a direct comparison of the performance of ground, beaded and silica-grafted mips in hplc and...

Biosensors and Bioelectronics 20 (2004) 1098–1105

A direct comparison of the performance of ground, beadedand silica-grafted MIPs in HPLC and Turbulent Flow

Chromatography applications

Robert E. Fairhurstb,∗, Christophe Chassainga, Richard F. Venna, Andrew G. Mayesb

a Pfizer Global R&D, Ramsgate Road, Sandwich, Kent CT13 9NJ, UKb 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 2004Available online 11 March 2004

Abstract

Spherical molecularly imprinted polymers (MIPs) specific to the�-blocker propranolol have been synthesised using two different approachesand compared to traditional ground monolithic MIPs in HPLC and TFC applications. TFC is a LC technique used for rapid extraction ofcompounds 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 sphericalsilica particles using a surface-bound initiator species. Synthesis of both beaded and silica-grafted MIPs was more practical than using thetraditional grinding method and yields of spherical particles of the required size between 80 and 100% were routinely achieved. Under HPLCconditions, 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 flow properties and peak shape than the ground material. Silica-grafted MIP showed some separation in five ofthe drugs and a large improvement in peak shape and analysis times compared with both ground and beaded MIPs. The materials preparedwere also used in extraction columns for Turbulent Flow Chromatography (TFC). Although no imprinting effect was observed under typicalTFC conditions, beaded polymer materials showed promise for use as TFC extraction columns due to the good flow properties and cleanextracts 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 highlycrosslinked polymers with recognition towards a tar-get molecule or class of molecules. This is achieved by‘imprinting’ a molecule within the polymer during synthesisby covalent or, more commonly, non-covalent interactionsbetween the imprint molecule and polymer (Sellergren,2001a). The expanding interest in MIPs has led to use ina 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) andsensor technology (Haupt and Mosbach, 2000), where theirdurability and ease of preparation makes them an attrac-

∗ Corresponding author.E-mail address: [email protected] (R.E. Fairhurst).

tive alternative to biomolecules such as proteins. The rigidand insoluble nature of monolithic MIPs, however, oftenmeans long preparation times and can adversely affect theproperties of the materials. Amongst the techniques used toaddress this problem have been suspension polymerisation(Mayes and Mosbach, 1996), multi-step swelling (Hosoyaand Frechet, 1993) and grafting directly to a suitable sup-port (Rückert et al., 2002; Schweitz, 2002; Nakayama et al.,2002). In this study, three types of MIPs have been preparedand compared for their ability to retain and enantiomericallyseparate a number of drugs in the�-blocker class underHPLC and Turbulent Flow Chromatography (TFC) condi-tions. TFC is a relatively new technique used for rapid ex-traction and analysis of drugs from biological fluids (Ayrtonet al., 1997; Chaissang et al., 2001). The solvent frontprofile observed with TFC is of a plug nature rather thanparabolic (Pretorius and Smuts, 1966). The formation ofeddies promoted cross channel mass transfer and increases

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R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 1098–1105 1099

the diffusion of small drug molecules into pores containingreverse-phase material. Separation of plasma proteins frombound drug is achieved by size-exclusion (60 Å pore size)and slow diffusion of proteins into pores. As a result, TFCallows the rapid passage of large biomolecules through thecolumn with simultaneous retention of small analytes.

2. Experimental

2.1. Chemicals

Ethylene glycol dimethacrylate (EDMA), methacrylicacid (MAA), 2,2′-azobis(isobutyronitrile) (AIBN), sodiumdiethyldithiocarbamate (DTC), 2,2-dimethoxy-2-phenylace-tophenone (DPA), (R, S, R/S)-propranolol, acebutolol,atenolol, nadolol and pindolol were purchased from Aldrich,Dorset, UK. Perfluoro-1,3-dimethylcyclohexane (PMC) wasobtained from Apollo, Cheshire, UK and toluene was fromFisher, Leicestershire, UK. 4-Chloromethylphenyltrimeth-oxysilane (CPTS) was purchased from Lancaster, Lan-cashire, UK. Alprenolol, carvedilol, metoprolol and ox-prenolol were all provided by Pfizer Global R&D. Nucleosilspherical silica (5�m, 120 Å pore size, 200 m2 g−1) waspurchased from Phenomenex, Cheshire, UK and Silicyclespherical silica (40–75�m, 120 Å pore size, 280 m2 g−1)was purchased from Silicycle, Quebec, Canada. Polymericsurfactant for bead production was synthesised as describedelsewhere (Mayes and Mosbach, 1996).

