Journal of Chromatography A, 1107 (2006) 165–174

Application of cellulose and amylose arylcarbamates as chiralselectors in counter-current chromatography

Eva Pereza,b, Maria J. Santosa,b, Cristina Minguillona,b,∗a Institut de Recerca Biomedica-Parc Cientıfic de Barcelona (IRB-PCB), Josep Samitier, 1-5, E-08028 Barcelona, Spain

b Laboratori de Quımica Farmaceutica, Facultat de Farmacia, Universitat de Barcelona, Avda. Diagonal s/n, E-08028 Barcelona, Spain

Received 25 July 2005; received in revised form 16 December 2005; accepted 19 December 2005Available online 18 January 2006

Abstract

Here we studied the applicability of cellulose and amylose tris(3,5-dimethylphenylcarbamate) as chiral selectors for the separation of enantiomersby counter-current chromatography (CCC). A variety of organic/aqueous biphasic solvent systems and eight racemic analytes of acidic, neutral, andbasic nature were tested for this purpose. Classical elution mode and pH-zone-refining displacement conditions were used. Partial enantioseparationof pindolol and warfarin was achieved in methyl isobutyl ketone (MIBK)/aqueous solution and methyltert-butyl ether (MTBE)/aqueous solution,r ed. However,r mes©

K ; pH-zone-r

1

ptabbouscccpt

B+

per-sep-

ble at

itionlventtionarymustad-nt of

ldFre-ftenhileobile

t mayow-

phaseobileent,ised

0d

espectively. For these two racemates enantiomeric excess values from 84% to 97% were achieved under the best conditions testesolution by CCC was not achieved for propranolol, naproxen andN-(3,5-dinitrobenzoyl)-(±)-leucine, easily separated in HPLC by the saelectors.2005 Elsevier B.V. All rights reserved.

eywords: Counter-current chromatography (CCC); Centrifugal partition chromatography (CPC); Cellulose arylcarbamate; Amylose arylcarbamateefining; Enantiomer separation; Warfarin; Pindolol

. Introduction

In recent years, the need for enantiomerically pure com-ounds has led to the development of enantioselective separation

echniques, both at analytical and preparative scales[1]. Mostnalytical enantiomer separations can now be performed eithery HPLC or CE. Regarding preparative enantioseparations[2],atch HPLC[3] combined with multiple injection or continu-us simulated moving bed chromatography (SMB)[4] is widelysed in spite of elevated solvent consumption and costly chiraltationary phases (CSPs) and equipment. In this regard, counter-urrent chromatography (CCC)[5] and its modalities, such asentrifugal partition chromatography (CPC)[6], liquid–liquidhromatographic separation techniques especially adapted toreparative purposes[7], are alternatives for preparative enan-

iomer separations. Nevertheless, the extent of their applicability

∗ Corresponding author at: Institut de Recerca Biomedica-Parc Cientıfic dearcelona (IRB-PCB), Josep Samitier, 1-5, E-08028 Barcelona, Spain. Tel.:34 93 403 71 07; fax: +34 93 403 71 04.

is dependent on the availability of chiral selectors (CSs), eximental conditions and technical devices, which make thearation of a broad range of enantiomeric compounds feasia competitive level.

The separation of enantiomers by CCC involves the addof a suitable CS to one of the phases of the biphasic sosystem used. The phase containing the CS is used as staphase. To be applicable for preparative purposes, the CSbe fairly soluble in this solvent or solvent mixture as the loability of the chromatographic system depends on the amouCS involved in the separation[8,9]. In addition, the CS shoumaintain its enantiorecognition capacity in the liquid phase.quently the CS is introduced in the more lipophilic phase, ocomprised of an organic solvent or mixture of solvents, wthe more hydrophilic, often an aqueous solution, acts as a mphase. In the former case, the polarity of the organic solvenincrease the solubility of the CS in the stationary phase. Hever, separation of phases and the stability of the stationarymay be affected because of partition of the CS to the mphase[10]. Moreover, depending on the nature of the solvthe enantiorecognition phenomenon may also be comprom

E-mail address: cminguillon@pcb.ub.es (C. Minguillon). [9,11]. This effect, added to the intrinsically restricted applica-

021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2005.12.061

166 E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174

Fig. 1. Chemical structures of the polysaccharide derivatives tested as chiral selectors (CSs) and the racemic compounds used in the study.

tion domain of some of the CSs used up to now, may account forthe low number of chiral separations by CCC reported to date[12,13].

Among the CSs employed in CSPs for HPLC polysaccharidederivatives are widely used because of their broad applicationdomain, both in normal-[14] and reversed-phase conditions[15,16], and their high loadability[14]. These properties makethem especially suited for HPLC preparative enantioseparationsand are also desirable for CSs to be used in CCC/CPC. Fromthe variety of polysaccharide derivatives used in HPLC, 3,5-dimethylphenylcarbamates of cellulose and amylose (Fig. 1),which are CSs in the well-known Chiralcel OD and ChiralpakAD CSPs, respectively, are among the most applied. Studieson these two derivatives[17] have provided detailed informa-tion about the enantiorecognition capacity of these materialsand some insight into their mechanism of chiral discrimina-tion [18,19]. In this study, these two derivatives were used toevaluate the feasibility of their application to the separation ofenantiomers by CPC. We tested the enantioselectivity of thesetwo derivatives in the separation of several racemic drugs byCPC using binary organic/aqueous solvent systems consistingof methyl isobutyl ketone (MIBK) or methyltert-butyl ether(MTBE) and aqueous solutions. We considered classical elutionmode, analogous to reversed-phase conditions in HPLC (station-ary phase more lipophilic than the mobile phase). In addition,because of the ionisable nature of some of the racemates, thes hrom

