Novel method to prepare polystyrene-based monolithic columns for chromatographic and electrophoretic separations by microwave irradiation

Available online at www.sciencedirect.com

Journal of Chromatography A, 1188 (2008) 43–49

Novel method to prepare polystyrene-based monolithic columns for chromatographic and electrophoretic separations by microwave irradiation Yu-Ping Zhang a,∗ , Xiong-Wen Ye b , Meng-Kui Tian a , Ling-Bo Qu b,∗ , Seong-Ho Choi c , Anantha Iyengar Gopalan d , Kwang-Pill Lee d a Henan Institute of Science and Technology, Xinxiang 453003, China Department of Chemistry, Zheng Zhou University, Henan 450052, China c Department of Chemistry, Hannam University, Daejeon 305-811, South Korea d Department of Chemistry Education, Kyungpook National University, Daegu 702-701, South Korea b

Available online 26 November 2007

Abstract Microwave irradiation can provide a viable alternative to the traditional means such as ultraviolet light and thermal initiation for the preparation of monolithic capillary columns. Polystyrene-based monolithic stationary phases were prepared in situ in fused-silica capillaries and simultaneously in vials. The column permeability, electrophoretic and chromatographic behavior were evaluated using pressure-assisted capillary electrochromatography (pCEC), capillary electrochromatography (CEC) and low pressure liquid chromatography (LPLC). With an optimal monolithic material, the largest theoretical plates for preparing the column could be close to 18,000 plates/m for thiourea in the mode of pCEC. Furthermore, the influence of the composition of the porogenic solvents (toluene/isooctane) on the morphology of organic-based monoliths [poly(styrene-divinylbenzenemethacrylic acid)] was systematically studied with mercury intrusion porosimetry and scanning electron microscopy. The monoliths which were prepared with a high content of isooctane had a bigger pore size and better permeability, and hence resulted in a faster separation. © 2007 Elsevier B.V. All rights reserved. Keywords: Microwave irradiation; Monolithic columns; Pressure-assisted electrochromatography; Low pressure liquid chromatography; Polymerization

1. Introduction Monolothic columns with continuous and porous structures are expected to posses unique properties such as fast separation, high-linear flow velocities and nonrequirement of frits [1–3] and production of such columns is one of the most required competitive chromatographic column technologies. Mostly, the organic polymer-based monolithic columns are prepared ‘in situ’ through UV or thermal polymerization routes [4–7]. The term ‘photopolymerization’ generally refers to the use of electromagnetic radiation for the initiation of polymerization. Although ultraviolet light and thermal initiation are mainly used for this purpose, polymerization can also be induced by ionizing radiation (e.g., electron beam, ␥- and X-ray), infrared, microwave or ultrasound [8–11], etc. Monolithic stationary phases prepa-

∗

Corresponding authors. Tel.: +86 373 3040861; fax: +86 373 3040861. E-mail address: [email protected] (Y.-P. Zhang).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.10.068

red from hydrophobic, hydrophilic or charged monomers can be employed for a wide range of applications. Polystyrene-based stationary phases have been proved to be an excellent stationary phase with outstanding chemical stability in a broad range of pH values and used widely as matrix for separation columns [12,13]. Xiong et al. [14] and Jin et al. [15] have reported on the preparation of a ternary, negatively charged, porous polymer monolithic column. The column consists of polymers of styrene (used as the monomer), divinyl benzene (cross-linker) and methacrylic acid [support to generate electroosmotic flow (EOF)] and prepared in toluene and/or isooctane as porogenic solvent(s) using azobisisobutyronitrile as the initiator. Recently, Huang et al. also fabricated two kinds of polystyrene-based monolithic columns for the separation of a few acidic analytes by capillary electrochromatography [16,17]. The capillaries mentioned in the above studies must be submerged in a water bath maintained at 70 ◦ C for several hours. Such a heating process used for the activation purpose presents a few disadvantages. A long activation is needed due to the slow convection of heat. The preparation