MAA was distilled under vacuum before use. EDMA waswashed three times with 0.5 M NaOH then passed througha plug of MgSO4. Both were then stored in a refrigeratorover 4 Å sieves. Toluene was stored over 4 Å sieves at roomtemperature. Before imprinting, the hydrochloride salt of(S)-propranolol was extracted into dichloromethane (DCM)from 0.5 M NaOH, evaporated to dryness and stored in therefrigerator. All other chemicals were used as received.

2.2. Equipment

2.2.1. HPLCEmpty stainless-steel columns (150 mm× 4.6 mm i.d.)

were obtained from Supelco, PA, USA. HPLC columnswere packed in methanol at approximately 2000 psi usingan Alltech Model 1666 slurry packer. Columns packed withbeaded and silica-grafted materials were fitted with 0.5�mfrits and ground monolithic polymer columns with 2�mfrits all from Supelco, UK. The standard, achiral columnused as a comparison was a 150 mm×4.6 mm i.d. HiChromS5-CN (HiChrom, Berkshire, UK). HPLC analysis was per-formed using a Jasco AS-950-10 Intelligent Autosampler fit-ted with a 250�l loop and a Jasco PU-98 Intelligent HPLCpump. The column was maintained at 40◦C using a JonesChromatography Model 7990 column temperature regula-tor and detection was performed using a Shimadzu SPD-6Asingle 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 70:30acetonitrile: phosphate buffer (20 mM, pH 5.1) was used(Haginaka and Sakai, 2000).

2.2.2. TFCTurbulent flow conditions are achieved using high flow

rates of low viscosity solvents in micro-bore columns packedwith particles of a large diameter. A guide to the flow char-acteristics of a mobile phase in a packed column is given bythe Reynolds number,Re = (µDp)/η, whereµ is the lin-ear velocity of the mobile phase,Dp the average diameter ofthe stationary phase particles andη the mobile phase kine-matic viscosity. Turbulent flow is described by a Reynoldsnumber greater than 1. Large particles are therefore used toencourage turbulent flow conditions whilst simultaneouslylowering backpressure on the column, which is particularlyimportant due to the high flow rates required.

This study uses TFC in dual column mode, which is per-formed in three stages. The first is the sample load where thesample containing the drug of interest is loaded onto the ex-traction column using an aqueous mobile phase. Lipophilicdrug molecules are retained on the column and polar mate-rials are eluted to waste. Reverse flow through the extractioncolumn using a high organic phase then takes the drugs ontoan analytical column and through a detector of choice. Fi-nally, the extraction column is re-equilibrated with aqueousphase ready for the next load.Fig. 1shows a valve diagram ofthe TFC system andTable 2gives the standard protocol usedfor all TFC work throughout this study. The TFC systemconsisted of HP1100 series binary and isocratic pumps anda 2300 HTLC valve module both from Cohesive Technolo-gies, Buckinghamshire, UK. Previously used TFC columnsfrom Cohesive Technologies (50 mm×1 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 forthis study.