2

2

W3 ny)A tely3 ICNB o-c Liq-

uid phases for CPC and HPLC were prepared from analyti-cal reagent-grade sodium monohydrogenphosphate and sodiumdihydrogenphosphate and Milli-Q water, together with HPLC-grade solvents. Racemic pindolol, propranolol, warfarin andnaproxen were purchased from Sigma (St Louis, MO, USA).trans-Stilbene oxide (TSO), 2,2,2-trifluoro-1-(9-anthryl)ethanol(TFAE) and N-(3,5-dinitrobenzoyl)-(±)-leucine (DNB-Leu)were obtained from Aldrich (Steinheim, Germany). Oxazepamwas a gift from Sanofi (Massy, France).

2.2. Apparatus

1H NMR spectra were recorded at 70◦C on a Varian (PaloAlto, CA, USA) GEMINI-300 Spectrometer with pyridine-d5

as solvent. Elemental analyses were determined by theServeisCientıfico-Tecnics de la Universitat de Barcelona (Spain) ina Thermo Electron apparatus model EA 1108 using standardconditions. The CPC experiments were performed in a HPCPCmodel LLB-M (EverSeiko, Tokyo, Japan), a bench centrifuge(30 cm× 45 cm× 45 cm) with a stacked circular partition diskrotor with a volume of 190 mL (measured experimentally).This apparatus was connected between the manual injector(Rheodyne), equipped with a 2.4 mL loop, and the detectorto a conventional HPLC system HP 1100 (pump, autosampler,UV detector, and chromatographic data station software) (Agi-l as3 wask rpmf wasd ac-t per-i sedb com-p PCw 600Ep diodea urew ns.T col-

o-called pH-zone-refining mode, a type of displacement catography, was also studied.

. Experimental

.1. Reagents

Microcrystalline cellulose Avicel (approximated M0 kg/mol) was purchased from Merck (Darmstadt, Germamylose (linear component of starch containing approxima00 glucose units per molecule) was obtained from ofiomedicals (Aurora, OH, USA). 3,5-Dimethylphenyl isyanate was obtained from Aldrich (Steinheim, Germany).

-

.

ent Technologies, Palo Alto, CA, USA). The flow rate wmL/min for the CPC runs and the centrifuge rotationept at 1200 rpm for MIBK-containing systems or at 1100or MTBE-containing systems. The elution of the analytesirectly monitored by UV detection (254 nm). However, fr

ions of the eluate (3 mL) were collected throughout the exment (Gilson FC 203B fraction collector) and later analyy enantioselective HPLC to determine the enantiomericosition during the elution. The fractions collected from Cere analysed in an HPLC system consisting of a Watersump, a Waters 717 autosampler and a Waters 996 photorray detector (Waters, Milford, MA, USA). The temperatas maintained at 25◦C during the HPLC and the CPC ruhe pH of the mobile phases and that of the fractions

E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174 167

lected from CPC were measured with a Crison pH-meter (Alella,Spain).

2.3. Preparation of the polysaccharide derivatives

Avicel for CS1 or amylose for2 (0.5 g, equivalent to 3.1 mmolof glucose units) was suspended in toluene (20 mL) and themixture was dried azeotropically for 2 h using a Dean-Starkwater trap. The remaining solvent was removed under reducedpressure and the solid was suspended in dry pyridine (25 mL)and treated with 1.8 g (12.1 mmol) of 3,5-dimethylphenyl iso-cyanate. After stirring under reflux for 16 h, the dimethylphenyl-carbamate derivatives1 and 2 were isolated as the insolublefraction in methanol and thoroughly washed in hot ethanolto remove theN,N′-bis(3,5-dimethylphenyl)urea formed as aby-product[20]. Further dissolution in chloroform and repre-cipitation in methanol allowed us to obtain the CSs1 and 2,which were characterized by their1H NMR spectra and elemen-tal analyses. The substitution degree per glucose unit (DS) wascalculated from the elemental analyses following the procedurepreviously described, which considers the experimental error inthe determination[21]. Analysis calculated for (C33H37N3O8)n

(DS: 3.00): C, 65.66; H, 6.18, N, 6.96. CS1, found: C, 65.18;H, 6.10; N, 6.88; DS: 2.77± 0.13. CS2, found: 65.67, H, 6.18;N, 6.96; DS: 2.92± 0.08.

2

l elut Ma te pH( akent asesw S wa

ol-v werm nces acif ) and acidf uppp addt

2

2in a

s sol-v mMa ntrod f thC ed at 0 mia ther to a

5 mL volume volumetric flask. The filtered liquid was not a clearsolution. The solvent was evaporated and the resulting residuewas dried under vacuum and weighed.