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of UV-transparent coated capillaries by photopolymerization is relatively more expensive in comparison to the preparation of conventional polyimide coated capillaries, yet the use of microwave heating for the coating of capillaries seems to have the advantages of being volumetric, direct, selective and instantaneously controllable [18,19]. In the case of microwave heating, the molecules that are microwave absorptive receive the radiation and hence a maximum proportion of molecules (excepting the molecules which are in the deep) contribute to the reaction and the coating process. Herein, we are reporting on the preparation of porous monolithic columns based on polymers of styrene (St), divinyl benzene (DVB) and methacrylic acid (MAA) using microwave irradiation. To the best of our knowledge, this is the first attempt of this sort. The porous properties of the monoliths (St–DVB–MAA) prepared by varying the conditions such as different ratios of St, DVB and MAA, volume ratios of pore forming solvents, etc., were analyzed. The chromatographic and electrophoretic behavior of the studied monolithic columns (St–DVB–MAA) were comparatively evaluated through capillary electrochromatography (CEC), pressure-assisted CEC (pCEC) and low pressure-driven liquid chromatography (LPLC) in this work. Baseline separation of some typical neutral compounds could be obtained for a few of the selected model compounds (thiourea, benzene, toluene, ethyl benzene, biphenyl and naphthalene) using the prepared monolithic columns under the optimal conditions. The present strategy of using microwave heating for the preparation of monolithic columns offers an alternative to the traditional methods for the preparation of monoliths. 2. Experimental 2.1. Instrumentation All CEC experiments were performed on a Agilent 3D CE system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector and the capability to apply up to 12 bar pressure to one or both ends of the capillary. The rinse of all prepared monolithic columns was carried out using a HP1100 Series HPLC system (Agilent Technologies) equipped with a quaternary pump. Microwave irradiation step was carried out in a home made microwave oven (Midy Co., Guangdong, China) with a microwave output power of 700 W and a frequency of 2450 Hz. Porosity data were obtained by using PM-33-11 Poremasters (Quantachrome Instruments, FL, USA) for low-pressure and high-pressure analysis, respectively. An FEI QUANTA 200 Scanning Electron Microscope (Philips-FEI, The Netherlands) was used to study the morphology of the monolith. A capillary with the monolith was sectioned into 10 mm segments. These segments were sputtered with gold prior to scanning electron microscopy (SEM) analysis. 2.2. Materials and chemicals Fused-silica capillaries (75 ␮m inside diameter, 375 ␮m outside diameter) were purchased from Yongnian Ruipu Optic Fiber Plant (Yongnian, Hebei Province, China). DVB was from

Tokyo Chemical Industry (Tokyo, Japan); methacryloxypropyltrimethoxysilane (MPTMS), St, MAA, acetonitrile (ACN), ammonium acetate, thiourea, benzene, toluene, ethylbenzene, biphenyl and naphthalene were purchased from Beijing Bailingwei Chemical Reagent Co. and Tianjing Chemical Reagent Co., China. Distilled water was obtained from a super-purification system (Danyangmen Corp., Jiangshou, China). The buffers used in the experiments were prepared with various ratios of 50 mM ammonium acetate, water and ACN, all solutions were degassed with ultrasonication and filtered through a membrane (0.45 ␮m) before use. In a typical chromatographic and electrophoretic experiment, aromatic hydrocarbon compound was dissolved in methanol and injected for peak identification. 3. Results and discussion 3.1. Preparation of the monolithic column Initially, the capillary was pretreated to create binding sites for the attachments of the polymeric materials to the walls of the capillary. Thus, before filling the reactants into the capillary, it was pretreated using the following procedure. Firstly, the capillary column with a length of 40 cm was rinsed with 1 M NaOH for 30 min and then with 0.1 M HCI for 30 min. Subsequently, the capillary was flushed with H2 O for about 30 min and it was dried by passing nitrogen gas. The purpose of capillary pretreatment is to increase the concentration of surface silanol groups. Since silanol groups on the capillary surface represent the principal binding sites for in situ created polystyrene-based stationary phases, higher concentration of these binding sites on the capillary surface would facilitate the coating formation of highly secured organic-based stationary phases through chemical bonding with the capillary inner walls. Monolithic capillary columns were fabricated in situ in 75 ␮m polyimide-coated capillaries whose internal walls had been modified by [3-(methacryloyloxy)propyl]trimethoxysilane solution through a procedure described elsewhere [20,21]. Thereby, Si O Si C bonds were formed between the capillary wall and the reactive methacryloyl groups which are available for subsequent attachment of reactant to the wall. Then the capillaries were partially filled with the polymerization mixture, which consists of 1.0 mL monomer (St, 33.3%, v/v), crosslinker (DVV,33.3%,v/v), MAA(33.3%) for generating EOF, and 4.0 mL porogenic solvents with various ratio of toluene and isooctane. Furthermore, AIBN (about 1.0%, w/v, of the monomer) was added to produce free radicals for the initiation of the polymerization of ethylenically unsaturated monomers. The partial filling of the capillary was achieved as follows: it should be noted that the liquid levels in the capillary could be observed under the daylight lamp when the solution was slowly forced through a syringe into the capillary. Hence, the liquid level in the capillary could be carefully controlled to precision into any of the designated position and the monolith length could be measured with a ruler. The compositions of polymerization mixture used for the preparation of monoliths are listed in Table 1. In a series of experiments, the ratio of the volume fractions of the monomer mixture (V1 ) to the porogenic solvent (V2 ) was

