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On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths
Journal of Chromatography A, 1217 (2010) 5389–5397
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths Ivo Nischang ∗ , Oliver Brüggemann Institute of Polymer Chemistry, Johannes Kepler University Linz, Welser Strasse 42, A-4060 Leonding, Austria
a r t i c l e
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Article history: Received 15 February 2010 Received in revised form 2 June 2010 Accepted 8 June 2010 Available online 12 June 2010 Keywords: Adsorption Copolymerization Gel porosity Nano-LC Partition Plate height Porous polymer monolith Reversed phase Retention
a b s t r a c t Macroporous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases were synthesized in the confines of 100 m I.D. fused-silica capillaries via a free radical copolymerization of mono and divinyl monomeric precursors in the presence of porogenic diluents. These columns were used in order to determine their suitability for the reversed-phase separation of small molecules in isocratic nano-LC mode. Carefully designed experiments at varying realized phase ratio by a terminated polymerization reaction, as well as content of organic modifier in the mobile phase, address the most significant parameters affecting the isocratic performance of these monoliths in the separation of small molecules. We show that the performance of methacrylate-based porous polymer monoliths is strongly affected by the retention factor of the analytes separated. A study of the porous and hydrodynamic properties reveals that the actual nature of the partition and adsorption of the small analyte molecules between mobile and stationary (solvated) polymer phases are most crucial for their performance. This is due to a significant gel porosity of the polymeric stationary phase. The gel porosity reflects stagnant mass transfer zones restricting their applicability in the separation of small molecules under conditions of strong retention. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The beginnings of macroporous polymeric monolithic stationary phases in analytical column format for liquid chromatographic applications were in the late 1980s and the early 1990s [1–3]. This unique type of material has proven advantageous for certain applications, in particular to that of protein and peptide separations in gradient mode. Capillary formats of monolithic columns emerged in the second half of the 1990s with the increased interest in miniaturization of separation formats, in particular to that of micro and nano-liquid chromatography (LC), as well as capillary electrochromatography (CEC) [4–7]. Subsequently, they quickly found a new variety of applications such as gas chromatography [8], solid phase extraction [9], catalysis [10], enzymatic reactors [11], as well as microfluidic mixers [12,13]. They also possess excellent miniaturization potential [14–16]. Free radical polymerization reactions of acrylate/methacrylate monomers in porogenic diluents can be conveniently carried out by both heat-initiated or light-initiated free radical polymerization using suitable initiators for the fabrication of porous adsorbents [17–19]. This makes them particularly
∗ Corresponding author. Tel.: +43 732 6715 4766; fax: +43 732 6715 4762. E-mail address: [email protected] (I. Nischang). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.06.021
interesting for use in microfluidic devices and for a variety of bioanalytical applications in the analysis of minute amounts of sample [16]. The relatively simple free radical polymerization processes are easy to implement [17–19] and the majority of control variables for the porous properties of the resultant monolithic stationary phases, are well identified and studied [20]. In turn, little is known about the microscopic topology of the pore space and its impact on the actual heterogeneity of flow and consequently dispersion of analytes in due course of separation [21]. The current application of macroporous polymer monoliths is mainly performed in the separation of proteins and (larger) peptides in gradient mode. Porous polymer monoliths typically show a poor performance in the separation of small molecules under more normal chromatographic conditions in isocratic mode [16,19,21]. The origins of this are discussed frequently in the literature and may include the lack of mesopores with a narrow distribution providing necessary surface area, the existence of micropores, and their structural in-homogeneity leading to flow dispersion [19,21]. Despite these rational reasons the polymer swells in hydro-organic solvents. This swelling causes a non-uniform gel porosity which is absent in the dry state of the polymer. The non-uniform gel structure of styrene-co-divinylbenzene based polymer beads, characterized as stationary phase in size exclusion chromatography, has been noted as early as 1985 [22]. However, the resultant gel porosity is not
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detrimental in the separation of large molecules, which do not enter the swollen gel structure. Attempts to improve the efficiencies in small molecule separation include tailoring of macropore morphologies to be more similar to silica-based monoliths [23,24]. Although some improvements in their efficiency have been observed, this approach in the fabrication of porous polymer monoliths has not proven too promising [23]. The monoliths still lack the desired and welldefined mesopores. In the search for higher surface areas, Bonn’s research group used polymerization reaction time to tailor the porous properties of macroporous polymer monoliths based on styrene chemistry [25,26]. The increased surface area with terminated polymerization reactions was reported to aid small molecule separations. This was attributed to a significant population of small pores [25,27]. Another attempt published most recently is that of the fabrication of a generic precursor column from a three monomer mixture of styrene, vinylbenzylchloride, and divinylbenzene which allowed the post-polymerization hyper-cross-linking reaction, leading to a large surface area monolithic capillary column. The authors reported the existence of nanopores/mesopores in the monolith structure [28]. Most of the presented data in the separation of small molecules have been obtained under gradient elution conditions [25,29,30]. A systematic investigation of both the retention and efficiency in the transport of small analytes, particularly in isocratic elution mode, has rarely been reported [31–35]. Often the presented plate height or efficiency data are reported at weak retention, or at low linear chromatographic velocities [28,35]. Huo et al. have found a significant retention-dependent plate height for polyaromatic hydrocarbons on hydrophobic methacrylate-based monoliths in LC and CEC [33]. The retention factors ranged from 0.1 to 1.25 only. The poor performance has been attributed to the unfavorable surface diffusion at a low volume fraction of stagnant mobile phase. The authors highlighted the solute specific plate heights. Our current study was initiated by the poor performance of porous polymer monoliths, in particular methacrylate-based, for the separation of small molecules under nano-LC conditions employing isocratic elution. These materials are particularly well suited for the use in microfluidic separation platforms as they enable convenient photo-initiated, as well as thermally-initiated polymerization [12,17,18]. A more efficient performance in the transport of small molecules is therefore required to enlarge the array of applications. We selected a typical macroporous monolith based on poly(butyl methacrylate-co-ethylene dimethacrylate) obtained by polymerization of mono and divinyl monomer in a suitable porogenic diluent at an optimized polymerization temperature and initiator concentration [15]. This communication reports on the effect of polymerization time on porous, hydrodynamic, and mass transfer properties for the isocratic separation of a homologous series of alkylbenzenes. A special emphasis lies on chromatographic retention and hydrodynamic dispersion.
