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Journal of Chromatography A, 1345 (2014) 115–127

Contents lists available at ScienceDirect

Journal of Chromatography A

j o ur na l ho me page: www.elsev ier .com/ locate /chroma

hiral �-cyclodextrin functionalized polymer monolith for the directnantioselective reversed phase nano liquid chromatographiceparation of racemic pharmaceuticals�

arwa Ahmed, Ashraf Ghanem ∗

hirality Program, Biomedical Science, University of Canberra, Canberra, Australian Capital Territory (ACT) 2601, Australia

r t i c l e i n f o

rticle history:eceived 3 January 2014eceived in revised form 1 April 2014ccepted 7 April 2014vailable online 16 April 2014

eywords:-Cyclodextrinolymer monolith

a b s t r a c t

2,3,6-Tris(phenylcarbamoyl)-�-cyclodextrin-6-methacrylate was prepared and used as a functionalmonomer for the preparation of new �-cyclodextrin (�-CD) functionalized polymer monoliths. Thepolymer monoliths were prepared via the copolymerization of �-CD methacrylate and ethylene gly-col dimethacrylate in different ratios in situ in fused silica capillary (internal diameter 150 �m). Theeffect of functional monomer/cross linker concentration on the chiral separation of different classesof pharmaceuticals namely; �- and �-blockers, antiinflammatory drugs, antifungal drugs, dopamineantagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuret-ics, antihistaminics, anticancer drugs and antiarrhythmic drugs was investigated. Baseline separation was

hiral separationano-LCeversed phase chromatography

achieved for propranolol, ifosfamide, alprenolol, tertalol, 1-indanol, tebuconazole, o-methoxymandelicacid, celiprolol and cizolertine under reversed phase conditions with mobile phase composed of methanoland water, using nano liquid chromatography. The method provides more economical analysis underenvironmentally benign conditions. The prepared capillary columns showed good mechanical stabilityand good repeatability (run-to run and batch-to batch).

Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.

. Introduction

Pharmaceutical enantiomers have distinctive stereoselectiveinding interactions with the biological receptors and conse-uently enantiomers of a single drug may be considerablyifferent in their pharmacokinetic and pharmacodynamic prop-rties. Therefore, regulatory authorities such as the US Food andrug administration (FDA) recommends complete pharmacolog-

cal and toxicological evaluation of each individual enantiomerven if the drug product will be marketed as a racemate [1].onsequently, pharmaceutical companies are now shifting towardhe development of single pure enantiomer drugs via prepara-ive chiral separation techniques rather than the time-consuming

hiral syntheses [2]. This in turn created an immense need for

robust, reliable, high throughput, environmentally benign and

� Presented at the 40th International Symposium on High Performance Liquidhase Separations and Related Techniques (HPLC 2013 Hobart), Hobart, Tasmania,ustralia, 18–21 November 2013.∗ Corresponding author. Tel.: +61 02 6201 2089/+61 417350889.

E-mail address: ashraf.ghanem@canberra.edu.au (A. Ghanem).

ttp://dx.doi.org/10.1016/j.chroma.2014.04.023021-9673/Crown Copyright © 2014 Published by Elsevier B.V. All rights reserved.

economically feasible separation technique of chiral pharmaceut-icals to enable their availability on a commercial scale [3,4].

Therefore, miniaturization – as a trend in modern analyti-cal chemistry – has evolved to reduce the total analysis time,reagents consumption and to handle small samples. This resultedin the development of nano-liquid chromatography (nano-LC)[5]. Nano-LC is one of the microfluidic techniques where reduc-tion in both column inner diameter (10–300 �m) and flow rate(200–1000 nL/min) are combined [6]. The limited amount of sta-tionary phases required to pack the capillary columns offers greatadvantages especially when expensive chiral stationary phases(CSPs) are used [7]. However, packing the column with small par-ticles (usually 3–5 �m diameter) requires inserting retaining fritswithin the capillaries rendering the technically challenging pro-cedure complicated, non-reproducible and sometimes affects theproperties of the packing material [5]. Thus, the use of monolithicmaterials eliminates the need for retaining frits and hence providesa good alternative to packed capillary columns.

