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Journal of Chromatography A, 1217 (2010) 5389–5397

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

n the separation of small molecules by means of nano-liquid chromatographyith methacrylate-based macroporous polymer monoliths

vo Nischang ∗, Oliver Brüggemannnstitute of Polymer Chemistry, Johannes Kepler University Linz, Welser Strasse 42, A-4060 Leonding, Austria

r t i c l e i n f o

rticle history:eceived 15 February 2010eceived in revised form 2 June 2010ccepted 8 June 2010vailable online 12 June 2010

eywords:dsorption

a b s t r a c t

Macroporous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases weresynthesized in the confines of 100 �m I.D. fused-silica capillaries via a free radical copolymerization ofmono and divinyl monomeric precursors in the presence of porogenic diluents. These columns wereused in order to determine their suitability for the reversed-phase separation of small molecules inisocratic nano-LC mode. Carefully designed experiments at varying realized phase ratio by a terminatedpolymerization reaction, as well as content of organic modifier in the mobile phase, address the mostsignificant parameters affecting the isocratic performance of these monoliths in the separation of small

opolymerizationel porosityano-LCartitionlate heightorous polymer monolitheversed phase

molecules. We show that the performance of methacrylate-based porous polymer monoliths is stronglyaffected by the retention factor of the analytes separated. A study of the porous and hydrodynamicproperties reveals that the actual nature of the partition and adsorption of the small analyte moleculesbetween mobile and stationary (solvated) polymer phases are most crucial for their performance. Thisis due to a significant gel porosity of the polymeric stationary phase. The gel porosity reflects stagnantmass transfer zones restricting their applicability in the separation of small molecules under conditionsof strong retention.

etention

. Introduction

The beginnings of macroporous polymeric monolithic station-ry phases in analytical column format for liquid chromatographicpplications were in the late 1980s and the early 1990s [1–3]. Thisnique type of material has proven advantageous for certain appli-ations, in particular to that of protein and peptide separations inradient mode. Capillary formats of monolithic columns emergedn the second half of the 1990s with the increased interest in minia-urization of separation formats, in particular to that of micro andano-liquid chromatography (LC), as well as capillary electrochro-atography (CEC) [4–7]. Subsequently, they quickly found a new

ariety of applications such as gas chromatography [8], solid phasextraction [9], catalysis [10], enzymatic reactors [11], as well asicrofluidic mixers [12,13]. They also possess excellent miniatur-

zation potential [14–16]. Free radical polymerization reactionsf acrylate/methacrylate monomers in porogenic diluents can beonveniently carried out by both heat-initiated or light-initiatedree radical polymerization using suitable initiators for the fabri-ation of porous adsorbents [17–19]. This makes them particularly

∗ Corresponding author. Tel.: +43 732 6715 4766; fax: +43 732 6715 4762.E-mail address: Ivo.nischang@jku.at (I. Nischang).

021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2010.06.021

© 2010 Elsevier B.V. All rights reserved.

interesting for use in microfluidic devices and for a variety of bio-analytical applications in the analysis of minute amounts of sample[16]. The relatively simple free radical polymerization processes areeasy to implement [17–19] and the majority of control variablesfor the porous properties of the resultant monolithic stationaryphases, are well identified and studied [20]. In turn, little is knownabout the microscopic topology of the pore space and its impacton the actual heterogeneity of flow and consequently dispersionof analytes in due course of separation [21]. The current appli-cation of macroporous polymer monoliths is mainly performedin the separation of proteins and (larger) peptides in gradientmode.

Porous polymer monoliths typically show a poor performancein the separation of small molecules under more normal chro-matographic conditions in isocratic mode [16,19,21]. The originsof this are discussed frequently in the literature and may includethe lack of mesopores with a narrow distribution providing neces-sary surface area, the existence of micropores, and their structuralin-homogeneity leading to flow dispersion [19,21]. Despite theserational reasons the polymer swells in hydro-organic solvents. This

swelling causes a non-uniform gel porosity which is absent inthe dry state of the polymer. The non-uniform gel structure ofstyrene-co-divinylbenzene based polymer beads, characterized asstationary phase in size exclusion chromatography, has been notedas early as 1985 [22]. However, the resultant gel porosity is not

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390 I. Nischang, O. Brüggemann / J. C

etrimental in the separation of large molecules, which do not enterhe swollen gel structure.

