1 Review 1 2 KenHosoya2 Polymer ...download.xuebalib.com/5n0fwRAeZqld.pdf · The polymer-based...

J. Sep. Sci. 2009, 32, 341 – 358 H. Aoki et al. 341

Hiroshi Aoki1

Nobuo Tanaka1

Takuya Kubo2

Ken Hosoya2

1Division of Applied Science forFunctionality, Graduate Schoolof Science & Technology, KyotoInstitute of Technology, Kyoto,Japan

2Graduate School ofEnvironmental Studies, TohokuUniversity, Sendai, Japan

Review

Polymer-based monolithic columns in capillaryformat tailored by using controlled in situpolymerization

This review introduces to the readers our new perspectives of polymer-based mono-lithic column with a high performance for small solutes such as drug candidates,illustrating the fabrication of LC columns in capillary. First, we briefly reviewed thestatus quo of polymer-based monolithic columns, comparing with silica monoliths.The miniaturization of LC system with higher throughput (shorter analytical time)was stressed conceptually, along with a fine permeable bicontinuous monolithicstructure with submicron domain size (skeletal thickness + pore size) for higher per-formance. Second, from these perspectives, our column preparation was described,while our specially designed porogenic solvents were introduced as a controller ofthe monolithic morphology via reaction-induced phase separation. Specifically,monolithic columns were exemplified in two polymer formats, that is, one mono-lith prepared by free radical polymerization of glycerin 1,3-dimethacrylate, GDMA,and the other prepared by stepwise polymerization of newly introduced multifunc-tional epoxy and diamino monomers. Both monolithic columns in capillary formatdemonstrated a fine bicontinuous structure, affording a good compatibility of theefficiency (H) and permeability (D). Especially, the epoxy-based column showed anexcellent separation impedance, E (=H2/D). Our micro-HPLC data were discussedalong with a prototyped wired chip device.

Keywords: Morphology / Micro-HPLC / Polymer-based monolith / Phase separation / Porogen /

Received: September 8, 2008; revised: November 12, 2008; accepted: November 18, 2008

DOI 10.1002/jssc.200800508

1 Introduction

This review article centers upon the preparation of poly-mer-based monolith in capillary format for microscaleseparation such as micro-HPLC. The polymer-basedmonolithic columns are differentiated from the silica-based monolithic and packed columns in terms of wideravailability of monomers and solvents for preparation.The monomers commonly applied are hydrophobic sys-tems such as methacrylate, styrene/divinylbenzene, andhydrophilic acryl amides. The column preparation is per-formed by a free radical polymerization, thermally initi-ated or photoinitiated, while the separation media in

particulate format are polymerized in an aqueous phaseby suspension polymerization [1]. Polymer-based mono-liths are advantageous over the silica monoliths due tothe chemical stability at higher alkali conditions and thealleviation of end capping of residual silanols (silylation,pKa of the residual silanols is around 4 [2]).

The polymer-based monolithic columns were chroma-tographically investigated with compressed aqueouspolyamide beds (gel chromatography) in the late 1980sby Hjert�n et al. [3, 4]. The polymer monolithic columnshave been investigated for HPLC and CEC applicationssince the early 1990s [5–7]. The concept of polymer-basedmonolith was claimed in the patent by Frechet and Svec

Correspondence: Dr. Hiroshi Aoki, Division of Applied Sciencefor Functionality, Graduate School of Science & Technology,Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto606-8585, JapanE-mail: [email protected]: +(81)-75-601-6930

Abbreviations: ADVN, 2,29-azobis(2,4-dimethylvaleronitrile);BACM, 4-[(4-aminocyclohexyl)methyl]cyclohexylamine; AIBN,2,29-azobisisobutyronitrile; ATRP, atom transfer radical poly-

i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

merization; carboxy-TEMPO, 4-carboxy-TEMPO; EDMA, ethylenedimethacrylate; DPB, deuterated polybutadiene; GDMA, glycer-in 1,3-dimethacrylate; HILIC, hydrophilic interaction chroma-tography; HMPP, 2-hydroxy-2-methyhlpropiophenone; LSCM, la-ser scanning confocal microscopy; MW, molecular weight; PB,polybutadiene; PS, polystyrene; RAFT, reversible addition-frag-mentation chain transfer polymerization; SEC, size exclusionchromatography; SFRP, stable free radical polymerization; TEM-PO, 2,2,6,6-tetramethylpiperidinyl-1-oxy; TEPIC, tris(2,3-epoxy-propyl)isocyanurate

342 H. Aoki et al. J. Sep. Sci. 2009, 32, 341 – 358

[8] as a chromatographic column with a specified bimo-dal pore (double pore) size distribution. The columns pre-pared in situ were exemplified by the free radical poly-merization of vinyl or methacrylate monomers in a poro-genic solvent selected from alcohols, aromatic hydrocar-bons, ethers, or their mixtures. The bimodal pore size dis-tribution was specified in the said patent with smallersize pores ranging less than 200 nm and larger size poresmore than 600 nm. Svec later formulated a mix porogencomposed of 1-propanol, 1,4-butandiol, and water, whichis applied widely to monolithic column preparation [9–11].

The extensive reviews of polymer-based and silica-based monoliths have been timely published in Journalof Separation Science, JSS (more information availableon JSS site at http://www3.interscience.wiley.com/jour-nal/76510662/home/2259_mostaccessed_2006.html).The status of the polymer monolithic column was cov-ered including applications [11], while as to silica-basedmonolithic column, the recent trend was reviewed bydescribing the commercial monoliths [12]. The polymer-based monolithic columns were comparatively discussedwith those of silica [13]. The monolithic media both insilica and polymer formats were extensively discussedinvolving mathematical and physicochemical analysesas well as polymerization and surface functionality [14].The monolithic column in capillary is increasinglyimportant as a hyphenated method coupled to MS, dueto the reduced raw chemicals and sample amount, andhigher sensitivity owing to sample enrichment [15]. Thisreview is not intended to compile an extensive overviewof polymer-based monoliths. Rather than that, we aregoing to introduce some new perspectives of polymer-based monolith through the discussion about the reac-tion induced phase separation while describing ourongoing investigation of polymer-based monolithic col-umn for micro-HPLC.

To the best of our knowledge, the polymer-basedmonolithic columns reported in the relevant literaturesseem to be mostly characterized by their agglomeratedglobule structures, although the said agglomeratedmonolithic structure was recently commented on refer-ring to a bicontinuous structure [11, 16]. (“Bicontinuous”is a term relative to local plane curvature [17, 18]. The rel-ative topics of geometrical definitions will be reviewedlater in Section 2.1). Those columns are conventionallyprepared by using vinyl monomers such as poly(styrene-co-divinylbenzene) (PS-DVD) and adding a large amountof a poor solvent with the volume fraction well over 0.5(off critical point). The amount of solvent addition isempirically decided dependent upon a requisite liquidflow in column. Such an asymmetric composition of thepolymerization mixture results in a monolithic struc-ture characterized by agglomerated globules. For exam-ple, the globule grows rapidly in its size due to the misci-

bility with monomer and mutual aggregation. Thisagglomerated monolithic structure is not macroscopi-cally ordered and virtually not mesoporous. The presentstatus of polymer-based monolithic column is thus lim-ited in terms of its performance, especially, with regardsto small solute application. In contrast, a bicontinuousmonolithic structure has been successfully realized insilica monoliths prepared by sol–gel process, in whichthe polymerization proceeds rather in a controlled andstepwise manner due to the slow hydrolysis reaction ofsilicone alkoxide [19]. Our review proceeds with the saidaggregated globule monolithic structure as an issue forinvestigation.

First, our review begins with the brief literature surveyon the subjects relative to the polymer-based monolithiccolumn preparation. Our short survey is involved mainlyin this decade since the year of 2000. The citation ofsome key papers before the year of 2000 is also included.The following subjects are specifically explored, namely,monolithic porous structure, living radical polymeriza-tion for monolithic architecture, and the controlled mor-phology by polymerization-induced phase separation.The quantitative studies on monolithic structures areincluded, referring to the recent topics of image analysissuch as laser scanning confocal microscopy (LSCM) andtransmission electron microscopy (TEM). A new conceptof phase separation, “viscoelastic phase separation” [20–22], will be reviewed in terms of the bicontinuous mono-lithic structure. According to this viscoelastic phase sep-aration mechanism, even in the case where the solventphase is a major component (solvent volume fraction ismore than 0.5), provided that the asymmetric dynamicsbetween polymer and solvent phase should exist, thepolymer phase might become continuous matrix,whereas the solvent phase might be a discrete phase inthe said polymer matrix. Interestingly, the viscoelasticphenomenon during phase separation was predictedwith regards to the sol–gel process of silica monolithswith a bicontinuous structure [23, 24]. As to the applica-tion of polymer-based monolithic columns, the recentdevelopments pertaining to microscale separation arereviewed briefly by the survey of the subjects such asminiaturized LC system, LC hyphenation to MS, andsecond dimensional (2-D) analysis.