1100 R.E. Fairhurst et al. / Biosensors and Bioelectronics 20 (2004) 1098–1105

in methanol and transferred to an empty 250 mm× 5 mmi.d. column with a male end adapter. The empty TFC col-umn was fitted directly to the end of this column and thematerial was pumped at 9 ml min−1 followed by 150 ml ofmethanol. 5�g ml−1 propranolol in plasma was prepared byspiking 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 microlitersof 1 M monochloroacetic acid solution in methanol:water(10:90) was then added and the solution was centrifuged fora further 30 min at 4◦C. Two hundred microliters of this so-lution was then injected onto the column. ‘Clean’ solutionswere also injected onto the system. These were 5�g ml−1

solutions of the�-blocker in acetonitrile:water (70:30). Thestandard set up was using a C18 silica or reference polymerbeads 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 detectorand a Merck Hitachi Lachrom L-7480 fluorescence detectorin series.

2.2.3. Suspension polymerisationSolutions were homogenised before polymerisation

using an IKA-Werke Ultra-Turrax T8 homogeniser. AnIKA-Labortechnik Eurostar Digital stirrer was used tomaintain the emulsion throughout polymerisation. Jacketedreaction vessels and stainless-steel stirrer paddles were cus-tom built. The neck of the flask where the paddle enteredwas sealed with a teflon 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 intensitybefore being applied.

2.3. Methods

2.3.1. Imprinted polymersA standard 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 themethods. In the case of a bulk polymerisation the initiatorwas AIBN, for beaded polymers the initiator used was DPAand no initiator was added to the silica-grafted mixture.Reference polymers were synthesised and washed in exactlythe same way as their imprinted counterparts, but containedno (S)-propranolol. Polymers were washed according to amethod described previously (Andersson, 1996).

2.3.2. Monolithic polymersA standard imprinting mixture using AIBN as the initia-

tor was held in a reaction vessel maintained at 25◦C bypassing heated water through the jacket. The mixture wasthen subjected to UV irradiation from a distance of 5 cmfor approximately 24 h. The polymer was then ground byhand and size-selected using steel sieves. Particles between38 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 verysmall particles and used for HPLC.

2.3.3. Beaded polymersDPA, EDMA, MAA and (S)-propranolol were all taken

up in toluene. To this was added 20 ml PMC saturated withtoluene and polymeric surfactant. The amount of surfactantadded depended on the final use of the beads: 25 mg for SPEand 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 vesselheld at 25◦C by a water heater/recirculator. The mixture waspurged with argon for 5 min and then stirred at 2000 rpmfor 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 filtration, washed withcopious quantities of acetone and dried under high vacuum.PMC was collected to recycle. Beads made using 25 mgsurfactant were passed through sieves and the 38–75�mfraction was collected. Beads made using 95 mg surfac-tant were subjected to three 60 min sedimentation cycles inmethanol.

2.3.4. Silica-grafted polymersThis was synthesised in two parts. Firstly, spherical sil-

ica gel was modified with a DTC-type free-radical initia-tor species, the MIP was then grafted to the silica via theinitiator.

2.3.5. Modification of silica with DTC-type initiatorBoth type of silica particles (5�m and 40–75�m) were

treated in the same way during synthesis. Silica (0.70 g) wasfirst added to a 5% solution of CPTS in toluene (7 ml). Themixture was sealed and stirred for approximately 40 h at55◦C. The silica was collected by filtration and washed withtoluene followed by acetone and dried under high vacuumthen transferred to a 2% DTC solution in THF (3.5 ml).The silica suspension was stirred for 4 h at 40◦C then againfiltered and washed with THF, water and finally methanolbefore being dried under high vacuum.

2.3.6. Polymer grafting onto silicaInitiator-modified silica (0.65 g) was added to a standard

imprinted polymer mixture with a magnetic stirrer bar in ajacketed reaction vessel. The mixture was then subjected tothree freeze–thaw cycles to remove oxygen. After the finalthaw, argon was introduced to the vessel. The vessel washeld at 25◦C using a water heater/recirculator and UV wasapplied from a distance of approximately 5 cm for 60 min.The mixture was stirred constantly during polymerisationand a positive pressure of argon was maintained through-out. After polymerisation, silica was collected by filtration,washed with DCM and dried under high vacuum. The im-print mixture was collected and recycled to be used again.


Elemental analysis was used to determine the amount ofpolymer bound to the surface.