The introduction of non-particulate monolithic stationary

phases in nano-LC offered great advantages; the capillary columnscan be prepared via in situ polymerization technique and asmentioned above they do not require frits and hence thisavoids frits-associated problems such as permeability, fragility,

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16 M. Ahmed, A. Ghanem / J. Ch

on-specific interactions and manufacture problems [8]. More-ver, the monolithic capillary columns are highly permeable whichllows the flow of mobile phase at adequate column back pres-ure with reasonably short equilibration time and this in turn has

great emphasis on the analysis time which is essential for indus-rial applications [9]. It is worth pointing out that few monolithicolumns (sizes up to 8 L) are already commercially available for thereparative and industrial separation of large biomolecules such aslasmid DNA, proteins and protein aggregates [10]. Nonetheless, tohe best of our knowledge, there are no available monolithic CSPsor industrial applications.

Monoliths were first introduced in early 1990s and since thenave become a good alternative for established beaded supports11–15]. They are continuous uniform beds composed of one sin-le piece of material with a network of wide, flow carrying poresflow-through pores) and narrow diffusion pores (mesopores) [9].he properties of the monolith vary from highly porous to highlyense depending on the nature of the constructing material. Nowa-ays, two main types of monolithic capillary columns are available,amely; polymer based and silica based, monoliths [16].

Polymer-based monoliths are prepared via in situ copolymer-zation of one or more monovinyl monomer with a divinyl crossinker in suitable porogenic solvents (binary or trinary) in a sin-le step process [9]. The polymer is formed within the capillaryia thermal or photo-initiated free radical polymerization to form

continuous porous bed. Polymer monoliths are not only consid-red for the separation of macromolecules (proteins and peptides)ut also for small organic molecules. Tremendous efforts have been

nvested to increase the surface area of the polymer monolith tonsure adequate separation of small organic molecules [17,18].

The ease and speed of preparation of polymer-based mono-iths compared to the technically challenging procedures forilica-monoliths preparation made polymer monoliths an attrac-ive tool in the field of separation science. To introduce chiralityn polymer-based monoliths, the chiral selector (CS) is copoly-

erized as a functional monomer [19] or immobilized after theonolith preparation [20,21] in a one pot polymerization pro-

edure or post-polymerization surface modification of reactiveroups, respectively. Several categories of CSs are available fornantioselective separation of racemates, for example, molecularlymprinted polymers, ligand exchange, brush-type, macrocyclicntiobiotics-based, protein/glycoprotein-based, cellulose/amyloseerivatives and cyclodextrin(s) derivatives [22].

@@Cyclodextrins (CDs) are torch-like cyclic oligosaccharidesonsists of six, seven or eight d-glucopyranose units connected via-1,4-linkage, this unique shape provides three points of interac-

ion required for the chiral recognition mechanism [23]. The nativeDs structure is a hollow cone with secondary hydroxyl groupsat the second- and third-positions of the individual glucose units)ligned at the “mouth” or wider opening of the CD cavity, whilehe primary hydroxyl groups (at the 6-position) are located at thepposite narrower rim. Since the polar hydroxyl groups are onhe outside of the molecule, the cavity inside the CD is relativelyydrophobic while the outside of the CD is hydrophilic. There-

ore, CD can form inclusion complexes with a variety of moleculesn aqueous solution or in a solid state with organic, inorganic

olecules or ions [24]. CDs are widely explored in chiral separa-ion of racemic compounds where they are either used as mobilehase additives [25] or CSPs [26,27].

The derivatization of the CD hydroxyl groups significantly alterD enantioselectivity, derivatization is performed to improve solu-ility in certain medium, providing additional points of interaction

o improve CD enantiorecognition, optimizing the formation ofnclusion complexes or immobilizing CD as a CS on solid supportsuch as silica or polymer monoliths [28]. Commercial CD HPLColumns have been available since 1983; their enantioselectivities

ogr. A 1345 (2014) 115–127

depend on the coverage, the binding chemistry to the solid sup-port, CD cavity size and the derivative groups incorporated in theCD structure [28]. In fact, most commercially available CD CSPs arederived from �-CD. It is also worth mentioning that the presenceof modified phenylcarbamates in the sugar structure may produceCSPs with different enanatiorecongnition abilities depend on thenature and positioning of the groups on the phenyl ring [29,30].