Second, with the said literature survey as our back-ground for discussion, the review proceeds to introduceour recent investigation of the newly developed polymer-based monoliths with a bicontinuous structure in the fol-lowing two polymer formats. One monolith is preparedby the free radical polymerization of glycerin 1,3-dimeth-acrylate, GDMA [25–27], the other monolith by the step-wise polymerization of a newly introduced trifunctionalepoxy, tris(2,3-epoxypropyl)isocyanurate (TEPIC), and di-amine compound, 4-[(4-aminocycohexyl)methyl]cyclo-hexylamine (BACM) [28–31]. The column performance is


J. Sep. Sci. 2009, 32, 341 – 358 Liquid Chromatography 343

basically discussed in terms of the compatibility of per-meability (D) and efficiency (plate height, H), expressed inseparation impedance, E (=H2/D) [32].

As to GDMA, a standard PS (polystyrene) of ultra highmolecular weight (MW) solution in chlorobenzene ischosen as a porogen so that a bicontinuous structuremight be induced and evolved via the said viscoelasticphase separation. This PS porogenic solution is alsoexpected to afford a sharp bimodal pore size by thestaged phase separation of porogen itself, namely, theearly separation of the standard high MW PS moleculesin uniform giant clusters and the later solvent moleculeseparation [25, 26, 33]. OH group in the GDMA structurepresumably enhances the crosslinking reaction throughthe molecular restriction via hydrogen bonding at earlierstage of the gelation [34, 35].

Next, as to the said TEPIC-BACM epoxy-based system,PEG of lower MW is selected as a porogen. The stepwisereaction of the epoxy proceeds consecutively via theepoxy ring opening reaction with the amine and formsOH groups [36]. These OH groups can be assumed toenhance the epoxy affinity with PEG and delay the phaseseparation affording a resultant bicontinuous structure.OH groups might make the monolithic surface hydro-philic. Hydrophobic interaction chromatography (HIC)or hydrophilic interaction chromatography (HILIC) [37,38] were investigated, while the wired chip device wasprototyped using a long TEPIC-BACM-based capillary coil[30].

2 Short survey of polymer-based monolith

2.1 Porous structure of polymer-based monolith

The design concept of monolith is the realization of thehierarchical porous structure with mesopores and mac-ropores (according to IUPAC definition, minipore isdefined in size as less than 2 nm, mesopore is defined as2–50 nm, and macropore as larger than 50 nm [33]). Themacropores work as flow through channels for mobilephase liquid, whereas the mesopores afford the retentivesites for solutes with a loading capacity in adsorptionchromatography. The pore surface area should be suffi-ciently large enough, while larger minipore (less than2 nm) surface tends to increase the specific retentivitytoward smaller solutes, which might cause larger tailing[39, 40].

As mentioned in Section 1, the preparation of poly-mer-based monoliths has been commonly carried out byadding a large amount of a porogenic solvent more than50 vol%, in order to prepare a macroporous structure forflow in column. Whereas, it is only in the isometric caseof the solvent volume fraction equal to 0.5 where a bicon-tinuous structure could be induced via the phase separa-tion [17]. The polymer segregation or demixing during

the polymerization-induced phase separation can be gen-erally described by physicochemical theories of polymersolution such as Flory–Huggins free energy of mixingdescribed in the classical polymer science books [41–43].Under such solvent rich (asymmetrical composition) con-dition, the van der Waals attraction of the growing poly-mer chains overcomes their mutual steric hindrance,leading to polymer phase as discrete droplets dispersedin the solvent matrix as the polymerization proceeds.Polymer-based monoliths thus prepared reveal a coarsemacroporous structure of agglomerated globules withits smaller pore surface. The application of such macro-porous polymer monoliths has been confined mainly tothe bioseparation of large molecules such as protein ortryptic digests [44, 45].

As to the silica monolith preparation, the sol–gelmethod originally reported by Nakanishi and Soga [19]in the early 1990s is commonly applied. The silica mono-lith with a well-defined bicontinuous structure is con-ventionally prepared by the hydrolytic polymerization ofalkoxysilanes such as tetramethoxysilane (TMOS) inaqueous acetic acid as hydrolytic catalyst while using adispersing agent such as PEG. Mesopores can be formedseparately after the polycondensation of silica by solventexchange with a basic solvent such as ammoniumhydroxide at higher pH and following heat treatment[46]. The pore size control can be thus independently per-formed mesoscopically and macroscopically with a resul-tant bimodal pore size distribution. Minakuchi et al. [47]successfully realized silica monolith as HPLC columnwith a well-ordered bicontinuous structure by sol–gelprocess, coworking with Nakanishi. The sharp bimodalpore size distribution was shown in the range from meso-pore to macrospore. Their silica monolith demonstrateda covalently frozen bicontinuous structure with its skele-ton size of 1 lm, mesopore size of 14 –25 nm, mesoporesurface area of 170–370 m2/g, and the relatively high effi-ciency of 10–20 lm toward small solutes such as aro-matic hydrocarbons. The separation impedance wasreported as around 500 [48]. Some formats of silica mono-liths in disk and rod have been already commercialized[49].

The performance of a commercial silica-based mono-lithic column was comparably examined by Leinweber etal. [50] with a commercial packed particulate column byassuming the monolith as equivalent packed column. Itwas demonstrated that the silica monolithic columncould be virtually mimicked as a packed column withpellicular particle of its overall diameter of 15 lm con-taining a surface diffusive layer of 1.5 lm. Their simula-tion indicated that the silica monolith might beextremely permeable and enhance convection. The silicacapillary with a bicontinuous skeletal structure of 8–10 lm through-pores and 2 lm thick skeletons shows agood performance (plate height, H around 10 lm) for



small molecules and is well compatible with 5 lm silicaparticle packed column [51]. However, with regards tothe separation efficiency, the silica monoliths still seemto lag behind the newly developed silica packed columnwith tiny particles of sub-2 lm size [52]. Whereas, thetemperature control is essential for a packed columnwith extremely small size particles due to the increasedcolumn pressure [53]. This limited efficiency of silicamonoliths is owing to the difficulty in miniaturizing thedomain size (defined as the addition of through-pore sizeand skeletal thickness). Moreover, the relatively largethrough-pores of the silica monolith might increase theA term of the van Deemter equation due to the slow masstransfer in mobile phase as well [32, 51]. The domain sizewas reported to be decreased by reducing the concentra-tion of PEG [51], with 2 lm domain size as the lowestlimit.

Guiochon [14] discussed the structural heterogeneityof the polymer-based column in the radical directioncompounding with the heterogeneity of the axial direc-tion. The A term (eddy diffusion term of van Deemterequation) contribution to the efficiency of polymer-basedmonoliths was examined in HPLC and CEC mode. Accord-ing to his opinion, the scenario favorably attributing tothe flat plug flow profile in CEC might need a littleinsight with the structural heterogeneity taken into con-sideration. Analytically, a monolith column can be virtu-ally considered as a bundle of microopen tubes [54], or,alternatively, as an equivalent packed column [32, 50].The domain size can be considered virtually as an equiva-lent microtube size, or equivalent particle size, respec-tively. The smaller domain size results in lower perme-ability at higher column back pressures, offsetting thegained higher efficiency, while larger domain size leadsto lower efficiency, offsetting the gained higher perme-ability. This compatibility of the permeability, D, withthe efficiency, H, can be expressed in the separationimpedance, E (E = H2/D). A slight decrease in percentageorder of the variability of the domain size could improvethe column efficiency in a magnitude of order as well[54].

