Synthesis and characterization of polyrotaxane-based polymeric continuous beds for capillary...

Ž .J. Biochem. Biophys. Methods 50 2002 217–232www.elsevier.comrlocaterjbbm

Synthesis and characterization ofpolyrotaxane-based polymeric continuous beds for

capillary electrochromatography

O. Kornysovaa, E. Machtejevasa, V. Kudirkaitea, U. Pyellb,ˇ ˙A. Maruskaa,)ˇ

a Department of Chemistry, Vytautas Magnus UniÕersity, Vileikos 8, LT-3035 Kaunas, Lithuaniab Department of Chemistry, Philipps UniÕersitat Marburg, Hans-Meerwein-Strasse,¨

D-35032 Marburg, Germany

Received 15 July 2001; received in revised form 20 July 2001; accepted 26 July 2001

Abstract

The continuous bed technique with its attractive features, such as fritless design, one-step insitu synthesis, low back pressure and no need for pressurising the electrode vessels to suppressbubble formation was applied to form polyrotaxane-based stationary phases for capillary elec-

Ž .trochromatography CEC . Rotaxanes are synthesized from two classes of substances, namelylinear reactive monomers and inert cyclic compounds. Upon polymerisation, a gel forms with the

Ž .cyclic molecules mechanically immobilized see Fig. 1 . We have employed this simple approach,using charged derivatives of cyclodextrins in order to introduce charged groups into continuousbeds and thus render them appropriate for electrochromatography. The self-assembly of supra-molecular structures to form rotaxanes during the synthesis of the continuous beds is treated. Theelectroosmotic and chromatographic properties of the various polyrotaxane-based stationaryphases synthesized are discussed, as well as the synthesis of the continuous beds, including how toaffect their porosity and its influence on the efficiency of the electrokinetic separation. Theapplicability of the rotaxane-based continuous bed is demonstrated by separation of modelcompounds by reversed- and normal-phase chromatography. A separation of enantiomers is alsopresented. This experiment is of particular interest because it indicates that the interaction with the

Ž .cavity of b-cyclodextrin b-CD is not a fundamental requirement for enantioseparations.q2002Elsevier Science B.V. All rights reserved.

Keywords: Rotaxanes; Self-assembly; Stationary phase; Continuous bed; Capillary electrochromatography;Enantioseparation; Electroosmotic mobility

) Corresponding author. Fax:q370-7-45-13-81.Ž .E-mail address: audrius [email protected] A. Maruska .ˇ–

0165-022Xr02r$ - see front matterq2002 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-022X 01 00233-0

( )O. KornysoÕa et al.rJ. Biochem. Biophys. Methods 50 2002 217–232ˇ218

1. Introduction

Rotaxanes are supramolecular structures, consisting of two moieties, one axial andone cyclic. They are mechanically and irreversibly linked, which is in contrast tocommon host–guest complexes. Cyclodextrins, porphyrins, crown ethers, calixarenesand other macrocyclic molecules can be used for the synthesis of rotaxanes. The

w x w xthreading axes can consist of aliphatic, aromatic 1 chains with chiral 2 or non-chiralw xproperties. The threads can be symmetric or asymmetric 3 . The formation of double

w xencircled and double stranded pseudorotaxanes was recently demonstrated 4 . Thestability of rotaxanes, i.e. the prevention of dethreading can be ensured by end capping

w x w xwith different bulky ligands, for instance, dendrites 5 , fullerenes 6 , calixarenes andw xblocking group initiators 7 .