Attempts to improve the efficiencies in small molecule sepa-ation include tailoring of macropore morphologies to be moreimilar to silica-based monoliths [23,24]. Although some improve-ents in their efficiency have been observed, this approach in

he fabrication of porous polymer monoliths has not proven tooromising [23]. The monoliths still lack the desired and well-efined mesopores. In the search for higher surface areas, Bonn’sesearch group used polymerization reaction time to tailor theorous properties of macroporous polymer monoliths based ontyrene chemistry [25,26]. The increased surface area with termi-ated polymerization reactions was reported to aid small moleculeeparations. This was attributed to a significant population of smallores [25,27]. Another attempt published most recently is thatf the fabrication of a generic precursor column from a threeonomer mixture of styrene, vinylbenzylchloride, and divinylben-

ene which allowed the post-polymerization hyper-cross-linkingeaction, leading to a large surface area monolithic capillary col-mn. The authors reported the existence of nanopores/mesopores

n the monolith structure [28].Most of the presented data in the separation of small molecules

ave been obtained under gradient elution conditions [25,29,30].systematic investigation of both the retention and efficiency in

he transport of small analytes, particularly in isocratic elutionode, has rarely been reported [31–35]. Often the presented plate

eight or efficiency data are reported at weak retention, or at lowinear chromatographic velocities [28,35]. Huo et al. have found

significant retention-dependent plate height for polyaromaticydrocarbons on hydrophobic methacrylate-based monoliths in LCnd CEC [33]. The retention factors ranged from 0.1 to 1.25 only. Theoor performance has been attributed to the unfavorable surfaceiffusion at a low volume fraction of stagnant mobile phase. Theuthors highlighted the solute specific plate heights.

Our current study was initiated by the poor performancef porous polymer monoliths, in particular methacrylate-based,or the separation of small molecules under nano-LC conditionsmploying isocratic elution. These materials are particularly welluited for the use in microfluidic separation platforms as theynable convenient photo-initiated, as well as thermally-initiatedolymerization [12,17,18]. A more efficient performance in theransport of small molecules is therefore required to enlarge therray of applications. We selected a typical macroporous mono-ith based on poly(butyl methacrylate-co-ethylene dimethacrylate)btained by polymerization of mono and divinyl monomer in auitable porogenic diluent at an optimized polymerization tem-erature and initiator concentration [15]. This communicationeports on the effect of polymerization time on porous, hydrody-amic, and mass transfer properties for the isocratic separation ofhomologous series of alkylbenzenes. A special emphasis lies on

hromatographic retention and hydrodynamic dispersion.

. Experimental

.1. Chemicals and materials

Ethylene dimethacrylate (EDMA), butyl methacrylate (BuMA),-propanol, 1,4-butanediol, azobisisobutyronitrile (AIBN), 3-trimethoxysilyl)propyl methacrylate, uracil, alkylbenzenes, andPLC-grade acetonitrile were purchased from Sigma–Aldrich

Vienna, Austria). Prior to use, monomers EDMA and BuMA wereurified by running them over basic alumina to remove the

nhibitors. Water was purified on a Milli-Q Reference water purifi-ation system from Millipore (Vienna, Austria). Sample solutions atypical concentrations of 20 �g/ml uracil, 2 �g/ml of benzene and

togr. A 1217 (2010) 5389–5397

that of five alkylbenzenes were always prepared in running mobilephases containing various volume percentages of acetonitrile inwater (v/v). Polyimide coated fused-silica capillaries of 100 �m I.D.were purchased from Optronis (Kehl, Germany).

2.2. Column fabrication

The capillaries were surface-vinylized as recently reported [17].After several rinsing steps with hydrochloric acid and sodiumhydroxide to re-activate the inner surface of the capillaries, theywere flushed with ethanol. Then a solution of 20% (v/v) 3-(trimethoxysilyl)propyl methacrylate in ethanol at an apparent pHvalue of 5 (adjusted using acetic acid) was pumped through eachcapillary for 2 h using a syringe pump (KD Scientific, Holliston, MA,USA). After surface-vinylizing via silane, the capillaries were rinsedwith acetone, dried under a stream of nitrogen, and then were readyto use as a mold.

The surface-vinylized capillaries were filled with a polymeriza-tion mixture containing 24% BuMA, 16% EDMA, 34% 1-propanol,26% 1,4-butanediol and 1% AIBN (all w/w) [15,17]. Prior to this fill-ing, the polymerization mixture was purged with nitrogen for atleast 10 min to remove all dissolved oxygen. Polymerization wascarried out at 60 ◦C for varying polymerization times to obtain rigidmacroporous polymer monoliths. A minimum polymerization timeof 0.5 h was required to obtain rigid, coalesced and wall adheredmacroporous polymer in the capillary allowing for the maximumflow rate of 2 �l/min at a stable backpressure. After polymeriza-tion, the seals were removed, a piece of capillary at the inlet andoutlet end was cut to achieve a length of 20 cm, and they werewashed with 250 �l acetonitrile using a syringe pump. The washvolume was collected in HPLC-vials for later monomer conversionstudies. Column repeatability for nano-LC was tested at selectedpolymerization times including all of the above-mentioned stepsfor four columns each. Error bars in the respective graphs reflectthe standard deviation of their measured properties.