3. Results and discussion

3.1. HPLC

Nine �-blockers were tested on the columns packed withthe three MIP materials. A summary of the results can beseen inTable 1. Unsurprisingly, the imprint molecule, pro-pranolol was the most strongly retained and effectively enan-tioseparated of all the�-blockers on each column. The mostretained compounds were generally also the most enan-tioseparated. An interesting observation is that carvedilol(which contains three aromatic rings) is very highly retainedon all polymer materials, yet is the least separated of thecompounds, whereas the greatest enantioseparation (afterpropranolol) is observed for pindolol, which is structural

Table 1Data from HPLC analysis of the�-blockers tested on imprinted and reference MIP materials

Compound Imprinted Reference

tr〈R〉 tr〈S〉 α Rs W0.5〈R〉 tr k W0.5〈R〉Ground

Acebutolol 10.4 20.0 2.1 0.8 2.2 7.1b 1.0b 2.1b

Alprenolol 20.1a 96.8a 4.3a 1.0a 7.4a 13.0b 2.7b 4.1b

Atenolol 9.3 19.8 1.6 0.7 2.1 7.2b 1.0b 2.5b

Carvedilol 53.8 74.5 1.4 0.4 11.7 13.7b 2.8b 4.1b

Metoprolol 19.6 53.1 2.9 0.8 5.1 9.8b 1.8b 3.1b

Nadolol 8.3 16.6 2.3 0.7 1.8 7.1b 1.0b 2.5b

Oxprenolol 21.7 56.7 2.7 1.0 5.4 10.5b 2.0b 3.6b

Pindolol 17.6a 74.4a 4.5a 0.9a 7.1a 11.3b 2.2b 4.1b

Propranolol 69.0 nd nd nd 42.5 16.7b 4.1b 6.6b

Silica-graftedAcebutolol 12.0 12.0 1.0 0.0 5.4 5.4 3.2 3.3Alprenolol 18.6 28.7 1.6 0.5 4.5 10.3 7.0 3.7Atenolol 12.7 12.7 1.0 0.0 5.9 7.8 5.0 4.8Carvedilol 34.1 34.1 1.0 0.0 12.2 14.5 10.5 6.7Metoprolol 16.4 18.4 1.2 0.1 7.1 9.5 6.5 3.7Nadolol 9.3 9.3 1.0 0.0 5.6 4.7 2.6 3.1Oxprenolol 16.9 20.5 1.4 0.3 5.4 7.8 4.9 2.8Pindolol 18.9 37.3 2.1 0.6 5.6 6.1 3.7 2.2Propranolol 37.4 82.7 2.3 1.1 10.7 8.8 6.0 3.5

BeadsAcebutolol 6.3 11.6 2.4 0.4 2.5 2.8 0.8 2.5Alprenolol 10.0a 49.4a 5.8a 1.0a 5.9a 4.4 1.4 1.5Atenolol 5.6 11.0 2.5 0.3 2.6 2.6 0.4 0.8Carvedilol 27.7 38.0 1.4 0.1 19.9 7.2 3.4 2.6Metoprolol 11.0 30.6 3.1 0.6 5.5 3.2 0.9 1.0Nadolol 5.4 9.2 2.1 0.2 2.3 2.8 0.4 0.8Oxprenolol 12.5 34.2 3.0 0.7 6.1 3.7 1.1 1.2Pindolol 18.8 94.0 3.9 1.0 11.6 3.9 1.1 1.3Propranolol 20.1a 150.0a 7.7a 0.8a 13.3a 4.4 3.6 0.8

tr is the retention time (tr〈R〉 and tr〈S〉 of the (R)- and (S)-enantiomers, respectively),k the retention factor,α the separation factor of the two enantiomers,Rs the resolution of the enantiomers andW0.5 the peak width at half height (W0.5〈R〉 is the width of the (R)-enantiomer).α andRs on reference materialswere 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 (whichdoes increase the opportunity for interaction with imprintsites), size and shape similarity with the imprint moleculeis ultimately required for chiral recognition. Compound re-tention is also affected subtly by imprinting on silica. Thepolar character of the silica surface favours retention of themore polar atenolol over acebutolol and enhances retentionof pindolol over alprenolol in the imprinted material. Thiscould be due to the silanol groups of the silica backboneplaying a small part in the imprinting process.

