Monolithic columns reproducibility

Figure 4.9 Total ion current chromatogram and extracted MRM chromatograms for eight pesticides in 10 mL of river water (River Parthe, February 10, 2001, Leipzig, Germany), analyzed by on-line turbulent flow extraction and analysis using a monolithic column. Reproduced from Asperger et al, J. Chromatogr. A (2002), 960, 109, copyright (2002), with permission from Elsevier...

FIGURE 7.1 Scanning electron micrographs of a polystyrene-divinylbenzene monolithic column prepared in a 20-pm fused silica capillary tube (reproduced from the reference, Ivanov et al. (2003), with permission from American Chemical Society). 

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). 

Kele, M., Guiochon, G. (2002). Repeatability and reproducibility of retention data and band profiles on six batches of monolithic columns. J. Chromatogr. A 960, 19 -49. 

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... 

The main bottleneck in the further development of CEC is related with the state of the art of the column manufacturing processes and the robustness of the columns/instrumentation. Moreover, evidence to demonstrate reproducibility of separations from column to column still has to be established. The formation of bubbles in the capillaries due to the Joule heating and variations in EOF velocity on passing from the stationary phase through the frit and into the open tube is still very challenging in packed column CEC. A way to overcome this problem is to use monolithic columns or apply open tubular CEC [108]. Currently, many efforts are placed in improving column technology and in the development of chip-CEC [115] as an attractive option for lab-on-a-chip separations. 

Kele, M. and Guiochon, G. Repeatability and Reproducibility of Retention Data and Band Profiles on Six Batches of Monolithic Columns,/. Chromatogr., 960 19—49,2002. 

FIGURE 9 Scanning electron micrograph of monolithic silica-based capillary column. (Reproduced with permission from reference 55.)... 

Monolithic columns are comparatively easy to prepare. This is particularly true for capillary columns, which are known to be tedious to pack with particles. Furthermore, the reproducibility of microcolumn packing is low. 

However, silica monoliths and organic polymers both exhibit very advantageous chromatographic characteristics enhanced mass transfer characteristics, high reproducibility, and versatile surface chemistry, which make monolithic column attractive for a variety of forward-looking applications. 

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. 

Tests of the reproducibility of retention times, retention factors, separation selec-tivities, and column efficiencies for our methacrylate monolithic capillary columns are summarized in Table 6.2. This table shows averaged data obtained for 9 different analytes injected 14 times repeatedly every other day over a period of 6 days, as well as for 7 different capillary columns prepared from the same polymerization mixture. As expected, both injection-to-injection and day-to-day reproducibilities measured for the same column are very good. Slightly larger RSD values were observed for col-umn-to-column reproducibility. While the selectivity effectively did not change, larger differences were found for the efficiencies of the columns. 

Fig. 10.16. Gradient electrochromatogram of derivatized neutral steroids. Macroporous monolithic column 350 (250) mm x 100 pm i.d. gradient mobile phase, acetonitrile-water-240 mM ammonium formate buffer, pH 3 (30 60 5-65 30 5 v/v/v) field strength, 600 V/cm injection, 100 V/cm for 10s detection, laser-induced fluorescence (excitation at 325 nm, emission at >495 nm). Reproduced with permission from Que et al. [76],...

Recendy, rapid separation of enantiomers by CEC had been carried out in less than 1 min [86], and the high reproducibility of the separation was consistent in over 30 runs. Batch-to-batch reproducibility was reported to be within 5% relative standard deviation as measured by the migration time of an unretained solute using nine different monolithic columns, and run-to-run reproducibility of within 1% relative standard deviation [87]. 

Advances in column technology have improved the selectivity, stability, and reproducibility of LC analytical columns. For example, analytical columns are packed with a variety of stationary phases, providing enormous versatility in the separation process. This section describes (1) column dimensions, (2) particulate column packings, (3) monolithic column packings, and (4) the use of guard columns. 

Chemistry and material sciences are key disciplines for the development of advanced and more specific adsorbents. The stability and reproducibility of chromatographic columns was significantly increased by the introduction of spherical instead of irregular stationary phases. Recently, another step forward was made by the development of high efficiency monolithic columns with a rather low pressure drop. Future, further improvements, which include surface activation and internal pore structures of stationary phases, should help to tailor stationary phases for certain applications. But, besides the need for more specific and efficient solid phases, their cost is often a major problem for the widespread application of preparative chromatography. 

Numerous studies have been made characterizing the performance of silica-based rod columns [69], demonstrating the high level of reproducibility of analytical retention data [68] and of isotherm data [70], showing that the thermodynamic properties of the interactions between various solutes and chemically bonded Cig silica were very similar whether the silica support was made of particulate or monolithic material [71]. It has also been shown that the mass transfer kinetics was very similar for particulate and monolithic columns [72-74]. Even the satu-... 

Figure 16.12 Comparison of the experimental (symbols) overloaded elution band profiles of a mixture of is -chjnnotrypsinogen and horse-cytochrom c on a monolithic column of ion- exchange resin and calculated profiles. Top mobile phase 50 mM buffer, pH = 6.0 Bottom same mobile phase + 80 mM Na+. Left calculations made with the RD model. Right calculations made with the TD model. Reproduced with permission from S. Chose, S. M. Cramer,. Chromatogr. 928 (2001) 13 (Figures 5 and 6).

Two speciflc experimental improvements are on the horizon. First, a two-loop set-up will replace the existing one-loop set-up for the collection of pi fractions. In the new set-up, one loop will collect the fraction from the CIEF capillary while the contents of the second loop are transferred to the storage loop. In this way, CIEF can run in a continuous mode with improved reproducibility of pi measurement over the stop and go mode. Second, a monolithic column will replace the standard packed column in RPLC. Since monolithic columns typically achieve efficiencies of 25 000, chromatographic resolution can be increased while significantly shortening the analysis time. ... 

A pepsin microreactor was developed using a sol-gel monolithic column photo-polymerized within a fused silica capillary [6]. The column was used for on-line ESI CE/MS. Although monolithic microreactors are fast and efficient, the process of their preparation may require more than 24 h and can be difficult to reproduce. 

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