Bulk polymerizations were carried out in glass vials of 2 ml vol-ume of the respective polymerization mixture. Bulk samples wereSoxhlet extracted for 24 h with acetonitrile and dried in a vacuumoven at 40 ◦C over night. Bulk porous properties were determinedvia nitrogen adsorption.

2.3. Equipment

Chromatographic measurements were performed on a DionexUltimate 3000 nano-LC system (Dionex GmbH, Vienna, Austria),incorporating a flow splitter, a flow sensor, and a flow controlvalve ensuring exact flow rates. Experiments with a nano-flow sen-sor from Upchurch Scientific (Oak Harbor, WA, USA) behind thenanofluidic LC-setup has shown reliability of the measured flow.For permeability measurements, capillaries were connected to theinjector and their pressure drop at variable flow rates was recorded.After subtracting the system pressure, the pressure drop generatedby monolithic columns was obtained. The linear slope of backpres-sure against flow rate was used for determination of permeability.

Injection was achieved by a Vici Valco Cheminert 4 nl inter-nal sample loop injection valve (Bartelt GmbH, Vienna, Austria)switched in line with the flow path. Switching for injectionwas timed with the data acquisition of the instrument by themacro-based instrument software. Extra-column volumes in thenano-LC-setup arose from the detector cell volume of 3 nl andthe transfer capillary which was connected to the column outlet

with zero-dead-volume connections (Postnova Analytics GmbH,Landsberg, Germany). The total post-column volume was 113 nl.UV-detection was carried out at 254 nm.

Scanning electron micrographs were obtained using a Cross-beam 1540 XB electron microscope (Carl Zeiss SMT AG,

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I. Nischang, O. Brüggemann / J. C

berkochen, Germany). Nitrogen adsorption experiments wereealized with a Micromeritics TriStar II Surface Area and Porositynstrument (SY-LAB Geräte GmBH, Neu-Purkersdorf, Austria).

Monomer conversion was determined with a 1290 InfinityPLC system (Agilent Technologies, Vienna, Austria) using a Zorbaxclipse Plus C18 column (2.1 × 50 mm, packed with 1.8 �m par-icles). The mobile phase composition was 50% (v/v) acetonitrilen water and analysis was performed at 0.2 ml/min. The injectionolume was 20 �l of appropriately diluted wash volume from theapillaries. The linear response of the detector in the concentrationange required allowed quantitative determination of peak areasf (non-)reacted mono and divinyl monomer. This allowed a studyf monomer specific conversion by taking the calculated capillaryolume into account.

. Results

Initial experiments were performed with the poly(butylethacrylate-co-ethylene dimethacrylate) monoliths obtained by

olymerization at 60 ◦C for 48 h. This polymerization time leadso a complete conversion of monomeric precursors [15,17]. Fig. 1ahows the elution of a mixture of uracil as non-retained tracer, ben-

ig. 1. Elution of a homologeous series of alkylbenzenes on porous monolithicoly(butyl methacrylate-co-ethylene dimethacrylate) columns in a 100 �m I.D.apillary. Conditions: isocratic elution, flow rate: 1.6 �l/min, mobile phase: 50%v/v) acetonitrile in water. Injection volume: 4 nl. (a) Monolith obtained after aolymerization time of 48 h, linear chromatographic velocity: uo = 4.6 mm/s, col-mn pressure: �P = 3.92 MPa. (b) Monolith obtained after a polymerization timef 30 min of an identical polymerization mixture, linear chromatographic velocity:o = 3.6 mm/s, column pressure: �P = 1.14 MPa. Peaks: (1) uracil, (2) benzene, (3)oluene, (4) ethylbenzene, (5) propylbenzene, (6) butylbenzene, (7) pentylbenzene.

togr. A 1217 (2010) 5389–5397 5391

zene and five alkylbenzenes at a high flow rate of 1.6 �l/min andin isocratic mode. This resulted in a linear chromatographic veloc-ity of uo = 4.6 mm/s. The unsuitability for the separation of smallmolecules is striking and all retained analytes elute as one broad,barely defined signal. The eluting retained components show unac-ceptable dispersion. In turn, a monolith polymerized for just 30 minwith a terminated polymerization reaction shows an excellent sep-aration of the alkylbenzenes at the same flow rate (Fig. 1b) spanningretention factors from k′ = 1 for benzene to k′ = 6 for pentylbenzene.The monolith affords well-resolved symmetrical peak shapes for allretained analytes. Due to a higher porosity of the polymer mono-lith with a terminated polymerization reaction, the elution time ofuracil is higher than that of a monolith resulting from a completepolymerization reaction. This results in a linear chromatographicvelocity of uo = 3.6 mm/s (Fig. 1b).