2. Experimental 2.1. Chemicals and materials Ethylene dimethacrylate (EDMA), butyl methacrylate (BuMA), 1-propanol, 1,4-butanediol, azobisisobutyronitrile (AIBN), 3(trimethoxysilyl)propyl methacrylate, uracil, alkylbenzenes, and HPLC-grade acetonitrile were purchased from Sigma–Aldrich (Vienna, Austria). Prior to use, monomers EDMA and BuMA were purified by running them over basic alumina to remove the inhibitors. Water was purified on a Milli-Q Reference water purification system from Millipore (Vienna, Austria). Sample solutions at typical concentrations of 20 g/ml uracil, 2 g/ml of benzene and
that of five alkylbenzenes were always prepared in running mobile phases containing various volume percentages of acetonitrile in water (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 sodium hydroxide to re-activate the inner surface of the capillaries, they were flushed with ethanol. Then a solution of 20% (v/v) 3(trimethoxysilyl)propyl methacrylate in ethanol at an apparent pH value of 5 (adjusted using acetic acid) was pumped through each capillary for 2 h using a syringe pump (KD Scientific, Holliston, MA, USA). After surface-vinylizing via silane, the capillaries were rinsed with acetone, dried under a stream of nitrogen, and then were ready to use as a mold. The surface-vinylized capillaries were filled with a polymerization mixture containing 24% BuMA, 16% EDMA, 34% 1-propanol, 26% 1,4-butanediol and 1% AIBN (all w/w) [15,17]. Prior to this filling, the polymerization mixture was purged with nitrogen for at least 10 min to remove all dissolved oxygen. Polymerization was carried out at 60 ◦ C for varying polymerization times to obtain rigid macroporous polymer monoliths. A minimum polymerization time of 0.5 h was required to obtain rigid, coalesced and wall adhered macroporous polymer in the capillary allowing for the maximum flow rate of 2 l/min at a stable backpressure. After polymerization, the seals were removed, a piece of capillary at the inlet and outlet end was cut to achieve a length of 20 cm, and they were washed with 250 l acetonitrile using a syringe pump. The wash volume was collected in HPLC-vials for later monomer conversion studies. Column repeatability for nano-LC was tested at selected polymerization times including all of the above-mentioned steps for four columns each. Error bars in the respective graphs reflect the standard deviation of their measured properties. Bulk polymerizations were carried out in glass vials of 2 ml volume of the respective polymerization mixture. Bulk samples were Soxhlet extracted for 24 h with acetonitrile and dried in a vacuum oven at 40 ◦ C over night. Bulk porous properties were determined via nitrogen adsorption. 2.3. Equipment Chromatographic measurements were performed on a Dionex Ultimate 3000 nano-LC system (Dionex GmbH, Vienna, Austria), incorporating a flow splitter, a flow sensor, and a flow control valve ensuring exact flow rates. Experiments with a nano-flow sensor from Upchurch Scientific (Oak Harbor, WA, USA) behind the nanofluidic LC-setup has shown reliability of the measured flow. For permeability measurements, capillaries were connected to the injector and their pressure drop at variable flow rates was recorded. After subtracting the system pressure, the pressure drop generated by monolithic columns was obtained. The linear slope of backpressure against flow rate was used for determination of permeability. Injection was achieved by a Vici Valco Cheminert 4 nl internal sample loop injection valve (Bartelt GmbH, Vienna, Austria) switched in line with the flow path. Switching for injection was timed with the data acquisition of the instrument by the macro-based instrument software. Extra-column volumes in the nano-LC-setup arose from the detector cell volume of 3 nl and the 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 Crossbeam 1540 XB electron microscope (Carl Zeiss SMT AG,
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Oberkochen, Germany). Nitrogen adsorption experiments were realized with a Micromeritics TriStar II Surface Area and Porosity Instrument (SY-LAB Geräte GmBH, Neu-Purkersdorf, Austria). Monomer conversion was determined with a 1290 Infinity UPLC system (Agilent Technologies, Vienna, Austria) using a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, packed with 1.8 m particles). The mobile phase composition was 50% (v/v) acetonitrile in water and analysis was performed at 0.2 ml/min. The injection volume was 20 l of appropriately diluted wash volume from the capillaries. The linear response of the detector in the concentration range required allowed quantitative determination of peak areas of (non-)reacted mono and divinyl monomer. This allowed a study of monomer specific conversion by taking the calculated capillary volume into account. 3. Results Initial experiments were performed with the poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths obtained by polymerization at 60 ◦ C for 48 h. This polymerization time leads to a complete conversion of monomeric precursors [15,17]. Fig. 1a shows the elution of a mixture of uracil as non-retained tracer, ben-
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zene and five alkylbenzenes at a high flow rate of 1.6 l/min and in isocratic mode. This resulted in a linear chromatographic velocity of uo = 4.6 mm/s. The unsuitability for the separation of small molecules is striking and all retained analytes elute as one broad, barely defined signal. The eluting retained components show unacceptable dispersion. In turn, a monolith polymerized for just 30 min with a terminated polymerization reaction shows an excellent separation of the alkylbenzenes at the same flow rate (Fig. 1b) spanning retention factors from k = 1 for benzene to k = 6 for pentylbenzene. The monolith affords well-resolved symmetrical peak shapes for all retained analytes. Due to a higher porosity of the polymer monolith with a terminated polymerization reaction, the elution time of uracil is higher than that of a monolith resulting from a complete polymerization reaction. This results in a linear chromatographic velocity of uo = 3.6 mm/s (Fig. 1b). Fig. 2 shows the actual k of alkylbenzenes against number of alkyl carbon atoms for the two porous polymer monoliths. This was done at a lower flow rate where the alkylbenzenes could also be resolved on the column polymerized for 48 h. It is evident, that the slope of k in both cases is linear and very similar for both a monolith derived from a complete and a terminated polymerization reaction. This indicates a similar selectivity in the separation at an overall reduced retention. The absolute values of retention decrease by a factor of more than two. It could be explained by the lower phase ratio of the porous polymer monolith derived from a terminated polymerization reaction. It results in an overall lower amount of converted monomer incorporated in the polymer backbone. However, these results show that while we observe a practically similar selectivity for the elution of alkylbenzenes (Fig. 2), they are much more dispersed in a column obtained from 48 h polymerization time (Fig. 1a). Consequently, they are not resolvable or apparently observed in the chromatogram at high linear chromatographic velocity. These initial experiments were our guidance for a deeper investigation of the influence of polymerization time on the porous and hydrodynamic properties as well as retention-based performance of the monoliths. 3.1. Monomer conversion Table 1 shows the results of conversion studies in identical, previously vinylized 100 m I.D. fused-silica capillaries which were realized by quantitative HPLC. It becomes clear that the divinyl
Fig. 1. Elution of a homologeous series of alkylbenzenes on porous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns in a 100 m I.D. capillary. 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 a polymerization time of 48 h, linear chromatographic velocity: uo = 4.6 mm/s, column pressure: P = 3.92 MPa. (b) Monolith obtained after a polymerization time of 30 min of an identical polymerization mixture, linear chromatographic velocity: uo = 3.6 mm/s, column pressure: P = 1.14 MPa. Peaks: (1) uracil, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene, (6) butylbenzene, (7) pentylbenzene.
Fig. 2. Retention factor k against number of alkyl carbon atoms of alkylbenzenes for poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths obtained after 30 min polymerization time () and a complete conversion at 48 h (䊉). Flow rate: 0.2 l/min, mobile phase: 50% (v/v) acetonitrile in water.
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Table 1 Monomer specific conversion in a 100 m I.D. vinylized fused-silica capillary by analyzing of remaining monomers from a defined wash volume. Polymerization time, h
EDMAa , % conversion
BuMAa , % conversion
0 0.5 1 2 3 6
0 29.97 73.81 97.78 99.60 99.99
0 13.55 48.24 84.24 94.84 99.69
a Monomer specific conversion is related to the total initial amount of monomer in the polymerization mixture prior to polymerization.
Table 2 Impact of polymerization time on porous properties of monoliths determined by nitrogen adsorption performed for three samples each with average values and coefficient of variation in brackets. Polymerization time, h
BET surface areaa , m2 /g
BJH pore volumeb , ml/g
0.5 3 48
5.85 (2.47%) 1.28 (2.06%) 1.15 (1.59%)
0.0168 (3.38%) 0.0024 (3.19%) 0.0018 (1.27%)
a Total surface area after Brunauer–Emmet–Teller based on pores between 1.7 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 monomer EDMA shows higher conversion at identical polymerization times than that of the monovinyl monomer BuMA indicating the higher degree of cross-linking in the early stages of the polymerization reaction. For example, shortly after phase separation (0.5 h) almost 30% of the divinyl monomer EDMA is converted to the polymeric stationary phase while only 13–14% of monovinyl monomer BuMA has been converted. At polymerization times higher than 3 h, conversion of both divinyl monomer EDMA and BuMA approach completeness. However, conversion for EDMA approaches completeness much earlier than that of BuMA. It is therefore evident that the divinyl monomer EDMA is incorporated in the polymer nuclei (and consequently resulting monolith) at a higher rate in the early stages of the polymerization reaction.
Nitrogen adsorption is extensively used for determination of porous properties of monoliths in their dry state. However, it only has limited relevance since it requires different mold dimensions for porous polymer formation than that of a capillary-sized mold. Despite this limited relevance it allows a useful estimate of expected total surface areas of porous polymeric materials and the existence of a permanent mesoporous pore space at least qualitatively [36]. Table 2 shows the results of nitrogen adsorption experiments. The low surface areas are striking and typical for macroporous methacrylate-based monoliths [31]. The smaller size of the formed highly cross-linked globules in early stages of the polymerization and rapid phase separation explains the increased
Fig. 3. Scanning electron micrographs of the cross-section of porous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns obtained after a polymerization time of 30 min (left) and 48 h (right). (a) Complete cross-section, (b) bulk region, and (c) wall region.