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Table 1 Composition of polymerization mixture and condition for the preparation of polystyrene-based monolithic columns (MC) MC

1 2 3 4 5 6 7 8 9

Monolith length (cm)

Porogenic solvent (V2 ) Toluene

Isooctane

15 10 15 10 15 10 6 10 6

1/3 1/3 1/2 1/2 2/3 2/3 2/3 1 1

2/3 2/3 1/2 1/2 1/3 1/3 1/3 0 0

Ko × 10−14 (m2 )

7.7 9.5 12 7.6 – – 0.9 – 0.5

kept constant (1:4, v/v). While the volume ratios of the binary pore-forming solvents (toluene/isooctane) were varied in our experiments. The mixture was homogeneously mixed through sonication for 20 min to obtain a homogeneous mixture, and then purged with nitrogen for 10 min before filling into the capillaries. After the pretreated capillary was partially filled with the mixture to a set position and sealed the capillary was sealed at both ends with glue and rubber stoppers and the monomer filled capillary was then irradiated for about 15 min in a home microwave oven using an output power of 350 W. Upon microwave irradiation, free radical polymerization occurs and the carbon-carbon double bonds were destroyed, the molecule itself becomes highly reactive and links itself to another highly reactive molecule leading to the formation of very long macromolecules Thus, the liquid monomer filled in the capillary changes into a solid polymer that can have properties totally different from the liquid monomer. Before CEC experiments, the capillaries were flushed with mobile phase for 30 min. A preconditioning step was performed by applying a stepwise increase in voltage up to 30 kV over the column, until a stable current was observed. Simultaneously with the polymerization in capillaries, by adopting a similar procedure, a polymerization was carried out with the same mixture in a glass vial. The formed polymer was cut into small pieces with a razor blade, and Soxhlet extraction was carried out with methanol for 24 h. After drying at 50 ◦ C for 4 h, mercury intrusion porosimetry and scanning electron microscope experiments were performed on the monolithic materials. 3.2. Column performance study with pCEC, CEC and LPLC According to the theory of nucleation and phase separation, polymerization reaction time has an important influence on the pore and channel size, and specific area of monoliths [22,23]. In the present study, the polymerization within the capillary was carried out for a shorter duration of 15 min by using the microwave irradiation and hence longer polymerization time is not required as in traditional coating methods. Furthermore, no additional derivatizations on the surface of monolith surface are required. After columns 1–9 in Table 1 were prepared, they were connected to the HPLC pump for flushing. Columns 5, 6 and 8 prepared with a higher proportion of toluene in the porogenic

Fig. 1. Schematic diagram of pCEC, CEC and LPLC operation modes.