Fig. 2 shows the actual k′ of alkylbenzenes against number ofalkyl carbon atoms for the two porous polymer monoliths. This wasdone at a lower flow rate where the alkylbenzenes could also beresolved on the column polymerized for 48 h. It is evident, that theslope of k′ in both cases is linear and very similar for both a monolithderived from a complete and a terminated polymerization reaction.This indicates a similar selectivity in the separation at an over-all reduced retention. The absolute values of retention decreaseby a factor of more than two. It could be explained by the lowerphase ratio of the porous polymer monolith derived from a termi-nated polymerization reaction. It results in an overall lower amountof converted monomer incorporated in the polymer backbone.However, these results show that while we observe a practicallysimilar selectivity for the elution of alkylbenzenes (Fig. 2), theyare much more dispersed in a column obtained from 48 h poly-merization time (Fig. 1a). Consequently, they are not resolvable orapparently observed in the chromatogram at high linear chromato-graphic velocity. These initial experiments were our guidance fora deeper investigation of the influence of polymerization time onthe porous and hydrodynamic properties as well as retention-basedperformance of the monoliths.

3.1. Monomer conversion

Table 1 shows the results of conversion studies in identical, pre-viously vinylized 100 �m I.D. fused-silica capillaries which wererealized by quantitative HPLC. It becomes clear that the divinyl

Fig. 2. Retention factor k′ against number of alkyl carbon atoms of alkylbenzenesfor poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths obtained after30 min polymerization time (�) and a complete conversion at 48 h (�). Flow rate:0.2 �l/min, mobile phase: 50% (v/v) acetonitrile in water.

5392 I. Nischang, O. Brüggemann / J. Chromatogr. A 1217 (2010) 5389–5397

Table 1Monomer specific conversion in a 100 �m I.D. vinylized fused-silica capillary byanalyzing of remaining monomers from a defined wash volume.

Polymerization time, h EDMAa, % conversion BuMAa, % conversion

0 0 00.5 29.97 13.551 73.81 48.242 97.78 84.243 99.60 94.846 99.99 99.69

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Table 2Impact of polymerization time on porous properties of monoliths determined bynitrogen adsorption performed for three samples each with average values andcoefficient of variation in brackets.

Polymerization time, h BET surface areaa, m2/g BJH pore volumeb, ml/g

0.5 5.85 (2.47%) 0.0168 (3.38%)3 1.28 (2.06%) 0.0024 (3.19%)

48 1.15 (1.59%) 0.0018 (1.27%)

a Total surface area after Brunauer–Emmet–Teller based on pores between 1.7

experiments. The low surface areas are striking and typical for

Ft

a Monomer specific conversion is related to the total initial amount of monomern the polymerization mixture prior to polymerization.

onomer EDMA shows higher conversion at identical polymeriza-ion times than that of the monovinyl monomer BuMA indicatinghe higher degree of cross-linking in the early stages of the polymer-zation reaction. For example, shortly after phase separation (0.5 h)lmost 30% of the divinyl monomer EDMA is converted to the poly-eric stationary phase while only 13–14% of monovinyl monomer

uMA has been converted. At polymerization times higher than 3 h,onversion of both divinyl monomer EDMA and BuMA approachompleteness. However, conversion for EDMA approaches com-leteness much earlier than that of BuMA. It is therefore evident

hat the divinyl monomer EDMA is incorporated in the polymeruclei (and consequently resulting monolith) at a higher rate inhe early stages of the polymerization reaction.

ig. 3. Scanning electron micrographs of the cross-section of porous monolithic poly(butyion time of 30 min (left) and 48 h (right). (a) Complete cross-section, (b) bulk region, and

and 100 nm.b Barrett–Joyner–Halenda cumulative specific pore volume based on pores

between 1.7 and 100 nm.

