Evanescent wave-initiated photopolymerisation as a new way to create monolithic open-tubular capillary columns: use as enzymatic microreactor for on-line protein digestion

Evanescent wave-initiated photopolymerisation as a new way to create monolithic open-tubular capillary columns: use as enzymatic microreactor for on-line protein digestion
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  Evanescent wave-initiated photopolymerisation as a new way to createmonolithic open-tubular capillary columns: use as enzymatic microreactor foron-line protein digestion Silvija Abele, † ab Petr Smejkal, † ac  Oksana Yavorska, a Franti  sek Foret ‡ * c  and Mirek Macka ‡ * a Received 5th October 2009, Accepted 18th January 2010First published as an Advance Article on the web 25th January 2010 DOI: 10.1039/b920789aEvanescent wave-initiated photopolymerisation in an optically waveguiding PTFE-coated fused silica capillary using light-emittingdiode as a light source, is established here as a way to fabricatemonolithic porous layer open-tubular capillary columns with apotentialincapillaryseparationmethods;applicationoftheobtainedopen-tubular columns as enzymatic microreactors for on-lineprotein digestion is demonstrated. Evanescent wave (EW)-initiated photopolymerisation inside fusedsilica capillaries functioning as light-waveguides and illuminated atoneendaxiallywithasinglelight-emittingdiode(LED)isestablishedhereasawaytoattainmonolithicporouslayeropen-tubular(PLOT)capillarycolumnsforcapillaryreactors,nano-liquidchromatography(nano-LC), capillary electrochromatography (CEC) and relatedseparation methods.Monolithic materials have been acknowledged as a major tech-nological breakthrough in column technology since the invention of chromatography by Tsvet more than a century ago. 1 Monolithicmaterials for chromatography as an alternative separation media toclassical particulate materials for packed columns were introduced inthe 1990s: organic monoliths in 1992 by Svec and Fr  echet 2 andmonolithic silica columns in 1996 by Tanaka and co-workers. 3 Sincethen monolithic supports have been used as stationary phases inchromatography 4,5 and for immobilisation of enzymes. 6–8 Organicpolymers are popular supports for enzyme immobilisation due to thewide variety of available chemistries and formats. 6 Open-tubular capillary (OTC) columns used in chromatography,in contrast to columns fully filled with a packing material ora monolith, are open tubes with the stationary phase on the innercapillarywalls.Porouslayeropen-tubular(PLOT)capillarycolumns,first suggested by Golay in the late 1950s, 9,10 represent a subgroup of OTC columns, in which the inner capillary wall is coated witha porous material.Apart from separations, monolithic OTC and PLOT capillarycolumns could be used as a solid support for on-line enzymaticreactors, which offer advantages over the enzymatic digestion of proteins in solution in terms of automated on-line operation andfaster digestion. 6,8 The first OTC microreactors in plain fused silicacapillaries were reported in the 1990s. 11,12 While the strength of OTCmicroreactors is in their simplicity and the ability to flush relativelyeasily the open capillary, they suffer from a low available surfacecomparedtotheexisting‘full’(acrossthecapillarylumen)monolithicreactors, where the enzyme is covalently bound to the monolithicsurface. 13–15 Monolithic porous layer OTC reactors have a potentialto combine the advantages of both types offering on-line fast diges-tion on an increased surface of a monolithic layer-coated OTCmicroreactor. In this work, demonstration of the EW photo-polymerised OTC columns as enzymatic microreactors is selected asthe initial application of these columns.Photo-induced polymerisation (photopolymerisation) was intro-duced already at the early stages of the organic monolith’s develop-ment as a better alternative of their synthesis in comparison tothermally-induced polymerisation. 16 Polymerisation initiated byultraviolet (UV) light irradiating perpendicularly the capillary (ormicrofluidic chip)mould filled with monomer mixture using differentUV light sources has been studied extensively. 