mixture and with longer monolithic length, created a high back pressure and thus were unsuitable for further chromatographic evaluation. Electrochromatographic and electrophoretic performance of other columns were evaluated in modes of pCEC, CEC and LPLC. Fig. 1 shows a visual illustration of the three operation modes. In the mode of p-CEC, the pressure was applied only at the sample inlet and a separation with a simultaneous operation of voltage was simultaneously added between the inlet and outlet. In this mode, an EOF caused by the voltage is superimposed on a pressure-induced hydrodynamic flow. Low pressure and EOF were simultaneously created for accelerating the velocity, which could improve the separation efficiency and also to avoid bubble formation. For the CEC mode, a pressure of 10 bar was applied at both ends and a separation voltage was simultaneously operated between the capillary inlet and outlet. For the LPLC or low pressure-driven mode, a pressure of 10 bar was only applied at the inlet (the maximum limit of the Agilent 3D CE system is 12 bar), without loading a high voltage between the capillary inlet and outlet. The column performances were evaluated by evaluating the monolith permeability in terms of retention time. A reversed-phase mechanism was observed for the separation of analyte in the three modes using the columns, 1–4, 7 and 9. The partitioning of solution between the mobile and stationary phases is the main cause for the retention of the model compounds. The trend noticed in the elution order of the columns is similar to that of reversed-phase chromatography. The analytes with larger higher molecular weight or more hydrophobic analytes were eluted later as compared to the analytes with smaller lower molecular weight or more hydrophilic analytes. Morphology of the monolithic material has a profound effect on the column performance. Hence, it is essential to optimize the composition of polymerization mixture. Here, toluene and isooctane, the binary porogenic solvents used for the preparation of monoliths in our experiment are expected to perform the dual roles, as a pore template and a solubilizer [16,17,23]. The permeability of the analytes depends on the pore size, the ratio of toluene and isooctane. While preparing the columns, the composition

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Fig. 2. Separation of model compounds using MC 1 in the modes of pCEC, CEC and LPLC. Injection: 5 kV, 3 s; 200 nm; 20 ◦ C; buffer, 50 mM ammonium acetate:water:acetonitrile = 1:4:5 (v/v/v); pH 7.12. p-CEC running with 1.0 MPa (inlet) +20 kV; CEC running with 1.0 MPa (both) +20 kV; LPLC running with 10 bar (inlet). Peak identification: 1, thiourea; 2, benzene; 3, toluene; 4, ethyl benzene; 5, biphenyl; 6, naphthalene.

of St, DVB and MAA was kept constant, and while the volume ratio between toluene and isooctane was varied. It was observed that columns prepared with the increase of toluene volume fraction, showed difficulty for the permeation of the analyte. Only for a few appropriate volume ratios of the two solvents, the columns showed adequate permeability because of the pore sizes. A porous polymeric material with larger pores was formed for a 1:2, v/v, ratio of toluene to isooctane, see Fig. 2. Baseline separation of all model compounds could be achieved with a retention time of 5.45, 9.20 and 12.49 min, respectively, for pCEC, CEC and LPLC modes. For the monolith prepared within the column for a volume ratio of 1:1 for the porogens, faster retention times of 3.35, 6.96 and 6.28 min, respectively, were observed for the three modes (Fig. 3). At this condition, the polarity of the toluene determines the pores formation and hence the monolith may be macroporous. Further, the monoliths

Fig. 3. Separation of model compounds using MC 3 in the modes of pCEC, CEC and LPLC. Operation condition and peak identification of three modes are the same as Fig. 1.

Fig. 4. Separation of model compounds using MC 9 in the modes of pCEC, CEC and LPLC. Operation condition and peak identification of three modes are the same as Fig. 1.

prepared with a higher toluene proportion did not have good permeability and hence the separation was not good. The monolith prepared with toluene alone as porogen resulted high back pressure and had a much longer retention time (over 100 min) in the LPLC mode, see Fig. 4. The effect of monolithic length in the capillary on the retention time was studied for different lengths (15, 10, 6 and 0 cm from the window to the inlet). In the absence of monolithic material within the capillary, no resolution is possible in the mode of capillary zone electrophoresis. As the monolithic length decreases, a gradual decrease in resolution and a shorter retention time were observed naturally. However, to our surprise, baseline separation of the test mixture was still achieved for the column, MC 7, with a length of 6 cm in Fig. 5. It should also be noted that baseline separation could still be achieved for MC 2 and MC 4 in the three modes (not shown). The monoliths prepared in the presence of similar volume ratios of porogenic solvents but with a longer monolithic length (columns MC 5 and MC 6) were easily blocked during the operation. A constant current is expected throughout the column irrespective of the length of

Fig. 5. Separation of model compounds using MC 7 in the modes of pCEC, CEC and LPLC. Operation condition and peak identification of three modes are the same as Fig. 1.