The domain size measured by SEM in a pseudo 3-Dimage involves some ambiguity in its definition. Thedimensional sizes such as skeletal thickness and poresize are widely variable from the view point of anobserver (dependent upon view coordinate). Therefore, itwould be useful to review more appropriate scales inde-pendent of the observer's view point. Jinnai et al. [17, 18]measured the time-evolved morphology of polymer-based monolith and silica-based monolith by usingLSCM. The polymer-based monolith was prepared usingthe mixture of deuterated polybutadiene (DPB) and poly-butadiene (PB) in benzene. The silica gel was sintered andtreated with Fluorescein for fluorescence, and madetransparent in toluene/chloroform for laser transpar-

ency. The 2-D digital images of monolith, which wereincrementally sliced along the longitudinal axis, werepiled up as 3-D image data. The local curvature of themonoliths was measured utilizing parameters of differ-ential geometry such as Gauss curvature, K. The sinteredsilica monolith and DPB/PB polymer-based monolithshowed K values located mostly in a negative region(K a0). The interface curvature of the silica and DPB/PBpolymer monoliths was hyperbolic and bicontinuousalthough some droplet feature was contained (K A0). Thedrying/sintering effect of the silica monolith was dis-cussed. As to the silica monolith, Kanamori et al. [24]investigated the deformation of phase separated hybridgel (methyltrimethoxysilane, MTMS) in narrow confine-ment under the wall effect by using LSCM. The viscoelas-tic phase separation of siloxane–sol–gel system wascommented. Aksimentiev and Moorthi [55] studied thespinodal decomposition of hypothetical polymer mix-ture, using computer simulation for polymer-basedmonolith including a thermal noise. The domain sizewas estimated as periodicity of solvent phase (pore).

Tanaka and Araki [22] investigated the viscoelasticphase separation with a binary polymer solution. Their3-D space simulation topologically demonstrated as asnap shot in a time-evolved manner the morphology ofpolymer phase via viscoelastic phase separation. It wasindicated that polymer phase might show a transientnetworklike morphology via viscoelastic phase separa-tion, even if the polymer phase should be a minor compo-nent (solvent rich). The viscoelastic modulus and stresseswere considered as influential dynamical factors in apolymer phase under the asymmetric dynamics of thepolymer/solvent [20]. Gzil et al. [56] analyzed a virtualmodel of silica monolith based on tetrahedral skeletonmodel (TSM) utilizing computation fluid dynamics foroptimum column efficiency. TEM technique was appliedto 3-D macroporous analysis of polymer-based monolithas an alternative to mercury intrusion method [57]. Themonolith was embedded in lead-methacrylate for con-trast enhancement before being microtomed. Some ofour authors examined the porous skeletal structure ofsilica monolith column for HPLC three-dimensionally byusing LSCM [58]. We recently measured the mesopore ofepoxy-based monolith using small angle X ray scattering(SAXS), while comparing to BET data as well [59].

2.2 New polymerization system for polymer-basedmonolith

The polymer-based monolith is conventionally preparedby free radical polymerization thermally or photochemi-cally initiated, although redox system was reported [60].Since the radical polymerization proceeds rapidly at itsearly stage, the large concentration of propagating radi-cals causes the transfer reaction or bimolecular termina-



tion at the initial stage of the polymerization. This resultsin the distribution of network size which might lead tostructural inhomgeneity. The radical polymerization ofliving nature has been investigated as the alternative tothe free radical polymerization of monolithic column.There are three main types of the radical polymerizationin living mode such as atom transfer radical polymeriza-tion (ATRP) (or transient –metal-mediated polymeriza-tion), stable free radical polymerization (SFRP) (or nitro-xide-mediated polymerization), and reversible addition-fragmentation chain transfer polymerization (RAFT).

The ATRP system is composed of alkyl halide as initia-tor, transition metal complex such as Cu(I)Br as catalyst,and amine ligand as additive for solvating the catalystand controlling the redox potential of the metal [61].ATRP polymerization of PEG dimethacrylate was investi-gated, compared with the conventional free radical poly-merization with BPO (benzoyl peroxide) [62]. It was dem-onstrated that ATRP proceeded in living mode withoutautoacceleration at the early stage of polymerization(conversion a40%). At the later stage, the increased diffu-sion resistance restricted the mobility of catalyst/ligand,transferring ATRP to the free radical polymerization.ATRP system of ethylene dimethacrylate (EDMA) andmethyl methacrylate (MMA) was monitored using ESR(electron spin resonance) on line from the beginning ofthe polymerization [63]. The ATRP in living mode trans-ferred to the free radical polymerization due to the diffu-sion-controlled deactivation of MMA radicals as the poly-merization progressed.

As to SFRP, the porous monolith of PS-DVD was pre-pared in situ using carboxyfunctional TEMPO (4-carboxy-2,2,6,6-tetramethylpiperidinyl-1-oxy) by Virklund et al.[64]. The SFRP polymer monolith was prepared combinedwith a binary porogen of PEG and 1-decanol. The sizeexclusion chromatography (SEC) data showed the fractio-nation of PS MW standards, while the monolith surfacemodified with 3-sulfopropyl methacrylate demonstratedthe cation exchange separation for the protein mixture.The morphology revealed by SEM seems to be still globu-lar. Kanamori et al. [65] prepared a divinylbenzene (DVB)-based monolith with a well-ordered bicontinuous struc-ture by using TEMPO-mediated living radical polymeriza-tion. Their polymerization was performed with TEMPOand dibenzoylperoxide (BPO) as initiation system. Thepolymerization was performed stepwise, first, at 958C,then, at 1208C. The phase separation was controlled withdimethylsiloxane (DMS, called a phase separator) in1,3,5-trimethylbenzene (TMB) as solvent. The polymeryield was almost 100% using acetic anhydride as an acti-vator. HPLC data were not disclosed at the time of thepublication.

As to RAFT, wider range of monomers, reaction condi-tions, and narrower polydispersity of MW were featured[66]. Liu et al. [67] investigated molecular imprinted mono-

lithic column (150 mm64.6 mm id) prepared in situ byusing RAFT. The polymerizing system was methyl acrylateacid (MAA) as functional monomer, EDMA as crosslinkingagent, and enrofloxacin (Enro) as template (fluoroquino-lone antibiotic for veterinary use). The porogen was themixture of dichloromethane and 1-dodecanol (8:2) withRAFT agent (dibenzyltrithiocarbonate, DBTCC) and 2,29-azobisisobutyronitrile (AIBN). The separation of enroflox-acin and lomenfloxacin was demonstrated on a selectedcolumn whose globule structure was homogeneous andshowed the lowest separation impedance.

Buchmeiser [68] discussed the pertinent topics of meta-thesis polymerization for polymer monolith. The meta-thesis polymerization is in principle a ring opening poly-merization of norbornene proceeding in living mode.The monolithic support preparation was demonstratedwith NBE (norborn-2-ene) copolymer initiated by ruthe-nium-based Grubbs type initiator in porogenic solventcomposed of toluene and 2-propanol. The example of apolymer monolith prepared by metathesis polymeriza-tion was reported by Mayer et al. [69]. The monoliths weremeasured by using HPLC/ESI-MS. The peak resolution ofoligodeoxynucleotides was demonstrated. The morphol-ogy of these polymer-based monoliths was globular,while the globule size was controllable by the polymer-ization conditions.

A suspension polymerization was applied to polymermonolith preparation back in the late 1980s [70]. Thismonolith was a highly microcellar monolith called poly-HIPE (highly internal phase emulsion), based on styrene/DVB emulsion polymerization with water phase dis-persed in monomer matrix. The polyHIPE monolith withits surface modified by functional monomers via ATRPhas been recently reported [71] for increasing the loadingcapacity. Some new investigation can be cited withregards to the polymer-based monolithic column basedon a conventional free radical polymerization. Ueki et al.[72] prepared polymer monolith capillaries by usingmethacrylate ester monomer of various alkyl chain (car-bon number, C2–18) with EDMA as crosslinking agent.C18 alkyl chain monomer (n-octadecyl methacrylate) wasutilized with the mix solvent of isoamyl alcohol/1,4-buta-nediol as a porogenic solution to prepare the permeablecolumn of the efficiency (l66 lm for alkylbenzenes) atthe high flow rate near 100 mm/s. Their monolithicstructure is still composed of about 5 lm size aggregatedglobules.

The polymer monolith incorporated with carbonnanotube (CNT) was investigated in lHPLC and lCEC [73].The polymerization mixture involved vinylbenzylchlor-ide and EDMA with 2-propanol containing soluble singlewall CNT and formamide as a porogen. The peptide sep-aration was obtained on the monolithic capillary of75 lm id in CEC mode with the plate height from 17.7 to22.4 lm.