17–19 Photopolymerisedmonoliths of various chemistries with applications in ion exchangeandreversed-phaseLC 2 andCEC 17,20 havebeenreportedoverthelastdecade.Photopolymerisations of monoliths using a single UV or visibleLED as the light source, as a simple alternative to classical lightsources, has been recently introduced by the authors and demon-strated in both fused silica capillaries and microfluidic chips. 21,22 Incomparison to the various other light sources, which are mostlybulky, heat-producing and expensive, LEDs offer several advantagesincludingsmallsize,compatibilitywithminiaturisation,robustnessof thesolid-statelighttechnology,lowcosts,longlife time,andlowheatgeneration.Different techniques of coating have been used for the preparationofOTCand PLOTcolumns,suchas insitu emulsion polymerisation,dynamiccoating,staticcoatingandothers. 9 Formationofathinlayerof polymer inside the capillary using UV irradiation perpendicular tothe capillary axis while rotating the capillary has been described bySvec and co-workers. 23 EW-initiated photopolymerisation is estab-lished here as a new and simple way to attain monolithic PLOTcapillary columns.EWphotopolymerisationisinducedbytheevanescentfieldcreatedat the inner wall of a transparent polytetrafluoroethylene-coated(outside coating) fused silica (PTFE-FS) capillary illuminated axiallyandactingasalight-waveguide.Sofar,photopolymerisedmonolithicOTC columns could be only prepared in the classical transverseilluminationmodebyrotatingthecapillary, 23 whichisonlyapplicable a Dublin City University, Irish Separation Science Cluster and School of Chemical Sciences, Glasnevin, Dublin 9, Ireland. E-mail:; Fax: +353 700 5503; Tel: +353 700 5611 b University of Latvia, Faculty of Chemistry, 19 Boulevard Rainis, LV-1586 Riga, Latvia. E-mail: c Institute of Analytical Chemistry ASCR, v.v.i., Veverˇ´  ı  97, 60200 Brno,Czech Republic. E-mail: † Authors contributed equally to this work.‡ Both authors claim an equal intellectual contribution to the work. This journal is  ª  The Royal Society of Chemistry 2010  Analyst  , 2010,  135 , 477–481 | 477 COMMUNICATION | Analyst  for relatively short pieces of capillary that can be rotated, with thehere introduced EW photopolymerisation as a simple convenientalternative. Photopolymerisation using evanescence field without thehere presented wave guiding has been previously suggested formicrofabrication purposes to obtain submicrometre-scale polymercoatings on mostly planar surfaces, 24,25 but to the authors’ bestknowledgepolymersynthesisincapillarieshasnotbeenconductedsofar by EW photopolymerisation.The here introduced EW-initiated photopolymerisation is experi-mentally a straightforward procedure (Fig. 1).After the desired section of a PTFE-FS capillary is filled with thepolymerisationmixture,UVlightfromanLEDenteringthecapillaryat one end and propagating axially within the fused silica body of thecapillary initiates the photopolymerisation at the inner capillary wall.The light undergoes total reflection within the fused silica capillarymaterial due to the differences in refractive indices of the capillarymaterial ( n FS  ¼  1.47 26,27 ), PTFE as the outside capillary coating( n PTFE  ¼  1.35 28 ) and the polymerisation mixture (typically solutionsin aliphatic alcohols,  n z 1.45 calculated using the  n  values for eachcomponent of a typical photopolymerisation solution 29 ). Since theevanescent field penetrates the solution inside the capillary only toa depth comparable to the wavelength of the irradiation, the pho-topolymerisation process starts at the inner surface of the capillary.The evanescent field is responsible for decomposition of the photo-initiator into radicals which initiate the polymerisation. 25 The createdradicals gradually move due to diffusion into the bulk solutioninitiatingthepolymerisationalsoatlargerdistancesthanthedepthof the evanescent field and the polymerisation will propagate towardsthe capillary centre. Since diffusion is relatively slow in liquids, thethickness of the fabricated polymer layer can be controlled by theexperimental parameters – light intensity, irradiation time andmonomer concentration. With longer exposure time the polymermonolith will be ultimately formed across the whole capillary lumen.In this work, polymethacrylate OTC monoliths were fabricatedby photopolymerisation of methacrylic monomers conducted inPTFE-FS capillaries using a single low-cost 365 nm UV-LED asa light source. 