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monoliths. Thus, from the data it is inferred that only the mobile phase, and not the monolithic material, influences the conductance in the modes of p-CEC and CEC. In a partially packed column, there are differences in the conductivity between the packed and open segments exhibiting different conductances, and hence, different electric field strengths are produced in the column. This behavior leads to a mismatch of the local electroosmotic flow velocities between the packed and open sections, which ultimately causes a decrease in column efficiency. However, to our surprise, the electric field strength is nearly invariant in the entire portion of partially filled polystyrene-based column. It follows that the electroosmotic flow velocity is constant throughout the column, so that broadening effects from velocity variations are not expected to be predominant. The largest theoretical plates for the prepared columns could be close to 18,000 plates/m for thiourea in the mode of pCEC. The flow-through properties of the monolithic columns were comparatively evaluated by determining the chromatographic permeability (Ko ) of the column [20], which is defined as Ko =

uηL P

where u is the linear velocity, η is the viscosity of the mobile phase, L is the column length and P is the pressure drop across the column. It is shown in Table 1 that the permeability Ko of polystyrene-based monolith was comparatively estimated to be in the range of 0.5 ∼ 12 × 10−14 m2 (Table 1). Similar Good permeability was noticed for MC 1–4. Comparatively lower chromatographic permeability values were observed for MC 7 and 9. Furthermore, MC 5, 6 and 8 were easily blocked in the procedure of rinse. 3.3. Tailoring the morphology of monoliths It is strictly required to control of the morphology of the monolithic stationary phase to obtain a generic porous monolithic material that provides a good separation efficiency and a low resistance to flow. The latter is of prime importance, since it enables easy flushing of the column with liquids that are used in the subsequent separation in the modes of pCEC, CEC and LPLC. The key variables that could allow the control of the pore size are the percentage of cross-linking monomer (DVB) and the composition of the porogenic solvent. The composition of porogenic solvent in the polymerization mixture could be conveniently adjusted to control the pore size distribution because it does not change in the total amount of the monomers in the polymerization process. In the present study, the influence of the composition of porogenic solvents on the morphology of the resulting material was studied. Monoliths were prepared in situ in the fused-silica capillaries and in bulk as well by microwave initiation. Previous researchers have used different ratios of St, DVB and MAA for the preparation of the monoliths following a similar recipe, which was developed by several groups [14–17]. But in our experiment, the ratio of the weight fractions of the monomers and the solvent in the polymerization mixture was kept constant, but the composition of pore-forming solvent was varied in order to adjust systematically the average size

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Fig. 6. Typical pore-size distribution of polystyrene-based monoliths determined by mercury-intrusion porosimetry. 1, Toluene; 2, toluene:isooctane = 2:1; 3, toluene:isooctane = 1:1; 4, toluene:isooctane = 1:2.