2.3 Polymer monolith morphology via controlledphase separation

The viscoelastic phase separation is examined theoreti-cally and experimentally by Tanaka and Araki [20–22].They observed the time-evolved morphology of thebinary mixture of 20% w/w PS (Tg = 1008C) and 80% poly-(vinylether) (PVE, Tg = –238C) by using a phase contrastmicroscopy. The phase separation of this system wasinduced by temperature jump from room temperatureup to 1238C. The transient bicontinuous 3-D networkstructure of PS was found in a time-evolved manner withthe domain size of several micrometer. The evolution ofa bicontinuous structure was described by noting theasymmetric dynamics of the constituent polymer mole-cules (PS-rich phase is slower in dynamics due to Tg close-ness to the quench temperature). Some Japanese patenthas been published with regards to the preparation ofthe porous polyimide membrane with its pore size andinterpore distance controlled via viscoelastic phase sep-aration, which was induced by the solvent substitutionduring the film casting in a coagulation bath [74]. Thepolyimide film 23 –28 lm thick with through-pores ofaverage size ranging from 0.13 to 0.2 lm was preparedfor battery separator and microfiltration.

Macintyre and Sherrington [33] examined the porousstructure of poly(divinylbenzene) resin while utilizingthe staged phase separation of the porogenic mixture ofpoly(propylene glycol) (PPG) or PDMS with toluene ascoporogen [33]. The suspension polymerized poly(divi-nylbenzene) particulate resin showed a sharp bimodalpore size distribution. The staged phase separation wasdescribed by the earlier separation of PDMS or PPG asmacropore former and the later separation of toluene asminipore former.

Super critical carbon dioxide (scCO2) was investigatedas pressure adjustable porogen for methacrylate-basedmonolith [75]. The monolith was prepared with trime-thylolpropane trimethacrylate (TRIM)-based monolithwith scCO2 by adding AIBN as an initiator. The monomerconversion was 95–100%, while the total pore volumewas estimated to be 0.87 cm3/g. For more environmentalfriendliness, Hebb et al. [76] examined a porous cross-linked polymer monolith prepared by TRIM with 1,1,1,2-tetrafluoroethane (R134a) as a porogen. The global warm-ing potential, GWP is 0.29 with R134a (1 for CO2). Themacroporous polyacrylamide (pAAm) monoliths wereexamined as a hydrophilic monolith for bioseparation[77]. The plain monolith of pAAm and the modifiedmonolith (epoxy-pAAm) with epoxy group were preparedwith N,N9-methyl methacrylate in a deionized water fro-zen at –128C as a cryogenic porogen. These monolithsseem to be highly macroporous and spongy with thepores of 10 –100 lm.

2.4 Application of polymer-based monolith tomicroscale separation

The microchip designs for lHPLC and lCEC systems havebeen studied utilizing polymer monolith. As to LC sys-tems in a microchip, one of the key technologies is a pho-tolithographic patterning of microchannels widely prac-ticed in IC industries such as printed wiring boards [78].Another key technology is EOF through the tiny chan-nels, in which a pressure-driven flow cannot be practi-cally implemented due to very high backpressures forthe flow in tiny microchannels. The CEC on polymer-based monolith is recently reviewed [79]. A polymer-based monolith in lchip was exemplified [80] in lCECchip, which was prepared by UV irradiation with a mixedmonofunctional acrylate-based monolith being cast inchannels. The separations of the bioactive peptides andamino acids labeled with fluorogenic dye were achievedby laser-induced (Kr+laser) fluorescence detection withthe efficiency of 600000 plates/m (6 lm) in 40 s.

The post genome research such as combinatorial andproteome analysis is stimulating the development ofhigher throughput of analysis system with a large peakcapacity more than 300 [81]. A comprehensive 2-D HPLCwas investigated by applying a silica monolithic columnof a high permeability and efficiency as first and seconddimension of orthogonal or different selectivities, anddemonstrated as a highly integrated separation systemof higher throughput for combinatorial analysis [82, 83].The microchip fabrication for 2D-HPLC was disclosed in apatent based on polymer monolith prepared by usingmonomers selected among the vinyl polymer systemssuch as styrene/DVB, alkyl methacrylate, or glycidylmethacrylate/EDMA [84]. This separation system is com-posed of stacked layers, namely, first substrate for ion-exchange (strong cation exchange) and second substratefor RP separation. The stacked chip device contains anelectrospray microfabricated on chip (polymer mono-lith/multiple-nozzle electrospray-device) hyphenatedwith mass spectrometer.

The nanocapillary column with 20 lm id was preparedbased on PS-divinylbenzene for LC-ESI-MS system for thetryptic peptide digests by Ivanov et al. [85]. Their SEMshowed still the globular monolithic structure filled in acapillary. Toll et al. [86] reported their offline 2-D separa-tion of the tryptic digestion of ten proteins preparingpoly(styrene-divinylbenzene)-based capillary columns.RP-HPIPC (ion-pair-RP HPLC) at high pH as the first dimen-sion and RP-HPIPC at low pH as the second dimensionwas hyphenated with ESI-MS/MS in positive detectionmode. Their SEM showed still a globular monolithicstructure filled in a capillary. PS-DVB polymer monolithswere prepared with a porogenic mixture of THF and de-canol for separating oligonucleotides, peptides, and pro-



teins [87, 88]. The separation of a synthetic oligonucleo-tide standard, dT16, was obtained with the minimumheight of 8.6 lm (116280 m – 1).

2.5 Brief summary

The application of poymer-based monoliths to micro-scale separation is now extensively investigated withregard to micro-HPLC or CEC on chip by utilizing photo-lithography. The nanocapillary with the size less than50 lm id has become increasingly important for the inte-gration with MS. Most of the polymer-based columnspresently reported reveal basically their agglomeratedglobule structure, demonstrating better performance inCEC mode rather than in pressure-driven HPLC. Theapplication of polymer-based monoliths centering on PS/DVB format seems to be mainly confined to larger solutessuch as proteins or tryptic digests. However, the applica-tion of the polymer monolith to small solutes is stillrequired in the manufacturing fields such as the racemicresolution with higher throughput for drug preparation[89, 90]. GC/MS system utilizes an open tubular column(OTC, Golay column) with its length of 100 m or moreand affords extremely high efficiency well over 105 platesand excellent low separation impedance less than 100[91]. Polymer-based monolith should have more con-trolled monolithic structure with the compatibility ofthe efficiency and permeability for higher performancein a shorter analytical time. Our discussion now proceedsto the investigation of polymer-based monolith with awell-ordered bicontinuous structure.

3 Novel approach to polymer-basedmonolith

Our investigation of polymer-based monolith with abicontinuous structure is exemplified in two differentformats, poly-GDMA and trifunctional epoxy (TEPIC)-di-amine (BACM) system as follows.

3.1 Poly(glycerin 1,3-dimethacrylate)-basedmonolith

3.1.1 Polymerization system of GDMA

Our investigation of poly-GDMA started with the basicgelation study using dynamic light scattering (DLS) [34,35]. DLS results indicated that the crosslinking reactionof GDMA might proceed under the stronger molecularrestriction at earlier stage of the polymerization due tothe hydrogen bonding. The poly-GDMA monolith pre-pared in a hydrophobic solvent like toluene demon-strated a three dimensionally bicontinuous sponge-likestructure characterized by homogeneously dispersedmacropores of 1–2 lm size. These hydrogen bonds pre-

sumably decreased the mutual distance between car-bon–carbon double bonds (CH2=C(CH3)– , isopropenyl) ofthe monomer at earlier stage of polymerization [34, 35,92]. It was expected that poly-GDMA could be effectivelypolymerized by photoinitiated in situ polymerization dueto the said closeness of C=C bonds (GDMA is a product ofShin Nakamura Kagaku, Wakayama, Japan, molecularweight, 228, purity, 100%).

Next, our investigation was carried out to evolve abicontinuous monolithic structure of poly-GDMA byselecting a porogenic solution. Our initial examinationdemonstrated that poly-GDMA showed a bicontinuousstructure with standard ultra high MW PS solution inchlorobenzene utilized as a porogen [25–27]. More spe-cifically, this highly viscous porogen presumablyinduced the asymmetric dynamics due to the “swift” sep-aration of the solvent phase (chlorobenzene) and “slowseparation” of the polymer phase “dragging” giant PSclusters. It is presumed that the polymer phase, which is“left behind by the solvent”, might become viscoelasticgel of its temporal bulk modulus decaying in a relaxationtime. The relaxation of the viscoelastic bulk modulusmight lead to a transient sponge-like structure with sol-vent holes dispersed as discrete phase. This transientlyevolved continuous structure might gradually shrink itsvolume as the transient bulk modulus decays, andbecome networklike.

