Monolithic stationary phases fabrication

Monolithic stationary phases have to be regarded as the first substantial further development of HPLC columns, as they present a single particle separation medium, made up of porous polymer. As a consequence of their macroporous structure, they feature a number of advantages over microparticulate columns in terms of separation characteristics, hydrodynamic properties, as well as their fabrication ... 

Due to the fact that thermally initiated free radical copolymerization is by far the most routinely employed method for fabrication of organic monolithic stationary phases, the pore formation mechanism is discussed for this particular kind of polymerization. 

The transfer of macroscale chromatographic methods to chip-based formats, although advantageous, is not trivial, and imaginative solutions to the problems of stationary-phase introduction are required. It seems likely that future chromatographic chip systems will incorporate monolithic stationary phases due to the ease of their fabrication and localization within microchannels. Other nanoporous materials may also be of use. 

The application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

Despite the advantages of CEC over CE and HPLC, particle-packed columns are plagued with problems such as the difficulty in the preparation of frits to retain the stationary phase and bubble formation that results in current leakage and EOF breakdown. These problems set the pace for the development of column technology to overcome the problems associated with particle-packed columns and to improve on the speed of separation of analytes in mixtures. The fabrication of a continuous porous rod (monoliths), not requiring any frits and ensuring a constant and uniform current flow to give a stable EOF has so far proved a potential development for microseparations. ... 

An alternative to sintering frits, which deserves mention here, is to form frits via UV photopolymerization of a glycidyl methacrylate and trimethylolpropane trimethacrylate solution (UV radiation, 365 nm for 1 hour) [135]. The photopolymerization process is similar to that used in the fabrication of monolithic columns (Chapters 5 and 6). Frits fabricated with this method have shown to be reproducible since there is no sintering of packing material, weakening of the capillary column by removal of the polyimide coating and/or alteration of the stationary phase at the frit are avoided. 

A wide variety of approaches are currently being used in the fabrication and technology of columns for capillary electrochromatography (CEC). Continuous polymer bed, or monolithic columns (see Section 3.4), manufactured by in-situ polymerization within the columns, have been used in numerous application areas and have been shown to be highly efficient. In a second approach, a sol-gel process is employed to form a silica xerogel within the capillary, followed by bonding of the stationary-phase group alternatively, the separation medium itself may be polymerized in situ. 

An integrated microfabricated system composed of a proteolytic reactor and chromatographic column with direct interface to ESl-MS was reported by Carlier et al. [133] The system is represented in Fig. 11 and was fabricated from SU-8. The chromatographic end of the chip was terminated with a nano-ESl interface. The digestion module was composed of trypsin covalently attached to a monolithic polymer, which was also used to prepare a hydrophobic stationary phase for the separation of peptides prior to MS analysis. Monoliths were made in situ by photopolymerizing ethylene glycoldimethacrylate (EDMA) monomers in the presence of lauryl methacrylate (LMA) or butyl methacrylate (BMA) crosslinkers. 

CEC columns are generally made of fused-silica tubing, usually packed with the appropriate stationary phase. Today, the most commonly used CEC columns have i.d. of 100 p,m or less, with 50 and 75 p,m i.d. being the most popular. The stationary phase is retained in the column by two frits. Column designs can be categorized into two major types OT columns and packed structures, which include packed columns, monolithic columns, and microfabricated stractures (open or continuous beds). Packed capillary columns are most commonly used, as has been demonstrated in numerous papers [9-11]. They can be subdivided into three different categories columns packed with particles, columns with continuous beds fabricated in situ creating a rod-like monolithic structure, and columns with immobilized or entrapped particulate materials. 

---