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surface area at a terminated polymerization reaction [16,25,27]. Most of the drop in surface area and total pore volume occurs between 0.5 and 3 h polymerization time, i.e. after phase separation. However, the total pore volume of pores from 1.7 to 100 nm indicates the almost complete absence of (meso)pores in the polymer globules in their dry non-swollen state. This strongly confirms assumptions made recently [33]. 3.3. Microscopic structure Scanning electron micrographs of the porous polymer monoliths based on poly(butyl methacrylate-co-ethylene dimethacrylate) resulting in the separations shown in Fig. 1, are shown in Fig. 3. In both cases, these graphs indicate features typically observed for macroporous polymer monoliths. These features include the existence of relatively large flow through channels in the micrometer size as well as the globular topology of the phase-separated polymer. However, differences for varying polymerization times are discernable. The individual size of the inter-adhered polymer globules differs significantly and the porous structure with a terminated polymerization reaction shows much smaller features than that at a complete conversion. Overall the polymer monolith derived from a terminated polymerization is more porous with a smaller individual feature size (left hand side). The phase separated inter-adhered polymer globules increasingly lose their individuality and further polymerization leads to larger clusters. The result is a coarse monolithic polymer structure (right hand side). The disappearance of smaller cavities between these clusters is discernable at increased polymerization time. This explains the lower surface area found for the porous polymer at a complete conversion (Table 2 and Fig. 3). Close observation of the wall shows the occurrence of more dense and extended layers of polymer at a complete polymerization reaction (Fig. 3c). This layer has its origin in pendent methacrylate groups at the confining wall allowing chain growth during free radical polymerization. It avoids shrinking and enables covalent attachment of the monolith to the wall [15–17]. 3.4. Porous and hydrodynamic properties In order to relate the different microscopic topology presented in Fig. 3 to the hydrodynamic flow properties of the columns, we studied the superficial velocity-based hydrodynamic permeability kp,f of the monoliths in dependence of polymerization reaction time [21]: kp,f =
L u P sf
(1)
where L is the length of the column, P is the pressure drop, is the mobile phase viscosity, and usf is the superficial velocity. usf is referred to as the ratio of volumetric flow rate FV and the crosssectional area A of the column. The ratio of superficial velocity usf and linear chromatographic velocity uo (calculated from the residence time of non-retained uracil in nano-LC experiments), was used to determine the total porosity εt of the monolith [21]: εt =
usf uo
(2)
Fig. 4 shows a plot of kp,f and εt determined for monoliths in capillaries obtained at different polymerization times before the reaction was terminated. In Fig. 4a a trend to lower permeabilities is found with an increase in polymerization reaction time while rapidly approaching values close to that found at a complete polymerization reaction (average value of four independently prepared columns indicated by the dashed line). The higher values of permeability with a terminated polymerization reaction have their origin
Fig. 4. Hydrodynamic and porous properties of monolithic poly(butyl methacrylateco-ethylene dimethacrylate) columns in the confines of a 100 m I.D. capillary elucidated by nano-LC in dependence of polymerization reaction time. Mobile phase: 50% (v/v) acetonitrile in water. (a) Superficial velocity-based hydrodynamic permeability kp,f (Eq. (1)), (b) total porosity εt (Eq. (2)) measured with uracil as non-retained, totally permeating solute. Error bars reflect standard deviation from average values of four independently prepared and tested columns.
in a lower conversion of monomeric precursors and a much higher porosity as seen in Fig. 3b (left hand side). At the initial stages of the polymerization reaction, highly crosslinked nuclei are formed, that slowly form polymer globules, which rapidly phase separate in the poor porogenic solvents and become fixed in a continuous polymer globule structure (e.g. 0.5 h polymerization time in Table 1). Both new nuclei and phase separated globules are swollen with the remaining monomers of the polymerization mixture [27,37]. The proceeding polymerization largely takes place in the polymeric globules and in their vicinity. As typical for free radical polymerizations, further nucleation, chain growth, and cross-linking of existent polymer leads to macroporous coalesced and coarse globular structures (Fig. 3 right hand side). This leads to a drop in total porosity at further polymerization. The porosity values asymptotically approach that at a complete polymerization reaction (Fig. 4b). Fig. 5 shows the plate height curves of non-retained uracil for poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths resulting in the separations shown in Fig. 1 and the corresponding SEM images in Fig. 3. They most closely reveal the dynamics of flowdispersion through the macroporous polymer based monoliths shortly after phase separation to that after a complete polymerization reaction. We observe a typical shape of these curves with a minimum plate height of 15 m in both cases. This explains
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Fig. 5. Plate height curves of non-retained tracer uracil using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns obtained after a polymerization time of 30 min () and 48 h (). Mobile phase: 50% (v/v) acetonitrile in water.
the similar porous flow through structure of the monoliths with a micrometer-sized average channel diameter. The monoliths slowly lose their efficiency in fluid transport at increased linear flow velocities. The minimum value of the plate height compares well to that typically observed for macroporous methacrylate-based polymer monoliths under non-retained conditions [32,38,39]. It is also comparable to that of 13.7 m (73,000 plates/m) reported by Urban et al. for a 600 m2 /g hyper-cross-linked poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic capillary column [28]. The insensitivity of our reported plate height on the obvious difference in porous and hydrodynamic properties of the monoliths prepared in this work (Figs. 3 and 4) suggests the limited accessibility of structure-flow-transport relationships by using non-retained tracer. 3.5. Separation efficiency for retained analytes While the non-retained uracil only samples the heterogeneity of flow in the macroporous structure, Fig. 6 shows the plate height
Fig. 6. Plate height curves for non-retained tracer uracil () and retained benzene (), toluene (䊉), ethylbenzene (), propylbenzene (), butylbenzene (), and pentylbenzene () for the monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerized at 30 min polymerization time. Mobile phase: 70% (v/v) acetonitrile in water.
Fig. 7. Effect of chromatographic retention k on the plate height curves for retained analytes using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerized at 30 min polymerization time. (a) Plate height curve for nonretained tracer uracil () and retained benzene at a k = 0.4 () and 1 (䊉), (b) plate height curve for non-retained tracer uracil () and retained pentylbenzene at a k = 1.2 (♦) and 6 (), respectively.
curves additionally for retained benzene, and that of five alkylbenzenes. This is done with 70% (v/v) acetonitrile in the mobile phase and 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 -values from 0.37 to 1.2 significantly increases, in particular observed in the steeper slopes of the plate height curves at increased linear chromatographic velocities. This indicates significant mass transfer resistance originating from stagnant mass transfer zones in the porous structure. Interestingly, a decreased acetonitrile content in the mobile phase of 50% (v/v) results in even steeper slopes of the plate height curves for retained alkylbenzenes (Fig. 7). The determined plate heights reach up to 60 m for the least retained component benzene at k = 1 (Fig. 7a) and up to 100 m for the last eluting component pentylbenzene at k = 6 (Fig. 7b). This behavior practically contrasts that observed for typical silica-based materials with an almost retention-independent plate height [38]. The plate height at several linear chromatographic velocities uo 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 strongest retained analytes have the highest plate height, i.e. the lowest efficiency. Indeed, the curves show that the height equivalent to a theoretical plate displays a transition from the plate height observed for only slightly retained analyte (with a k close to zero) to
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Fig. 8. Plate height of the recorded elution bands of alkylbenzenes using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerized at 30 min polymerization time in dependence of the retention factor k of the individual retained analytes and at different linear chromatographic velocities. Mobile phase: 50% (v/v) acetonitrile in water (solid symbols), and 70% (v/v) acetonitrile in water (open symbols). Symbols: uo = 0.43 mm/s (squares), uo = 0.86 mm/s (circles), uo = 1.29 mm/s (triangles), uo = 1.73 mm/s (hexagons), uo = 2.19 mm/s (pentagons).