The said ultra high MW standard PS solution in chloro-benzene (solubility parameter d = 19.4 MPa1/2, GDMA,d = 20.5 MPa1/2 [35]) was formulated by utilizing standardPS with MW between 3000000 and 8400000 and its poly-dispersity of 1.03–1.17. PS concentration was 1–3% w/vto the solvent. The volume fraction of PS solution was65–75% to the monomer. The PS cluster size in chloro-benzene in the MW range said above was estimated to bein the range of 80–130 nm as a radius of gyration [25].

Our attention now focuses on the photoinitiated sys-tem for more homogeneous bicontinuous morphologyof poly-GDMA. The viscoelastic phase separation is a phe-nomenon appearing at earlier stage of phase separation[20]. Therefore, the transient bicontinuous structureshould be chemically frozen timely before the coarsen-ing of the structure. The inherent issues of thermal prep-aration such as heat and generated nitrogen from azoinitiator decomposition should be avoided. In terms ofthe initiator efficiency, the thermal decomposition ofazo initiators is generally slow. For example, in the caseof AIBN, the half time of life is about 10 h at 658C [93].The thermal initiator decomposition is obstructed due to“cage effect” [94] in a viscous solution as well. The meritsof the photoinitiated polymerization can be cited such asthe polymerization at lower temperatures, the independ-ent controllability of light intensity and irradiationtime, and the easy use of volatile solvents at ambient tem-perature. Our preparation of poly-GDMA monolith by



photoinitiated in situ polymerization are detailed else-where [27].

2-Hydroxy-2-methylpropiophenone or 2-hydroxy-2-methyl-1-phenyl-propan-1-one (abbreviated as HMPPhereafter) was applied as a photoinitiator. HMPP is classi-fied into Norrish TYPE 1 photoinitiator in liquid at roomtemperatures [95]. The process under “yellow room” (UVcut illumination) environment was not required in ourordinary laboratory operation. HMPP is photo frag-mented at the a bond to the carbonyl group into initiat-ing species, benzyl and ketyl radicals (a cleavage). Itsabsorbance band ranges from 220 to 380 nm with a mod-erate sensitivity to visible light. The characteristics ofthis photoinitiator is its high rate constant of the a cleav-age well over 1010 s – 1, effective enough to prevent thequenching of triplet excited state by atmospheric oxygen[96]. HMPP was added to the said formulated porogenicPS solution with 1–3 mol% to the equivalent of the C=Cbond of GDMA (l114). UV irradiation was performed at365 nm (near ultraviolet, radiation energy l328 kJ/mol)[97].

Some recent papers are additionally cited as to thepolymer morphology induced by photopolymerizationincluding viscoelastic phase separation [97, 98]. They aremainly concerned with the basic morphological analysisof the polymer blends such as interpenetrating network(IPN). The polymer monolith capillary prepared by UVirradiation was reported already in the late 1990s as amolecularly imprinted column for enantiomeric separa-tion in CEC of antagonist [99]. The polymer monolithsprepared by UV-initiated polymerization have beenexamined for the application to a microchip device as l-TAS, micrototal analytical system [100, 101]. Also, thepolymer monolith was photoinitiated in situ polymeriza-

tion (diethylene glycol dimethacrylate in a porogen suchas methanol and 2-propanol) by using a strong radiationlike Co60 c ray without any initiator [102]. The polymermonolith was prepared in multiple microchannel on sili-con wafer, using photolithography by UV and EB (elec-tron beam) [103]. The polymerizing system was the mix-ture of HEMA (2-hydroxyethyl methacrylate) and EDMAwith methanol and n-hexane as a mixed porogenic solu-tion.

Figure 1 exemplifies a bimodal pore size distributionof poly-GDMA monolith thermally prepared in our ini-tial investigation [25, 26]. The macropore size distribu-tion became strikingly sharp at 1–2 lm in diameter,while the sharp mesopore peak approximately at 3–4 nm in diameter was located. Poly-GDMA monolith pre-pared by UV showed a pore surface around 170 m2/g [27].

3.1.2 lHPLC analysis of poly-GDMA capillarycolumn

The UV transparent TeflonTM coated fused-silica capillaryof 100–200 lm id was treated with a silane couplingagent (3-acryloxypropyl) trimethoxysilane for adhesionof the inner wall. UV irradiation at 365 nm was perform-ed in a black box at 208C for 24 h. The capillary waswashed after polymerization by using methanol and THFconsecutively before drying.

First, the structures of UV and thermally preparedcapillary sections are compared in Fig. 2a (thermally pre-pared capillary with 2,29-azobis(2,4-dimethylvaleroni-trile) (ADVN) as initiator) and 2b (UV prepared capillarywith HMPP as photoinitiator), respectively. Both UV andthermally prepared capillaries were fabricated usingalmost the same polymerizing solution (GDMA/PS solu-tion = 30:70–33:67 v/v, PS MW = 8420000, 1–2% solu-


Figure 1. Pore size distribution ofpoly-GDMA prepared in situ withmonodisperse ultra high MW poly-mer (poly(styrene)) porogenic solu-tion. Mesopore distribution by BETand macropore distribution by Hgintrusion were combined; porogen,PS solution in chlorobenzene, PSMW = 3840000, PS concentra-tion = 0, 1, 3, and 5% w/v; polymer-ization, at 608C for 24 h with AIBN10 mg/mL. Reproduced from ref.[25] with permission of Elsevier,copyright 2006.


tion in chlorobenzene). According to Fig. 2a, the poly-GDMA capillary prepared thermally shows the mono-lithic structure much coarser due to the globule aggrega-tion, whereas Fig. 2b reveals the structure of poly-GDMAcapillary prepared by UV irradiation with its bicontinu-ous network-like morphology. The poly-GDMA capillarymonolith prepared by UV initiation was morphologicallymuch more homogeneous, compared with the poly-GDMA capillary prepared thermally. The thermally pre-pared sample seems to be affected morphologically dueto the confinement in a capillary. The extended sectionalimages of the UV prepared poly-GDMA capillary, Figs. 3a

(inner wall interface, 65000), 3b (middle of the section,65000), and 3c (middle section, 68000), demonstratethe network-like structure of poly-GDMA with the fineskeletal thickness of around 1 lm.

The structure of poly-GDMA capillary prepared by UVirradiation was further examined under different poly-merizing conditions (GDMA/ultra high MW PS solu-tion = 25:75 v/v, PS MW = 6770000, 2% solution in chlor-obenzene, HMPP, 3%) as shown in Fig. 4. The volume frac-tion of monomer to ultra high MW PS solution was moreasymmetric (porogen content increased to 75 vol%) thanin the case of Fig. 2b (67 vol%). Figures 4a (62500) and 4b(65000) show the extended SEM images of poly-GDMAcapillary section prepared with the said highly asymmet-ric composition of the polymerization mixture. TheseSEM images revealed the unique bicontinuous structurecharacteristic of the three dimensionally closely inter-connected polymer skeletons. Poly-GDMA capillary witha well-defined bicontinuous structure could be thusreproduced even under the highly asymmetric composi-tion of the mixture of GDMA/porogen (porogenic solu-tion was highly rich). When the amount of porogen iswell over 50 vol%, the monolith structure of poly-GDMAbecomes much coarser in a solvent like toluene [25, 26,35]. Accordingly, the bicontinuous structures of thesepoly-GDMA samples seem to be unconventional from thestand point of the ordinary phase separation via spinodaldecomposition.

Some chromatograms were obtained on poly-GDMAcapillary column as shown in Fig. 5. Figure 5 comparesthe elution profile of acetophenone on UV prepared poly-GDMA capillary (with PS MW = 8420000, 2% w/v solutionas a porogen, GDMA 30 vol% with 1 mol% HMPP) andthat of the poly-GDMA capillary prepared thermally(with PS MW = 6770000, 3% w/v solution as a porogen,GDMA 33 vol% with ADVN), respectively. The linear veloc-ity was almost the same in both cases. According to Fig.5, the profile of acetophenone on the UV capillary ismuch sharper as compared with the capillary preparedthermally in spite of the longer effective length of the UVcapillary (215 mm with UV prepared column, and155 mm with thermally prepared column). The relevantchromatogram data are tabulated in Table 1. Accordingto Table 1, the UV capillary demonstrates its muchhigher efficiency and permeability, compared with thoserelevant values of the thermally prepared capillary. Theretention factor is a little larger on the thermally pre-pared column presumably due to the slightly larger sta-tionary phase volume.