The purpose of this study is to provide a facile one pot prepara-tion method for new �-CD functionalized polymer monoliths in situin capillary format for the chiral separation of different classesof pharmaceutical racemates using nano liquid chromatography.It is worth mentioning that the preparation of �-CD-based CSPsvia the post-modification procedure is tedious, time consumingand of poor reproducibility [31]. However, the one pot strategyis a potential approach to simplify columns preparation, improvereproducibility and allow higher CS loadability. Nonetheless, opti-mization of the polymerization mixture is crucial to minimizeshielding of the CD in the entrapment procedure [32]. Few reportshave demonstrated higher enantioselectivities of the one pot pro-cedure compared to the post-modification approach [31–34]. Tothe best of our knowledge, the one pot environmentally benignand economically feasible preparation procedure has not been pre-viously reported for applications in nano liquid chromatography.

2. Experimental

2.1. Reagents and materials

Ethylene glycol dimethacrylate (EDMA, 98%), 3-(trimethoxysilyl)propyl methacrylate (98%), 1-propanol (99%),1,4-butanediol (99%), methacryloyl chloride (97%), phenylisocyanate (>98%), trifluoroacetic acid (TFA, ≥99.5%), sodiumhydroxide and hydrochloric acid were purchased from Aldrich(Milwaukee, WI, USA). �-Cyclodextrin was purchased from TCA(Tokyo, Japan). Acetone (AR grade), ethanol (HPLC grade) werepurchased from BDH (Kilsyth, Vic., Australia). Methanol (HPLC)grade was purchased from Scharlau (Sentmenat, Spain). Allother reagents were of the highest available grade and used asreceived. The fused-silica capillaries (150 �m internal diameter)were purchased from Polymicro Technologies (Phoenix, AZ, USA).2,2′-Azobis(isobutyronitrile) (AIBN) were obtained from Wako(Osaka, Japan). All water used for dilutions and experiments waspurified by Nanopure Infinity water system (NJ, USA). Racemicanalytes were mostly purchased from sigma Aldrich.

2.2. Preparation of the monolithic columns

2.2.1. Preparation of ˇ-CD functionalized monomer2,3,6-Tris(phenylcarbamoyl)-�-CD-6-methacrylate was pre-

pared as shown in Fig. 1 (cf. Fig. 1); �-CD (0.42 g, 0.37 mmol)was dissolved in 9 mL dry pyridine, methacryloyl chloride (40 �L,0.38 mmol) was added dropwise. The solution was stirred at roomtemperature for 6 h, then excess (1.9 mL) phenyl isocyanate wasadded dropwise and the solution was refluxed for 1 day. After evap-oration of the solvent under vacuum, the product was purified bycolumn chromatography using silica gel to give a white solid (yield90%). 1HNMR (300 MHz, DMSO): ı = 1.9 (s, 3H, CH3), 4.1 (t, 1H, H4),4.3 (m, 2H, H6), 4.5 (t, 1H, H2), 4.8 (m, 1H, H5), 4.9 (d, 1H, H1), 5.5(m, 1H, H3), 6.9 (dd, 2H, vinyl), 7–7.6 (m, 10H, aromatic), 9.4 (s, 2H,NH).

2.2.2. Preparation of the porous polymer monoliths in fused silicacapillaries

The surface modification in fused silica capillaries (150 �mID) was done by using the same procedure of Schaller et al.

M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127 117

n of 2

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pccbmptt1ppv4ars

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The Chromeleon Chromatography Management System version7.00 was used for the data processing and control of the HPLCsystem.

Table 1Monomers composition in the prepared monoliths and their porosity values.

Column �-CD monomer EDMA (wt%) εT (%)

Fig. 1. Schematic diagram showing the preparatio

35]. Briefly, the fused silica capillaries were rinsed using a Har-ard syringe pump (Harvard Apparatus, Holliston, MA, USA) and

250 �L gas-tight syringe (Hamilton Company, Reno, NE, USA)ith acetone and water, activated with 0.2 mol/L NaOH for 30 min,ashed with water till neutral, then with 0.2 mol/L HCl for 30 min,

insed with water and ethanol. A 20% (w/w) solution of 3-trimethoxysilyl)propyl methacrylate in 95% ethanol adjusted toH 5 using acetic acid was pumped through the capillaries at aow rate of 0.25 �L/min for 1 h. The capillary was then washedith acetone and dried with a stream of nitrogen and left at room

emperature for 24 h. The short (∼25 cm in length) surface modi-ed capillary was filled by Harvard syringe pump with the degassedolymerization mixture at 0.25 �L/min using the syringe pump.