Ž . w xSelf-assembly of rotaxanes can be spontaneous entropy-controlled 8 and tem-Ž . w xplate-directed enthalpy-controlled process 9–11 . Template-directed synthesis can be

w x w xbased on donorracceptor, hydrophobic interactions 12 and hydrogen bonds 9,10 .w xPolyrotaxanes can be formed as side chain polyrotaxanes 13 , main chain rotaxanes

w x14,15 . Preparation of mechanically linked, branched and cross-linked polyrotaxanes isw x Ž .possible 16 . Recently, the synthesis of side chain poly methacryl-rotaxanes with more

w xthan one ring of cyclodextrin per side chain was reported 17 . Pseudorotaxanes differfrom common rotaxanes in that their axes have bulky blocking groups only at one end.

w xGibson et al. 18 reported formation of polyrotaxanes using conventional free-radicalpolymerization of acrylic monomers in the presence of a crown ether. Harada et al.w x w x w x19,20 , Steinbrunn et al. 21 and Weickenmeier and Wenz 22 demonstrated thepossibilities to form polyrotaxanes from cyclodextrines threaded on different polymers,such as polyethylene glycol, polypropylene glycol, polymethyl vinyl ether, polyamide,polypropylene, polyisobutylene and polyesters.

Nano-sized rotaxane assemblies are of great interest due to their special structure andproperties and the potential application as molecular devices for photoswitching or

w x Ž .electron switching 23 . Macrocyclic molecules cyclodextrins, crown ethers are widelyapplied as additives or as chiral selectors covalently attached to chromatographic and

w xelectrophoretic matrices for the separation of enantiomers 24–26 . To our knowledge,the application of rotaxane structures as stationary phases for chromatography has notbeen published previously.

The objectiÕe of this study. The purpose of this preliminary investigation was to studythe use of rotaxane structures as stationary phases for capillary electrochromatographyŽ .CEC . The synthesis of these phases is simple, since it involves only one step. The

Ž .conventional stationary phases are synthesized in two or more steps: 1 synthesis of theŽ . Ž .matrix, 2 matrix activation and 3 attachment of the specific ligands. For instance,

Ž .capillaries with monolithic continuous beds of different functionality can be tradition-Ž .ally synthesized via synthesis of acrylic polymer matrix with epoxy-groups step 1 and

Ž . w xsubsequent attaching of ligands of interest by chemical reaction in situ step 2 27,28 .Ž .The matrix activation step 2 is more difficult to perform. Since the derivatization is

accomplished in the capillary, it is not always easy to achieve uniform experimentalconditions along the capillary, for instance, constant concentrations of the derivatization

( )O. KornysoÕa et al.rJ. Biochem. Biophys. Methods 50 2002 217–232ˇ 219

agent and the catalyst, injected separately, which also may cause low chromatographicefficiency of the bed.

2. Materials and methods

2.1. Materials

Ž . X XAmmonium peroxodisulfate APS andN,N,N ,N -tetramethylethylenediamineŽ . Ž .TEMED were purchased from Reanal Finechemical Budapest, Hungary . Piperazine

Ž . Ž .diacrylamide PDA , methacrylamide MA and a 30% vinyl sulfonic acid sodium saltŽ .aqueous solution were obtained from Fluka Chemie Buchs, Switzerland .N-isopropyl

Ž .acrylamide, sulfatedb-cyclodextrin b-CD and neutralb-CD were from Aldrich Chem.Ž .Milwaukee, WI, USA . 6-aminob-CD was from Advanced Separation TechnologiesŽ . Ž . Ž .USA . Racemic ibuprofen was from Sigma St. Louis, MO, USA . Methanol MeOH ,

Ž . Ž .glacial acetic acid HOAc , triethylamine TEA , methacryloxypropyl trimethoxysilaneŽ . Ž .Silan A 174 , ammonium sulfate were from E. Merck Darmstadt, Germany . Organic

Žsolvents used within the study were of HPLC grade. Fused silica capillaries 50 and 100. Žmm I.D., 375mm O.D. were obtained from Polymicro Technologies Phoenix, AZ,

.USA .

2.2. Apparatus

ŽAll CEC experiments were carried out using a Model UVM II detector Pharmacia,.Sweden modified by installing ball lenses for on-capillary detection, a Model DA-30

Ž .high voltage source SpectroVision, USA . For washing and conditioning the continuousbeds, a 2-ml stainless-steel loop filled with water or mobile phase was connected to the

Ž .capillary and a Model 110A single piston HPLC pump from Altex Texas, USA . TheŽ .pump was operated at constant pressure 120 bars .