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Fig. 9. Impact of volume percentage of acetonitrile in water (v/v) as the mobile phase on the retention factor k of a homologeous serious of alkylbenzenes using a monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) column obtained after 30 min polymerization time for benzene (), toluene (), ethylbenzene (), propylbenzene (), butylbenzene (), and pentylbenzene ().
that of strongly retained analyte (with a k larger three). In this case band broadening is controlled by arguments based on a distribution of the analytes between mobile liquid phase and (swollen) stationary polymer phase. The steepest transition is observed between k -values below 0.5 to a k = 3, while approaching plateau values at higher values of the retention factor. The graph also shows that an increased acetonitrile concentration allows achievement of lower plate heights, i.e. higher efficiency. This is associated with a significantly reduced retention of the alkylbenzenes (open symbols). The curves at both 70 and 50% (v/v) of acetonitrile in the mobile phase practically coincide, once equivalent k -values are obtained. This excludes significant effects of swelling on the performance of this monolith at different volume percentages of acetonitrile in the mobile phase. Interestingly, for all flow rates plate height values approach that of the non-retained tracer at retention factors approaching zero. The retention factor of the analytes for a variety of volume percentages of acetonitrile (v/v) in the mobile phase is shown in Fig. 9. It displays a linear decrease at increasing volume percentages of acetonitrile. It is the strongest for that of the strongest retained component pentylbenzene and significantly reduces the selectivity in the separation of alkylbenzenes at increased acetonitrile concentrations. Here, efficiencies come more close to each other due to low retention (Fig. 8). Fig. 9 confirms the typical reversed phase nature of the retention mechanism based on adsorption and partitioning [40]. 3.6. Impact of polymerization time on separation efficiency While we have demonstrated that porous polymer monoliths can be prepared with acceptable performance by a terminated polymerization reaction (Figs. 1b and 6) and involving separation conditions of strong retention (Fig. 8), we now focus our attention on the impact of polymerization reaction time on both retention factor k , as well as height equivalent to a theoretical plate. Fig. 10a shows the actual impact of polymerization reaction time in the capillary mold on the performance of macroporous polymer monoliths for both non-retained tracer uracil and three retained alkylbenzenes. This is done using 70% (v/v) acetonitrile in the mobile phase and at a linear chromatographic velocity of
Fig. 10. Impact of polymerization time on the plate height of non-retained tracer uracil and that of three retained analytes, as well as on the retention factor, using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns. Linear chromatographic velocity: uo = 0.5 mm/s, mobile phase: 70% (v/v) acetonitrile in water. (a) Plate height, (b) retention factor. Symbols: uracil (), benzene (), ethylbenzene (), and pentylbenzene (♦). Error bars reflect standard deviation from average values of four independently prepared and tested columns.
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uo = 0.5 mm/s (realized by proper adjustments in flow rate). The associated plate height for retained analytes is smallest at a minimum polymerization time of 30 min used in this work. Under this condition the retention-dependency of the plate height is also minimized and very similar plate heights for both non-retained and only slightly retained analytes are observed. Increased polymerization times lead to a significant increase in both retention (Fig. 10b) and plate height (Fig. 10a). At polymerization times exceeding 3 h plate heights of more than a 100 m at retention factors of 0.92–3 are observed. Interestingly, the increase in k levels off at polymerization reaction times exceeding 3 h. This indicates the advanced stage of the polymerization reaction and a monolith coming close to its final chemical composition (see Table 1). However, the increased band broadening at increased polymerization times undermines the similar selectivity for the elution of alkylbenzenes. This, at some point, results in a loss of resolution. The same effect was already demonstrated in Figs. 1 and 2 with an almost 10 times higher linear chromatographic velocity. Most importantly, polymerization reaction time has no detrimental influence on the band dispersion of the non-retained tracer uracil. It reflects the actual flow heterogeneity in the porous structure (Figs. 10a and 5). 3.7. Discussion We report the fabrication of a typical macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith by a copolymerization reaction involving free radical initiation, nucleation, globule formation, phase separation and completion of the reaction. It is well known that the copolymer structure of any polymer derived from initiation, propagation, cross-linking, and termination reactions depends at every moment of the reaction on the relative comonomer concentrations and on their reactivity [41]. Our homogeneous single phase polymerization mixture comprises the monovinyl monomer BuMA and divinyl monomer EDMA in alcoholic porogenic diluents and initiator. The higher degree of functionality in divinyl monomer EDMA leads to higher reactivities than that of BuMA for a given free radical concentration [42]. In due course of nucleation the divinyl monomer EDMA is incorporated in the polymer nuclei at a rate higher than that of the monovinyl monomer BuMA. As the polymerization reaction progresses (including new initiation, and growth of the size of polymer nuclei) the degree of cross-linking in the polymer network (nuclei that slowly form globules) changes considerably. The initial high degree of cross-linking in nuclei, leading to globule formation, results in a rapid phase separation in the early stages of the polymerization reaction [16]. At this stage of polymerization the individual coalesced polymer globules in the polymer matrix have a high degree of cross-linking (Table 1) [27,42]. Polymerization, and hence globule growth, takes place after phase separation in the phase separated globules and their close vicinity since the remaining monomers are thermodynamically good solvents for the polymer. The globule growth is shown in Fig. 3. Also, at this stage of the polymerization the divinyl monomer EDMA available for further cross-linking reactions is depleted (Table 1) [42,43]. Consequently, the globule becomes softened by slightly cross-linked chains. It is therefore suggested, that the globule shows a crosslink density distribution [42]. The outer regions of the globules do not contain a permanent uniform network of small pores with a defined size. This is indicated by our nitrogen adsorption experiments (Table 2). Such polymer monoliths swell when in contact with the hydro-organic solvents used in nano-LC. The resultant gel-like interfaces allow the permeation of small hydrophobic molecules. The diffusion-limited transport of small molecules in the gel matrix with non-uniform gel porosity is additionally hindered by polymer chain movement and adsorptive interactions. The resulting increased dispersion can be rationalized in the steep
slopes of the plate height at increased mobile phase velocities. This is even seen with a terminated polymerization reaction (Fig. 7). At an advanced stage of the polymerization, large heterogeneous globular structures with a significant amount of gel porosity are formed. As a consequence the increased band dispersion for retained analytes slowly deteriorates the separation (Fig. 10a) and results in a totally unsuitable material for small molecule separation at high flow speed and retention (Fig. 1a). 4. Conclusions Results presented in this study strongly suggest, that the apparently small surface area of typical porous polymer monoliths is not responsible for their poor performance in the separation of small molecules. Surface area provides capacity, retention, and consequently selectivity. Retention and selectivity are observed with low surface area porous polymer monoliths incorporating a simple monomer such as BuMA. We have shown that the poly(butyl methacrylate-co-ethylene dimethacrylate) monolith provides sufficient methylene selectivity in the separation of a homologeous series of alkylbenzenes. However, this selectivity is undermined by the strong band dispersion for retained analytes. This is attributed to the significant amount of gel porosity in the polymeric skeleton and particularly on its surface. The gel porosity stems from the monolith preparation procedure. The preparation involves copolymerization, rapid phase separation followed by further chain growth with only slight cross-linking. This leads to heterogeneous (globule scale) polymer phases with varying gel porosity and originates from a radial crosslink density distribution in globules. This is typical for macroporous copolymer networks derived from free radical polymerization and being controlled only to a limited extent. The resulting gel porosity, again not present in the dry polymer, introduces strong mass transfer resistance for retained small analytes reflected in the broad analyte bands observed. Our experiments indicate that the actual microheterogeneity of the individual polymer globs (size and shape reflected in a certain macropore size distribution) has little influence on the poor performance obtained for retained small analytes. The non-retained tracer uracil shows acceptable dispersion for a material of such high permeability. More controlled chain growth and cross-linking reactions are desirable to keep the gel porosity to a minimum. This may be possible by controlled polymerization techniques for more uniform cross-linking throughout the globules. This would also allow more controlled phase separation and termination of the polymerization reaction at the desired stage and would drastically increase control over the whole process. Hopefully our study will spark further interest in the study of morphological aspects, as well as globule-scale crosslink homogeneity which is important to minimize band broadening for small retained analytes. Reported plate heights appear most meaningful at sufficiently high retention factors. Acknowledgement The authors thank Dr. Ian Teasdale, Institute of Polymer Chemistry at Johannes Kepler University Linz, for proofreading of the manuscript. References [1] [2] [3] [4] [5] [6] [7]