Our chromatographic data on poly-GDMA column pre-pared by UV are further detailed elsewhere [27] includingthe separation of planar aromatic hydrocarbon (PAH)and hydrophobic retention. The peak separation of PAHswas relatively good with the asymmetry of about 1.1. Themethylene unit selectivity, or the contribution of one


Figure 2. SEM comparison of poly-GDMA capillaries pre-pared in situ by thermally initiated and photoinitiated poly-merization (62500). (a) Thermally initiated with ADVN,20 mg/mL (l1 mol%), at 608C for 24 h, GDMA/ultra high MWstandard PS (2% w/v, MW = 8420000) in chloroben-zene = 30:70 v/v, (b) photoinitiated with HMPP, 3 mol% perthe equivalent of C=C bond of monomer, GDMA/ultra highMW standard PS (1% w/v, PS MW = 8420000) solution inchlorobenzene = 33:67 v/v; UV irradiation at 365 nm with twoUV lamps (8 W64, 1820 mW/cm2 at 50 mm from the source)for 24 h in a black box at 208C. Reproduced from ref. [27]with permission of Wiley-VCH, copyright 2008.


methylene unit to the retention was approximately 1.27,showing a relatively moderate hydrophobic retention ofpoly-GDMA column as compared to PS particles [11]. Asmentioned in Section 2.1, a slight decrease in percentageorder of the variability of the domain size might improvethe column efficiency in a magnitude of order [54]. Themore uniform domain size distribution is expected toafford a sharper elution peak and higher efficiency. Moresolutes including larger molecules such as proteins witha faster elution shall be examined as well.

3.2 Epoxy-based monolith

Nonradical polymerization system such as stepwise poly-merization has been rarely applied to the preparation ofpolymer monolith except some example of suspensionpolymerization [104]. This epoxy resin-based system isexplained as our another approach to polymer-basedmonolith with a well-ordered bicontinuous structure.

3.2.1 Polymerization system of epoxy resin

The reaction-induced phase separation of the multifunc-tional epoxy with diamino compounds was recently


Figure 3. Extended SEM images of poly-GDMA capillaryprepared in situ by UV irradiation. UV irradiation at 365 nmfor 24 h in a black box at 208C; photoinitiator, HMPP,3.0 mol% per the equivalent of the C=C bond of GDMA;GDMA/ultra high Mw standard PS (PS MW = 8420000, 1%w/v) solution in chlorobenzene = 33:67 v/v; (a) interface nearthe capillary wall (65000); (b) middle of the section (65000);(c) middle of the section (68000). Reproduced from ref. [27]with permission of Wiley-VCH, copyright 2008.

Table 1. Comparison of chromatogram on poly-GDMA capillaries by UV-initiated and thermally initiated polymerization

Capillary L6id (mm) Retention factor k9 Width at half height min Plate height lm K N 10 – 14 m2

By UV irradiation 21560.1 0.36 0.90 75 3.39Thermally prepared 15560.1 0.48 1.40 175 2.31

Note: The column preparation and HPLC conditions are shown in Fig. 5. Solute, acetophenone reproduced from ref. [27] withpermission of Wiley-VCH, copyright 2008.


detailed in our papers [29, 30]. At the beginning of ourexperiment, the formulation of monomers and poro-genic solvent was basically investigated with regards tothe morphology of the epoxy resin. First, the polymeriza-tion condition was examined by using difunctional bis-Aepoxy (BADE, bisphenol A diglycidyl ether, or, 2-[(4-{1-methyl-1-[4-(2-oxiranylmethoxy) phenyl] ethyl}phenoxy)-methyl]oxirane) and BACM as amine. The polymerizationwith stochiometric ratio of epoxy/amine (2:1) did not pro-

ceed homogeneously due to highly increased viscosity ofthe mixture. The feed of epoxy to amine was controlledto 2.77 while a volume fraction of a porogenic solventand polymerization temperature being taken into con-sideration. Under the optimized polymerization condi-tion, our bis-A-epoxy-based system afforded a well-ordered bicontinuous monolith. The polymerizationtemperature and the MW of PEG as a porogen were mosteffective in controlling the monolithic structure, whilethe porogen to monomer ratio did not show a morpho-logical effect on the monolith so significantly.

Next, our investigation focused on the system of thetrifunctional epoxy and diamine. The epoxy monomer istris(2,3-epoxypropyl) isocyanurate (TEPIC, racemic, andchiral), whereas the diamine is BACM (BACM is the prod-uct of Tokyo Kasei, Tokyo, Japan. TEPIC is the donationfrom Nissan Chemicals, Tokyo, Japan). The chemicalstructures of the epoxy and diamino compounds areshown in Fig. 6. The ratio of epoxy to amine was opti-mized by checking the morphology with SEM. The feed


Figure 4. Extended SEM images of poly-GDMA capillaryprepared in situ by UV irradiation with highly asymmetricpolymerization mixture (porogen rich in the composition = 75vol%). The morphology of poly-GDMA capillary sectionrevealed 3-D bicontinuous monolithic structure characteristicof the skeletons interconnected closely each other. Thebicontinuous monolithic structure was reproduced even withthe highly asymmetric composition of the porogen. UV irradi-ation at 365 nm for 24 h in a black box at 208C; photoinitiator,HMPP, 3.0 mol% per the equivalent of the C=C bond;GDMA/ultra high MW standard PS (PS MW = 6770000, 2%w/v,) solution in chlorobenzene = 25:75 v/v; (a) 62500; (b)65000. Reproduced from ref. [27] with permission of Wiley-VCH, copyright 2008.

Figure 5. The elution profiles of acetophenone obtained onpoly-GDMA capillaries prepared in situ thermally and by UVirradiation. Broken line: poly-GDMA capillary prepared ther-mally with ADVN 20 mg/mL; 155 mm6100 lm id; GDMA/ultra high MW standard PS (MW = 6770000, 3% w/v) solu-tion in chlorobenzene = 33:67 v/v; t0, 10.55 min; tR,15.62 min; k9 = 0.48; plates, N = 886; 100 nL/min (0.28 mm/s); 1.1 MPa; solid line: poly-GDMA capillary prepared by UVirradiation at 365 nm with HMPP, 1.0 mol% per the equiva-lent of C=C bond of GDMA; GDMA/ultra high MW standardPS (2% w/v, PS MW = 8420000) solution in chloroben-zene = 30:70, v/v; capillary size, 215 mm6100 lm id; t0,15.62 min; tR = 21.21 min; k9 = 0.36; plates, N = 2908; mobilephase, 50% ACN at ambient temperature, 100 nL/min(0.26 mm/s); 1.2 MPa; acetophenone 0.1 mg/mL; detectionat 214 nm with off-column adaptor, 200 mm650 lm havinga window defined 10 cm from the inlet. The elution profile issharper on UV prepared column than on thermally preparedone. Reproduced from ref. [27] with permission of Wiley-VCH, copyright 2008.


ratio of epoxy to amine was selected around 2.8 whilethe volume fraction of a porogenic solvent and polymer-ization temperature being considered.

Poly(ethylene) glycol of lower MW was applied as poro-gen. The MW of PEG was decided by investigating themonolithic structure. The solubility of PEG (a solubilityparameter of PEG l11 MPa1/2 [59]) was found to affect themiscibility with the monomers and the phase separatedmorphology via spinodal decomposition [59]. Although aporogenic solvent should be miscible with the mono-mers at an initial state of polymerization, it must be apoor solvent inducing the phase separation as the poly-merization proceeds. The selection of PEG was importantin terms of defined monolithic structure. PEG wasselected among lower MW samples in the range ofaround 200 [29, 30, 59].