Three polymer-based monolithic capillary columns were pre-ared via in situ copolymerization of binary monomer mixturesonsisted of �-CD-derived functional monomer and EDMA as arosslinker along with three porogens namely; 1-propanol, 1,4-utanediol and water in the presence of 1 wt% AIBN (with respect toonomers). The filled capillaries were then sealed with a septum,

laced in 70 ◦C water bath for 18 h for the polymerization reac-ion to take place. The unreacted monomers were removed fromhe monolithic columns by pumping methanol at a flow rate of00 �L/h for 4 h before being conditioned with water and mobilehase, both for 1 h at 30 �L/h. The ratios of the monomers to theorogens were kept 40% and 60%, respectively (guided by our pre-ious investigations) [33]; the ratios of the porogens were fixed as8% 1-propanol, 6% 1,4-butanediol and 6% water, all percentagesre w/w. Whilst the ratio of �-CD derived monomer varied withespect to EDMA, the prepared columns monomers composition ishown in Table 1.

.2.3. Evaluation of columns total porosity

Monoliths total porosity was determined using nano HPLC flow

ethod where the void volume of unretained marker (uracil) waseasured using methanol as mobile phase [19]. The eluted vol-

me of methanol was collected in a sealed vial to avoid errors due

,3,6-tris(phenylcarbamoyl)-�-CD-6-methacrylate.

to mobile phase evaporation and weighed out in a given time at0.3 �L/min flow rate. The weight was then converted to volumeusing methanol density and the total porosity was calculated usingthe following equation:

εT = V

�r2u× 100

where εT is the total porosity, V (m3/s) is the mobile phase vol-ume, r (m) is the inner radius of the empty capillary, u (m/s) isthe linear velocity of the mobile phase which is determined by theunretained compound uracil. The linear velocity is calculated bydividing the effective length of the column by the retention time ofuracil.

2.3. Instrumentation

The nano liquid chromatographic system was from Dionex Cor-poration (UltiMate® 3000 capillary LC system) which featuredan integrated flowsplitter connected to a continuously monitoredflow meter and control valve to maintain a constant flow ratein the range from 0.1 to 1.0 �L/min. It also comprised a binarypump, a vacuum degasser, an autosampler and a UV detector.

®

(wt%)

A1 10 30 24.5 ± 0.75A2 20 20 22 ± 2A3 30 10 Too hard to pump through

118 M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127

aratio

2

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Fig. 2. Schematic diagram showing the prep

.4. Standard solutions and sample preparation

Stock solutions of the racemic analytes at concentrations of mg/mL in filtered HPLC grade methanol were prepared. Prior to

njection, the stock solutions were further diluted 10× and filteredhrough Sartorius Minisart RC 15 0.2 �m pore size filters (Goettin-en, Germany).

Tested compounds: ˇ-blockers: alprenolol, celiprolol, meto-rolol, pindolol, propranolol, acebutolol, carbuterol, bufuralol,ertatolol, atenolol. ˛-Blockers: naftopidil. Antiinflammatoryrugs: ketoprofen, ibuprofen, naproxen, flurbiprofen, indo-rofen, cizolirtine, cizolirtine carbinol, carprofen, etodolac,esmethylcizolirtine, glafenine. Antifungal drugs: hexaconazole,yproconazole, tebuconazole, miconazole, diniconazole, sulcona-ole. Dopamine antagonists: eticlopride. Norepinephrine-dopamineeuptake inhibitor: nomifensine. Catecholamines: arterenol,ormetanephrine. Sedative hypnotics: aminoglutethimide, phenyl-lutethimide, N-acetylaminoglutethimide, 4-bromogluthethimide,entobarbital, p-hydroxyphenobarbital. Diuretics: etozoline. Anti-istaminics: chlorpheneramine. Anticancer drugs: ifosamide.ntiarrhythmic drugs: tocainide. Miscellaneous: 1-acenaphthenol,-methoxy mandelic acid, 4-hydroxy-3-methoxymandelic acid,-indanol, 1-(2-chlorophenyl)ethanol, 1-phenyl-2-propanol.hemical structures of the investigated racemates are listed inable 2.

.5. HPLC conditions

The mobile phase A and B consisted of 0.1% TFA in water (v/v) andethanol (v/v), respectively. For all samples, the injected volume

as 0.2 �L. Preliminary UV analyses were performed at several dif-

erent wavelengths (219–270 nm) for each compound, in order toelect the optimum wavelength for all the analytes and best utilise

single wavelength UV detector.

n of �-CD functionalized polymer monolith.