2.3. Preparation of capillary columns

The fused silica capillaries were pretreated with methacryloxypropyl trimethoxysilanein order to attach covalently the methacryloyl groups to the capillary inner wall, as

w xdescribed previously 29 . The synthesis of polyrotaxane-based continuous beds is basedon in situ polymerisation of selected comonomers in the presence of macrocyclicmolecules. In Fig. 1, a general scheme of the supramolecular structure formed is shown.Different polyrotaxane-based acrylic continuous beds were synthesized in surface-activated fused silica capillaries of 50 and 100mm I.D. Sulfatedb-CD and carboxylatedb-CD were used as macrocyclic compounds for the preparation of negatively chargedpolyrotaxanes. 6-Aminob-CD and hydroxypropyltrimethylammoniumb-CD were em-ployed for the synthesis of positively charged polyrotaxanes. Polyrotaxane-based contin-

Žuous beds were prepared as follows, if not otherwise stated. The comonomers 0.125 g. Ž .of PDA, 0.075 g of MA and 0.1 g of IPA , ab-CD derivative 0.12 g and 0.064 g of

Ž .ammoniumsulfate were dissolved in 1 ml of 50 mM sodium phosphate buffer pH 7.0 .


Fig. 1. The polyrotaxane structure.

Ž . Ž .The total concentration of the comonomers in this mixture %T was 21.4% wrv andŽ . Ž .the cross-linker concentration %C was 41.7% wrw . Following degassing 40ml of a

Ž . Ž .10% vrv aqueous solution of TEMED and 40ml of a 10% wrv aqueous solution ofAPS were added. The polymerisation mixture was immediately sucked into the capillar-ies using a water jet pump. The polymerisation was left to proceed overnight. The nextday, the continuous beds were washed with distilled water at 120 bars by means of anHPLC pump.

2.4. Electrochromatographic characteristics

Ž .To characterise the chromatographic beds we used the 1 retention factor

ks t y t rt , 1Ž . Ž .R 0 0

Ž .where t is peak elution time,t marker elution time, 2 selectivityR 0

ask rk , 2Ž .2 1

Ž .where k and k are retention factors for the second and first peaks, respectively, 32 1

resolution

R s2 t y t r w qw , 3Ž . Ž . Ž .s R 2 R1 b1 b2

where t and t are elution times for the second and first peaks,w and w areR2 R1 b1 b2Ž .peak-widths at the baseline for first and second peaks, 4 efficiency

2Ns16 t rw . 4Ž . Ž .R b


The zeta potential of the beds was calculated using the Helmholtz–Smoluchowskiequation:

´´ z0Õ s E 5Ž .eo

h

where:Õ —velocity of electroosmotic flow,Õ sm E; m selectroosmotic mobility,eo eo eo eow 2 x w xm rV s ; Eselectric field strength, Vrcm ; ´ selectric permittivity of vacuum,0

y12 w x8.85=10 , A srV m ; ´sdielectric constant of the mobile phase;zszeta potentialw x w xof the stationary phase, mV ;hsviscosity of mobile phase, Pa s . The electroosmotic

velocity and the chromatographic efficiency were evaluated, using acetone as neutralmarker.

2.5. Self-assembly

Since the specific ligands are not covalently bound, but attached to the polymericmatrix via rotaxane formation, some data of the self-assembling of these nanostructuredformations were collected. For instance, the relative number of the different ionogenicmacrocyclic molecules in the polyrotaxanes was estimated through measurement of theelectroosmotic flow, the magnitude of which is determined by the density of theavailable immobilized charged groups.

2.6. Permeability of the beds

ŽThe hydrodynamic permeability of the beds was measured at constant pressure 60.bars for capillary columns of 100mm I.D., 26-cm long, using water as mobile phase.

The effluent was collected in Eppendorf vials for 10 to 30 min and weighed on ananalytical balance.