3.2. Surface area of the stationary phase

Nitrogen adsorption is extensively used for determination ofporous properties of monoliths in their dry state. However, itonly has limited relevance since it requires different mold dimen-sions for porous polymer formation than that of a capillary-sizedmold. Despite this limited relevance it allows a useful estimate ofexpected total surface areas of porous polymeric materials and theexistence of a permanent mesoporous pore space at least qual-itatively [36]. Table 2 shows the results of nitrogen adsorption

macroporous methacrylate-based monoliths [31]. The smaller sizeof the formed highly cross-linked globules in early stages of thepolymerization and rapid phase separation explains the increased

l methacrylate-co-ethylene dimethacrylate) columns obtained after a polymeriza-(c) wall region.

hromatogr. A 1217 (2010) 5389–5397 5393

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Fig. 4. Hydrodynamic and porous properties of monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns in the confines of a 100 �m I.D. capillaryelucidated by nano-LC in dependence of polymerization reaction time. Mobilephase: 50% (v/v) acetonitrile in water. (a) Superficial velocity-based hydrodynamic

I. Nischang, O. Brüggemann / J. C

urface area at a terminated polymerization reaction [16,25,27].ost of the drop in surface area and total pore volume occurs

etween 0.5 and 3 h polymerization time, i.e. after phase separa-ion. However, the total pore volume of pores from 1.7 to 100 nmndicates the almost complete absence of (meso)pores in the poly-

er globules in their dry non-swollen state. This strongly confirmsssumptions made recently [33].

.3. Microscopic structure

Scanning electron micrographs of the porous polymer mono-iths based on poly(butyl methacrylate-co-ethylene dimethacry-ate) resulting in the separations shown in Fig. 1, are shown in Fig. 3.n both cases, these graphs indicate features typically observed for

acroporous polymer monoliths. These features include the exis-ence of relatively large flow through channels in the micrometerize as well as the globular topology of the phase-separated poly-er. However, differences for varying polymerization times are

iscernable. The individual size of the inter-adhered polymer glob-les differs significantly and the porous structure with a terminatedolymerization reaction shows much smaller features than that atcomplete conversion. Overall the polymer monolith derived fromterminated polymerization is more porous with a smaller individ-al feature size (left hand side). The phase separated inter-adheredolymer globules increasingly lose their individuality and furtherolymerization leads to larger clusters. The result is a coarse mono-

ithic polymer structure (right hand side). The disappearance ofmaller cavities between these clusters is discernable at increasedolymerization time. This explains the lower surface area found forhe porous polymer at a complete conversion (Table 2 and Fig. 3).lose observation of the wall shows the occurrence of more densend extended layers of polymer at a complete polymerization reac-ion (Fig. 3c). This layer has its origin in pendent methacrylateroups at the confining wall allowing chain growth during freeadical polymerization. It avoids shrinking and enables covalentttachment of the monolith to the wall [15–17].

.4. Porous and hydrodynamic properties

In order to relate the different microscopic topology presentedn Fig. 3 to the hydrodynamic flow properties of the columns, wetudied the superficial velocity-based hydrodynamic permeabilityp,f of the monoliths in dependence of polymerization reaction time21]:

p,f = L

�P�usf (1)

here L is the length of the column, �P is the pressure drop, � ishe mobile phase viscosity, and usf is the superficial velocity. usf iseferred to as the ratio of volumetric flow rate FV and the cross-ectional area A of the column. The ratio of superficial velocity usfnd linear chromatographic velocity uo (calculated from the res-dence time of non-retained uracil in nano-LC experiments), wassed to determine the total porosity εt of the monolith [21]:

t = usf

uo(2)

Fig. 4 shows a plot of kp,f and εt determined for monoliths inapillaries obtained at different polymerization times before theeaction was terminated. In Fig. 4a a trend to lower permeabilities

s found with an increase in polymerization reaction time whileapidly approaching values close to that found at a complete poly-erization reaction (average value of four independently prepared

olumns indicated by the dashed line). The higher values of perme-bility with a terminated polymerization reaction have their origin

permeability kp,f (Eq. (1)), (b) total porosity εt (Eq. (2)) measured with uracil asnon-retained, totally permeating solute. Error bars reflect standard deviation fromaverage values of four independently prepared and tested columns.

in a lower conversion of monomeric precursors and a much higherporosity as seen in Fig. 3b (left hand side).

At the initial stages of the polymerization reaction, highly cross-linked nuclei are formed, that slowly form polymer globules, whichrapidly phase separate in the poor porogenic solvents and becomefixed in a continuous polymer globule structure (e.g. 0.5 h poly-merization time in Table 1). Both new nuclei and phase separatedglobules are swollen with the remaining monomers of the poly-merization mixture [27,37]. The proceeding polymerization largelytakes place in the polymeric globules and in their vicinity. As typicalfor free radical polymerizations, further nucleation, chain growth,and cross-linking of existent polymer leads to macroporous coa-lesced and coarse globular structures (Fig. 3 right hand side). Thisleads to a drop in total porosity at further polymerization. Theporosity values asymptotically approach that at a complete poly-merization reaction (Fig. 4b).