3. Results and discussion

3.1. Polymer monoliths: Preparation and characterization

Three polymer monoliths were prepared via in situ copolymer-ization of 2,3,6-tris(phenylcarbamoyl)-�-CD-6-methacrylate as afunctional monomer and EDMA as a cross linker in the pres-ence of ternary porogenic system composed of 1-propanol (48%),1,4-butanediol (6%) and water (6%) (cf. Fig. 2). The ratio of themonomers to the porogens was fixed at 40:60, respectively; thisratio was selected to provide monoliths with good balance ofpermeability, surface area and mechanical stability [33,36]. Nev-ertheless, the ratio of the functional monomer to the cross linkervaried in attempt to study the effect of CS concentration and/orcolumn porosity on the enantioselective separation (cf. Table 1).

3.1.1. Evaluation of columns performanceThe quality of the polymer monoliths was evaluated as a func-

tion of the column total porosity (εT) and efficiency (in termsof separation factor and resolution Rs). It is well establishedthat total monolith porosity gives only partial characterization forthe monolith while better characterization is determined by theaverage pores size [37]. However, mesopores of organic polymerskeleton swell upon exposure to organic solvents which makesmonolith porosity in dry state largely differs from that of the solventexposed counterpart [38]. Therefore, monoliths total porosity wasdetermined using nano HPLC flow method as previously describedin Section 2.2.3. εT = −V/�r2u × 100 Measured εT values for thecolumns A1 and A2 are listed in Table 1.

From Table 1, it was observed that �-CD methacrylate/EDMAweight ratio determined column porosity since the porogens com-position is kept constant. It is well established that increasing the

content of the crosslinker EDMA leads to decreased column poros-ity [39]. However, it was observed that increasing the functionalmonomer 2,3,6-tris (phenylcarbamoyl)�-CD methacrylate led toless permeable columns with smaller total porosity. Moreover, the

M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127 119

Table 2Chemical structures of the investigated racemates and their suppliers.

Class Compound name Structure Supplier

�-Blockers Alprenolol andl-alpenolol

3B ScientificCorporation, USA

Celiprolol andS-celiprolol, carbuterol,tertatolol

American CustomChemicals Corp.,USA

Atenolol, acebutolol,metoprolol,propranolol, bufuralol,pindolol

Sigma (St. Louis,MO, USA)

�-Blockers Naftopidil Sigma (St. Louis,MO, USA)

120 M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127

Table 2 (Continued)

Class Compound name Structure Supplier

Anti-inflammatories

Ketoprofen, ibuprofen,naproxen, carprofen,etodolac, glafenine,flurbiprofen,indoprofen

Sigma (St. Louis,MO, USA

Desmethylcizolirtine,cizolirtine,S-cizolirtine, cizolirtinecarbinol

American CustomChemicals Corp.,USA

M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127 121

Table 2 (Continued)

Class Compound name Structure Supplier

Antifungals Hexaconazole,cyproconazole,tebuconazole,miconazole,diniconazole,sulconaole

3B ScientificCorporation, USA

Dopamineantagonists

Eticlopride American CustomChemicals Corp.,USA

Norepinephrine-dopaminereuptakeinhibitors

Nomifensine Sigma (St. Louis,MO, USA

122 M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127

Table 2 (Continued)

Class Compound name Structure Supplier

Sedativehypnotics

Aminoglutithimide,phenylglutethimide,4-bromoglutethimide,N-acetylglutethimide,pentobarbital, p-hydroxyphenobarbital

American CustomChemicals Corp.,USA

Diuretics Etozoline American CustomChemicals Corp.,USA

Antihistaminics Chlorpheniramine Sigma (St. Louis,MO, USA

Anticancers Ifosfamide Sigma (St. Louis,MO, USA

Antiarrhythmics Tocainide Sigma (St. Louis,MO, USA

Catecholamines Arterenol Sigma (St. Louis,MO, USA

M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127 123

Table 2 (Continued)

Class Compound name Structure Supplier

Miscellaneous 1-Acenaphthenol,o-methoxy mandelicacid, 1-indanol,4-hydroxy-3-methoxymandelic acid,1-(2-chlorophenyl)ethane,1-phenyl-2-propanol

Sigma (St. Louis,MO, USA

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p

olumn was imperviuos upon using high concentation of the �-D functional monomer which impeded the flow of the mobilehase, this is probably attributed to the agglomeration of the poly-er at high CS concentration and formation of lumps inside the

olumn which was confirmed by SEM (cf Fig. 5). This finding ofeduced coloumn porosity with high functional monomer and lowrosslinker ratio could be explained in terms of the high molecu-ar mass of the CS, a similar observation of reduced electroosmotic

obility was prviously reported in CEC at high �-CD concnetrations a consequence of its effect on pore size [40].