All imprinted materials showed greater retention of thenine�-blockers tested than the corresponding reference ma-terials. Ground and beaded MIPs both showed some degreeof enantioseparation for all the�-blockers tested, whereasthe silica-grafted material showed separation for only fiveout of the nine racemic mixtures. Separation and resolu-tion factors were still comparable with result in a recently


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‘fines’).

published study using spherical material made by themulti-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 verylong retention times and poor peak shape. Poor peak shapesare generally obtained with imprinted materials due to thevery strong retention of the analyte and their operation inthe non-linear region of the adsorption isotherm. They arealso made worse by the irregular shape of the particlesfrom the grinding and sieving method. Due to the resultingbackpressure, larger particle sizes are required resulting infurther peak broadening. Both silica-grafted and beaded ma-terials showed an improvement over the ground material inthis area. The morphology of the three materials is shown inFig. 2. Imprinted beaded material (diameter 4.0 ± 1.8�m)showed slightly improved peak shapes over ground materialand backpressure approximately 20% that of the ground andsilica-grafted materials. Imprinted silica-grafted material(5.0 ± 0.8�m in diameter) showed a similar backpressureto imprinted ground material (10.0 ± 4.3�m), but also avastly improved peak shape to both the ground and beadedparticles. Although the improvement in peak shape usingsilica-grafted MIP was largely due to poorer recognition (asshown 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 littleor no polymerisation on the surface of the silica and esti-mates of the volume of polymer present are very similar tothe total pore volume of the silica. This suggests that poly-merisation is limited to the pores, hence reducing the access

Table 2TFC 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 BBackflush 10 R/W A F/D BElute 720 F/D B F/D BRe-equilibrate 30 F/W A F/D B

The flow rates were 1 ml min−1 through the analytical column and 5 ml min−1 through the extraction column throughout. F stands for flow in the forwarddirection (direction of arrows inFig. 1) and R stands for reverse flow. W stands for flow to waste and D to the detector. Composition A is water with0.01% TFA and B is acetonitrile:water (70:30) with 0.01% TFA.

to the imprint sites, but still maintaining the external shapeof the silica. This is not the case for the beaded material inwhich polymerisation is not confined to pores since spheri-cal beads are composed entirely of imprinted polymer. As aresult, beaded MIPs showed much better resolution of enan-tiomers than the silica-grafted material for all the�-blockerstested. However a 22% increase in average peak width andextended retention times was observed. The backpressureon the beaded polymer columns for HPLC was approxi-mately 20% of the one observed for the purchased 5�mspherical particle column, which suggests that the polymermaterials are much more porous than silica. This meant thatthe beaded materials could be easily used at higher flowrates 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-graftedand ground polymer materials as extraction columns forTFC. In both cases, the backpressures on the columns weretoo high and caused the system to leak. Study of thesematerials as extraction columns for TFC application wastherefore discontinued. Pressure readings on the beaded ma-terials, however, were found to be similar to traditionalC18 silica extraction columns, which is most likely due tothe highly porous structure of the beads. Both imprintedand non-imprinted beads were assessed for their extrac-tion ability under TFC conditions. The standard procedurefor a TFC extraction is shown inTable 2. Leaching oftemplate molecule from the imprinted beads was tested by