2.5.2. Determination of racemate distributionPrevious to the CPC study, the qualitative distribution of

CSs and racemates in the biphasic solvent systems was deter-mined by means of liquid–liquid extraction experiments. Theorganic/aqueous solvent systems were prepared in advance andallowed to equilibrate overnight. Two milliliters of the lowerphase was added to test tubes containing 5 mg of the corre-sponding CS in 2 mL of the upper organic phase. Then, 1 mgof the appropriate racemic compound (Fig. 1) was added to thebiphasic system followed by 2 min of vortexing. The distribu-tion of racemate and selector in the two phases was analysed byTLC (hexane/ethyl acetate, 2:8). All the solvent systems testedretained the CS exclusively in the organic phase. The solventsystems that permitted the partition of the racemate were con-sidered for the development of the CPC experiments.

2.6. CPC experimental conditions

To obtain a maximum retention of stationary phase for thesolvent systems, rotation speed and flow rate were previouslyoptimised[6,7]. Thus, the rotation speed was first set at 1300 rpmwhile the flow rate was slowly increased. During this opera-t rota-t et at3 rpmf E-c e con-d wasa

hichw phase( wasc f thee

itionsw (3:2)a mplesf aryp gan.

at2 ue-o r, thee iose-l er tod dataw erep

2

ousf con-t lka-

.4. Preparation of solvent systems

The biphasic solvent systems to be used in the classicaion mode were prepared by mixing MIBK or MTBE and 50 mqueous sodium phosphate buffer solution of the appropria2.0, 6.0, 7.0, 8.0 and 9.0) in a separatory funnel. Once shhe mixtures were allowed to equilibrate for 16 h. The phere separated, filtered and degassed before use. The Cdded to the organic lower-density phase.

The MIBK or MTBE and Milli-Q water to be used as sent systems in the pH-zone-refining displacement modeixed and allowed to equilibrate in a separatory funnel. O

eparated, convenient amounts of retainer (trifluoroaceticor acidic racemates and diethylamine for basic racematesisplacer (ammonia for acidic racemates and hydrochloric

or basic racemates) agents were added to the organichase and aqueous lower phase, respectively. The CS was

o the lipophilic lower-density phase.

.5. Selection of the solvent systems

.5.1. Incorporation of CSs into solventsThe maximum amount of CS that can be suspended

olvent was determined. Six milliliters of the chosen organicent, previously equilibrated in a separatory funnel with 50queous sodium phosphate buffer solution (pH 7.0), was iuced in a test tube with screw cap. Increasing amounts oSs were added to this solvent until a solid residue remain

he bottom. The suspension obtained was sonicated for 3nd allowed to stand for a minimum of 3 h. Five milliliters ofesulting suspension was filtered through glass-fibre filter

-

,

as

e

dd

ered

-etn

ion pressure increases and is controlled by reducing theion speed. When the flow rate of the mobile phase was smL/min, the optimal centrifuge rotation speed was 1200

or MIBK-containing solvent systems and 1100 rpm for MTBontaining systems (back pressure: 72–75 bar). Under thesitions the retention of the stationary phase in the centrifugeround 75%.

The CSs were incorporated to the organic upper phase, was used as stationary phase. The volume retained of this

Vst) and the amount of CS involved in the separationalculated from the volume displaced at the beginning oxperiment.

Samples to be processed under classical mode condere injected in a mixture mobile phase/stationary phasefter the solvent system had reached a steady state. Sa

or pH-zone-refining conditions were injected in the stationhase immediately before pumping of the mobile phase be

The elution was directly monitored by UV detection54 nm. The content of MIBK or MTBE that saturated the aqus mobile phase did not hinder analyte detection. Howeveluate was collected in fractions of 3 mL and a further enant

ective HPLC analysis of the fractions was performed in ordetermine the enantiomeric content during elution. Theseere used to build elution profiles for each run. All runs werformed at least twice.

.7. Analysis of the fractions collected

A liquid–liquid extraction was performed on the aqueractions collected during the CPC experiments. Fractionsaining basic analytes (pindolol or propranolol) were a

168 E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174

Table 1Analytical conditions for CPC control

Racemate k′1

a α RS Mobile phase

Pindolol 2.37 2.29 1.92 Hept/2-PrOH/DEA (70:30:0.2)Propranolol 3.31 2.13 2.09 Hept/2-PrOH/DEA (90:10:0.2)Warfarin 3.35 2.16 1.99 Hept/2-PrOH/TFA (90:10:0.5)Naproxen 11.60 1.15 1.60 Hept/2-PrOH/TFA (98:2:0.5)DNB-leucine 2.33 1.98 1.52 Hept/2-PrOH/TFA (80:20:0.5)

a Column: CSP-100B in[22].

linized with diethylamine and 1 mL of a mixture heptane/2-propanol (8:2) was added. Fractions containing acidic racemates(warfarin, naproxen, or DNB-Leu) were first acidified withhydrochloric acid and 1 mL of the mixture heptane/2-propanol(8:2) was then added. After vortexing, the organic phase wastransferred to a vial and analysed by HPLC.

An enantioselective column (15 cm× 0.46 cm) containingcellulose 3,5-dimethylphenylcarbamate bonded to allyl silicagel as CS was used to evaluate the enantiomeric content of theeluted fractions (CSP-100B in[22]). Suitable HPLC normal-phase conditions for baseline separation of all the racemateswere determined (Table 1). The elution order in the CPC experi-ments was established on the basis of the elution order in HPLCon cellulose 3,5-dimethylphenylcarbamate-based CSPs[23,3].