.1.2. Scanning electron microscopy (SEM)Scanning electron microscopy (SEM) was used to study the

ffect of variation of the functional monomer/cross linker ration the morphology of the prepared monolith. Columns A1 and2 showed homogenous porous structure with interconnectinghannels allowing the flow of mobile phase under low column back-ressure (cf. Figs. 3 and 4). On the other hand, A3 showed muchner structure with clusters of interconnected globules (cf. Fig. 5).3 resisted the flow of mobile phase through and hence, it wasxcluded from further investigations.

.1.3. Columns mechanical stabilityThe polymer monoliths were tested for mechanical stability by

umping a mixture of methanol/water (80:20 v/v) at varying flow

Fig. 3. Scanning electron micrograph of A1 at 500× and 25,000×, res

rate (0.1–1 �L/min). The pressure drop as in function of the mobilephase flow rate was linear indicating that the packing monolith didnot collapse under high pressure values (cf. Fig. 6). The overlay plotsof the backpressure versus the mobile phase flow rate are shownin Fig. 6, A1 and A2 columns demonstrated good stability over thepressure ranges confirmed by excellent linear velocity at pressurerange 20–250 bar. However, linearity was lost for A2 column at highflow rates (0.9 and 1 �L/min) which is three times higher than theflow rate used for the enantioselctive separation in this study.

3.2. Columns repeatability

In order to investigate the repeatability of the prepared columns(the ability to prepare equally performing columns at differ-ent locations [41]); two columns were prepared on the sameday using the same polymerization mixture to test column-to-column repeatability. Moreover, batch-to-batch repeatability wastested by preparing three different batches at different daysusing identical polymer mixtures. Run-to-run, column-to-columnand batch-to-batch repeatability in terms of percentage relativestandard deviation values are shown in Table 3. Propranolol was

selected to check columns performance in terms of repeatabilityas it was baseline resolved on both columns. As shown in Table 3,reproducibility of retention times of both propranolol peaks wassatisfactory. Run-to-run repeatability using one column was also

pectively, showing homogenous porous monolithic structure.

124 M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127

Fig. 4. Scanning electron micrograph of A2 at 500× and 25,000×, respectively, showing homogenous porous monolithic structure.

Fig. 5. Scanning electron micrograph of A3 at 500× and 25,000×, respectively,

frmai

acceptable resolution (Rs 1–1.5) was achieved for diniconazole,

TRT

Fig. 6. Column backpressure versus flow rate.

avorable. The relative standard deviations of the retention timesanged between 0.36% and 4.4%, these results suggest that these

onolithic capillary columns can be used for reproducible routine

nalysis. It is worth mentioning that the acceptable %RSD values forntra-batch and inter-batch are 2.5% and 15%, respectively [42].

able 3un-to-run, column-to-column and batch-to-batch repeatability of the monolithic columFA) 10:90 v/v, UV: 254 nm, flow rate: 0.3 �L/min.

Column Analyte Run-to-run (n = 5)

Retention time (min) (%RSD)

Peak 1 Peak 2

A1 Propranolol 13.01 (0.8) 13.6 (0.79)

A2 Propranolol 13.2 (1.05) 13.85 (0.56)

showing impervious monolith with separation from the capillary walls.

3.3. Enantioseparation of different classes of pharmaceuticalracemates using polymer monoliths

A1 and A2 columns were investigated for the enantioselectivenano liquid chromatographic separation of a set of different classesof racemic pharmaceuticals namely: �-blockers, �-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists,norepinephrine-dopamine reuptake inhibitors, catecholamines,sedative hypnotics, diuretics, antihistaminics, anticancer drugsand antiarrhythmic drugs. The choice of compounds was arbi-trary and guided by our previous investigations [33]. Initially,the enantioselective separation was investigated using the morepolar solvent acetonitrile, mobile phase composed of acetoni-trile and water mixture ranged from 10–90% (v/v) was tested.No enantioselective separation was observed under this con-dition. However, when aqueous methanol-based mobile phasewas used, baseline separation (Rs ≥ 1.5) was achieved forpropranolol, ifosfamide, alprenolol, tertalol, 1-indanol, tebucona-zole, o-methoxymandelic acid, celiprolol and cizolertine while

ketoprofen, metoprolol and 6-hydroxyflavanone (cf. Figs. 7–10).It was observed that increasing the percentage of water inthe mobile phase increased the retention and improved the

ns comparing the retention times of propanol, mobile phase: methanol/water (0.1%

Column-to-column (n = 2) Batch-to-batch (n = 3)