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths Ivo Nischang ∗ , Oliver Brüggemann Institute of Polymer Chemistry, Johannes Kepler University Linz, Welser Strasse 42, A-4060 Leonding, Austria
a r t i c l e
i n f o
Article history: Received 15 February 2010 Received in revised form 2 June 2010 Accepted 8 June 2010 Available online 12 June 2010 Keywords: Adsorption Copolymerization Gel porosity Nano-LC Partition Plate height Porous polymer monolith Reversed phase Retention
a b s t r a c t Macroporous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases were synthesized in the confines of 100 m I.D. fused-silica capillaries via a free radical copolymerization of mono and divinyl monomeric precursors in the presence of porogenic diluents. These columns were used in order to determine their suitability for the reversed-phase separation of small molecules in isocratic nano-LC mode. Carefully designed experiments at varying realized phase ratio by a terminated polymerization reaction, as well as content of organic modifier in the mobile phase, address the most significant parameters affecting the isocratic performance of these monoliths in the separation of small molecules. We show that the performance of methacrylate-based porous polymer monoliths is strongly affected by the retention factor of the analytes separated. A study of the porous and hydrodynamic properties reveals that the actual nature of the partition and adsorption of the small analyte molecules between mobile and stationary (solvated) polymer phases are most crucial for their performance. This is due to a significant gel porosity of the polymeric stationary phase. The gel porosity reflects stagnant mass transfer zones restricting their applicability in the separation of small molecules under conditions of strong retention. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The beginnings of macroporous polymeric monolithic stationary phases in analytical column format for liquid chromatographic applications were in the late 1980s and the early 1990s [1–3]. This unique type of material has proven advantageous for certain applications, in particular to that of protein and peptide separations in gradient mode. Capillary formats of monolithic columns emerged in the second half of the 1990s with the increased interest in miniaturization of separation formats, in particular to that of micro and nano-liquid chromatography (LC), as well as capillary electrochromatography (CEC) [4–7]. Subsequently, they quickly found a new variety of applications such as gas chromatography [8], solid phase extraction [9], catalysis [10], enzymatic reactors [11], as well as microfluidic mixers [12,13]. They also possess excellent miniaturization potential [14–16]. Free radical polymerization reactions of acrylate/methacrylate monomers in porogenic diluents can be conveniently carried out by both heat-initiated or light-initiated free radical polymerization using suitable initiators for the fabrication of porous adsorbents [17–19]. This makes them particularly
∗ Corresponding author. Tel.: +43 732 6715 4766; fax: +43 732 6715 4762. E-mail address: [email protected] (I. Nischang). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.06.021
interesting for use in microfluidic devices and for a variety of bioanalytical applications in the analysis of minute amounts of sample [16]. The relatively simple free radical polymerization processes are easy to implement [17–19] and the majority of control variables for the porous properties of the resultant monolithic stationary phases, are well identified and studied [20]. In turn, little is known about the microscopic topology of the pore space and its impact on the actual heterogeneity of flow and consequently dispersion of analytes in due course of separation [21]. The current application of macroporous polymer monoliths is mainly performed in the separation of proteins and (larger) peptides in gradient mode. Porous polymer monoliths typically show a poor performance in the separation of small molecules under more normal chromatographic conditions in isocratic mode [16,19,21]. The origins of this are discussed frequently in the literature and may include the lack of mesopores with a narrow distribution providing necessary surface area, the existence of micropores, and their structural in-homogeneity leading to flow dispersion [19,21]. Despite these rational reasons the polymer swells in hydro-organic solvents. This swelling causes a non-uniform gel porosity which is absent in the dry state of the polymer. The non-uniform gel structure of styrene-co-divinylbenzene based polymer beads, characterized as stationary phase in size exclusion chromatography, has been noted as early as 1985 [22]. However, the resultant gel porosity is not
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detrimental in the separation of large molecules, which do not enter the swollen gel structure. Attempts to improve the efficiencies in small molecule separation include tailoring of macropore morphologies to be more similar to silica-based monoliths [23,24]. Although some improvements in their efficiency have been observed, this approach in the fabrication of porous polymer monoliths has not proven too promising [23]. The monoliths still lack the desired and welldefined mesopores. In the search for higher surface areas, Bonn’s research group used polymerization reaction time to tailor the porous properties of macroporous polymer monoliths based on styrene chemistry [25,26]. The increased surface area with terminated polymerization reactions was reported to aid small molecule separations. This was attributed to a significant population of small pores [25,27]. Another attempt published most recently is that of the fabrication of a generic precursor column from a three monomer mixture of styrene, vinylbenzylchloride, and divinylbenzene which allowed the post-polymerization hyper-cross-linking reaction, leading to a large surface area monolithic capillary column. The authors reported the existence of nanopores/mesopores in the monolith structure [28]. Most of the presented data in the separation of small molecules have been obtained under gradient elution conditions [25,29,30]. A systematic investigation of both the retention and efficiency in the transport of small analytes, particularly in isocratic elution mode, has rarely been reported [31–35]. Often the presented plate height or efficiency data are reported at weak retention, or at low linear chromatographic velocities [28,35]. Huo et al. have found a significant retention-dependent plate height for polyaromatic hydrocarbons on hydrophobic methacrylate-based monoliths in LC and CEC [33]. The retention factors ranged from 0.1 to 1.25 only. The poor performance has been attributed to the unfavorable surface diffusion at a low volume fraction of stagnant mobile phase. The authors highlighted the solute specific plate heights. Our current study was initiated by the poor performance of porous polymer monoliths, in particular methacrylate-based, for the separation of small molecules under nano-LC conditions employing isocratic elution. These materials are particularly well suited for the use in microfluidic separation platforms as they enable convenient photo-initiated, as well as thermally-initiated polymerization [12,17,18]. A more efficient performance in the transport of small molecules is therefore required to enlarge the array of applications. We selected a typical macroporous monolith based on poly(butyl methacrylate-co-ethylene dimethacrylate) obtained by polymerization of mono and divinyl monomer in a suitable porogenic diluent at an optimized polymerization temperature and initiator concentration [15]. This communication reports on the effect of polymerization time on porous, hydrodynamic, and mass transfer properties for the isocratic separation of a homologous series of alkylbenzenes. A special emphasis lies on chromatographic retention and hydrodynamic dispersion.