of polystyrene-based monoliths by changing the percentage of toluene with (a polarity of 2.4) and isooctane with (a polarity of 0.1). On increasing the polarity of the mixture, by increasing the toluene volume fraction, monoliths with smaller pores resulting in a strong decrease of the average pore size were obtained. An explanation for this observation could be as follows [24,25]: The pores in the polymer are formed during the polymerization. During the polymerization with a mixture of ST, DVB and MA mixture (St–DVB–MAA) with isooctane and or toluene as the porogen solvent, the combined volume of St, DVB and MAA was kept as 20% of the total polymerization mixture in order to impart pore stabilization and rigidity to the polymer structure and also to prevent pore collapse. The porogens such as toluene and/or isooctane, is/are chosen to control the pore size distributions. The monoliths are macroporous when the solubility parameter of porogen is nearer to the solubility parameter of the polymer. Accordingly, the proportion of pores is necessarily limited. Fig. 6 shows pore-size distribution curves for these monolithic materials in Table 1. With the decrease of vol.% of toluene volume in the total proportion of porogenic solvents, the pore size increased apparently. All these monoliths exhibit unimodal pore-size distributions with no pores smaller than 200 nm. The pore size at the apex of the distribution curve could be controlled for a wider range by adjusting the isooctane/toluene ratio. A decrease in the toluene content in the pore-forming solvents from 100 to 30% leads to an increase in pore size from 279 to 8883 nm. It should be noted that mercury-intrusion porosimeter was used and the monoliths prepared in larger glass tubes with a microwave irradiation time of 15 min. The environment for the preparation of monoliths distinctly varies between “in situ” in the capillary and “bulk” in the glass vials. Since the different environment in tube and in capillary has a distinct effect on the morphology, the pore size of the bulk monolith determined by mercury porosimetry may not correspond to that of a monolith prepared in capillaries. Therefore, mercury porosimetry data can be used as the general trend than considering as the absolute pore size of the actual chromatographic monoliths [26]. The SEM micrographs in Fig. 7 revealed a close association between the polymers and the capillary walls, which is to be expected if covalent bonds are indeed formed between the

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Fig. 7. SEM images of the porous monolithic columns (see Table 1 for detailed description of porogenic composition). The scale bar corresponds to 75 ␮m (10 or 20 ␮m in the inset).

walls and the polymer. Denser monolithic beds (MC 7 and 9) are less permeable and higher pressures are needed to drive the liquid flow through [27,28] the column. The monoliths (MC 1–4) had a low flow resistance and this may presumably be attributed to availability of macroscpic pores with a pore size in the range of 4327–8883 nm. Superficially, column permeability may not seem to play a dominant role irrelevant to the CEC separation since EOF is the predominant driving force in a CEC to propel the mobile phase through the column without requiring mechanical pressure [29]. But, in the case of MC 9 with too dense monolithic bed, the last analyte took an elu-

tion time over 30 and 60 min, respectively, in the modes of pCEC and CEC for the separation. However, columns with high permeability have significant advantages especially in pCEC operation, LPLC operation, sample injection or quick flushing of the capillary during column regeneration or equilibration. Thus, the preparation of monoliths with a suitable pore size is still the key for obtaining efficient separation of capillary LPLC and CEC. The macroporous monolithic structures facilitate the mobile phase flow through the pores, and thereby, promote effective solute/stationary phase interactions by bringing them together. Effective solute transport mechanism that may ope-

Y.-P. Zhang et al. / J. Chromatogr. A 1188 (2008) 43–49

rate within this monolithic structure due to mobile phase flow through the macropores together with the flat flow profile of EOF is the main reason for the high speed and separation efficiency in pCEC. 4. Conclusions An easy and method for the preparation of polystyrene-based monolithic columns using microwave initiation has been firstly attempted with the polymerization time to be shortened from 24 h (thermal initiation) to 15 min. Importantly, bubble formation is not observed during any of the chromatographic and electrophoretic runs. The microwave prepared monoliths are well suited for the separation of a variety of neutral compounds by both voltage-driven and pressure-driven systems. Our intention in this report is simply to demonstrate that microwave initiation is feasible and facile for making the monoliths of St–DVB–MAA. The present method offers a simple alternative to the tedious one. It exhibited good potential instead of the traditional thermal and UV light initiations. The main advantage of microwave irradiation as an energy source is its short reaction time, ease of the preparation required and lower expense. Monoliths can probably be developed for micro-HPLC, HPLC and even for preparative HPLC columns (not steel). However, further intensive studies are needed and are partly ongoing in order to better understand the microwave heating for the monolith preparation in terms of generation and distribution of the radiation energy within the narrow capillaries, energetic of the interactions between the monoliths and energy, reaction speed, morphological control, etc. Acknowledgements The financial supports by Joint Research Project under the KOSEF (F01-2006-000-10119-0) and KRF-2006-C00001, South Korea) and NSFC (China) Cooperative Program (No. 20611140646), Program for New Century Talents in University of Henan Province (2006HNCET-01) and Program for Backbone Teacher in Henan Province of China are gratefully acknowledged.

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