Retention time (min) (%RSD) Retention time (min) (%RSD)

Peak 1 Peak 2 Peak 1 Peak 2

13 (0.39) 13.6 (0.36) 13.1 (1.07) 13.7 (0.92)12.9 (4.4) 13.8 (1.08) 13.3 (0.23) 13.9 (1.07)

M. Ahmed, A. Ghanem / J. Chromatogr. A 1345 (2014) 115–127 125

Fig. 7. Chromatograms (a–c) showing the UV traces of the enantioselective nano-lc separation of alprenolol on A2 capillary column (150 �m ID, 25 cm length). (a) Racemicalprenolol, (b) (S)-alprenolol, (c) co-injected (S)-alprenolol with racemic alprenolol: (S) + (R,S), Mobile phase: methanol/water (0.1% TFA) 10:90 v/v, UV: 240 nm, flow rate:0.3 �L/min.

Fig. 8. Enantioselective nano-lc separation of racemic celiprolol on A2 capillary col-umn (150 �m ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90 v/v,UV: 254 nm, flow rate: 0.3 �L/min.

Fig. 9. Enantioselective nano-lc separation of racemic propranolol on A2 capillarycolumn (150 �m ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90v/v, UV: 254 nm, flow rate: 0.3 �L/min.

126 M. Ahmed, A. Ghanem / J. Chromat

Table 4Chromatographic parameters, separation and resolution factors for the baselineresolved compounds, using mobile phase composed of methanol/water mixtures(0.1% TFA), flow rate: 0.3 �L/min.

Column Separated compound Separation factor (˛) Resolution (Rs)

A1

Propranolol 1.39 2.22Ifosfamide 1.44 1.65Diniconazole 1.65 1.26Ketoprofen 1.46 1.32Tertatolol 1.67 2.561-Indanol 1.76 2.46

A2

Propranolol 1.52 2.53Metoprolol 1.54 1.23Tebuconazole 1.45 2.56-Hydroxyflavanone 1.67 1.18o-Methoxymandelic acid 1.71 1.58Cizolertine 1.72 2.62Alprenolol 1.89 2.2

efT

alsgHpnaC[(soHpivlec

Fcv

Celiprolol 1.52 1.93Tertatolol 1.65 2.56

nantioseparation. Separation (˛) and resolution (Rs) factorsor the baseline/acceptable resolved compounds are listed inable 4.

It is worth mentioning that the enantioselective separation waschieved under reversed phase conditions which allow the use ofess costly solvents and provide easier sample preparation fromerum or plasma [43]. The baseline-resolved racemates investi-ated in this study were previously resolved using conventionalPLC under normal phase conditions using n-hexane-based mobilehases. Not only to mention the health hazards associated with-hexane use but also the high analysis cost. Taking alprenolols an example, it was previously separated on amylose-basedSP (particle packed, 0.46 cm ID) under normal phase conditions44,45]. Taking the column dimensions and mobile phase flow rate1 mL/min) into consideration, the chiral analysis for one run con-umes 15 mL of the environmentally unfriendly solvents. On thether hand, running the same analysis under reversed phase nanoPLC conditions consumes less than 500 nL of water-based mobilehase. The �-CD-based capillary monolithic column is 10.000 less

n internal diameter and operates with one million time less solvent

olume than the conventional column. Consequently, it consumesess solvent and produces faster and reproducible separations. Thelution order of the enantiomers was determined using the opti-ally pure individual form; the individual enantiomer was injected

ig. 10. Enantioselective nano-lc separation of racemic cizolirtine on A2 capillaryolumn (150 �m ID, 25 cm length). Mobile phase: methanol/water (0.1% TFA) 10:90/v, UV: 270 nm, flow rate: 0.3 �L/min.

ogr. A 1345 (2014) 115–127

as such in a single run and co-injected with the racemate (racemicenriched) in another run (cf. Fig. 7).