4,4 0 -Bis( N  , N  -dimethylamino)benzophenone, alsocalled Michler’s ketone (MK), was used as the photo-initiator as itsabsorbance spectrum matches well the emission spectrum of the365 nm LED. 21 The validity of the EW photopolymerisation in a capillary actingas a light-waveguide is demonstrated by using a bent capillary for thesynthesis of monoliths as shown in Fig. 2.A desired portion of the capillary length (11 cm from the total of 25 cm) was filled with the polymerisation mixture (capillary forcewas used for simplicity; alternatively, a nano-flow pump could beused). Photopolymerisation was then induced by the UV-LED fromthe opposite (empty) end of the capillary. By using a bent capillary, itisobviousthatnodirectlightpenetrationthroughthecapillarylumenintothepolymerisationmixtureispossible,whiletheUVlightiswaveguidedthroughthefusedsilicacapillarybodytothesectionwherethemonomer mixture is filled inside the capillary. Here the polymerisa-tion is initiated by evanescent waves at the inner capillary wallsresulting in a monolithic open-tubular capillary. In this experimentwith the setup shown in Fig. 2, an 11 cm long open-tubular monolithwas obtained.The key role of the PTFE coating for the light-waveguidingproperties of the PTFE-coated fused silica due to its refractive indexbeing lower than that of fused silica has been proven by a controlexperiment (results not shown) in a polyimide-coated fused silicacapillaryofidenticaldimensions.Nowaveguidingcould beobservedin polyimide-coated capillary as the refractive index of polyimide( n PI  ¼  1.70 30 ) is higher than that of fused silica, and therefore nooccurrence of any polymer layer on the inside of the capillary wasobserved even after a prolonged exposure.Having proven the principle ofEWphotopolymerisation, the bulkof the monolith synthesis in this work was conducted using straight(unbent) capillaries (25, 50 or 100  m m i.d.) in a horizontal position.Although axial illumination along the capillary length is used tophoto-initiate the polymerisation, it is possible to gain a spatialcontrolofthecreatedmonolithpositioninthecapillary.Thepositionofoneendofthefabricatedmonolithcanbecontrolledbythelevelof monomer solution in the capillary during its filling, simply by leavingthe remainder of the capillary empty (several centimetres long). Theposition of the monolith at the other end can be controlled by fillingthe capillary in the solvent after a desired capillary length has beenfilled with the polymerisation mixture. The solvent used in thepolymerisation mixture can be filled into the capillary following the Fig. 1  Photograph of the setup (top) and a scheme illustrating theprinciple of EW photopolymerisation at the inner surface of a fused silicacapillary using total reflection light-waveguiding and evanescent field(bottom). Fig. 2  Experimental setup for EW photopolymerisation in a bent fusedsilica capillary. Inset: SEM image (left) and optical photograph (right)obtained after photopolymerisation show the open-tubular polymermonolith at the inner capillary wall. 478 |  Analyst  , 2010,  135 , 477–481 This journal is  ª  The Royal Society of Chemistry 2010  polymerisationmixture(acoloureddyesolutionofthesolventcanbeused to better visualise the length of filling in the capillary). In thisway a monolith of desired length with sharp edges on both sides canbeobtainedinadesiredsectionofthecapillaryasillustratedinFig.3.Using glycidyl methacrylate (GMA) and ethylene dimethacrylate(EDMA) as monomers, 21 monolithic open-tubular capillary columnswith a polymer layer thickness varying between 2 and 25  m m werecreated as shown in Fig. 3. These are comparable values to those of most commercial PLOT columns, with their thickness ranging from5 to 50  m m. 9 It is obvious that the thickness of the monolithic layerwill be a function of the irradiation time, the light intensity in thecapillary (LED power and light coupling efficiency) and monomerconcentration, 23 and the exact role of these parameters is the subjectof ongoing research.The attenuation of the light radiation along an ideal waveguide of a length of tens