3. Results and discussion

3.1. Changes in the morphological structure of the continuous beds to adjust theefficiency and the back pressure

The separation efficiency is very dependent on the morphology of the continuousbeds since they consist of polymeric skeleton with flow channels in between the chains.The lowest efficiency was observed with beds, which had the highest hydrodynamic

Ž .permeability, i.e. lowest back pressure Fig. 2a and c , as expected. These results arew xconsistent with earlier data for continuous beds 30 .

It is known, that the phase separation process and the formation of the polymericcontinuous bed are highly dependent on the ionic strength in the comonomer solutionw x31 . An increase in ionic strength increases the hydrophobic interaction between theacrylic comonomers dissolved in the aqueous buffer—a finding that can be employed tochange the morphology of the continuous beds. A polymeric skeleton with broadchannels is formed, when the copolymerisation is carried out at high ionic strengths.Conversely, at low ionic strength more homogeneous continuous beds with narrowerchannels are created, which reduces eddy diffusion, increases the separation efficiencyand promotes the mass transfer in the mobile and stationary phases, i.e. the third term in


the van Deemter equation is reduced. However, excessively narrow channels maysignificantly increase the back pressure. A very high back pressure requires access to anHPLC pump for conditioning of the bed. Electroosmotic conditioning is another

Žalternative observe that an air bubble can be removed from the capillary only by a.hydrodynamic, not electroosmotic flow .

As shown in Fig. 2, the increase of the ammonium sulfate concentration from 16 upto 80 mgrml in the comonomer solution dramatically improved not only the permeabil-ity of the bed, but also resulted in a 50% higher electroosmotic flow. The tendenciesobserved here can be described by the abovementioned changes in the morphology ofthe continuous beds. It should be emphasized that the beds have a permeability so highthat they can be employed also for pressure-driven chromatography. In electrochro-

Ž . Ž . Ž . Ž .Fig. 2. Dependence of a permeability in terms of flow rate at 60 bars , b electroosmotic properties and cefficiency on the concentration of ammonium sulfate in the polymerisation mixture.


matography, the permeability should be low in order to get a high efficiency, but not solow that air bubbles cannot be removed by a hydrodynamic flow.

3.2. InÕestigation of self-assembly in polyrotaxanes

The influence of the hydrophobicity of monomers on polyrotaxane formation wasevaluated, assuming that template-directed synthesis is based mainly on the hydrophobicinteractions of the hydrophobic monomers and the hydrophobic cavity of the cyclodex-trin derivatives. To increase the hydrophobicity of the acrylic skeleton, the concentration

Ž .of IPA the nonpolar comonomer was increased in the comonomer mixture keeping theŽ .total concentration for IPA and MA the more polar comonomer constant.Ž .Interestingly, the permeability in terms of the flow rate at 60 bars and the

Želectroosmotic mobility have maxima at about the same concentration of IPA Fig. 3a.and b . One explanation, among others, may be that the porosity of the bed changes with

the concentration of the IPA, and if so, this change will affect both these parameters

Ž . Ž . Ž .Fig. 3. Dependence of a permeability, b electroosmotic properties and c efficiency on the concentration ofisopropylacrylamide in the polymerisation mixture.


Ž .similarly: the electroosmotic flow in a channel pore starts to decrease when thechannels become so small that the double layers overlap; the large channels responsiblefor the hydrodynamic flow are so wide that the electroosmotic flow in these channels isroughly constant and, therefore, one cannot expect so drastic changes in electroosmobil-ity as in permeability, which is in some agreement with the data presented in Fig. 3a andb.

Another plausible explanation could be that the incorporation of the chargedb-CDincreases with the IPA concentration up to a saturation value. The observation that the

Ž . Ž .efficiency does not decrease Fig. 3c as the permeability increases Fig. 3a , as is theŽ .case in Fig. 2c compare with Fig. 2a is difficult to explain.

No doubt, the results suggest that the self-assembling of polyrotaxanes is a processmore complex than is entrapment.

Ž . Ž . Ž .Fig. 4. Dependence of a permeability, b electroosmotic properties and c efficiency on the concentration ofsulfatedb-cyclodextrin in the polymerisation mixture.


