Monolithic silica columns

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). 

---

Van Nederkassel, A. M., Aerts, A., Dierick, A., Massart, D. L, Vander Heyden, Y. Fast separations on monolithic silica columns method transfer, robusmess and column ageing for some case smdies. /. Pharm. Biomed. Anal. 2003, 32, 233-249. 

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). 

FIGURE 7.3 Scanning electron micrographs of monolithic silica prepared from sol-gel methods, (a) monolithic silica prepared from TMOS in a test tube, and monolithic silica columns prepared from a mixture of TMOS and MTMS, (b) in a 50-pm fused silica capillary, (c) in a lOO-pm fused silica capillary, and (d) in a 200-pm fused silica capillary tube (reproduced from the reference, Motokawa et al. (2002), with permission from Elsevier). 

Disadvantages of monolithic silica columns include the labor-intensive preparation of individual columns with possible reproducibility problems, limited availability, and relatively short retention caused by the smaller amount of silica existing in a column... 

Correlation was found between domain size and attainable column efficiency. Column efficiency increases with the decrease in domain size, just like the efficiency of a particle-packed column is determined by particle size. Chromolith columns having ca. 2 pm through-pores and ca. 1pm skeletons show H= 10 (N= 10,000 for 10 cm column) at around optimum linear velocity of 1 mm/s, whereas a 15-cm column packed with 5 pm particles commonly shows 10,GOO-15,000 theoretical plates (7 = 10—15) (Ikegami et al., 2004). The pressure drop of a Chromolith column is typically half of the column packed with 5 pm particles. The performance of a Chromolith column was described to be similar to 7-15 pm particles in terms of pressure drop and to 3.5 1 pm particles in terms of column efficiency (Leinweber and Tallarek, 2003 Miyabe et al., 2003). Figure 7.4 shows the pressure drop and column efficiency of monolithic silica columns. A short column produces 500 (1cm column) to 2500 plates (5 cm) at high linear velocity of 10 mm/s. Small columns, especially capillary type, are sensitive to extra-column band... 

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). 

Monolithic silica columns with various surface derivatization, including ion exchange (Xie et al., 2005), and chiral functionalities (Chen et al., 2002 Lubda et al., 2003 Chankvetadze et al., 2004), as well as protein-immobilized monoliths (Kato et al., 2005) have been reported. The latter will be an important part of an integrated multidimensional separation/identification system. 

PEAK CAPACITY INCREASE BY USING MONOLITHIC SILICA COLUMNS IN GRADIENT ELUTION... 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

Utilizing the difference in selectivity between a monolithic silica-C18 column (2nd-D) and another particle-packed column of C18 phase (lst-D), 2D HPLC separation was shown mainly for basic compounds and other species (Venkatramani and Zelechonok, 2003). The authors also reported other examples of reversed-phase 2D HPLC, using amino- and cyano-derivatized particle-packed columns for 2nd-D separation. The combination of normal-phase separation for the 1 st-D and reversed-phase separation on monolithic Ci g column for the 2nd-D was reported (Dugo et al., 2004). The use of a microbore column and weak mobile phase for the lst-D and a monolithic column for the 2nd-D was essential for successful operation. Improvement in the 2D separation of complex mixtures of Chinese medicines was also reported (Hu et al., 2005). Following are practical examples of comprehensive 2D HPLC using monolithic silica columns that have been reported. 

Simple and comprehensive 2D HPLC was reported in a reversed-phase mode using monolithic silica columns for the 2nd-D separation (Tanaka et al., 2004). Every fraction from the lst-D column, 15cm long (4.6 mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles (5 pm), was subjected to the separation in the 2nd-D using one or two octadecylsilylated (Cig) monolithic silica columns (4.6 mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to lOmL/min with separation time of 30 s that provides fractionation every 15-30s for the lst-D, which is operated near the optimum flow rate of 0.4-0.8 mL/min. The 2D-HPLC systems were assembled, as shown in Fig. 7.6, so that the sample loops of the 2nd-D injectors were back flushed to minimize band broadening. 

When two monolithic silica columns were used for two sets of 2nd-D chromatographs (Fig. 7.6c) separating each fraction of the lst-D effluent alternately,... 

Chen, Z.L., Uchiyama, K., Hoho, T. (2002). Chemically modified chiral monolithic silica column. J. Chromatogr. A 942, 83-91. 

Lubda, D., Cabrera, K., Nakanishi, K., Lindner, W. (2003). Monolithic silica columns with chemically bonded b-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals. Anal. Bioanal. Chem. 377, 892-901. 

Motokawa, M., Kobayashi, H., Ishizuka, N., Minakuchi, H., Nakanishi, K., Jinnai, H., Hosoya, K., Ikegami, T., Tanaka, N. (2002). Monolithic silica columns with various skeleton size and through-pore size for capillary liquid chromatography. J. Chromatogr. A 961, 53-63. 

---