Acrylamide-based monoliths

The first hydrophilic monoliths based on acrylamide chemistry were based on copolymerization of acrylic acid and (V,/V -methylene bis(acrylamide) in the presence of an aqueous buffer as porogen [66], Shortly after, the first hydrophobic capillary support for hydrophobic interaction chromatography was fabricated by the substitution of acrylic acid by butyl methacrylate, whereas the monomer 

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Hoegger, D.Freitag, R. (2001). Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography. J. Chromatogr. A 914, 211-222. 

Since acrylamide-based monoliths generally represent polar support materials, they are predominately also used for separation in normal-phase mode. For that purpose, monolithic polymers were prepared by polymerization of mixtures containing piperazine diacrylamide as cross-linking agent and methacrylamide, A-isopropylacrylamide or 2-hydroxyethyl methacrylate, and vinylsulfo-nic acid as monomers. 

Hoegger and Freitag [32] also prepared acrylamide-based monoliths using polymerization in aqueous solutions. However, their typical polymerization mixture contained a much higher concentration of monomers (up to 29%) including piperazine diacrylamide 111 (52% in respect to total monomers), dimethylacrylamide lfi, and 2-acrylamido-2-methyl-l-propanesulfonic acid 6 dissolved in an aqueous phosphate buffer pH 7. 

The acrylamide-based monolith originally developed by Hjerten for CEC capillary columns was very recently used by the same group to fill a 30.6 cm long 40 mm wide serpentine channel etched in flat quartz substrate shown in Fig. 6.8 [33], The channel... 

Although both reproducible preparation and operation of CEC columns are extremely important issues that will further stimulate the development and the acceptance of this technique, only a few groups have reported data on column-to-col-umn, run-to-run, and day-to-day reproducibility of monolithic capillary columns. Palm and Novotny showed reproducibility data for migration times tr, efficiencies, and retention factors for a number of analytes on acrylamide-based monoliths [35], The relative standard deviations (RSD) were smaller for run-to-run compared to day-to-day measurements. For example, the average run-to-run RSD for 6 analytes was... 

Ibuprofen, indoprofen, fenoprofen, ketoprofen, suprofen, diclofenac, metenamic acid Acrylamide-based monoliths [174]... 

Hjerten s research group described the very fast separations (less than 100 s) of standard proteins and peptides on acrylamide-based monolithic capillary columns derivatized with Cig ligands. 

Hoegger and Freitag modified the Hjerten s procedure and prepared a variety of monolithic acrylamide-based CEC columns [118]. Their approach allowed them to adjust both rigidity and porous properties of the monoliths and to achieve excellent separations of model compounds as well as selected pharmaceuticals. 

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. 

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... 

Continuous beds including methacrylate-based disks CIM QA from BIA Separations (Figure 3.22) and SWIFT from Isco, acrylamide UNO monoliths from Bio-Rad, Ultimate polystyrene divinylbenzene disks from LC-Packings, cellulose-based Seprasorb monoliths from Sepragen and silica units Conchrom from Conchrom or Chromolith from Merck are also... 

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. 

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... 

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. 

Replacement of the hydrophilic acrylamide by the more hydrophobic N-iso-propylacrylamide, in combination with the pre-functionalization of the capillary with (3-methacryloyloxypropyl) trimethoxysilane, afforded a monolithic gel covalently attached to the capillary wall. A substantial improvement in the separations of aromatic ketones and steroids was observed using these fritless hydrogel columns, as seen by the column efficiencies of 160,000 found for hydrocortisone and testosterone [92]. The separations exhibited many of the attributes typical of reversed-phase chromatography and led to the conclusion that, in contrast to the original polyacrylamide-based gels, size-exclusion mechanism was no longer the primary mechanism of separation. 

In addition, the GMA/EDMA copolymer proved to serve as a basic unit for the fabrication of highly permeable bioreactors in capillary format. Trypsin immobilization after epoxide ring opening with diethylamine and attachment of glutaraldehyde is mentioned as the probably most prominent example [64], The immobilization of trypsin was also carried out using another class of reactive monolithic methacrylate polymer, which is based on 2-vinyl-4,4-dimethylazlactone, acrylamide, and ethylene dimethacrylate [65]. In contrast to GMA/EDMA, trypsin can directly be immobilized onto this kind of monolith, as the 2-vinyl-4,4-dimethylazlactone moieties smoothly react with weak nucleophils even at room temperature. 

Hjerten and his research group [41] introduced monolithic columns based on acrylamides as chromatographic separation media in the late 1980s. Xie et al. [42] prepared rigid porous polyacrylamide-co-butylmethacrylate-co-A,A -methylene-bis-acrylamide monolithic column for hydrophobic interaction chromatography. They also prepared polyacrylamide-co-At, M-methylene-bis-acrylamide monolithic rods and smdied the effect of polymerization conditions on rods morphology. 

The monoliths are prepared both from organic (mainly based not only on polystyrene, acrylamides, acrylates, or methacrylates but also on imprinted polymers) and inorganic materials (based on sUica, zirconia, titania, or aluminium oxide) as continuous... 

Hjerten et al. [124] introduced the monolithic stationary phases based on acrylamides in the late 1980s. The cross-linked polyacrylamide can be directly synthesized within the mold by a one step free-radical chain polymerization. Acrylamide, methacrylamide, or piperazine diacrylamide are often employed as monomers, while V,A -methylene-bis-acrylamide is used as a cross-linker. 2-acrylamido-2-methylpropane sulphonic acid, vinylsulphonic acid, butyl methacrylate, or stearoyl methacrylate are usually added to the polymerization mixture to provide charge and functional groups [114]. 

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]. 

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