2.8. Recovery of CSs and enantiomers after the CPC runs

The enantiomers from the pH-zone-refining up-scale experiments were recovered by neutralization of the aqueous elutefractions, extraction with methylene chloride and drying undervacuum. The enantiomeric excesses of the samples recovereas well as the yield of the recovering process, were determined

Once the elution of the analytes was complete, the stationarphase was displaced from the stopped centrifuge by pumpinthe mobile phase in ascending mode. The organic solution cont undev y tha thiss atura ery.

2

reda ingt olole ing

amounts of pindolol and warfarin were injected up to the satura-tion of the chromatographic system (RS < 1.0). The loadabilitystudies were performed in the conditions indicated inTable 2.

3. Results and discussion

The success of a CCC/CPC enantioseparation is highlydependent on the choice of the solvent system, which must beadapted to the CS and also to the racemate to be resolved. Thesolubility of the CS may be a suitable starting point in the searchfor an appropriate solvent system. In addition, high solubility ofthe CS in the stationary phase is convenient from the viewpoint ofpreparative purposes[8,9]. The polysaccharide derivatives thatwere examined in this study as CSs are fairly soluble in tetrahy-drofuran (THF) and in chlorinated (Cl3CH or Cl2CH2) solvents.However, when a simple binary biphasic organic/aqueous sol-vent system, constituted by the mixture of an organic solvent andan aqueous buffer solution, is envisaged, the miscibility of THFin water prevents its use. We did not study chlorinated solventsbecause of technical limitations (incompatibility with certaincomponents in the CPC device) and environmental considera-tions. Therefore, the search for an appropriate organic solventto constitute the stationary phase of the biphasic solvent systemwas a key point in the development of this study. In fact, becauseof the macromolecular character of the CSs considered theirs utea eseC ber ofs lloidals th oft -b opyle ethyla at a4 Si IBK( tei Li Li ganicc diump

anics ithint iquide nd as pin-

TC

R ) k

P 0:60)WWW

V de

aining the CS was washed with water and concentratedacuum. The polysaccharide derivative was precipitated bddition of methanol, filtered and thoroughly washed witholvent. The selector was dried overnight at room tempernd under reduced pressure, which resulted in 90% recov

.9. HPLC loadability determination

Samples of racemic pindolol and warfarin were prepat concentrations ranging from 1 to 20 mg/mL, by solubiliz

he racemate in the convenient solvent (acetonitrile for pindthanol or a mixture MTBE/ethanol for warfarin). Increas

able 2onditions for HPLC loadability determination

acemate Column Flow rate (mL/min

indolol Chiralcel OD-RH (15 cm× 0.46 cm) 0.5arfarin Chiralpak AD-H (25 cm× 0.46 cm) 1.0arfarin Chiralpak AD-H (25 cm× 0.46 cm) 0.5arfarin Chiralpak IA (25 cm× 0.46 cm) 1.0

a Values determined after the injection of 3–10�g amounts of racemate. U

-d

d,.

yg-r

e

e

,

,

olubility in certain solvents is limited, which may constitdrawback for their application to CCC/CPC. However, thSs can be substantially swelled and suspended in a numolvents. Depending on the solvents, the suspensions/coolutions prepared were stable over a considerable lengime, thereby permitting their use. Thus, CS1 was not solule and could not be suspended in toluene or in diisoprther (DIPE). However, this selector was incorporated intocetate and MTBE, both previously saturated with water,.0 mg/mL concentration. The highest incorporation of C1

n an organic solvent was achieved in water-saturated M7.5 mg/mL). The amylose derivative2 was easier to incorporan organic solvents than1. Thus, concentrations of 14.2 mg/mn water-saturated MIBK, 7.6 mg/mL in MTBE and 6.0 mg/mn 1-butanol were attained. The first two were selected as oromponents for the solvent system, together with 50 mM sohosphate aqueous buffer solution.

Once the maximum incorporation of the CSs in the orgolvents was established, the distribution of the racemates whe proposed solvent systems was undertaken. Liquid–lxtraction experiments were performed with the CSs aeries of acidic (naproxen, warfarin, and DNB-Leu), basic (

′1

a α RS Mobile phase

1.58 2.28 7.77 ACN/sodium phosphate 50 mM pH 9.0 (41.06 4.52 11.27 Hept/2-PrOH/TFA (80:20:0.1)0.28 2.97 5.12 EtOH/TFA (100:0.1)0.97 2.36 5.68 MTBEw/TFA (100:0.1)

tection,λ 220 nm.

E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174 169

dolol and propranolol) and neutral analytes (oxazepam, TSOand TFAE) (Fig. 1), chosen from those racemates which werebetter resolved in HPLC on CSPs containing cellulose 3,5-dimethylphenylcarbamate. The CSs were totally retained in theorganic phase in all cases, while partition of the analytes wastuned by modifying the pH of the aqueous buffered phase. Theacidic analytes were retained in the organic phase when an aque-ous buffer solution with pH 2.0 was used, and their partitiontowards the aqueous phase increased with pH, as expected. Pin-dolol and propranolol underwent partition in all the systems andpH values tested, including the most acidic, except at pH 9.0,which caused retention in the organic phase. This result indi-cates the lipophilicity of these racemates even when ionised.TSO, TFAE and oxazepam were not partitioned in any of thebinary solvent systems tested. Therefore, tertiary biphasic sol-vent systems such as MIBK/acetone/sodium phosphate bufferand MIBK/acetonitrile/sodium phosphate buffer were consid-ered. Only oxazepam experienced partition in the two phasesof a MIBK/acetonitrile/sodium phosphate buffer 50 mM pH 7.0(3:1:3) system. At this point TSO and TFAE were discarded forCPC tests.