As noted in Introduction, the crosslinking reactionbetween TEPIC and BACM generates OH group via theepoxy ring opening reaction with the amine, in whichprimary amine and the resultant secondary amine con-secutively lose hydrogen–nitrogen bond. OH group inthe epoxy polymer strongly interacts with PEG duringthe phase separation as the polymerization proceeds.This presumably delays the phase separation and devel-ops a monolithic structure evolved in a controlled man-ner, leading to three dimensionally interconnected net-work-like structure of TEPIC/BACM epoxy resin. Our pre-liminary examination revealed that PEG with MW of 200was the most effective to prepare the TEPIC/BACM-basedmonolith with a well-controlled bicontinuous structure.The volume ratio of PEG was 0.8 to the monomer

(80 vol%). The total porosity was calculated as 0.82. Thepertinent subjects including SEC result are described indetails elsewhere [29]. The pore size measurement of BETis demonstrated in Table 2. BET mesopore data usingnitrogen and helium did not demonstrate any distinctivemesoporous surface [29, 61] whereas the column effi-ciency toward small solutes was excellent in HPLC mode.This is our interesting subject as described in Section3.2.2.

3.2.2 lHPLC analysis of epoxy resin-basedcapillary column

At first, long TEPIC-BACM-based capillary columns withits length of 1 m were prepared in situ for SEM andlHPLC. The reproducibility of the column preparationwas checked chromatographically as well. Next, a chipdevice was prototyped by packaging a wired long epoxy-based capillary in the insulating films. The chromato-graphic performance was measured using this wiredchip device by lHPLC.

The lHPLC analysis of the epoxy-based capillary col-umn is summarized as follows. The further details aredescribed elsewhere [30]. The capillary columns of1 m6100 lm id were prepared by using a fused-silicacapillary with the inner wall treated for adhesion (3-ami-nopropyltriethoxysilane). The polymerization was con-ducted normally at 65–808C for 12 h. The polymerizedcapillaries were washed with water and methanol con-secutively before drying in vacuum. With regards to thepolymerization temperature (65, 70, and 758C), SEMimages are shown in Fig. 7. According to Fig. 7, the epoxy-based (TEPIC-BACM) capillary section showed a largerdomain structure as the temperature was lowered. Thisstructural coarsening suggests that the structural freez-ing might occur at later stage due to delayed phase sep-


Table 2. BET mesopore data of TEPIC-BACM-based epoxy

BET specific surfacearea (m2/g)

Total porevolume (cm3/g)

Average porediameter (nm)

2.7 0.004 5.65

Reproduced from ref. [29] with permission of AmericanChemical Society-VCH, copyright 2008.

Figure 6. Chemical structures of monomers for epoxy-basedmonolith TEPIC BACM, 4-[(4-aminnocyclihexyl)methyl]cy-clohexylamine. Reproduced from ref. [29] with permission ofAmerican Chemical Society, copyright 2008.

Figure 7. SEM images of thecapillary section polymerized at65, 70, and 758C. The magnifica-tion is 62500. The polymerizationwas carried out at a prescribedtemperature for 24 h. I-1, 658C, ll-1, 708C, lll-1, 758C. Reproducedfrom ref. [30] with permission ofThe Japan Society for AnalyticalChemistry, copyright 2008.


aration as the polymerization temperature decreased.This result is reflected in Table 2. The permeability, D,became larger, while the plate height, H, decreased, asthe temperature increased (Table 3). Therefore, the per-meability and the efficiency seemed to crossover eachother somewhere around 70l758C (the separation impe-dance, E, showed the minimum at this temperaturerange). This indicates that the polymerization tempera-ture should be controlled carefully in order to preparethe column with a good reproducibility.

Table 4 shows the reproducibility of 25 columns pre-pared by using different preparation methods. In Table4, the method IV means that five original columns of 1 min length prepared at the same time were cut out sepa-rately (565) as a 20 cm long column. The method Vmeans that 20 cm long column was individually pre-pared as 25 separate batches. The method V9 means thatthe preparation was basically the same with the methodV, but performed under more stringently controlled con-ditions in terms of sample transfer and duration in eachoperation unit from monomer mixing through the poly-merization via capillary filling. Among these three prepa-

ration methods, the method V9 showed the lowest varia-bility (smallest RSDs around 2–8%) of the column proper-ties. Figure 8 shows SEM images of the epoxy-based(TEPIC-BACM) capillary section based on method V9. Thefine bicontinuous structure with its skeletal thickness innearly submicron size and through pore of around 2 lmcould be recognized. The expanded SEM image did notreveal any deficiency of the adhesion along the interfaceof the capillary inner wall and the epoxy resin. The chro-matogram of alkylbenzene homolog on TEPIC-BACMcapillary will be mentioned later as compared with thewired chip device in Fig. 12. A chiral separation of (R,S)-1,19-bi-2-naphthol was also demonstrated elsewhere on achiral monolith prepared with a chiral TEPIC (s,s,s) and achiral diamine (trans-1,2-cyclohexanediamine, CHD (s,s)[29].

Figures 9a and b show the separation of nucleosidesand nucleic acid bases (in aqueous ACN 60 vol%). Accord-ing to Fig. 9a, although some incomplete separated peakswere found, the overall separation was satisfactory. Fig-ure 9b shows the retention factor plotted as log k9 againstACN content from 60–90 vol% with regards to the sep-aration of nucleosides and nucleic acid bases. The log k9became larger as ACN content increased. This demon-strated a separation in HILIC mode presumably owing toOH groups distributed on the surface of TEPIC-BACM-based monolith.

Next, the performance of the epoxy-based capillarywas compared with that of a silica packed column withC18 particles (Mightysil RP-18, 25.0 cm64.6 mm id, par-ticle size, 4.660.3 lm) in Figs. 10a –c. H–u plot (plateheight vs. linear velocity curve, Fig. 10a), the backpres-sure versus linear velocity (Fig. 10b), and the separationimpedance versus linear velocity (Fig. 10c) are shown withbenzene as a solute as compared to C18 packed column,


Figure 8. SEM image of TEPIC-BACM capillary column filled atroom temperature and polymer-ized at 748C. Reproduction fromref. [30] with permission of TheJapan Society for Analytical Chem-istry, copyright 2008.

Table 3. The effect of polymerization temperature on columnefficiencya)

Columnno.

Polymeriza-tion temper-ature (8C)

Columnlength(cm)

H(lm)

K(m2)

E

I-1 65 95 10.8 26.5610 – 14 440II-1 70 95 5.5 10.7610 – 14 280III-1 75 95 6.1 5.2610 – 14 710

a) The solute utilized was benzene. Reproduced from ref.[30] with permission of The Japan Society for AnalyticalChemistry, copyright 2008.

Table 4. Data summary for reproducibility of capillary columns

Method N RSD (%) K (m2) RSD (%) E RSD (%) ka) RSD (%)

IV-totalb) 10 400 l 600 5.4 10.0610 – 14 l 0.5610 – 14 5.2 3520 l 420 12 0.25 l 0.02 6.5V-totalc) 10 100 l 800 15 15.3610 – 14 l 4.0610 – 14 20 2320 l 370 16 0.26 l 0.02 9.3V9-totald) 9100 l 200 2.3 97.3610 – 14 l 6.8610 – 14 7.0 500 l 40 8.4 0.35 l 0.01 4.0

a) Benzene was utilized as a solute. Reproduced from ref. [30] with permission of The Japan Society for Analytical Chemistry,copyright 2008.

b – d) Arithmetic average of 25 columns in each preparation method.


respectively. The mobile phase was ACN 60% with 20 mMsodium phosphate buffer at pH 7. The impedance of theTEPIC-based capillary column was lower (200 at the low-est) than that of the C18 packed column by almost 30times (6000 at the lowest), showing the excellent compat-ibility with efficiency and permeability.