or even hundreds of centimetres is negligible.However, since the capillary is not a perfect waveguide and bothoptical defects (internal surface roughness) and light absorptionthrough the evanescent field may cause a gradual attenuation of theradiation, the polymer properties may change along its length. Thiseffect, which may be important especially in capillaries of consider-able length (tens of centimetres to several metres) that would be usedinseparationmethods(CEC,LC,GC),willbeinvestigatedlater.Thefocus of this work was only on relatively short capillaries used asenzymaticreactors.Inthesecapillaries,althoughsomedecreaseinthepolymer layer thickness along the capillary length was observed, itcan be deemed unlikely to influence substantially the performanceof the OTC reactors as it will be primarily a relatively thin top layerof the monolith in contact with the inner solution that will beresponsible for most of the digestion.Already Golay suggested that capillary columns should havea uniform porous layer of support material at the walls of a tubularcolumn instead of a smooth inner wall. 31 PLOT columns studied byHorvath and co-workers and used in CEC separations of proteinsand peptides were obtained by polymerising a porous styrenicsupport layer at the surface of the inner capillary walls. 10 In general,porogenic solvents (typically aliphatic alcohols) are used to achievethe necessary porosity of the monolithic polymer. A mixture of decanol and cyclohexanol has been used as porogenic solvent for thepresent study. 21 This study indicates that an increased surface of theOTC polymer layer can be obtained with EW photopolymerisationas the presence of porogen in the polymerisation mixture is wellknown to necessitate formation of a porous monolith 2 (Fig. 3(A2,B1)).Ideally,theporosityofamonolithiccolumnisequaltotheamount(volume percentage) of porogen in the polymerisation mixture. Inreality, the porosity is usually larger than the porogen volume frac-tion because of an incomplete polymerisation of the monomers. 32 Inthe case of PLOT columns obtained by EW-initiated photo-polymerisation,theporosityoftheresultingpolymermayinprinciplealso be a function of the polymer layer thickness, which may benecessary to investigate for chromatographic application but isunlikely to play a significant role in the presented application as areactor.An increased surface of the polymer layer can bring principalbenefits for many applications including enzymatic microreactors,OTC columns for CEC, nano-LC and others. This presents a signif-icant advantage over the non-porous layers prepared previously bysurfacecoatings. 13,14 Asenzymaticmicroreactorscanbenefitfromtheincreased surface of the EW photopolymerised monolithic PLOTcapillarycolumnanapplicationinthisareawasinvestigated.Enzymeimmobilisation onto the continuous monolithic surfaces 6,13,14 or ontothe surface of narrow-bore fused silica capillary (10  m m i.d.) 33 hasbeen reported.In this study, pepsin A from porcine stomach was immobilisedon the monolithic surface (monolith layer of approx. 7  m m) of poly-(GMA- co -EDMA) PLOT columns fabricated by EW-initiatedphotopolymerisation and these obtained enzymatic capillary reactorswere used for on-line digestion of proteins and subsequent massspectrometry (MS) analysis of peptides. The MS spectrum of horsemyoglobin digested using the obtained PLOT enzymatic micro-reactor is shown in Fig. 4 together with the spectrum of horsemyoglobindigestedoff-lineandtheMSspectrumofmyoglobinpriorto its digestion.AsseeninFig.4,completedigestionofmyoglobincanbeobtainedusing only 6.1 cm long enzymatic PLOT reactor comparable to thatperformedoff-line.Thedecreaseddigestion/analysistimeisoneofthebenefits as only a few minutes were necessary for the digestion of myoglobin in the PLOT monolithic enzymatic reactor.The increaseddigestion speed clearly demonstrates the advantage of the porouspolymer layer for the enzyme immobilisation. The selection of pepsinfor this demonstration was based on the simplicity of the experi-mental MS setup. The low pH required for the pepsin activitycorresponds also to operational conditions of the electrospray ioni-zation (ESI) in the positive mode. Thus, the sample solution pumpedthrough the PLOT microreactor could be directly electrosprayedwithout the need for composition adjustment prior to the ESI stage.Complete protein analysis with identification of all peptides was notthe aim of this MS study focused on the proof-of-principle of the useof PLOT columns as enzymatic microreactors. Although the pepsinis not a very specific enzyme and its cleavage sites may change Fig. 3  SEM images illustrating the EW photopolymerised open-tubularmonolith in 100  m m i.d. PTFE-FS capillary with a monolithic layerthickness of approx. 