Styrene-Based Monoliths

Beside borosilicate and fused silica capillaries, PS/DVB monoliths have been fabricated within the confines of steel and PEEK tubings [52]. In order to increase the hydrophobic character of the supports, a Friedel-Crafts alkylation reaction was used for the attachment of Cig-moieties to the polymer surface. The derivatized material was demonstrated to be more retentive and to provide more efficient peptide separations compared with the original, nonderivatized monolith. 

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In contrast to the acrylamide and styrene-based monoliths that have largely been characterized by their chromatographic performances, extensive materials development and optimization have been performed for monolithic CEC capillaries prepared... 

Lv, Y, Lin, Z., Tan, T., and Svec, F. Preparation of porous styrenic-based monolithic layers for thin layer chromatography coupled with matrix-assisted laser-desorption/ ionization time of flight mass spectrometric detection, J. Chromatogr. A, 1316,154-159, 2013. 

Since the hydrophobicity of styrene- or alkyl methacrylate-based monolithic matrices is too high to make them useful for hydrophobic interaction chromatography, porous monoliths based on highly hydrophilic copolymers of acrylamide and methylenebisacrylamide were developed [70,135]. The hydrophobicity of the matrix required for the successful separations of proteins is controlled by the addition of butyl methacrylate to the polymerization mixture. The suitability of this rigid hydrophilic monolith for the separation of protein mixtures is demonstrated in Fig. 21, which shows the rapid separation of five proteins in less than 3 min using a steeply decreasing concentration gradient of ammonium sulfate. 

Three main types of polymer-based monoliths are polymethacrylate-based monoliths where methacrylate forms the major component of the monomers for polymerization, polyacrylamide-based monoliths where cross-linked polyacrylamide is synthesized directly within the capillary, and polystyrene-based monoliths that are usually prepared from styrene and 4-(chloromethyl) styrene as monomers and divinylbenzene (DVB) as the cross-linker. 

Fig. 6.16. SEM micrographs of poly(styrene-divinylbenzene) based monolithic capillary. (Reprinted with permission from [49]. Copyright 1999 Elsevier).

During the past 10 years, in addition to silica-based monoliths [12], a broad range of organic polymeric monoliths has been studied. Their most advantageous attribute is their chemical stability over a wide pH range. The most common organic monoliths were the results of methacrylate [13] and styrene [14] monomers. Some examples that confirm the utility of monolithic columns in IPC are described below. 

One can view th e monoliths as a single big porous particle. Thus, some of the preparation procedures use similar ingredients as the procedures used to make tnacroporous particles by suspension polymerization. Consequently, the structures of the monoliths are similar to the pore structure of macroporous particles, as can easily be seen by electron microscopy. Also similar chemistries are available, including styrene- ivinylbenzene and methacrylates, which have been proven to form sufBciently rigid structures to be useful in HPLC. But the tedmology of the formation of the monoliths is less constrained than the suspension polymerization used to form particles, and thus a broader range of chemistries is available. The classic monoliths were based on polyurethanes (20). Recently, silica-based monoliths were formed in a capillary (24). 

More recently, columns have been developed where the stationary phase is formed of a porous polymer network inside the capillary. These are called monolithic phases, and have emerged as an alternative to traditional packed bed columns for use in micro-HPLC. They hold many advantages over traditional packed bed columns, being easy to manufacture since the monolith is formed in situ, often via a one-step reaction process, and its properties such as porosity, surface area, and functionality can be tailored. Another major advantage is that they eliminate the need for retaining frits. These columns can be manufactured from a variety of materials, but the most common include sol-gel, methacrylate-based, acrylamide-based, and styrene-based polymeric structures. 

All monolithic disks reported so far were methacrylate-or styrene-based. Because of that, they have a rigid structure, which minimizes compression when high flow rates are apphed. This is confirmed by a Unear correlation between the pressure drop and the hnear velocity. For the commercially available CIM disks of 12 mm diameter and 3 mm thickness, it was found that there is a pressure drop of about 0.5 bar per each ml/min. A low-pressure drop is very important for working at high flow rates. Because the mass exchange is not a limiting factor, the gradient can be adjusted in a way to optimally match the thickness of the monohthic disk. In this way, efficient separations in a very short time can be obtained. The... 

A higher content of divinyl monomer directly translates into more cross-linked polymers in the early stages of the polymerization process and, therefore, lead to an earlier phase separation. This approach is predominandy used for the preparation of monoliths with very large surface areas [54]. Typical cross-linking monomers used today are ethylene dimethacrylate for methacrylate-based monoliths and divinylbenzene with styrenic monomers. 