2. Experimental 2.1. Chemicals and materials Ethylene dimethacrylate (EDMA), butyl methacrylate (BuMA), 1-propanol, 1,4-butanediol, azobisisobutyronitrile (AIBN), 3(trimethoxysilyl)propyl methacrylate, uracil, alkylbenzenes, and HPLC-grade acetonitrile were purchased from Sigma–Aldrich (Vienna, Austria). Prior to use, monomers EDMA and BuMA were purified by running them over basic alumina to remove the inhibitors. Water was purified on a Milli-Q Reference water purification system from Millipore (Vienna, Austria). Sample solutions at typical concentrations of 20 g/ml uracil, 2 g/ml of benzene and
that of five alkylbenzenes were always prepared in running mobile phases containing various volume percentages of acetonitrile in water (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 sodium hydroxide to re-activate the inner surface of the capillaries, they were flushed with ethanol. Then a solution of 20% (v/v) 3(trimethoxysilyl)propyl methacrylate in ethanol at an apparent pH value of 5 (adjusted using acetic acid) was pumped through each capillary for 2 h using a syringe pump (KD Scientific, Holliston, MA, USA). After surface-vinylizing via silane, the capillaries were rinsed with acetone, dried under a stream of nitrogen, and then were ready to use as a mold. The surface-vinylized capillaries were filled with a polymerization mixture containing 24% BuMA, 16% EDMA, 34% 1-propanol, 26% 1,4-butanediol and 1% AIBN (all w/w) [15,17]. Prior to this filling, the polymerization mixture was purged with nitrogen for at least 10 min to remove all dissolved oxygen. Polymerization was carried out at 60 ◦ C for varying polymerization times to obtain rigid macroporous polymer monoliths. A minimum polymerization time of 0.5 h was required to obtain rigid, coalesced and wall adhered macroporous polymer in the capillary allowing for the maximum flow rate of 2 l/min at a stable backpressure. After polymerization, the seals were removed, a piece of capillary at the inlet and outlet end was cut to achieve a length of 20 cm, and they were washed with 250 l acetonitrile using a syringe pump. The wash volume was collected in HPLC-vials for later monomer conversion studies. Column repeatability for nano-LC was tested at selected polymerization times including all of the above-mentioned steps for four columns each. Error bars in the respective graphs reflect the standard deviation of their measured properties. Bulk polymerizations were carried out in glass vials of 2 ml volume of the respective polymerization mixture. Bulk samples were Soxhlet extracted for 24 h with acetonitrile and dried in a vacuum oven at 40 ◦ C over night. Bulk porous properties were determined via nitrogen adsorption. 2.3. Equipment Chromatographic measurements were performed on a Dionex Ultimate 3000 nano-LC system (Dionex GmbH, Vienna, Austria), incorporating a flow splitter, a flow sensor, and a flow control valve ensuring exact flow rates. Experiments with a nano-flow sensor from Upchurch Scientific (Oak Harbor, WA, USA) behind the nanofluidic LC-setup has shown reliability of the measured flow. For permeability measurements, capillaries were connected to the injector and their pressure drop at variable flow rates was recorded. After subtracting the system pressure, the pressure drop generated by monolithic columns was obtained. The linear slope of backpressure against flow rate was used for determination of permeability. Injection was achieved by a Vici Valco Cheminert 4 nl internal sample loop injection valve (Bartelt GmbH, Vienna, Austria) switched in line with the flow path. Switching for injection was timed with the data acquisition of the instrument by the macro-based instrument software. Extra-column volumes in the nano-LC-setup arose from the detector cell volume of 3 nl and the 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 Crossbeam 1540 XB electron microscope (Carl Zeiss SMT AG,
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Oberkochen, Germany). Nitrogen adsorption experiments were realized with a Micromeritics TriStar II Surface Area and Porosity Instrument (SY-LAB Geräte GmBH, Neu-Purkersdorf, Austria). Monomer conversion was determined with a 1290 Infinity UPLC system (Agilent Technologies, Vienna, Austria) using a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, packed with 1.8 m particles). The mobile phase composition was 50% (v/v) acetonitrile in water and analysis was performed at 0.2 ml/min. The injection volume was 20 l of appropriately diluted wash volume from the capillaries. The linear response of the detector in the concentration range required allowed quantitative determination of peak areas of (non-)reacted mono and divinyl monomer. This allowed a study of monomer specific conversion by taking the calculated capillary volume into account. 3. Results Initial experiments were performed with the poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths obtained by polymerization at 60 ◦ C for 48 h. This polymerization time leads to a complete conversion of monomeric precursors [15,17]. Fig. 1a shows the elution of a mixture of uracil as non-retained tracer, ben-
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zene and five alkylbenzenes at a high flow rate of 1.6 l/min and in isocratic mode. This resulted in a linear chromatographic velocity of uo = 4.6 mm/s. The unsuitability for the separation of small molecules is striking and all retained analytes elute as one broad, barely defined signal. The eluting retained components show unacceptable dispersion. In turn, a monolith polymerized for just 30 min with a terminated polymerization reaction shows an excellent separation of the alkylbenzenes at the same flow rate (Fig. 1b) spanning retention factors from k = 1 for benzene to k = 6 for pentylbenzene. The monolith affords well-resolved symmetrical peak shapes for all retained analytes. Due to a higher porosity of the polymer monolith with a terminated polymerization reaction, the elution time of uracil is higher than that of a monolith resulting from a complete polymerization reaction. This results in a linear chromatographic velocity of uo = 3.6 mm/s (Fig. 1b). Fig. 2 shows the actual k of alkylbenzenes against number of alkyl carbon atoms for the two porous polymer monoliths. This was done at a lower flow rate where the alkylbenzenes could also be resolved on the column polymerized for 48 h. It is evident, that the slope of k in both cases is linear and very similar for both a monolith derived from a complete and a terminated polymerization reaction. This indicates a similar selectivity in the separation at an overall reduced retention. The absolute values of retention decrease by a factor of more than two. It could be explained by the lower phase ratio of the porous polymer monolith derived from a terminated polymerization reaction. It results in an overall lower amount of converted monomer incorporated in the polymer backbone. However, these results show that while we observe a practically similar selectivity for the elution of alkylbenzenes (Fig. 2), they are much more dispersed in a column obtained from 48 h polymerization time (Fig. 1a). Consequently, they are not resolvable or apparently observed in the chromatogram at high linear chromatographic velocity. These initial experiments were our guidance for a deeper investigation of the influence of polymerization time on the porous and hydrodynamic properties as well as retention-based performance of the monoliths. 3.1. Monomer conversion Table 1 shows the results of conversion studies in identical, previously vinylized 100 m I.D. fused-silica capillaries which were realized by quantitative HPLC. It becomes clear that the divinyl
Fig. 1. Elution of a homologeous series of alkylbenzenes on porous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns in a 100 m I.D. capillary. 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 a polymerization time of 48 h, linear chromatographic velocity: uo = 4.6 mm/s, column pressure: P = 3.92 MPa. (b) Monolith obtained after a polymerization time of 30 min of an identical polymerization mixture, linear chromatographic velocity: uo = 3.6 mm/s, column pressure: P = 1.14 MPa. Peaks: (1) uracil, (2) benzene, (3) toluene, (4) ethylbenzene, (5) propylbenzene, (6) butylbenzene, (7) pentylbenzene.
Fig. 2. Retention factor k against number of alkyl carbon atoms of alkylbenzenes for poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths obtained after 30 min polymerization time () and a complete conversion at 48 h (䊉). Flow rate: 0.2 l/min, mobile phase: 50% (v/v) acetonitrile in water.
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Table 1 Monomer specific conversion in a 100 m I.D. vinylized fused-silica capillary by analyzing of remaining monomers from a defined wash volume. Polymerization time, h
EDMAa , % conversion
BuMAa , % conversion
0 0.5 1 2 3 6
0 29.97 73.81 97.78 99.60 99.99
0 13.55 48.24 84.24 94.84 99.69
a Monomer specific conversion is related to the total initial amount of monomer in the polymerization mixture prior to polymerization.
Table 2 Impact of polymerization time on porous properties of monoliths determined by nitrogen adsorption performed for three samples each with average values and coefficient of variation in brackets. Polymerization time, h
BET surface areaa , m2 /g
BJH pore volumeb , ml/g
0.5 3 48
5.85 (2.47%) 1.28 (2.06%) 1.15 (1.59%)
0.0168 (3.38%) 0.0024 (3.19%) 0.0018 (1.27%)
a Total surface area after Brunauer–Emmet–Teller based on pores between 1.7 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 monomer EDMA shows higher conversion at identical polymerization times than that of the monovinyl monomer BuMA indicating the higher degree of cross-linking in the early stages of the polymerization reaction. For example, shortly after phase separation (0.5 h) almost 30% of the divinyl monomer EDMA is converted to the polymeric stationary phase while only 13–14% of monovinyl monomer BuMA has been converted. At polymerization times higher than 3 h, conversion of both divinyl monomer EDMA and BuMA approach completeness. However, conversion for EDMA approaches completeness much earlier than that of BuMA. It is therefore evident that the divinyl monomer EDMA is incorporated in the polymer nuclei (and consequently resulting monolith) at a higher rate in the early stages of the polymerization reaction.
Nitrogen adsorption is extensively used for determination of porous properties of monoliths in their dry state. However, it only has limited relevance since it requires different mold dimensions for porous polymer formation than that of a capillary-sized mold. Despite this limited relevance it allows a useful estimate of expected total surface areas of porous polymeric materials and the existence of a permanent mesoporous pore space at least qualitatively [36]. Table 2 shows the results of nitrogen adsorption experiments. The low surface areas are striking and typical for macroporous methacrylate-based monoliths [31]. The smaller size of the formed highly cross-linked globules in early stages of the polymerization and rapid phase separation explains the increased
Fig. 3. Scanning electron micrographs of the cross-section of porous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns obtained after a polymerization time of 30 min (left) and 48 h (right). (a) Complete cross-section, (b) bulk region, and (c) wall region.