3.4. Insights into the chiral discrimination mechanism

Various CD derivatives have been synthesized and immobilizedon silica or polymer surface either by physical adsorption or bycovalent bonding [23]. Whilst physical adsorption is considered aneasy and fast method to prepare CSPs, covalent bonding expandsthe window for diverse mobile phase usage and creates a morerobust CSP [46]. It is worth pointing out that most of the CD-based CSPs have been prepared via post-immobilization processesto bond the CD derivative to the solid supports. This procedure,nonetheless, results in robust and more stable CSP; it is time con-suming and offers less coverage of the CS compared to the one pottechnique [31]. Introducing phenyl carbamate moieties into �-CDprovides CDs with more points of interactions which significantlyimprove the enantiorecognition ability of the CD CSP.

It is well established that under reversed phase conditions,the formation of inclusion complexes within the CD cavity is themost predominant mechanism of retention and enantioselectiv-ity. Moreover, the presence of phenyl carbamate moieties on therim creates more points of interactions between the enantiomersand the CSP via hydrogen bonding, �–� bonding, dipole–dipolestacking, etc. which can enhance the selectivity towards some ana-lytes. Initial testing with mixture of acetonitrile and water as mobilephase in the range of 10–90% v/v did not furnish any enantioselec-tive separation. On the other hand, when methanol-based mobilephase was used, enantioselective separation was observed; thisunderlines the importance of solvent polarity in determining reten-tion and chiral separation mechanism in terms of the inclusioncomplex stability. The use of buffers in mobile phase was avoidedbecause of their negative effect on the life time of the capillarycolumns as well as its potential problems with nano-LC systems(e.g. precipitation into the pumps and valves) [47].

It was observed that 8 out of the 13 baseline/acceptable resolvedracemates were for chiral centers with secondary alcohols withadjacent electronegative nitrogen bearing alkyl side chain; thisunderlines the importance of these groups in providing three pointsof interactions which is required for the chiral recognition as theside chain on the chiral carbon is also thought to be concerned in thechiral recognition mechanism [48]. This observation was confirmedupon derivatization of the secondary alcohol to its correspondingacetate ester which led to the loss of its chiral discrimination by theCSP. It was also observed that the enantioselective separation wasmostly achieved at high water content mobile phase; this indicatesthat water improved the magnitude of interaction between theracemates and the CSP. Aqueous mobile phases allow the inclusionof the whole molecule or just its lipophilic part into the hydropho-bic cavity of CD, it has been previously reported that the resolutionof this kind of CSP is improved by increase in water content [48].We postulate that the enantioselective separation in this study wasmainly achieved via the formation of inclusion complexes withinthe CD cavity stabilized by hydrogen bonding, hydrophobic interac-tions and �–� interactions cause by phenyl carbamate moieties. Itis worth mentioning that column porosity did not play a major rolein the chiral recognition mechanism however; one would expectdifference in analytes’ retention time with large macropores whichwas not evident in this study.

4. Conclusions

Novel 2,3,6-tris(phenylcarbamoyl)-�-CD methacrylate-co-ethylene glycol dimethacrylate monolithic capillary columnswere prepared and found to be efficient for the chiral separation

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M. Ahmed, A. Ghanem / J. Ch

f different classes of pharmaceuticals. Separation and resolu-ion factors for the baseline resolved compounds ranged from

= 1.39–1.89 to Rs = 1.56–2.62, respectively. This shows that theuggested one pot procedure for the preparation of the new CSPs effective for the enantioselective separation of pharmaceuticalacemates and that the approach could be expanded to otherlasses of chiral selectors. However, optimization of the chiralelector functional monomer/crosslinker ratio is required forptimum enantioselectivity and to avoid columns blockade at highonomer concentrations. In the present study, the enantioselec-

ive separation was furnished under reversed phase conditionssing mobile phase composed of methanol and water whichliminate the use of drastic and expensive solvents. Consequently,he newly prepared capillary columns augment the scope of benigneparation technique where reversed phase analytical mode worksn tandem with nano liquid chromatography.

cknowledgments

The authors gratefully acknowledge Dr. Karsten Gömann, Cen-ral Science Laboratory, University of Tasmania (UTAS) for the SEMmaging, also the W.J. Weeden scholarship program at the Univer-ity of Canberra, Australia, for the PhD scholarship offered to Ms.arwa Ahmed.

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