Monolithic silica-based

Tolstikov, V.V., Lommen, A., Nakanishi, K., Tanaka, N., Fiehn, O. (2003). Monolithic silica-based capillary reversed-phase liquid chromatography/electrospray mass spectrometry for plant metabolomics. Anal. Chem. 75, 6737-6740. 

Although Fields already mentioned the possible preparation of monolithic silica-based CEC columns, the lack of experimental data leads to the assumption that this option has not been tested [111]. In fact, it was Tanaka et al. who demonstrated the preparation of monolithic capillary columns using a sol-gel transition within an open capillary tube [99,112]. The trick was in the starting mixture that in addition to tetramethoxysilane and acetic acid also includes poly(ethylene oxide). The gel formed at room temperature was carefully washed with a variety of solvents and heated to 330 °C. The surface was then modified with octadecyl-trichlorosilane or octadecyldimethyl-A N-dimethylaminosilane to attach the hy-... 

Fig. 18. Scanning electron micrograph of monolithic silica-based capillary column. (Reprinted with permission from [205]. Copyright 2000 American Chemical Society)... 

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]. 

Monolithic silica is the most recently introduced class of stationary phases for electrochromatography on microchips. They benefit from their very high surface area, adjustable pore size, and controllable surface chemistry. Functionalities required for the separation in reversed-phase mode are typically incorporated onto the silica monolith by including an appropriate silicon alkoxide in the precursor mixture or by silanization of the surface after the monolith has been formed. Monolithic silica-based columns are generally known to exhibit superior performance in HPLC separations of small molecules, which is atftibuted to the presence of mesopores entailing large surface areas. 

Hermessy et al. [66] describe a general procedure for the generation of peptide maps of proteins with monolithic silica-based columns. The use of reversed-phase monolithic sorbents has been demonstrated to significantly reduce separation times for peptide map analysis, thus enabling increased sample throughput (see Figure 7.15). 

In this study, chromatographic experiments were 10 times faster with the monolithic column and results were equivalent to those obtained with the silica-based columns. This approach could be further optimized with faster gradient since flow rate should be increased by a factor 3 or 7 compared to conventional Cig supports [61, 62] and gradient time reduced by the same factor [63] to fully exploit the potential of monolithic supports. 

Cabrera, K. Applications of silica-based monolithic HPLC columns./. Sep. Sci. 2004, 27, 843-852. 

The latest innovation is the introduction of ultra-thin silica layers. These layers are only 10 xm thick (compared to 200-250 pm in conventional plates) and are not based on granular adsorbents but consist of monolithic silica. Ultra-thin layer chromatography (UTLC) plates offer a unique combination of short migration distances, fast development times and extremely low solvent consumption. The absence of silica particles allows UTLC silica gel layers to be manufactured without any sort of binders, that are normally needed to stabilise silica particles at the glass support surface. UTLC plates will significantly reduce analysis time, solvent consumption and increase sensitivity in both qualitative and quantitative applications (Table 4.35). Miniaturised planar chromatography will rival other microanalytical techniques. 

It is of much interest to compare polymer monoliths with monolithic silica columns for practical purposes of column selection. Methacrylate-based polymer monoliths have been evaluated extensively in comparison with silica monoliths (Moravcova et al., 2004). The methacrylate-based capillary columns were prepared from butyl methacrylate, ethylene dimethacrylate, in a porogenic mixture of water, 1-propanol, and 1,4-butanediol, and compared with commercial silica particulate and monolithic columns (Chromolith Performance). 

Chromatographic use of monolithic silica columns has been attracting considerable attention because they can potentially provide higher overall performance than particle-packed columns based on the variable external porosity and through-pore size/skeleton size ratios. These subjects have been recently reviewed with particular interests in fundamental properties, applications, or chemical modifications (Tanaka et al., 2001 Siouffi, 2003 Cabrera, 2004 Eeltink et al., 2004 Rieux et al., 2005). Commercially available monolithic silica columns at this time include conventional size columns (4.6 mm i.d., 1-10 cm), capillary columns (50-200 pm i.d., 15-30 cm), and preparative scale columns (25 mm i.d., 10 cm). 

In a sense each monolithic column is unique, or produced as a product of a separate batch, because the columns are prepared one by one by a process including monolith formation, column fabrication, and chemical modification. Reproducibility of Chro-molith columns has been examined, and found to be similar to particle-packed-silica-based columns of different batches (Kele and Guiochon, 2002). Surface coverage of a Chromolith reversed-phase (RP) column appears to be nearly maximum, but greater silanol effects were found for basic compounds and ionized amines in buffered and nonbuffered mobile phases than advanced particle-packed columns prepared from high purity silica (McCalley, 2002). Small differences were observed between monolithic silica columns derived from TMOS and those from silane mixtures for planarity in solute structure as well as polar interactions (Kobayashi et al., 2004). 

Bushey, M.M., Jorgenson, J.W. (1990). Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62,161-167. Cabrera, K. (2004). Applications of silica-based monolithic HPLC columns. J. Sep. Sci. 27, 843-852. 

Leinweber, F.C., Lubda, D., Cabrera, K., Tallarek, U. (2002). Characterization of silica-based monoliths with bimodal pore size distribution. Anal. Chem. 74, 2470-2477. 

Current commercial silica-based columns have two important characteristics (1) they can produce efficiency similar to that of columns packed with 3.5 /tm particles and (2) they typically produce a pressure drop of half that caused by a column packed with 5 /tm particles.35 Monolithic columns have been shown to exhibit flat van Deemter curves, resulting in little loss of efficiency at high flow rates.36 As a result, high-throughput separations on conventional HPLC instruments can be achieved by increasing flow rate up to nine times (up to 9 ml/min) the usual rate in a conventional packed column. Cycle times for HPLC analysis as short as 1 min (injection-to-injection) have been reported by users of monolithic columns. Additional benefits of monolithic columns cited include... 

Reproducibility of monolithic columns has also been cited as a major concern because the monoliths are manufactured individually.34-35 An extensive study by Kele and Guiochon indicates that the reproducibility results of Chromolith columns were almost comparable to those from different batches of particle-packed columns.37 Other drawbacks of monolithic columns include weak reten-tivity for polar analytes,38 efficiency loss at high flow rates for larger (800 MW) molecules,39 and peak tailing, even for neutral non-ionizable compounds.36-38-40 Furthermore, silica-based monolithic... 

FIGURE 13.7 Scanning electron micrograph showing flow-through channels in silica-based monolithic rods. 

Excellent performance for the elution of another peptide, insulin (molecular weight 5800 g/mol), was also observed using silica-based monoliths. The efficiency of the monolithic column was much better than that of a column packed with beads, and did not change much even at high flow rates. 

The secondary structure, the mesopores, is similar to the internal structure of standard HPLC particles. This secondary structure provides the surface for retention. The standard pore size is in the order of 13 nm, resulting in a specific surface area of about 300 mVg. Due to the lower ratio of retentive structure to interstitial space, the retentivity of monoliths and the preparative loadability tends to be significantly lower than the retentivity and loadability of packed beds of 10-nm particles. Since the monolithic columns described here are made from silica, they can be derivatized in the same way and with the same technology as silica-based particles. Also, the useful pH range is the same as for silica-based particles. 

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