Table 1Zeta potential of polyrotaxane-based continuous beds

Ž .Derivatizedb-CD z mV

Carboxylatedb-CD y15Sulfatedb-CD y11Hydroxypropyltrimethylammoniumb-CD q136-Aminob-CD q12

Ž .Mobile phase: 50% vrv aqueous MeOH buffered with 0.1% TEMED and 0.05% acetic acid, pH 6,Ž .conductivity adjusted to 165mSrcm. A minus plus sign indicates that the electroosmotic flow was directed

Ž .toward the cathode anode .

3.3. Generation of electroosmotic flow in charged polyrotaxanes

ŽPreviously, we have shown that electroosmotic flow can be generated and its.direction reversed in polyrotaxane-based continuous beds, using cationic or anionic

w xderivatives ofb-CD 32 . In this study, the electroosmotic properties of the polyrotax-anes were studied in more detail. For instance, various amounts of differentb-CDderivatives were added to the comonomer solution before the initiation of the copoly-merization. As expected, the electroosmotic mobility increased linearly when theconcentration of sulfatedb-CD increased from 30 to 180 mgrml. Low permeabilityŽ . Ž . ŽFig. 4a gives high efficiency Fig. 4c —in agreement with Fig. 2a and c but not with

.Fig. 3a and c .The electroosmotic zeta potentials of polyrotaxanes with different ionogenic groups

Ž . Ž .are listed in Table 1. The values of the zeta potential Table 1 calculated from Eq. 5Žare somewhat lower compared to that obtained for silica gel matrices 25 mV in aqueous

. w xsolution of 0.01 M sodium nitrate 33 . The electroosmotic flow showed a lineardependence on the electric field up to 500 Vrcm, at different pH and in both aqueous

Ž .and nonaqueous mobile phases Fig. 5 , indicating that the thermal zone deformation in

Fig. 5. Dependence of the electroosmotic velocity on the electric field strength applied. 1—Polyrotaxane-basedcontinuous bed with sulfated CD, mobile phase: 100% MeOH; 2—polyrotaxane-based stationary phase with6-amino b-CD, mobile phase: 100% MeOH; 3—polyrotaxane-based continuous bed with 6-aminob-CD,mobile phase: 50% MeOH buffered with acetic acid and TEMED, pH 4.78. Neutral marker: acetone.


the buffer used is negligible at least up to 500 Vrcm for 100 I.D. and 375 O.D. capillarycolumns. No pressurizing of the electrode vessels was required to avoid air bubbles inthe capillary.

3.4. Electrochromatographic separations

ŽReversed-phase chromatography is an important separation method 80% of allw x.chromatographic analysis are performed in this mode 34 . Fig. 7 is an example of such

an analysis of four ester homologues of benzoic acid. The separation efficiency wasaround 140.000 theoretical platesrm. The fact that the retention increased with thehydrophobicity of the homologues is an indication that the separation took place underreversed-phase conditions.

However, the beds described herein can be employed also for normal-phase elec-trochromatography in nonaqueous mobile phases. The electrochromatogram in Fig. 8

Žshows such a separation in contrast to reversed-phase chromatography, the elution order.in this mode of chromatography considers the order of non-polarity . These two expe-

riments show that polyrotaxane-based continuous beds can be characterized as am-

Fig. 6. Reversed-phase electrochromatography of alkyl benzoates. Capillary: polyrotaxane-based continuousbed with 6-aminob-CD, L s12.1 cm, Ls16.8 cm. Mobile phase: 50% of aqueous MeOH buffered witheff

Ž .0.05% TEMED and 0.1% HOAc pH 5.1 . Voltage: 5 kV. Solutes in order of elution: methyl, propyl, butyland isoamyl benzoate. Efficiency calculated for third peak:Ns140 000 platesrm.


phiphilic stationary phases, since they have higher affinity to less polar or more polarcompounds depending on the mobile phase polarity. Cellulose- and silica-based adsor-

w x w xbents 35,36 and acrylic continuous beds 37 have the same dual properties.The universality of the polyrotaxane-based continuous beds is reflected not only in

the fact that they can be used for both reversed- and normal-phase chromatography, butŽ .also in the finding that they can be used as chiral selectors Fig. 8 .