Fig. 5 shows the plate height curves of non-retained uracil forpoly(butyl methacrylate-co-ethylene dimethacrylate) monolithsresulting in the separations shown in Fig. 1 and the correspondingSEM images in Fig. 3. They most closely reveal the dynamics of flow-

dispersion through the macroporous polymer based monolithsshortly after phase separation to that after a complete polymer-ization reaction. We observe a typical shape of these curves witha minimum plate height of 15 �m in both cases. This explains

5394 I. Nischang, O. Brüggemann / J. Chromatogr. A 1217 (2010) 5389–5397

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ig. 5. Plate height curves of non-retained tracer uracil using monolithic poly(butylethacrylate-co-ethylene dimethacrylate) columns obtained after a polymerization

ime of 30 min (�) and 48 h (©). Mobile phase: 50% (v/v) acetonitrile in water.

he similar porous flow through structure of the monoliths with aicrometer-sized average channel diameter. The monoliths slowly

ose their efficiency in fluid transport at increased linear flow veloc-ties. The minimum value of the plate height compares well to thatypically observed for macroporous methacrylate-based polymer

onoliths under non-retained conditions [32,38,39]. It is also com-arable to that of 13.7 �m (73,000 plates/m) reported by Urban etl. for a 600 m2/g hyper-cross-linked poly(styrene-co-vinylbenzylhloride-co-divinylbenzene) monolithic capillary column [28]. Thensensitivity of our reported plate height on the obvious differ-nce in porous and hydrodynamic properties of the monolithsrepared in this work (Figs. 3 and 4) suggests the limited accessibil-

ty of structure-flow-transport relationships by using non-retainedracer.

.5. Separation efficiency for retained analytes

While the non-retained uracil only samples the heterogeneityf flow in the macroporous structure, Fig. 6 shows the plate height

ig. 6. Plate height curves for non-retained tracer uracil (�) and retained ben-ene (�), toluene (�), ethylbenzene (©), propylbenzene (�), butylbenzene (�),nd pentylbenzene (�) for the monolithic poly(butyl methacrylate-co-ethyleneimethacrylate) columns polymerized at 30 min polymerization time. Mobile phase:0% (v/v) acetonitrile in water.

columns polymerized at 30 min polymerization time. (a) Plate height curve for non-retained tracer uracil (�) and retained benzene at a k′ = 0.4 (©) and 1 (�), (b) plateheight curve for non-retained tracer uracil (�) and retained pentylbenzene at ak′ = 1.2 (♦) and 6 (�), respectively.

curves additionally for retained benzene, and that of five alkylben-zenes. This is done with 70% (v/v) acetonitrile in the mobile phaseand a monolith obtained after a polymerization time of 30 min.The lowest plate heights are observed for the non-retained uracil.The plate height for retained alkylbenzenes spanning k′-valuesfrom 0.37 to 1.2 significantly increases, in particular observed inthe steeper slopes of the plate height curves at increased linearchromatographic velocities. This indicates significant mass trans-fer resistance originating from stagnant mass transfer zones inthe porous structure. Interestingly, a decreased acetonitrile con-tent in the mobile phase of 50% (v/v) results in even steeper slopesof the plate height curves for retained alkylbenzenes (Fig. 7). Thedetermined plate heights reach up to 60 �m for the least retainedcomponent benzene at k′ = 1 (Fig. 7a) and up to 100 �m for the lasteluting component pentylbenzene at k′ = 6 (Fig. 7b). This behaviorpractically contrasts that observed for typical silica-based materialswith an almost retention-independent plate height [38].

The plate height at several linear chromatographic velocitiesuo in dependence of k′-values of the analytes is shown in Fig. 8.This graph indicates that retention strongly influences the band

broadening of the individual analytes of the mixture. The strongestretained analytes have the highest plate height, i.e. the lowestefficiency. Indeed, the curves show that the height equivalentto a theoretical plate displays a transition from the plate heightobserved for only slightly retained analyte (with a k′ close to zero) to

I. Nischang, O. Brüggemann / J. Chromatogr. A 1217 (2010) 5389–5397 5395

Fig. 8. Plate height of the recorded elution bands of alkylbenzenes using mono-lithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerizedat 30 min polymerization time in dependence of the retention factor k′ of the indi-vpwu

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Fig. 9. Impact of volume percentage of acetonitrile in water (v/v) as the mobilephase on the retention factor k′ of a homologeous serious of alkylbenzenes using amonolithic poly(butyl methacrylate-co-ethylene dimethacrylate) column obtainedafter 30 min polymerization time for benzene (�), toluene (©), ethylbenzene (�),propylbenzene (�), butylbenzene (�), and pentylbenzene (�).