The incorporation of the CSs in the organic lower-density liq-uid phase of the solvent system determines the stationary role forthis phase. To establish the operating conditions to be used withthe solvent systems in the CPC experiments, parameters suchas rotation speed and flow rate of the mobile phase were opti-m se int lved( achc with-o ntio( theo nteda ion-i ntiow itiont andp aral-l usinp itha nienf tionH ioni es iC

3

. Coe n ot tiosel o bei olarr ity inc oas

Regarding polysaccharide derivatives, because of their macro-molecular nature, it is assumable that several recognition siteswill be present on the same molecule for a given analyte. On thebasis of NMR data, an average of one repeated phenylcarbamoy-lated glucose unit has been determined to be involved in therecognition of a given analyte, in a particular cellulose derivative[18]. Nevertheless, given the possible lack of general characterof this result, a molar ratio derivatized-glucose-units/analyte inthe order of 10 was considered adequate to avoid saturation ofthe chromatographic system. The same molar amount for allracemic drugs (pindolol, propranolol, warfarin and naproxen),0.16 mmol, was injected in all runs.

CS1 was used in a MIBK/50 mM sodium phosphate buffersolution solvent system at a concentration of 7.5 mg/mL. Apartial separation for pindolol enantiomers was obtained whenusing a pH 7.0 mobile phase. A selectivity factor (αCCC) valueof 1.39 was calculated on the elution profile. However, onlymoderate enantiomeric excess values (ee) of about 40% wereobtained at the maximal elution of each enantiomer (Fig. 2).The same phenomenon has been reported in the separation ofDNB-Leu when usingl-proline derivatives as CSs in a simi-lar MIBK/sodium phosphate buffer system[11]. Assuming thatthe separation of enantiomers by CCC/CPC is the result of twoprocesses: association of enantiomers with the CS in the sta-tionary phase and partition of the free fraction of the analyteto the mobile phase, several factors may account for the lacko s. Thec lec-t mayr ion-a n tot ss atw n-s n thes rtitione umt te wast eten-t ieved.

l int ts onw 0 and9 to thel theh ed. Ins asesc theirc e int

ss CSd sted,iis singa w-

ised to achieve the maximum retention of stationary phahe system and, therefore, the maximum amount of CS invoSection2.6). The non-stereoselective retention time for eompound was determined by performing blank CPC runs,ut CS, in several solvent systems and pHs. The long rete>350 min) observed for oxazepam, when applying to CPCnly conditions that afforded partition in test tubes, preveny further development with this racemate. With respect to

sable racemates, a direct relationship between pH and reteas observed, as expected, pH 7.0 affording the best cond

o perform the CPC runs for pindolol, pH 6.0 for propranolol,H 9.0 for warfarin and naproxen. Taking into account the p

elism between reversed-phase HPLC enantioseparationsolysaccharide derivatives[16] and the CPC experiments wnalogous CS, a low rate of ionisation would be more conve

or interaction with the CS and successful enantioseparaowever, solvent systems at pH values that produced a low

sation degree of the analytes caused long retention timPC, which limit the feasibility of the experiment.

.1. CPC runs in classical mode

The CSs were first tested in the classical elution modelution of the two enantiomers may arise either by saturatio

he chromatographic system or as a result of the low enanectivity in the conditions studied. The amount of racemate tnjected was set to avoid saturation of the CS. A maximum matio CS/analyte (1:1) was determined to be the limit capaclassical elution mode CCC/CPC[9]. This maximum molar ratipplies to CSs forming 1:1 complexes with enantiomers[24] andhowing high enantioselectivity for the analytes studied[25].

n

ns

g

t.-n

-f-

f the expected Gaussian shape for the enantiomer peakombination of low association constants with low enantioseivity (low difference between these association constants)esult in a relatively large fraction of free analyte in the statry phase, with a low enantiomeric excess, that will partitio

he mobile phase and will determine the enantiomeric excehich it is eluting. Moreover, MIBK is an organic solvent of coiderable polarity that allows the ionization of the racemate itationary phase at the pH applied, as observed in the paxperiments. To shift the ionization/unionization equilibriowards the presence of a single species, the same racemaested using a pH 2.0 mobile phase. Unfortunately, a short rion time was observed and enantioseparation was not ach

CS1 did not show enantioselectivity towards propranolohe solvent system buffered at pH 6.0 and the experimenarfarin and naproxen using basic mobile phases (pHs 8..0) also gave negative results. These findings, in addition

ack of separation of pindolol at pH 2.0, were attributed toigh ionisation degree of the analytes at the pH values testpite of affording reasonable elution times, these mobile phaused the ionisation of the analytes, which may hinderorrect interaction with the non-charged cellulose derivativhe stationary phase.