3.5. The separation mechanism

The experiments presented in Figs. 6 and 7 are of interest also from that view pointthat the beds have not been derivatized with ligands to achieve the desired separations.The matrix itself thus, warrants the separations, which very likely are due to so-calledAaromatic adsorptionB, a vague term because many interactions are involved, includingp–p interactions.

Cyclodextrins and their derivatives are widely used as chiral selectors in capillaryelectrophoresis, due to their high water solubility, UV transparency, commercial avail-

w xability and low cost 38–41 . The cavity of the cyclodextrin molecule is considered to bemain responsible for the enantio separations. Accordingly, it is somewhat unexpected

Fig. 7. Normal-phase electrochromatography of alkylbenzoates. Capillary: polyrotaxane-based continuous bedŽwith 6-amino b-CD, L s12.1 cm, Ls16.8 cm. Mobile phase: MeOH:Hexane:PropylOH 50:42.5:7.5eff

.vrv% , 0.05% TEMED, 0.05% HOAc. Voltage: 5 kV. Solutes in order of elution: isoamyl and methylŽ .benzoate the analytes were dissolved in methanol; first peak corresponds to methanol .


that the beds described herein have enantioselectivity, since the cavity is blocked by theŽ .axial unit in the rotaxane structure and thus not available for the enantiomers Fig. 8 .

Therefore, for some enantiomers, the outside of the cyclodextrin molecule and the rim ofthe cavity may be of more importance than generally claimed.

3.6. Reproducibility of some chromatographic characteristics and stability of the beds

Reproducibility data on run-to-run and day-to-day for retention time, selectivity,efficiency, resolution, and retention factor are presented in Table 2. For the evaluation ofrun-to-run reproducibility, eight consecutive electrochromatographic experiments wereperformed. In order to calculate day-to-day reproducibility, averaged data were collectedfor 3 days.

Run-to-run and day-to-day differences of retention time, retention factor and selectiv-Ž .ity were very similar and relative standard deviations RSD for these parameters did not

exceed 0.7%. Day-to-day variations were, as expected, somewhat higher than run-to-run

Fig. 8. Separation of ibuprofen enantiomers. Capillary: polyrotaxane-based continuous bed with neutralb-CDand vinylsulfonic acid,L s8.5 cm, Ls12.4 cm. Mobile phase: 50% aqueous MeOH buffered with 0.05%eff

HOAc and 0.05% TEMED, pH 5.8; conductivity adjusted to 165mSrcm. Voltage: 5 kV. Current: 3mA.Sample: racemic ibuprofen.


Table 2Reproducibility of some electrochromatographic properties of the polyrotaxane-based continuous beds

Parameter RSD%

Ž . Ž .Run-to-run ns8 Day-to-day ns3

Ž .Retention time acetone 0.28 0.69Ž .Retention factor acetone 0.23 0.44

Ž .Selectivity acetoneracetophenone 0.20 0.31Ž .Resolution acetoneracetophenone 4.4 6.9Ž .Efficiency acetone 7.6 11.2

Conditions: capillary columns,L s10.1 cm,Ls14.5 cm, 100mm I.D.; stationary phase: polyrotaxane-basedDŽ .continuous bed with 6-aminob-CD; mobile phase: 50% vrv aqueous MeOH buffered with 0.05% TEMED

Ž .and 0.1% acetic acid, pH 5.1, conductivity of which was adjusted with 50% vrv aqueous MeOH to 165mSrcm; UV detection at 254 nm; voltage 5 kV; sample injected: acetone and acetophenone, diffusion–extrac-tion-based injection performed by dipping the inlet of capillary column into the sample solution for 10 s.