Fig. 3. HPLC traces of pindolol through 150 mm× 4.6 mm i.d. columns packed with ground MIP (top, scale 0–150 min), beaded MIP (middle, scale0–90 min) and silica-grafted MIP (bottom, scale 0–120 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 columnonto the analytical column with the solvent front at thebeginning of the elute step (seeTable 2). The imprintedbeads showed leaching at a level of approximately 0.5 pgfrom every blank methanol injection. A variety of load,elute and re-equilibration steps were tested to encourageexpression of a molecular imprinting effect, but (R)- and(S)-enantiomers of propranolol were retained equally onboth imprinted and reference beads under all conditionstested. The aqueous nature of the mobile phase encouragesdeposition of the lipophilic analyte directly onto the station-ary phase due to non-specific hydrophobic interactions ratherthan 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 removedthe negative effect of template leaching. Recovery of pro-pranolol using a beaded extraction column compared witha traditional C18 silica column is shown inTable 3. Recov-ery was found to be significantly higher using the C18 sil-ica column, although the extract using the beaded materialwas noticeably cleaner (Fig. 4). Carry-over from a previousextraction was found to be minimal for both of the mate-rials (C18 = 0.010%, beads= 0.006%). Further analysismonitoring recovery of a number of�-blockers in organicsolution is shown inTable 4. Results showed that, for bothbeaded polymer and C18 silica extraction columns, recovery


Table 3Recovery of propranolol from reference beaded polymer and C18 silica extraction columns at 5 ml min−1 after injection of spiked plasma and carry-overin subsequent blank plasma washes (total mass of propranolol loaded onto column is 1000 ng)

Extractioncolumn

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 51C18 silica 37 725.7 7.9 1.6 73

Table 4Recovery of nine�-blockers as a percentage of mass loaded from acetonitrile:water (70:30) using a reference beaded polymer extraction column and aC18 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 36.9C18 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 caseof carvedilol (the most lipophilic of the�-blockers), how-ever, the beaded polymer column outperforms the C18 silicacolumn and extracts a higher percentage of the load. Thisproves that, under certain conditions, the very hydrophobicnature of the beaded polymers can show improved recover-ies over existing materials and gives scope for further de-velopment of beaded materials to ‘fine-tune’ the propertiesof the polymer (e.g. by using different monomers).

Fig. 4. Chromatograms obtained by injecting blank plasma onto a TFCC18 silica extraction column (top) and a beaded reference polymer extrac-tion column (bottom) following an injection of a sample containing a highconcentration of propranolol. Peak at∼10 min is propranolol carry-overand other signals are due to unidentified compounds from plasma. De-tection was using fluorescence (ex. 292 nm/em. 332 nm).

4. Conclusions

Ground monolithic imprinted polymer was still the best‘all-round’ performer for enantiomeric separations of anumber of �-blockers by HPLC. However, silica-graftedMIP provided vastly improved peak shape and completeenatiomeric resolution of a racemic mixture of the imprintspecies in a fraction of the time it would take using theground material. Synthesis of this material is also muchmore efficient and less laborious than ground polymer andtherefore provides a good alternative for bulk enantiomericseparations of racemic mixtures. Although synthesis of thesilica-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 besynthesised in bulk and stored in a dark place for at least 1month without degrading. Beaded material was the fastest,easiest and most efficient to synthesise, with preparationand synthesis complete within 2 h and yields of around90%. The resulting beads provided some degree of enan-tioseparation of all the�-blockers as well as a significantimprovement in peak shape over the ground materials. Thiswould therefore be a good method for rapid MIP synthesisand analysis. The very low backpressures on the columnalso meant that analysis of well separated compounds withlong retention times could be shortened by using higher flowrates. The beaded polymers were also effective in extract-ing propranolol from plasma using TFC and even showedimprovements in recovery over traditional C18 columns forthe most lipophilic compound tested (carvedilol). Extractsfrom plasma using the beaded materials also showed muchcleaner baselines than traditional C18 extraction columns.Although imprinting made no observable difference in theextraction of propranolol, cross-linked beaded materialshave shown much promise for use in turbulent flow extrac-tion and the high porosity of the material did not appearto increase the amount of endogenous biological materials(e.g. proteins) retained on the column.


Acknowledgements

The authors wish to thank EPSRC and Pfizer Global Re-search and Development for financial support of this work.Also to Dr. Elena Piletska and Prof. Sergey Piletksy for helpwith HPLC column packing and Stephen Bennett for assis-tance with SEM imaging.

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