Analogous experiments were performed with CS2 at theame concentration used with1 (7.5 mg/mL in MIBK/50 mModium phosphate buffer solution). Under these conditions,2id not display enantioselectivity for any of the racemates te

ncluding pindolol, which was partially resolved by CS1. Takingnto account the incorporation determined for CS2 in water-aturated MIBK, a series of experiments was performed uconcentration of 14 mg/mL of2 in the stationary phase. Ho

170 E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174

Fig. 2. Classical elution mode CPC. (I) Elution profiles corresponding to the separation of (a) 40 mg (0.16 mmol) of pindolol using CS1 (7.5 mg/mL); solvent systemMIBK/sodium phosphate buffer 50 mM pH 7.0; flow rate 3 mL/min;ω = 1200 rpm, descending mode; and (b) 50 mg (0.16 mmol) of warfarin using CS2 (7.5 mg/mL);solvent system MTBE/sodium phosphate buffer 50 mM pH 8.0; flow rate 3 mL/min;ω = 1100 rpm; descending mode. (II) Global elution profile showing the evolutionof the enantiomeric excess during the experiment. Left vertical axis, arbitrary absorbance units; right vertical axis, enantiomeric excess for thefirst eluting enantiomer(up) and the second eluting enantiomer (down). Horizontal axis, time (min).

ever, the higher concentration of CS did not result in improvedenantioselectivity.

The substantial incorporation of CS2 in water-saturatedMTBE (7.6 mg/mL) permitted the evaluation of this CS in CPCusing systems containing this solvent. Pindolol and propanolol,at pH values ranging from 6.0 to 8.0, and naproxen, at pH values8.0 and 9.0, were not resolved. A slight enantiomeric enrich-ment was observed for DNB-Leu. However, warfarin was nicelyresolved at pH 8.0 (αCCC: 1.39) and at pH 9.0 (αCCC: 1.66)(Fig. 2). Peaks were again constituted by a mixture of enan-tiomers although an enantiomeric excess of 80% was attainedat the maximum elution, considerably higher value than thatobtained for pindolol enantiomers with CS1 in the MIBK-containing system.

3.2. pH-zone-refining CPC

pH-zone-refining conditions refer to a type of displacementchromatography which was first described by Ito and co-workers[26] and is applicable to the separation of ionisable compounds.These conditions have also been applied to the separation ofenantiomers[27,9]. From the above described experiments, it isclear that an isocratic classical elution mode imposes constrainsregarding the pH value of the mobile phase for the attainmentof feasible retention times. pH-zone-refining displacement con-ditions were considered to overcome these limitations. Thesec anal ningo alyta ay bi n th

mobile phase produces the elution of the enantiomers as a func-tion of their affinity with CS in the conditions used.

The technique was first applied to CS1 at a concentrationof 7.5 mg/mL in the stationary phase of a MIBK/water solventsystem. The concentration of retainer agent was set in order tobe high enough to ensure the retention of the analyte injected(0.16 mmol) in the stationary phase. The concentration of thedisplacer agent was then optimised (Table 3). As expected,when the displacer concentration was reduced an increase inretention time was observed. In addition, enantiomer resolutionimproved. A nearly-baseline separation of pindolol enantiomerswas achieved and only a slight enantiomeric enrichment at thebeginning and the end of the run was detected for the structurallyclose propranolol. However, in spite of the lack of enantioselec-tivity of CS 1 for warfarin in classical eluting mode, the partialseparation of this racemate was achieved in these conditions(Fig. 3). In all cases, these results imply a neat improvementover the classical CPC eluting mode.

On the basis of the results provided by CS2 in the classi-cal elution mode, the pH-zone-refining displacement study forthis CS was performed in MTBE/water as a solvent system. A7.6 mg/mL concentration of CS2 was used in the stationaryphase and the retainer and displacer agents were the same thanthose used with CS1. Racemic pindolol, propranolol and war-farin were injected and the concentration of the displacer agentwas optimised in each case (Table 3). The attempts to resolvep ow-e ghe merice c-t e in

onditions imply the use of a retainer agent that holds theyte in its unionized form in the stationary phase at the beginf the experiment. The interaction between the neutral annd the CS should be promoted and enantioselectivity m

mproved. Subsequently, the displacer agent contained i

-

eee

ropranolol into its enantiomers failed in these conditions. Hver, CS2 allowed us to partially resolve pindolol, althounantiomers were recovered at only moderate enantioxcess values. Nevertheless, CS2 did not show enantioseleivity for this racemate when used in classical elution mod

E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174 171

Table 3CPC separations in pH-zone-refining displacement mode

Racemate Selector

CS1 (7.5 mg/mL) CS2 (7.6 mg/mL)

Disp Vst t1 t2 Disp Vst t1 t2

Pindolola 20 140 21 25 5 157 43 4610 150 38 42 2.5 151 53 645 143 40 47

Propranolola 10 134 40 43 5 149 54 595 130 44 47

Warfarinb 10 140 37 45 10 134 52 565 145 40 58 5 143 55 64

2.5 157 58 76

Flow rate 3 mL/min;ω = 1200 rpm for runs in MIBK and 1100 rpm for MTBE; descending mode;λ 254 nm. Disp, displacer agent (mM);Vst, volume of stationaryphase (mL);t1 andt2, elution time (min) for each enantiomer corresponding to the maximum intensity of the peak. Solvent system, MIBK/water for runs with CS1and MTBE/water for runs with CS2.

a Retainer agent, 10 mM DEA; displacer, HCl.b Retainer agent, 10 mM TFA, displacer, NH4OH.

comparable solvent system and CS concentration. The baselineseparation was achieved for warfarin enantiomers. Moreover,the enantiomeric excess of the enantiomers recovered attained90%, once again an improvement over the separation obtainedin classical eluting conditions (Fig. 3).