Žvariations. Remarkable variation of efficiency run-to-run RSDs7.6%, day-to-day.RSDs11.2% can be explained by the errors in manual evaluation of peak width

Žsimilar reproducibility values of efficiency were obtained for other non-rotaxane-based.capillary columns when the same microchromatographic system was used . The some-

what better reproducibility in resolution can be explained by the fact that the resolutionis proportional to the square root of the efficiency.

w xSvec et al. 42 investigated the reproducibility of the electrochromatographic proper-ties of methacrylate-based continuous beds. The data were collected for 3 days foracetone and three alkylphenones, injected 7–10 times a day. They obtained valuescomparable to ours for day-to-day RSDs for retention time, retention factor, selectivity

Ž .and efficiency 1.19%, 0.3%, 0.1% and 4.3%, respectively .The stability at extreme pH values of the polyrotaxane-based continuous beds

containing sulfatedb-CD was tested. No changes of the electroosmotic and chromato-graphic properties were observed within the pH range 4–9. A drop of the electroosmotic

Ž .flow more than four times was observed in alkaline media at pH 12.3 after 20 h,probably caused by hydrolysis of the amide bonds in the acrylic continuous bed, which

w xstarts at pH 12, when PDA is used as cross-linking agent 43 .

3.7. The adÕantage of the polyrotaxane-based continuous beds

Cyclodextrins with different ligands can be employed in a simple one-step procedureto prepare the desired polyrotaxane-based bed, as described herein. Many of the usefulcyclodextrin derivatives are commercially available. However, it is very simple to

Ž .derivatize cyclodextrins with any compound ligand containing an epoxide group andthen use them to prepare the desired polyrotaxanes. Alternatively, one can synthesize an

Ž .allyl acrylamide, acrylate component of a cyclodextrin containing the ligand andincorporate it as a comonomer into the continuous bed in the absence ofb-cyclodextrinŽ .in this case no polyrotaxane is formed . A combination of these two alternatives willlikely give an extremely high ligand density.


Different ligands can easily be incorporated into polyrotaxanes. Beds for mixed-modechromatography with known variable densities of the different ligands can, therefore, beprepared without difficulty. One should also recall that non-water-soluble compounds,for instance those containing C18 groups, cannot easily be incorporated into the standardcontinuous beds. This problem does not exist if these compounds are first coupled tohighly water-soluble cyclodextrins, which then are used for the synthesis of a polyrotax-ane-based beds.

Although not primarily designed to be enantioselective, the beds described hereinappeared to be useful for the separation of some enantiomers, which opens up thepossibility to study to which extent the outer surface of the cyclodextrin molecule and itsrim are responsible for the enantioselectivity.

Another advantage of the polyrotaxane derivatized continuous beds is that no ligandsare required for the separations of aromatic compounds, since all polymeric matriceshave this property.

It is known that the presence of cyclodextrins in the separation medium facilitates theseparation of alkyl homologues. It is, however an open question whether there is spaceenough for alkylchains to enter the cavity of the cyclodextrins in the polyrotaxane

Ž .structure. If so, the separations presented in Figs. 6 and 7 can partially be explained bysuch a mechanism.

4. Simplified description of the method and its potential applications

A method is described for polyrotaxane synthesis in order to induce desired physico-chemical properties, such as surface charge and adsorptive properties to the neutralpolymeric continuous beds to make them suitable for chromatographicrelectrochro-matographic separations. The polyrotaxane is formed by a simple one-step free-radicalpolymerisation of acrylic comonomers and cross-linker in the presence of a non-activatedmacrocyclic compound. Rotaxanes with different properties can be synthesized, using ageneral polymerisation procedure, by changing the chemical nature orrand the concen-tration, the degree of derivatization or substitution of the macrocyclic additive which ismechanically interlocked in the polyrotaxane structure.

The scope of the applicability of the approach described herein could be muchbroader than just coupling of specific ligands to different matrices for chromatography.The method is of general interest in material science, to design new materials withforeseen specific surface properties, for instance for fine tuning of these properties orgentle coupling of specific ligands to a polymeric matrix. Different format materialssuch as bulky porous monoliths, thin layers and plane surfaces can be formed by thistechnique.

Acknowledgements

Authors are grateful to Prof. S. Hjerten for the valuable discussions of this matter.´Ž .This study was financially supported by the Volkswagen Foundation Germany .


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