magnetic stirrer bar in a
jacketed reaction vessel. The mixture was then subjected to
three freezethaw cycles to remove oxygen. After the nal
thaw, argon was introduced to the vessel. The vessel was
held at 25

C using a water heater/recirculator and UV was

applied from a distance of approximately 5 cm for 60 min.
The mixture was stirred constantly during polymerisation
and a positive pressure of argon was maintained through-
out. After polymerisation, silica was collected by ltration,
washed with DCM and dried under high vacuum. The im-
print mixture was collected and recycled to be used again.
R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105 1101
Elemental analysis was used to determine the amount of
polymer bound to the surface.
3. Results and discussion
3.1. HPLC
Nine -blockers were tested on the columns packed with
the three MIP materials. A summary of the results can be
seen in Table 1. Unsurprisingly, the imprint molecule, pro-
pranolol was the most strongly retained and effectively enan-
tioseparated of all the -blockers on each column. The most
retained compounds were generally also the most enan-
tioseparated. An interesting observation is that carvedilol
(which contains three aromatic rings) is very highly retained
on all polymer materials, yet is the least separated of the
compounds, whereas the greatest enantioseparation (after
propranolol) is observed for pindolol, which is structural
Table 1
Data from HPLC analysis of the -blockers tested on imprinted and reference MIP materials
Compound Imprinted Reference
t
r
R t
r
S R
s
W
0.5
R t
r
k W
0.5
R
Ground
Acebutolol 10.4 20.0 2.1 0.8 2.2 7.1
b
1.0
b
2.1
b
Alprenolol 20.1
a
96.8
a
4.3
a
1.0
a
7.4
a
13.0
b
2.7
b
4.1
b
Atenolol 9.3 19.8 1.6 0.7 2.1 7.2
b
1.0
b
2.5
b
Carvedilol 53.8 74.5 1.4 0.4 11.7 13.7
b
2.8
b
4.1
b
Metoprolol 19.6 53.1 2.9 0.8 5.1 9.8
b
1.8
b
3.1
b
Nadolol 8.3 16.6 2.3 0.7 1.8 7.1
b
1.0
b
2.5
b
Oxprenolol 21.7 56.7 2.7 1.0 5.4 10.5
b
2.0
b
3.6
b
Pindolol 17.6
a
74.4
a
4.5
a
0.9
a
7.1
a
11.3
b
2.2
b
4.1
b
Propranolol 69.0 nd nd nd 42.5 16.7
b
4.1
b
6.6
b
Silica-grafted
Acebutolol 12.0 12.0 1.0 0.0 5.4 5.4 3.2 3.3
Alprenolol 18.6 28.7 1.6 0.5 4.5 10.3 7.0 3.7
Atenolol 12.7 12.7 1.0 0.0 5.9 7.8 5.0 4.8
Carvedilol 34.1 34.1 1.0 0.0 12.2 14.5 10.5 6.7
Metoprolol 16.4 18.4 1.2 0.1 7.1 9.5 6.5 3.7
Nadolol 9.3 9.3 1.0 0.0 5.6 4.7 2.6 3.1
Oxprenolol 16.9 20.5 1.4 0.3 5.4 7.8 4.9 2.8
Pindolol 18.9 37.3 2.1 0.6 5.6 6.1 3.7 2.2
Propranolol 37.4 82.7 2.3 1.1 10.7 8.8 6.0 3.5
Beads
Acebutolol 6.3 11.6 2.4 0.4 2.5 2.8 0.8 2.5
Alprenolol 10.0
a
49.4
a
5.8
a
1.0
a
5.9
a
4.4 1.4 1.5
Atenolol 5.6 11.0 2.5 0.3 2.6 2.6 0.4 0.8
Carvedilol 27.7 38.0 1.4 0.1 19.9 7.2 3.4 2.6
Metoprolol 11.0 30.6 3.1 0.6 5.5 3.2 0.9 1.0
Nadolol 5.4 9.2 2.1 0.2 2.3 2.8 0.4 0.8
Oxprenolol 12.5 34.2 3.0 0.7 6.1 3.7 1.1 1.2
Pindolol 18.8 94.0 3.9 1.0 11.6 3.9 1.1 1.3
Propranolol 20.1
a
150.0
a
7.7
a
0.8
a
13.3
a
4.4 3.6 0.8
t
r
is the retention time (t
r
R and t
r
S of the (R)- and (S)-enantiomers, respectively), k the retention factor, the separation factor of the two enantiomers,
R
s
the resolution of the enantiomers and W
0.5
the peak width at half height (W
0.5
R is the width of the (R)-enantiomer). and R
s
on reference materials
were zero; nd: data not available due to (S)-enantiomer being too broad to detect.
a
Run at 2 ml min
1
.
b
Run at 0.5 ml min
1
due to high backpressure.
similar to propranolol. This shows that, although hydropho-
bic interactions with the polymer backbone help to very ef-
fectively retain lipophilic molecules like carvedilol (which
does increase the opportunity for interaction with imprint
sites), size and shape similarity with the imprint molecule
is ultimately required for chiral recognition. Compound re-
tention is also affected subtly by imprinting on silica. The
polar character of the silica surface favours retention of the
more polar atenolol over acebutolol and enhances retention
of pindolol over alprenolol in the imprinted material. This
could be due to the silanol groups of the silica backbone
playing a small part in the imprinting process.