In simple non-enantioselective pH-zone-refining conditionsthe quick equilibrium protonation/deprotonation drives the elu-tion of compounds on the basis of their pKa values [28].This mechanism accounts for the square-shaped peaks com-monly observed in pH-zone-refining mode CCC/CPC separa-

F1ao

ig. 3. pH-zone-refining displacement mode: Elution profiles corresponding to(7.5 mg/mL); solvent system: MIBK/water or (II) CS2 (7.6 mg/mL); solvent systexis, arbitrary absorbance units; right vertical axis, pH. Horizontal axis, time (mverlayed.

the separation of 0.16 mmol of (a) pindolol, (b) propranolol, (c) warfarin using (I) CSm: MTBE/water. Flow rate 3 mL/min, descending mode,λ 254 nm. Left verticalin). On (I(a)) and (II(c)) the elution profiles corresponding to two different runs are

172 E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174

Table 4Up-scaling of CPC enantioseparations

Racemate mrac Vst CS/rac t1 t2 Recovery range 1 Yield1 ee1 Recovery range 2 Yield2 ee2

Pindolola (CS1: 7.5 mg/mL) 40 143 11.2 40 47 38–42 62.5 80.7 44–48 73.8 71.380 145 5.7 36 45 34–38 73.9 91.7 40–47 84.5 84.2

120 150 3.9 36 49 34–40 57.7 40.7 44–50 45.0 36.5

Warfarinb (CS2: 7.6 mg/mL) 50 157 12.4 58 76 56–66 82.7 90.4 69–77 71.4 90.0100 142 5.6 55 79 53–64 74.4 88.6 73–80 73.7 97.3150 130 3.4 58 143 55–75 55.6 75.0 125–150 70.0 77.1

mrac, mass of racemate (mg);Vst, volume of stationary phase (mL); CS/rac, molar ratio of derivatized glucose units/racemate;ti, elution time (min) for each enantiomercorresponding to the maximum intensity of the peak; recovery rangei, time frame (min) that afforded the recoveries indicated; yieldi, recovery of each enantiomer(%); eei, enantiomeric excess of the enantiomer recovered. Flow rate 3 mL/min; descending mode;λ, 254 nm.

a Solvent system, MIBK/water; retainer agent, 10 mM DEA, displacer, 5 mM HCl.ω, 1200 rpm.b Solvent system, MTBE/water; retainer agent, 10 mM TFA, displacer, 2.5 mM NH4OH. ω, 1100 rpm.

tions. However, round-shaped peaks were obtained for pindololand warfarin. Peaks similar to those obtained in this study haverecently been described[29] in anion-exchange displacementCPC, where an anion exchanger is added to the stationaryphase. The authors attributed the anomalous shape of peaksto slow mass transfer kinetics, in this particular case causedby ion-pair exchange. Analogously, in enantioselective pH-zone-refining conditions, the additional interaction with the CSmust be considered. The relative kinetics of the CS/enantiomersassociation equilibria with respect to that of the protona-tion/deprotonation process, which determines the elution, mayexplain the rounded shaped peaks observed with polysaccharide-derived CSs. Nevertheless, low molecular weight CSs do notproduce this phenomenon[9,27], indicating that in this lat-ter case the CS/enantiomers association permits mass transferkinetics comparable in magnitude to that resulting from the pro-tonation/deprotonation equilibrium.

3.3. Up-scaling pH-zone-refining CPC separations

Once the separation conditions were optimised for each CS-racemate pair, scale-up procedures were developed to determinethe loading capacity of the systems for the resolved analytesusing pH-zone-refining conditions (Table 4). For CS1, increas-ing amounts of pindolol were injected (Fig. 4) up to the satura-tion of the chromatographic system. When injecting 0.48 mmol( ura-t d int mgf ololp a-t it thr nan-t2 nalo redw was8 ts1 thee erie

A simple loading study by HPLC was performed on severalof the CSPs available in our laboratory and that contain CSsanalogous to CS1 and CS2 (Table 5). Thus, regarding the sep-aration of pindolol enantiomers, two different CSPs containingthe CS analogous to CS1 were used. Given the analogy of CPCusing organic/aqueous solvent systems with reversed-phase con-ditions the separation was performed on a Chiralcel OD-RHcolumn using acetonitrile/sodium phosphate buffer 50 mM (pH9.0) (40:60) as a mobile phase, one of the best conditions forthe separation of pindolol on this CSP[16]. Increasing amountsof pindolol were injected until the “touching-bands” separation(RS: 1.00) was attained. Five milligrams of this racemate wasresolved in a single run (RS: 0.94). In the search for condi-tions closer to those applied in CPC, an immobilized cellulose-derived phase containing 3,5-dimethylphenylcarbamate of cel-lulose (CSP-100B in[22]) was used. Nevertheless, MIBK asa mobile phase was too polar to produce the retention of theanalyte and selectivity.