The most widely used polymer monoliths are poly(styrene-co-divinylben-zene)-based monoliths and moderately polar monoliths based on methacrylic acid esters. While the first ones are strongly hydrophobic materials prepared by polymerization of styrene and its derivatives with divinylbenzene as the cross-linking agent [323,324], methacrylate-based monoliths are synthesized by polymerization of butyl methacrylate or other methacrylic acid esters with ethylene dimethacrylate as the cross-linking agent [325,326]. These types of monolithic media in capillary format are the main focus of a very detailed review by Urban and Jandera [327]. 

Table 7.1 shows the pore properties of several polymer monolithic columns prepared from styrene/DVB, methacrylates, and acrylamides along with the feed porosity and column efficiency, summarized from several recent publications. Some important points seem to be clearly shown in Table 7.1, especially by the comparison of the properties between methacrylate-based polymer monoliths and silica monoliths. 

Recent chromatographic data indicate that the interactions between the hydrophobic surface of a molded poly(styrene-co-divinylbenzene) monolith and solutes such as alkylbenzenes do not differ from those observed with beads under similar chromatographic conditions [67]. The average retention increase, which reflects the contribution of one methylene group to the overall retention of a particular solute, has a value of 1.42. This value is close to that published in the literature for typical polystyrene-based beads [115]. However, the efficiency of the monolithic polymer column is only about 13,000 plates/m for the isocratic separation of three alkylbenzenes. This value is much lower than the efficiencies of typical columns packed with small beads. 

Fig. 18a-c. Base peak chromatograms for the LC/MS analyses of a cytochrome c Lys-C digest (0.7 pmol injected) on a a poly(styrene-co-divinylbenzene) monolith-filled needle b Vydac C18-packed needle c Poros R2-packed needle. (Reprinted with permission from [128]. Copyright 1998 American Chemical Society)... 

Table 1.1 gives a comprehensive, albeit fragmentary, snmmary of investigated organic monolithic polymer systems (based on all different kinds of styrene, acrylate, methacrylate, (meth)acrylam-ide building blocks, as well as mixtnres thereof) together with their preparation conditions and ntilization as stationary phase. 

A monolithic silica-based CIS stationary phase was used under high flow rate condition (2 mL/min) without significant back pressure in IPC analysis of a recently discovered new drug candidate for the treatment of Alzheimer s disease [15]. Nanoscale IPC using a monolithic poly(styrene-divinylbenzene) (PS-DVB) nanocolumn coupled to nanoelectrospray ionization mass spectrometry (nano-ESl-MS) was evaluated to separate and identify isomeric oligonucleotide adducts. Triethylammonium bicarbonate was used as the IPR. Interestingly, the performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents CIS or PS-DVB [16]. 

A typical mobile-phase composition is an acetonitrile-water gradient with a fixed concentration of trifluoroacetic acid (TFA), formic, or acetic acid (typically 0.05-0.5%). TFA acts as an ion-pairing agent and masks secondary interactions with the silica-based stationary phase. TFA may significantly suppress the ESI response in positive-ion mode. To avoid this, either formic acid is preferred or a mixture of 0.02% TFA and 0.5% acetic acid can be used. Some silica-based RPLC materials can be used with a lower TFA concentration (PepMap ). Alternatively, poly(styrene-divinylbenzene) polymeric materials (PS-DVB) can be applied. With a monolithic PS-DVB column, only a small decrease in separation efficiency on the monolithic column was observed when the TFA concentration was reduced from 0.2%to0.05%[51]. 

Monohthic CEC columns are formed from both organic polymers and silica and the first appUcation involved a swollen hydrophilic polyacrylamide gel, similar to that used in capillary gd dectrophoresis [71]. Polymeric monoliths based on acrylamide, methacrylate and styrene, etc., are prepared in a mold by thermally or UV-initiated polymerization of the monomers, and new developments regarding their preparation have been reported in recent years [25]. 

Recently, the hydrogenation a mixture of toluene, styrene and 1-octene, representing a model feed for hydrotreating in the refining industry, was performed in monolith reactors [37]. One is a y-alumina monolith of diameter 1 cm and 15 or 30 cm long and the other is a more conventional cordierite monolith with a wall-coated layer of y-alumina. In both monoliths, the channels size is 1-2 mm and the catalyst is based on Ni. Substantial alkene conversions of more than 50% were observed in the small-channel reactors, which was attributed to the intensified mass-transfer rate generally measured in monolith reactors [16]. 

A polymeric monolith is a continuous porous polymeric rod made from a mixture of an initiator, monomers (including a cross-linking monomer), and a porogen (pore-forming solvent) that are polymerized in situ in a column. Tuning of the porous properties is typically achieved with a mixture of solvents such as toluene, THF, or decanol. The rationale for choosing an initiator depends on the mode of initiation and on solubility aspects. A common initiator is 2,2-azo-bis-isobutyronitrile (AIBN). Most polymerizations are radical polymerizations, activating radical formation either thermally [54] or with UV radiation [55]. Common monomers used in the preparation of polymer monoliths are styrene, (meth)acrylate, and acrylamide-based materials. The formation of the monolith... 

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