All imprinted materials showed greater retention of the
nine -blockers tested than the corresponding reference ma-
terials. Ground and beaded MIPs both showed some degree
of enantioseparation for all the -blockers tested, whereas
the silica-grafted material showed separation for only ve
out of the nine racemic mixtures. Separation and resolu-
tion factors were still comparable with result in a recently
1102 R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105
Fig. 2. SEM images of ground monolithic MIP (left), beaded MIP (centre) and silica-grafted MIP (right) for use in HPLC columns (before removal of
nes).
published study using spherical material made by the
multi-step swelling method (Haginaka and Sakai, 2000).
The traditional ground material showed the best overall res-
olution of enantiomers, but this was at the expense of very
long retention times and poor peak shape. Poor peak shapes
are generally obtained with imprinted materials due to the
very strong retention of the analyte and their operation in
the non-linear region of the adsorption isotherm. They are
also made worse by the irregular shape of the particles
from the grinding and sieving method. Due to the resulting
backpressure, larger particle sizes are required resulting in
further peak broadening. Both silica-grafted and beaded ma-
terials showed an improvement over the ground material in
this area. The morphology of the three materials is shown in
Fig. 2. Imprinted beaded material (diameter 4.0 1.8 m)
showed slightly improved peak shapes over ground material
and backpressure approximately 20% that of the ground and
silica-grafted materials. Imprinted silica-grafted material
(5.0 0.8 m in diameter) showed a similar backpressure
to imprinted ground material (10.0 4.3 m), but also a
vastly improved peak shape to both the ground and beaded
particles. Although the improvement in peak shape using
silica-grafted MIP was largely due to poorer recognition (as
shown by the much lower average resolution of -blockers),
it also means that high resolution of more strongly re-
tained compounds is possible in a relatively very short time
(Fig. 3). SEM images of silica-grafted material show little
or no polymerisation on the surface of the silica and esti-
mates of the volume of polymer present are very similar to
the total pore volume of the silica. This suggests that poly-
merisation is limited to the pores, hence reducing the access
Table 2
TFC conditions used throughout this study
Step Duration (s) Extraction column/loading pump Analytical column/eluting pump
Direction Composition Direction Composition
Load 15 F/W A F/D B
Backush 10 R/W A F/D B
Elute 720 F/D B F/D B
Re-equilibrate 30 F/W A F/D B
The ow rates were 1 ml min
1
through the analytical column and 5 ml min
1
through the extraction column throughout. F stands for ow in the forward
direction (direction of arrows in Fig. 1) and R stands for reverse ow. W stands for ow to waste and D to the detector. Composition A is water with
0.01% TFA and B is acetonitrile:water (70:30) with 0.01% TFA.
to the imprint sites, but still maintaining the external shape
of the silica. This is not the case for the beaded material in
which polymerisation is not conned to pores since spheri-
cal beads are composed entirely of imprinted polymer. As a
result, beaded MIPs showed much better resolution of enan-
tiomers than the silica-grafted material for all the -blockers
tested. However a 22% increase in average peak width and
extended retention times was observed. The backpressure
on the beaded polymer columns for HPLC was approxi-
mately 20% of the one observed for the purchased 5 m
spherical particle column, which suggests that the polymer
materials are much more porous than silica. This meant that
the beaded materials could be easily used at higher ow
rates to speed up analysis of strongly retained compounds,
making them much more versatile than the ground polymer.
3.2. TFC
Limitations were observed when using the silica-grafted
and ground polymer materials as extraction columns for
TFC. In both cases, the backpressures on the columns were
too high and caused the system to leak. Study of these
materials as extraction columns for TFC application was
therefore discontinued. Pressure readings on the beaded ma-
terials, however, were found to be similar to traditional
C
18
silica extraction columns, which is most likely due to
the highly porous structure of the beads. Both imprinted
and non-imprinted beads were assessed for their extrac-
tion ability under TFC conditions. The standard procedure
for a TFC extraction is shown in Table 2. Leaching of
template molecule from the imprinted beads was tested by
R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105 1103
Fig. 3. HPLC traces of pindolol through 150 mm 4.6 mm i.d. columns packed with ground MIP (top, scale 0150 min), beaded MIP (middle, scale
090 min) and silica-grafted MIP (bottom, scale 0120 min). Flow rates used were 2 ml min
1
through the ground and beaded MIP columns and 1 ml min
1
through the silica-grafted MIP column.