The resolution of 17 mg of warfarin was attained in a singlerun (RS: 0.80) when using the classical heptane/2-PrOH/TFA(80:20:0.1) mobile phase and that of 5 mg (RS: 0.91) in thepolar EtOH/TFA (100:0.1) on a Chiralpak AD-H column. Theimmobilized Chiralpak IA allowed us to test conditions simi-lar to those used in CPC. Water-saturated MTBE as a mobilephase permitted the separation of 8 mg of warfarin in a singlerun (R : 0.80). Although the differences in conditions (solventsi ato-g bilityr hus,g AD-Ha iallya p-a lvedil red.T essi-b itions (dis-s solids ecog-

120 mg) of pindolol, the elution profile showed a clear sation. Therefore, taking into account the amount of CS involvehe experiments and a maximum amount of 0.32 mmol (80or the racemate, a loadability of 71 mg of racemic pinder gram of CS1 (equivalent to 170 mmol/mol of deriv

ized glucose units) was calculated. These conditions permecovery of enantiomers in a yield ranging 74–84% with eiomeric excess values of 92% (R) and 85% (S). Regarding CS, the separation of warfarin was scaled-up to saturation. Agously to the CS1/pindolol pair, system saturation occurith amounts over 100 mg. Therefore, the loading capacity5 mg of racemic warfarin per gram of CS2. This represen60 mmol/mol of derivatized glucose units. In this case,nantiomers were recovered in a 74% yield with an enantiomxcess of 89% (S) and 97% (R).

)

e

-

c

Snvolved in the separations performed in the two chromraphic techniques) should not be overlooked, the loadaesults obtained in our hands were favourable to CPC. Tiven the dimensions of the columns used (Chiralpak IA or, 25 cm× 0.46 cm and Chiralcel OD-H, 15 cm× 0.46 cm) andssuming a 20% (w/w) content of CS in the CSPs commercvailable[30,31], the amount of CS involved in the HPLC serations can be estimated. The relative amount of CS invo

n the CCC separation of warfarin was 4–11 times (Table 5)ower than in HPLC, depending on the conditions compahis result can be rationalized taking into account the accility of the CS. Access of the enantiomers to the recognites in the CS is enhanced when the latter is incorporatedolved or suspended) in a liquid stationary phase than in atationary phase. In the latter case, only the most external r

E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174 173

Fig. 4. Up-scaling pH-zone-refining CPC: Elution profiles corresponding to the separation of increasing amounts of pindolol (solvent system: MIBK/water; stationaryphase: 10 mM DEA and 7.5 mg/mL of CS1; mobile phase: 5 mM HCl) and warfarin (solvent system: MTBE/water, stationary phase: 10 mM TFA and 7.6 mg/mLof CS2; mobile phase: 2.5 mM NH4OH). Flow rate 3 mL/min. Left vertical axis, arbitrary absorbance units; right vertical axis, pH. Horizontal axis, time (min).

Table 5Comparison of CPC and HPLC

CPC HPLC

CS1/pindololStationary phase CS1 (7.5 mg/mL), MIBK Chiralcel OD-RHMobile phase H2O (HCl 5 mM) pH 9 Na2HPO4/ACN (60:40)Loadability 71.1 mg/gCS (170 mmol/mol) 12.1 mg/gCS (29 mmol/mol)a

r CS/rac 5.8 34.6ee 92% (R), 85% (S) >95%Solvent consumption 1.9 L/g 2.5 L/g

CS2/warfarinStationary phase CS2 (7.6 mg/mL), MTBE Chiralpak AD-H Chiralpak AD-H Chiralpak IAMobile phase H2O (NH4OH 2.5 mM) Heptane/2PrOH/TFA(80:20:0.1) Ethanol/TFA (100:0.1) MTBEw/TFA (100:0.1)Loadability 85.1 mg/gCS (160 mmol/mol) 24.6 mg/gCS (48 mmol/mol)a 7.2 mg/gCS (14 mmol/mol)a 11.6 mg/gCS (22.5 mmol/mol)a

r CS/rac 6.2 20.9 71.3 44.5ee 89% (R), 97% (S) >95% >95% >95%Solvent consumption 2.5 L/g 1.4 L/g 1.6 L/g 2.1 L/g

a A 20% (w/w) has been assumed for the CS content in the commercially available CSPs.

nition sites will be accessible to the enantiomers. More studiesare in course to demonstrate this hypothesis.

4. Conclusions

Here we tested polysaccharide derivatives for the first timeas CSs in CCC. The results show the scope and limitations of

the applicability of these selectors in this context. Thus, propra-nolol, naproxen andN-(3,5-dinitrobenzoyl)-(±)-leucine, easilyseparated by the same selectors in HPLC, were not resolved byCCC in the conditions used. However, when the resolution wasachieved (pindolol, warfarin) less selector was needed in CCCfor a given amount of racemate. Nevertheless, enantiomers wererecovered with a lower enantiomeric excess than in HPLC.

174 E. Perez et al. / J. Chromatogr. A 1107 (2006) 165–174

Acknowledgements

Financial support from theMinisterio de Educacion y Cien-cia of Spain and from the European Regional DevelopmentFund (ERDF) (project number PPQ2003-00970) is gratefullyacknowledged. E.P. also thanks theMinisterio de Educacion yCiencia of Spain for a doctoral fellowship.

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