Fig. 10. Impact of polymerization time on the plate height of non-retained tracer

idual retained analytes and at different linear chromatographic velocities. Mobilehase: 50% (v/v) acetonitrile in water (solid symbols), and 70% (v/v) acetonitrile inater (open symbols). Symbols: uo = 0.43 mm/s (squares), uo = 0.86 mm/s (circles),

o = 1.29 mm/s (triangles), uo = 1.73 mm/s (hexagons), uo = 2.19 mm/s (pentagons).

hat of strongly retained analyte (with a k′ larger three). In this caseand broadening is controlled by arguments based on a distributionf the analytes between mobile liquid phase and (swollen) station-ry polymer phase. The steepest transition is observed between′-values below 0.5 to a k′ = 3, while approaching plateau values atigher values of the retention factor. The graph also shows that an

ncreased acetonitrile concentration allows achievement of lowerlate heights, i.e. higher efficiency. This is associated with a sig-ificantly reduced retention of the alkylbenzenes (open symbols).he curves at both 70 and 50% (v/v) of acetonitrile in the mobilehase practically coincide, once equivalent k′-values are obtained.his excludes significant effects of swelling on the performancef this monolith at different volume percentages of acetonitrile inhe mobile phase. Interestingly, for all flow rates plate height val-es approach that of the non-retained tracer at retention factorspproaching zero.

The retention factor of the analytes for a variety of volume per-entages of acetonitrile (v/v) in the mobile phase is shown in Fig. 9.t displays a linear decrease at increasing volume percentages ofcetonitrile. It is the strongest for that of the strongest retainedomponent pentylbenzene and significantly reduces the selectivityn the separation of alkylbenzenes at increased acetonitrile concen-rations. Here, efficiencies come more close to each other due to lowetention (Fig. 8). Fig. 9 confirms the typical reversed phase naturef the retention mechanism based on adsorption and partitioning40].

.6. Impact of polymerization time on separation efficiency

While we have demonstrated that porous polymer monolithsan be prepared with acceptable performance by a terminatedolymerization reaction (Figs. 1b and 6) and involving separa-ion conditions of strong retention (Fig. 8), we now focus ourttention on the impact of polymerization reaction time on bothetention factor k′, as well as height equivalent to a theoretical

late. Fig. 10a shows the actual impact of polymerization reac-ion time in the capillary mold on the performance of macroporousolymer monoliths for both non-retained tracer uracil and threeetained alkylbenzenes. This is done using 70% (v/v) acetonitrilen the mobile phase and at a linear chromatographic velocity of

uracil and that of three retained analytes, as well as on the retention factor, usingmonolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns. Linearchromatographic velocity: uo = 0.5 mm/s, mobile phase: 70% (v/v) acetonitrile inwater. (a) Plate height, (b) retention factor. Symbols: uracil (�), benzene (©), ethyl-benzene (�), and pentylbenzene (♦). Error bars reflect standard deviation fromaverage values of four independently prepared and tested columns.

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396 I. Nischang, O. Brüggemann / J. C

o = 0.5 mm/s (realized by proper adjustments in flow rate). Thessociated plate height for retained analytes is smallest at a mini-um polymerization time of 30 min used in this work. Under this

ondition the retention-dependency of the plate height is also mini-ized and very similar plate heights for both non-retained and only

lightly retained analytes are observed. Increased polymerizationimes lead to a significant increase in both retention (Fig. 10b) andlate height (Fig. 10a). At polymerization times exceeding 3 h plateeights of more than a 100 �m at retention factors of 0.92–3 arebserved. Interestingly, the increase in k′ levels off at polymeriza-ion reaction times exceeding 3 h. This indicates the advanced stagef the polymerization reaction and a monolith coming close to itsnal chemical composition (see Table 1). However, the increasedand broadening at increased polymerization times undermineshe similar selectivity for the elution of alkylbenzenes. This, at someoint, results in a loss of resolution. The same effect was alreadyemonstrated in Figs. 1 and 2 with an almost 10 times higher linearhromatographic velocity. Most importantly, polymerization reac-ion time has no detrimental influence on the band dispersion of theon-retained tracer uracil. It reflects the actual flow heterogeneity

n the porous structure (Figs. 10a and 5).