Although, as described in Section 3.2.1, the epoxy-based monolith is almost nonmesoporous on its surface,the reason why the monolith in capillary format demon-strated such high efficiency is an interesting subject. Oneplausible explanation is the existence of minipores func-tioning effectively as retentive site. Our SEC of TEPIC-BACM-based column indicated that minipores mightsurely exist as effective retentive site for small solutes[29]. As to SEC data, a little insight might be requiredwith the regards to the effect of a strong solvent like THF

dissolving PS standards. Another explanation might berelevant to chromatographic mode. As mentioned above,OH groups are presumably distributed as a function layeron the epoxy-based monolith. This OH-rich layer mightbe strongly interactive with a mobile phase and functionas a stationary phase of very small thickness in molecular


Figure 9. Separation of nucleic acid bases and nucleosideson TEPIC-BACM capillary, (a) chromatogram, (b) log k9 ver-sus acetonitrile content (vol%) nucleic acid bases, adenine,thymine, uracil, cytosine, guanine; nucleosides, adenosine,guanosine, uridine, cytidine; conditions: mobile phase, ACN/20 mM sodium phosphate buffer pH 7.0 = 60:40 v/v; pres-sure, 4.5 MPa; temperature, 208C; linear velocity, 0.58 mm/sin split flow; column size, 90.0 cm6100 lm id with off-col-umn, 9.0 cm650 lm id; detection at 254 nm. Reproducedfrom ref. [30] with permission of The Japan Society for Ana-lytical Chemistry, copyright 2008. Figure 10. Comparison of TEPIC-BACM capillary and C18

packed column. (a) Comparison of H–u plots betweenTEPIC-BACM capillary and C18 packed column; (b) compar-ison of P–u plots (pressure drop vs. linear velocity) betweenTEPIC-BACM capillary and C18 packed column; (c), com-parison of separation impedance E vs. linear velocity u onTEPIC-BACM capillary and C18 packed column conditions;mobile phase, ACN/20 mM sodium phosphate buffer atpH 7.0 = 60:40 v/v; temperature, 208C; column, TEPIC-BACM capillary, 90.0 cm6100 lm id; detection with off-col-umn, 9.0 cm650 lm at 210 nm; C18 packed column, Might-ysil RP-18, 25.0 cm64.6 mm id; solute, benzene. Repro-duced from ref. [30] with permission of The Japan Society forAnalytical Chemistry, copyright 2008.


order. This might lead to a high efficient partition chro-matography, rather than adsorption chromatography[105]. Further examination is still needed.

Finally, the wired chip device was prototyped. First, anopen fused-silica capillary of 100 lm id was wounded ina coil and sandwiched by sealing films (at Hitachi Chem-ical, Japan). Then, the TEPIC-BACM mixture in PEG wasfilled in the capillary coil and polymerized under thestrictly controlled conditions following the method V9mentioned in Table 4. The finished wired device is shownin Fig. 11. The total dimension of the device is 90 mm inwidth, 55 mm in height. The wound capillary in coil is95 cm long.

The chromatogram of alkylbenzene homolog could beobtained on this wired chip device. The chromatogramon the wired chip device is comparatively shown withthe original straight column in Figs. 12a (straight col-umn) and 12b (wired chip device). The chromatographicconditions were the same with each other (aqueous ACN60 vol%). The chip device showed the stable range ofplate number, 143000–122500 (H, 7–8 lm) with the sol-utes from benzene to hexylbenzene, whereas the originalstraight capillary demonstrated the theoretical platesranging from 153000 to 112000 (H, 6–8 lm). In terms ofthe efficiency, the wired chip device did not show any dif-ference at all from the straight original column beforewiring.

4 Conclusions

First, our survey indicated that the polymer-based mono-lithic columns reported in recent literatures reveal typi-cally an agglomerated globule structure. Polymer-basedmonolithic columns seem to be still confined to theapplications relevant to the separation of larger mole-

cules such as proteins and peptides, whereas the widerange of monomers and polymerization applicable areinherently advantageous with regards to the controlover the polymer-based monolithic structure for the sep-aration of small solutes. Recent developments of micro-chip device based on polymer monolith utilizing photo-lithography illustrate the versatility of polymer mono-lith in microscale separation.

As an alternative to the agglomerated polymer mono-lith, we proposed the preparation of a polymer-basedmonolith with a well-ordered bicontinuous structure.Although monolithic architecture based on living radi-cal polymerization was cited as another optionalapproach to a bicontinuous polymer monolith, ourreview still indicated that a bicontinuous structure couldbe evolved in a rather controlled manner by optimizingthe polymerizing conditions inducing phase separation.Our survey indicated that a transient bicontinuous con-tinuous structure could be induced by utilizing uncon-ventional dynamical effect such as viscoelastic phenom-enon during polymerization-induced phase separation.

Next, our preparation of polymer-based monolithiccolumns in two different polymer formats was intro-duced, that is, the one column based on poly-GDMA, andthe other on the TEPIC-BACM epoxy resin. As for poly-GDMA-based monolith, a fine bicontinuous structurecould be prepared in capillary format by utilizing UV-ini-tiated in situ radical polymerization, with an ultra highMW standard PS solution as a porogen. This fine network-like structure of poly-GDMA was attributed to a frozentransient bicontinuous structure via viscoelastic phaseseparation induced by the said standard PS porogenic sol-ution. The said PS porogenic solution was found to be aneffective porogen realizing a well-ordered bimodal sizedistribution of meso- and macropore as well. Poly-GDMAcapillary column with a bicontinuous structure could bereproducibly prepared by UV-initiated polymerizationeven in the case of the highly asymmetric polymeriza-tion mixture with porogen composition well over50 vol%. The poly-GDMA capillaries prepared by UV ini-tiation demonstrated higher efficiency and permeabilityas compared with poly-GDMA capillaries prepared ther-mally of the same bore size.

With regards to the epoxy resin-based monolith, ahigh performance capillary column could be preparedby utilizing the mixture of TEPIC/BACM with PEG of alower MW in hundreds as a porogen. The capillary col-umn with a well-ordered bicontinuous structure showedthe excellent compatibility of high efficiency and perme-ability, demonstrating the separation impedance in theorder of only a few hundreds. The optimum selectionand strict control of polymerizing temperature andoperational time for each process unit involving mono-mer mixing to polymerization, led to the column prepa-ration with a good reproducibility. The epoxy resin-based


Figure 11. Photograph of prototyped wired chip device col-umn. Reproduced from ref. [30] with permission of TheJapan Society for Analytical Chemistry, copyright 2008.


capillary of a long length (1 m) could be prepared repro-ducibly by regulating the said operational time frommixing to polymerization. RSD of the column perform-ance properties was well below 10%. The separation ofnucleosides and nucleic acid bases demonstrated HILIC

separation owing to OH groups on the surface of TEPIC-BACM epoxy-based monolith. A wired chip device wassuccessfully prototyped by using the said long straightepoxy-based capillary prepared under the optimizedprocess condition. This prototyped chip device demon-


Figure 12. Separation of alkylbenzene on original straight capillary column and wired chip device. (a) Separation of alkylbenzeneon TEPIC-BACM capillary column. Conditions: ACN/20 mM sodium phosphate buffer pH 7.0 = 60:40 v/v; detection at 210 nm;temperature, 208C; linear velocity, 0.58 mm/s in split flow; pressure, 4.5 MPa; column size, 95.0 cm6100 lm id with off-column,9.0 cm650 lm id; solutes, alkylbenzene, from benzene to hexylbenzene; theoretical plates, left to right, benzene, 152800; tol-uene, 153400; ethylbenzene, 147700; propylbenzene, 134400; butylbenzene, 134600; pentylbenzene, 118000; hexylbenzene,111800; (b) separation of alkylbenzene on wired chip device. Conditions: ACN/20 mM sodium phosphate buffer atpH 7.0 = 60:40 v/v; temperature, 208C; linear velocity, 0.56 mm/s in split flow; pressure, 6.5 MPa; column size, 95.0 cm6100 lmid; detection, with off-column 9.0 cm650 lm id at 210 nm; solutes, alkylbenzene, from benzene to hexylbenzene; theoreticalplates, left to right, benzene, 143000; toluene, 142500; ethylbenzene, 140000; ropylbenzene, 141300; butylbenzene, 133700;pentylbenzene, 121900; hexylbenzene, 122500. Reproduced from ref. [30] with permission of The Japan Society for AnalyticalChemistry, copyright 2008.


strated a high performance without any significant dif-ference from the original straight capillary. The platenumber was 143000–123000 (H, l7 lm) toward alkyl-benzene.

In conclusion, our polymer-based monolithic capilla-ries in two polymer formats, methacrylate-based (poly-GDMA) and epoxy-based (TEPIC-BACM), afforded reprodu-cibly a bicontinuous structure well ordered even in a nar-row confinement like capillary. More studies will berequired on the side of polymer-based monolith withregards to structural characteristics and physicochemi-cal properties as are implemented on the side of silicamonolith [106]. We expect more contribution to be madethrough the discussion on bicontinuous column versusagglomerated globule column.

We appreciate helpful comments during the course of thisresearch from Mr. Kuniaki Shimbo (Showa Denko K. K., Kawasaki,Japan). This work was financially supported in part by the Minis-try of Education, Science, Sport, and Culture of Japan (Grand-in-Aid for Basic Scientific Research, contract numbers 16350082 and19350086).

The authors have declared no conflict of interests.

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