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surface area at a terminated polymerization reaction [16,25,27]. Most of the drop in surface area and total pore volume occurs between 0.5 and 3 h polymerization time, i.e. after phase separation. However, the total pore volume of pores from 1.7 to 100 nm indicates the almost complete absence of (meso)pores in the polymer globules in their dry non-swollen state. This strongly confirms assumptions made recently [33]. 3.3. Microscopic structure Scanning electron micrographs of the porous polymer monoliths based on poly(butyl methacrylate-co-ethylene dimethacrylate) resulting in the separations shown in Fig. 1, are shown in Fig. 3. In both cases, these graphs indicate features typically observed for macroporous polymer monoliths. These features include the existence of relatively large flow through channels in the micrometer size as well as the globular topology of the phase-separated polymer. However, differences for varying polymerization times are discernable. The individual size of the inter-adhered polymer globules differs significantly and the porous structure with a terminated polymerization reaction shows much smaller features than that at a complete conversion. Overall the polymer monolith derived from a terminated polymerization is more porous with a smaller individual feature size (left hand side). The phase separated inter-adhered polymer globules increasingly lose their individuality and further polymerization leads to larger clusters. The result is a coarse monolithic polymer structure (right hand side). The disappearance of smaller cavities between these clusters is discernable at increased polymerization time. This explains the lower surface area found for the porous polymer at a complete conversion (Table 2 and Fig. 3). Close observation of the wall shows the occurrence of more dense and extended layers of polymer at a complete polymerization reaction (Fig. 3c). This layer has its origin in pendent methacrylate groups at the confining wall allowing chain growth during free radical polymerization. It avoids shrinking and enables covalent attachment of the monolith to the wall [15–17]. 3.4. Porous and hydrodynamic properties In order to relate the different microscopic topology presented in Fig. 3 to the hydrodynamic flow properties of the columns, we studied the superficial velocity-based hydrodynamic permeability kp,f of the monoliths in dependence of polymerization reaction time [21]: kp,f =
L u P sf
(1)
where L is the length of the column, P is the pressure drop, is the mobile phase viscosity, and usf is the superficial velocity. usf is referred to as the ratio of volumetric flow rate FV and the crosssectional area A of the column. The ratio of superficial velocity usf and linear chromatographic velocity uo (calculated from the residence time of non-retained uracil in nano-LC experiments), was used to determine the total porosity εt of the monolith [21]: εt =
usf uo
(2)
Fig. 4 shows a plot of kp,f and εt determined for monoliths in capillaries obtained at different polymerization times before the reaction was terminated. In Fig. 4a a trend to lower permeabilities is found with an increase in polymerization reaction time while rapidly approaching values close to that found at a complete polymerization reaction (average value of four independently prepared columns indicated by the dashed line). The higher values of permeability with a terminated polymerization reaction have their origin
Fig. 4. Hydrodynamic and porous properties of monolithic poly(butyl methacrylateco-ethylene dimethacrylate) columns in the confines of a 100 m I.D. capillary elucidated by nano-LC in dependence of polymerization reaction time. Mobile phase: 50% (v/v) acetonitrile in water. (a) Superficial velocity-based hydrodynamic permeability kp,f (Eq. (1)), (b) total porosity εt (Eq. (2)) measured with uracil as non-retained, totally permeating solute. Error bars reflect standard deviation from average values of four independently prepared and tested columns.
in a lower conversion of monomeric precursors and a much higher porosity as seen in Fig. 3b (left hand side). At the initial stages of the polymerization reaction, highly crosslinked nuclei are formed, that slowly form polymer globules, which rapidly phase separate in the poor porogenic solvents and become fixed in a continuous polymer globule structure (e.g. 0.5 h polymerization time in Table 1). Both new nuclei and phase separated globules are swollen with the remaining monomers of the polymerization mixture [27,37]. The proceeding polymerization largely takes place in the polymeric globules and in their vicinity. As typical for free radical polymerizations, further nucleation, chain growth, and cross-linking of existent polymer leads to macroporous coalesced and coarse globular structures (Fig. 3 right hand side). This leads to a drop in total porosity at further polymerization. The porosity values asymptotically approach that at a complete polymerization reaction (Fig. 4b). Fig. 5 shows the plate height curves of non-retained uracil for poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths resulting in the separations shown in Fig. 1 and the corresponding SEM images in Fig. 3. They most closely reveal the dynamics of flowdispersion through the macroporous polymer based monoliths shortly after phase separation to that after a complete polymerization reaction. We observe a typical shape of these curves with a minimum plate height of 15 m in both cases. This explains
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Fig. 5. Plate height curves of non-retained tracer uracil using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns obtained after a polymerization time of 30 min () and 48 h (). Mobile phase: 50% (v/v) acetonitrile in water.
the similar porous flow through structure of the monoliths with a micrometer-sized average channel diameter. The monoliths slowly lose their efficiency in fluid transport at increased linear flow velocities. The minimum value of the plate height compares well to that typically observed for macroporous methacrylate-based polymer monoliths under non-retained conditions [32,38,39]. It is also comparable to that of 13.7 m (73,000 plates/m) reported by Urban et al. for a 600 m2 /g hyper-cross-linked poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic capillary column [28]. The insensitivity of our reported plate height on the obvious difference in porous and hydrodynamic properties of the monoliths prepared in this work (Figs. 3 and 4) suggests the limited accessibility of structure-flow-transport relationships by using non-retained tracer. 3.5. Separation efficiency for retained analytes While the non-retained uracil only samples the heterogeneity of flow in the macroporous structure, Fig. 6 shows the plate height
Fig. 6. Plate height curves for non-retained tracer uracil () and retained benzene (), toluene (䊉), ethylbenzene (), propylbenzene (), butylbenzene (), and pentylbenzene () for the monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerized at 30 min polymerization time. Mobile phase: 70% (v/v) acetonitrile in water.
Fig. 7. Effect of chromatographic retention k on the plate height curves for retained analytes using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerized at 30 min polymerization time. (a) Plate height curve for nonretained tracer uracil () and retained benzene at a k = 0.4 () and 1 (䊉), (b) plate height curve for non-retained tracer uracil () and retained pentylbenzene at a k = 1.2 (♦) and 6 (), respectively.
curves additionally for retained benzene, and that of five alkylbenzenes. This is done with 70% (v/v) acetonitrile in the mobile phase and 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 -values from 0.37 to 1.2 significantly increases, in particular observed in the steeper slopes of the plate height curves at increased linear chromatographic velocities. This indicates significant mass transfer resistance originating from stagnant mass transfer zones in the porous structure. Interestingly, a decreased acetonitrile content in the mobile phase of 50% (v/v) results in even steeper slopes of the plate height curves for retained alkylbenzenes (Fig. 7). The determined plate heights reach up to 60 m for the least retained component benzene at k = 1 (Fig. 7a) and up to 100 m for the last eluting component pentylbenzene at k = 6 (Fig. 7b). This behavior practically contrasts that observed for typical silica-based materials with an almost retention-independent plate height [38]. The plate height at several linear chromatographic velocities uo 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 strongest retained analytes have the highest plate height, i.e. the lowest efficiency. Indeed, the curves show that the height equivalent to a theoretical plate displays a transition from the plate height observed for only slightly retained analyte (with a k close to zero) to
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Fig. 8. Plate height of the recorded elution bands of alkylbenzenes using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns polymerized at 30 min polymerization time in dependence of the retention factor k of the individual retained analytes and at different linear chromatographic velocities. Mobile phase: 50% (v/v) acetonitrile in water (solid symbols), and 70% (v/v) acetonitrile in water (open symbols). Symbols: uo = 0.43 mm/s (squares), uo = 0.86 mm/s (circles), uo = 1.29 mm/s (triangles), uo = 1.73 mm/s (hexagons), uo = 2.19 mm/s (pentagons).