loading a 200 l plug of methanol. Any propranolol leach-
ing from the system is carried from the extraction column
onto the analytical column with the solvent front at the
beginning of the elute step (see Table 2). The imprinted
beads showed leaching at a level of approximately 0.5 pg
from every blank methanol injection. A variety of load,
elute and re-equilibration steps were tested to encourage
expression of a molecular imprinting effect, but (R)- and
(S)-enantiomers of propranolol were retained equally on
both imprinted and reference beads under all conditions
tested. The aqueous nature of the mobile phase encourages
deposition of the lipophilic analyte directly onto the station-
ary phase due to non-specic hydrophobic interactions rather
than the dynamic exchange between mobile and stationary
phases which is required for selective binding of the ana-
lyte with the imprint sites. The reference beads were there-
fore studied instead of the imprinted ones, which removed
the negative effect of template leaching. Recovery of pro-
pranolol using a beaded extraction column compared with
a traditional C
18
silica column is shown in Table 3. Recov-
ery was found to be signicantly higher using the C
18
sil-
ica column, although the extract using the beaded material
was noticeably cleaner (Fig. 4). Carry-over from a previous
extraction was found to be minimal for both of the mate-
rials (C
18
= 0.010%, beads = 0.006%). Further analysis
monitoring recovery of a number of -blockers in organic
solution is shown in Table 4. Results showed that, for both
beaded polymer and C
18
silica extraction columns, recovery
1104 R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105
Table 3
Recovery of propranolol from reference beaded polymer and C
18
silica extraction columns at 5 ml min
1
after injection of spiked plasma and carry-over
in subsequent blank plasma washes (total mass of propranolol loaded onto column is 1000 ng)
Extraction
column
Backpressure
(bar)
Recovery from load or wash step Total recovery (%)
Load: spiked plasma (ng) Wash 1: blank plasma (pg) Wash 2: blank plasma (pg)
Beads 36 505.1 4.5 1.0 51
C
18
silica 37 725.7 7.9 1.6 73
Table 4
Recovery of nine -blockers as a percentage of mass loaded from acetonitrile:water (70:30) using a reference beaded polymer extraction column and a
C
18
extraction column
Extraction column Acebutolol Alprenolol Atenolol Carvedilol Metoprolol Nadolol Oxprenolol Pindolol Propranolol
Beads 7.9 18.0 0.1 68.6 5.4 12.7 16.7 13.1 36.9
C
18
silica 23.6 40.7 5.5 61.8 23.2 16.3 36.9 24.9 45.7
Lower recoveries than from plasma are expected due to the high organic content of the loading solution.
tends to improve with compound hydrophobicity. In the case
of carvedilol (the most lipophilic of the -blockers), how-
ever, the beaded polymer column outperforms the C
18
silica
column and extracts a higher percentage of the load. This
proves that, under certain conditions, the very hydrophobic
nature of the beaded polymers can show improved recover-
ies over existing materials and gives scope for further de-
velopment of beaded materials to ne-tune the properties
of the polymer (e.g. by using different monomers).
Fig. 4. Chromatograms obtained by injecting blank plasma onto a TFC
C
18
silica extraction column (top) and a beaded reference polymer extrac-
tion column (bottom) following an injection of a sample containing a high
concentration of propranolol. Peak at 10 min is propranolol carry-over
and other signals are due to unidentied compounds from plasma. De-
tection was using uorescence (ex. 292 nm/em. 332 nm).
4. Conclusions
Ground monolithic imprinted polymer was still the best
all-round performer for enantiomeric separations of a
number of -blockers by HPLC. However, silica-grafted
MIP provided vastly improved peak shape and complete
enatiomeric resolution of a racemic mixture of the imprint
species in a fraction of the time it would take using the
ground material. Synthesis of this material is also much
more efcient and less laborious than ground polymer and
therefore provides a good alternative for bulk enantiomeric
separations of racemic mixtures. Although synthesis of the
silica-grafted initiator species took in the order of 2 days,
subsequent MIP synthesis was possible within approxi-
mately 2 h. The silica-grafted initiator species could also be
synthesised in bulk and stored in a dark place for at least 1
month without degrading. Beaded material was the fastest,
easiest and most efcient to synthesise, with preparation
and synthesis complete within 2 h and yields of around
90%. The resulting beads provided some degree of enan-
tioseparation of all the -blockers as well as a signicant
improvement in peak shape over the ground materials. This
would therefore be a good method for rapid MIP synthesis
and analysis. The very low backpressures on the column
also meant that analysis of well separated compounds with
long retention times could be shortened by using higher ow
rates. The beaded polymers were also effective in extract-
ing propranolol from plasma using TFC and even showed
improvements in recovery over traditional C
18
columns for
the most lipophilic compound tested (carvedilol). Extracts
from plasma using the beaded materials also showed much
cleaner baselines than traditional C
18
extraction columns.
Although imprinting made no observable difference in the
extraction of propranolol, cross-linked beaded materials
have shown much promise for use in turbulent ow extrac-
tion and the high porosity of the material did not appear
to increase the amount of endogenous biological materials
(e.g. proteins) retained on the column.
R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 10981105 1105
Acknowledgements
The authors wish to thank EPSRC and Pzer Global Re-
search and Development for nancial support of this work.
Also to Dr. Elena Piletska and Prof. Sergey Piletksy for help
with HPLC column packing and Stephen Bennett for assis-
tance with SEM imaging.
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