25  m m (A1, A2) and 5  m m (B1, B2) and an opticalmicroscope image (A3) confirming a sharp edge of the monolith in thecapillary. Conditions: (A1, A2): 15 cm of 100  m m i.d. capillary filled withpolymerisation mixture with 5 cm capillary length from the LEDremaining empty, 30% GMA/EDMA, photo-initiator 1% MK, irradia-tion time 4 h, 10 cm of open-tubular monolith obtained; (A3): opticalimage of monolith in the capillary obtained with a Synchronized VideoMicroscope (LabSmith SVM340); (B): 8 cm of 100  m m i.d. capillary fullyfilled with polymerisation mixture, 30% GMA/EDMA, photo-initiator1% MK irradiation time 5 min, 1.2 cm of open-tubular monolithobtained. This journal is  ª  The Royal Society of Chemistry 2010  Analyst  , 2010,  135 , 477–481 | 479  depending on the experimental conditions, 33 the corresponding MSspectra in Fig. 4 reveal that most of the detected peptic fragments areidentical in the free solution and PLOT microreactor digests.In conclusion, EW photopolymerisation has been described hereas a new technique to fabricate polymer PLOT capillary columns.Such monolithic open-tubular capillary columns with an increasedsurface and porosity could find applications in OTC chromato-graphic separations and various flow-through reactors. MethacrylicPLOT columns obtained by EW-initiated photopolymerisation canbe successfully used as enzymatic reactors for on-line digestion of proteins as demonstrated here. Materials . 3-(Trimethoxysilyl)propyl methacrylate (TMSPM),glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA),cyclohexanol, decanol and 4,4 0 -bis( N  , N  -dimethylamino)benzophe-none(alsocalledMichler’sketone,MK),pepsinA(EC3.4.23.1)fromporcine stomach mucosa, myoglobin (horse), sodium cyanoboro-hydride, sodium periodate, hydrochloric acid, methanol, formic acid,sodium phosphate dihydrate and sodium phosphate monohydratewere purchased from Sigma Aldrich (Ireland). Polymicro Technol-ogies transparent polytetrafluoroethylene (PTFE)-coated fused silicacapillaries (25, 50, 100  m m i.d.) were purchased from CompositeMetal Services Ltd (West Yorkshire, UK). UV-LEDs (365 nmemission maximum, 1.4 mW optical power) were purchased fromRoithner Lasertechnik GmbH (Vienna, Austria). Pretreatment of fused silica capillaries . Silanisation of inner capil-lary walls with TMSPM in order to provide a covalent bondattachment of the formed monolith to the walls was conducted usingthe method introduced by the group of Svec 34 and is described indetail elsewhere. 21 EW-initiated photopolymerisation to create monolithic open-tubularcapillary columns . 15–25 cm-long previously silanised PTFE-coatedcapillary (25, 50 or 100  m m i.d.) was filled manually (by capillaryforce) with the polymerisation mixture, leaving 3–5 cm of capillarynot filled. For small i.d. capillaries (25  m m) the solution level can becontrolled using a microscope. The polymerisation mixture consistedof 60  m l of GMA, 60  m l of EDMA, 230  m l of cyclohexanol, 450  m l of decanol, 1.26 mg of MK; 15% GMA/EDMA (v/v) and 1% MK(w/w) relative to the monomers; in some experiments 30% of themonomer (v/v) was used. The capillary was then positionedhorizontally and a 365 nm light-emitting diode (LED) was placedhorizontally facing the empty end of the capillary. The photo-polymerisationwasstartedbytheLEDshiningontothecapillaryendat 20 mA for a specified time (5 min to several hours). After poly-merisation the residual monomer and solvents were flushed out fromthe capillary with methanol (1  m l/min) using an HPLC pump(Shimadzu LC-10AD) and the OTC monolith was dried usinga nitrogen stream (5 min). The thickness of the formed monolithiclayer around the inner capillary walls can be controlled by thepolymerisation time, LED power and monomer concentration. Visualisation . The thickness of the monolithic layer was estimatedbyScanningElectronMicroscopy(SEM,HitachiS3400).Allsampleswere gold-sputtered prior to imaging in order to minimize chargingand improve the image quality (contrast). A Synchronized VideoMicroscopeSVM340(LabSmith,CA,US)wasusedtoobtainopticalimagesofcapillariesafterpolymerisation to evaluatethesharpnessof the monolith edges in the capillaries. Pepsin A immobilisation on the surface of PLOT columns . Immo-bilisation of pepsin A was adopted and little modified from theprocedure described by Krˇenkov  a  et al  . 