.7. Discussion

We report the fabrication of a typical macroporous poly(butylethacrylate-co-ethylene dimethacrylate) monolith by a copoly-erization reaction involving free radical initiation, nucleation,

lobule formation, phase separation and completion of the reac-ion. It is well known that the copolymer structure of any polymererived from initiation, propagation, cross-linking, and terminationeactions depends at every moment of the reaction on the rela-ive comonomer concentrations and on their reactivity [41]. Ouromogeneous single phase polymerization mixture comprises theonovinyl monomer BuMA and divinyl monomer EDMA in alco-

olic porogenic diluents and initiator.The higher degree of functionality in divinyl monomer EDMA

eads to higher reactivities than that of BuMA for a given free rad-cal concentration [42]. In due course of nucleation the divinyl

onomer EDMA is incorporated in the polymer nuclei at a rateigher than that of the monovinyl monomer BuMA. As the polymer-

zation reaction progresses (including new initiation, and growthf the size of polymer nuclei) the degree of cross-linking in theolymer network (nuclei that slowly form globules) changes con-iderably. The initial high degree of cross-linking in nuclei, leadingo globule formation, results in a rapid phase separation in the earlytages of the polymerization reaction [16]. At this stage of polymer-zation the individual coalesced polymer globules in the polymer

atrix have a high degree of cross-linking (Table 1) [27,42].Polymerization, and hence globule growth, takes place after

hase separation in the phase separated globules and their closeicinity since the remaining monomers are thermodynamicallyood solvents for the polymer. The globule growth is shown in Fig. 3.lso, at this stage of the polymerization the divinyl monomer EDMAvailable for further cross-linking reactions is depleted (Table 1)42,43]. Consequently, the globule becomes softened by slightlyross-linked chains. It is therefore suggested, that the globulehows a crosslink density distribution [42]. The outer regions ofhe globules do not contain a permanent uniform network of smallores with a defined size. This is indicated by our nitrogen adsorp-ion experiments (Table 2). Such polymer monoliths swell when inontact with the hydro-organic solvents used in nano-LC. The resul-

ant gel-like interfaces allow the permeation of small hydrophobic

olecules. The diffusion-limited transport of small molecules inhe gel matrix with non-uniform gel porosity is additionally hin-ered by polymer chain movement and adsorptive interactions.he resulting increased dispersion can be rationalized in the steep

togr. A 1217 (2010) 5389–5397

slopes of the plate height at increased mobile phase velocities. Thisis even seen with a terminated polymerization reaction (Fig. 7). Atan advanced stage of the polymerization, large heterogeneous glob-ular structures with a significant amount of gel porosity are formed.As a consequence the increased band dispersion for retained ana-lytes slowly deteriorates the separation (Fig. 10a) and results in atotally unsuitable material for small molecule separation at highflow speed and retention (Fig. 1a).

4. Conclusions

Results presented in this study strongly suggest, that the appar-ently small surface area of typical porous polymer monoliths isnot responsible for their poor performance in the separation ofsmall molecules. Surface area provides capacity, retention, and con-sequently selectivity. Retention and selectivity are observed withlow surface area porous polymer monoliths incorporating a sim-ple monomer such as BuMA. We have shown that the poly(butylmethacrylate-co-ethylene dimethacrylate) monolith provides suf-ficient methylene selectivity in the separation of a homologeousseries of alkylbenzenes. However, this selectivity is undermined bythe strong band dispersion for retained analytes. This is attributedto the significant amount of gel porosity in the polymeric skele-ton and particularly on its surface. The gel porosity stems fromthe monolith preparation procedure. The preparation involvescopolymerization, rapid phase separation followed by further chaingrowth with only slight cross-linking. This leads to heterogeneous(globule scale) polymer phases with varying gel porosity and orig-inates from a radial crosslink density distribution in globules.This is typical for macroporous copolymer networks derived fromfree radical polymerization and being controlled only to a limitedextent. The resulting gel porosity, again not present in the dry poly-mer, introduces strong mass transfer resistance for retained smallanalytes reflected in the broad analyte bands observed.

Our experiments indicate that the actual microheterogeneity ofthe individual polymer globs (size and shape reflected in a certainmacropore size distribution) has little influence on the poor per-formance obtained for retained small analytes. The non-retainedtracer uracil shows acceptable dispersion for a material of such highpermeability. More controlled chain growth and cross-linking reac-tions are desirable to keep the gel porosity to a minimum. This maybe possible by controlled polymerization techniques for more uni-form cross-linking throughout the globules. This would also allowmore controlled phase separation and termination of the polymer-ization reaction at the desired stage and would drastically increasecontrol over the whole process.

Hopefully our study will spark further interest in the study ofmorphological aspects, as well as globule-scale crosslink homo-geneity which is important to minimize band broadening for smallretained analytes. Reported plate heights appear most meaningfulat sufficiently high retention factors.

Acknowledgement

The authors thank Dr. Ian Teasdale, Institute of Polymer Chem-istry at Johannes Kepler University Linz, for proofreading of themanuscript.

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