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Fig. 9. Impact of volume percentage of acetonitrile in water (v/v) as the mobile phase on the retention factor k of a homologeous serious of alkylbenzenes using a monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) column obtained after 30 min polymerization time for benzene (), toluene (), ethylbenzene (), propylbenzene (), butylbenzene (), and pentylbenzene ().
that of strongly retained analyte (with a k larger three). In this case band broadening is controlled by arguments based on a distribution of the analytes between mobile liquid phase and (swollen) stationary polymer phase. The steepest transition is observed between k -values below 0.5 to a k = 3, while approaching plateau values at higher values of the retention factor. The graph also shows that an increased acetonitrile concentration allows achievement of lower plate heights, i.e. higher efficiency. This is associated with a significantly reduced retention of the alkylbenzenes (open symbols). The curves at both 70 and 50% (v/v) of acetonitrile in the mobile phase practically coincide, once equivalent k -values are obtained. This excludes significant effects of swelling on the performance of this monolith at different volume percentages of acetonitrile in the mobile phase. Interestingly, for all flow rates plate height values approach that of the non-retained tracer at retention factors approaching zero. The retention factor of the analytes for a variety of volume percentages of acetonitrile (v/v) in the mobile phase is shown in Fig. 9. It displays a linear decrease at increasing volume percentages of acetonitrile. It is the strongest for that of the strongest retained component pentylbenzene and significantly reduces the selectivity in the separation of alkylbenzenes at increased acetonitrile concentrations. Here, efficiencies come more close to each other due to low retention (Fig. 8). Fig. 9 confirms the typical reversed phase nature of the retention mechanism based on adsorption and partitioning [40]. 3.6. Impact of polymerization time on separation efficiency While we have demonstrated that porous polymer monoliths can be prepared with acceptable performance by a terminated polymerization reaction (Figs. 1b and 6) and involving separation conditions of strong retention (Fig. 8), we now focus our attention on the impact of polymerization reaction time on both retention factor k , as well as height equivalent to a theoretical plate. Fig. 10a shows the actual impact of polymerization reaction time in the capillary mold on the performance of macroporous polymer monoliths for both non-retained tracer uracil and three retained alkylbenzenes. This is done using 70% (v/v) acetonitrile in the mobile phase and at a linear chromatographic velocity of
Fig. 10. Impact of polymerization time on the plate height of non-retained tracer uracil and that of three retained analytes, as well as on the retention factor, using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns. Linear chromatographic velocity: uo = 0.5 mm/s, mobile phase: 70% (v/v) acetonitrile in water. (a) Plate height, (b) retention factor. Symbols: uracil (), benzene (), ethylbenzene (), and pentylbenzene (♦). Error bars reflect standard deviation from average values of four independently prepared and tested columns.
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uo = 0.5 mm/s (realized by proper adjustments in flow rate). The associated plate height for retained analytes is smallest at a minimum polymerization time of 30 min used in this work. Under this condition the retention-dependency of the plate height is also minimized and very similar plate heights for both non-retained and only slightly retained analytes are observed. Increased polymerization times lead to a significant increase in both retention (Fig. 10b) and plate height (Fig. 10a). At polymerization times exceeding 3 h plate heights of more than a 100 m at retention factors of 0.92–3 are observed. Interestingly, the increase in k levels off at polymerization reaction times exceeding 3 h. This indicates the advanced stage of the polymerization reaction and a monolith coming close to its final chemical composition (see Table 1). However, the increased band broadening at increased polymerization times undermines the similar selectivity for the elution of alkylbenzenes. This, at some point, results in a loss of resolution. The same effect was already demonstrated in Figs. 1 and 2 with an almost 10 times higher linear chromatographic velocity. Most importantly, polymerization reaction time has no detrimental influence on the band dispersion of the non-retained tracer uracil. It reflects the actual flow heterogeneity in the porous structure (Figs. 10a and 5). 3.7. Discussion We report the fabrication of a typical macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith by a copolymerization reaction involving free radical initiation, nucleation, globule formation, phase separation and completion of the reaction. It is well known that the copolymer structure of any polymer derived from initiation, propagation, cross-linking, and termination reactions depends at every moment of the reaction on the relative comonomer concentrations and on their reactivity [41]. Our homogeneous single phase polymerization mixture comprises the monovinyl monomer BuMA and divinyl monomer EDMA in alcoholic porogenic diluents and initiator. The higher degree of functionality in divinyl monomer EDMA leads to higher reactivities than that of BuMA for a given free radical concentration [42]. In due course of nucleation the divinyl monomer EDMA is incorporated in the polymer nuclei at a rate higher than that of the monovinyl monomer BuMA. As the polymerization reaction progresses (including new initiation, and growth of the size of polymer nuclei) the degree of cross-linking in the polymer network (nuclei that slowly form globules) changes considerably. The initial high degree of cross-linking in nuclei, leading to globule formation, results in a rapid phase separation in the early stages of the polymerization reaction [16]. At this stage of polymerization the individual coalesced polymer globules in the polymer matrix have a high degree of cross-linking (Table 1) [27,42]. Polymerization, and hence globule growth, takes place after phase separation in the phase separated globules and their close vicinity since the remaining monomers are thermodynamically good solvents for the polymer. The globule growth is shown in Fig. 3. Also, at this stage of the polymerization the divinyl monomer EDMA available for further cross-linking reactions is depleted (Table 1) [42,43]. Consequently, the globule becomes softened by slightly cross-linked chains. It is therefore suggested, that the globule shows a crosslink density distribution [42]. The outer regions of the globules do not contain a permanent uniform network of small pores with a defined size. This is indicated by our nitrogen adsorption experiments (Table 2). Such polymer monoliths swell when in contact with the hydro-organic solvents used in nano-LC. The resultant gel-like interfaces allow the permeation of small hydrophobic molecules. The diffusion-limited transport of small molecules in the gel matrix with non-uniform gel porosity is additionally hindered by polymer chain movement and adsorptive interactions. The resulting increased dispersion can be rationalized in the steep
slopes of the plate height at increased mobile phase velocities. This is even seen with a terminated polymerization reaction (Fig. 7). At an advanced stage of the polymerization, large heterogeneous globular structures with a significant amount of gel porosity are formed. As a consequence the increased band dispersion for retained analytes slowly deteriorates the separation (Fig. 10a) and results in a totally unsuitable material for small molecule separation at high flow speed and retention (Fig. 1a). 4. Conclusions Results presented in this study strongly suggest, that the apparently small surface area of typical porous polymer monoliths is not responsible for their poor performance in the separation of small molecules. Surface area provides capacity, retention, and consequently selectivity. Retention and selectivity are observed with low surface area porous polymer monoliths incorporating a simple monomer such as BuMA. We have shown that the poly(butyl methacrylate-co-ethylene dimethacrylate) monolith provides sufficient methylene selectivity in the separation of a homologeous series of alkylbenzenes. However, this selectivity is undermined by the strong band dispersion for retained analytes. This is attributed to the significant amount of gel porosity in the polymeric skeleton and particularly on its surface. The gel porosity stems from the monolith preparation procedure. The preparation involves copolymerization, rapid phase separation followed by further chain growth with only slight cross-linking. This leads to heterogeneous (globule scale) polymer phases with varying gel porosity and originates from a radial crosslink density distribution in globules. This is typical for macroporous copolymer networks derived from free radical polymerization and being controlled only to a limited extent. The resulting gel porosity, again not present in the dry polymer, introduces strong mass transfer resistance for retained small analytes reflected in the broad analyte bands observed. Our experiments indicate that the actual microheterogeneity of the individual polymer globs (size and shape reflected in a certain macropore size distribution) has little influence on the poor performance obtained for retained small analytes. The non-retained tracer uracil shows acceptable dispersion for a material of such high permeability. More controlled chain growth and cross-linking reactions are desirable to keep the gel porosity to a minimum. This may be possible by controlled polymerization techniques for more uniform cross-linking throughout the globules. This would also allow more controlled phase separation and termination of the polymerization reaction at the desired stage and would drastically increase control over the whole process. Hopefully our study will spark further interest in the study of morphological aspects, as well as globule-scale crosslink homogeneity which is important to minimize band broadening for small retained analytes. Reported plate heights appear most meaningful at sufficiently high retention factors. Acknowledgement The authors thank Dr. Ian Teasdale, Institute of Polymer Chemistry at Johannes Kepler University Linz, for proofreading of the manuscript. References [1] [2] [3] [4] [5] [6] [7]
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