33 First, hydrolysis of theepoxygroupsatthemonolithicsurfacewasdonebyflushingof0.5MHClat80nl/min for18h.Grafting ofpepsinAwasperformed in thefivestepswhereaHamiltonsyringepump(KDScientific,MA,USA)was used for flushing: (1) flushing with 0.1 M sodium periodate for1 h, (2) rinsing with water for 5 min and (3) flushing with 50 mMacetate buffer (pH 4.50) for 5 min were all done at a flow rate of 300 nl/min, (4) flushing with a solution of pepsin A (dissolved in50 mM acetate buffer pH 4.50 containing 3 mg/ml of sodium cya-noborohydride) for 4 h was done at a flow rate of 30 nl/min, and (5)finally, the PLOT monolith was flushed at a flow rate of 300 nl/minfirst with 50 mM acetate buffer pH 4.50 (for 10 min) and then with1% formic acid (for 5 min). The obtained enzymatic reactor wasstored in the fridge at 6   C until it was used in MS analysis. Mass spectrometry analysis . A Mariner TOF mass spectrometer(Applied Biosystems, MA, USA) was used in all MS experiments.The measurements were carried out in a positive ion mode witha scan range of 415–1500  m / z . Nano-spray needles were prepared bysharpening and polishing of 8 cm-long polyimide-coated silicacapillary (10  m m i.d., 360  m m o.d.) with fibre optic polishing paper.The solution of off-line digested myoglobin was pumped byaHamiltonsyringepump(KDScientific,MA,USA)ataflowrateof 100 nl/min. The solution of myoglobin was pumped by an ABI 1400Solvent Delivery System (Perkin Elmer, MA, USA) and actual flowrates were measured with a Nano-Flowmeter N-565 (UpchurchScientifics, UT, USA).Off-line digestion of horse myoglobin in solution (0.05 mg/ml of horse myoglobin and 0.001 mg/ml of pepsin A diluted in 1% formicacid) was performed for 20 h at 37   C in an Eppendorf vial. On-linedigestion of horse myoglobin (0.05 mg/ml diluted in 1% formic acid)with subsequent MS detection of peptides was performed using a6.1 cm long monolithic PLOT column (obtained in PTFE-coatedfused silica capillary 100  m m i.d., 375  m m o.d.) with pepsin Aimmobilised on the monolithic surface. The linear flow rate throughthe PLOT monolithic column was 0.2 mm/s. Fig. 4  A schematic showing conditions for enzymatic digestion of horsemyoglobin in solution and in the poly(GMA- co -EDMA) PLOT mono-lithic column obtained by EW-initiated photopolymerisation and thecorresponding MS spectra: (a) spectrum of non-digested horsemyoglobin (0.05 mg/ml in 1% formic acid), (b) spectrum of myoglobindigested on-line by pepsin A immobilised on poly(GMA- co -EDMA)PLOT monolithic column (6.1 cm long column, made in 100  m m i.d.,375  m m o.d. PTFE-coated fused silica capillary), (c) spectrum of off-linedigested myoglobin (0.05 mg/ml horse myoglobin and 0.001 mg/ml of pepsin A diluted in 1% formic acid, digestion performed for 20 h at37   C). 480 |  Analyst  , 2010,  135 , 477–481 This journal is  ª  The Royal Society of Chemistry 2010  Measured spectra of myoglobin, off-line and on-line digestedmyoglobin were compared for changes in the digestion specificity.The authors gratefully acknowledge the financial support of theMarie Curie Excellence fellowship and grant MEXT-CT-2004-014361 (M.M., S.A.),the Enterprise Ireland Proofof ConceptgrantPC/2008/339 (S. A., O. Y., M. M.), KAN400310651, LC06023 andAV0Z40310501 of the Czech Republic (P. S., F. F.). Notes and references 1 M.Al-Bokari,D. CherrakandG. Guiochon,  J.Chromatogr.,A ,2002, 975 , 275.2 F. Svec and J. M. J. Fr  echet,  Anal. Chem. , 1992,  64 , 820.3 H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka and N. Tanaka, Anal. Chem. , 1996,  68 , 3498.4 F. Svec,  J. Sep. 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