Chromolith™ columns

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... 

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... 

An example of a high-speed application is shown in Figure 11. Here a rapid gradient separation of a few acids and esters is shown. A rapid gradient is executed at a high flow rate, 4mL/min, on a 100mm x 4.6mm Chromolith column. Seven compounds are separated in less than 3 min. 

One of the greatest challenges in monolith production is to increase the diameter. Presently, silica monoliths with a diameter of up to 50 mm can be produced. Commercial monolithic silica columns e.g. Chromolith columns of Merck KGaA possess a column permeability equivalent to a column packed with 15 xm particles, but show a column performance of a column packed with 5 xm beads. The major benefit of monolithic columns, however, is their robustness in use, their tailored pore structure and the tunable surface chemistry. 

Figure 3.41 Langmuir isotherm of 4-tert-butyl phenol measured by FA and by IM on two Chromolith columns. The inset illustrates the column-to-column reproducibility of the measurements. Reproduced with permission from A. Cavazzini, A. Felinger, G. Guiochon, J. Chromatogr. A, 1012 (2003) 139 (Fig. 1).

FIGURE 6.6 Merck Chromolith monolithic RPLC column at 1 mL/min (top) and 2 mL/min with various injection volumes. Protein standards A = aprotinin, B = cytochrome C, C = carbonic anhydrase. 

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). 

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). 

FIGURE 9.6 The peptide and small protein map from a 100 pL human plasma injection. Columns sample preparation SCX RAM analytical column chromolith performance RP-18, 100 x 0.1 mm I.D. Minute fractions were analyzed using MALDI-TOF MS. Fraction numbers correspond to the time scale. Dot size is related to signal intensity. 

FIGURE 17.10 Gradients for the separation of the FAE mixture on a monolithic column, stationary phase Chromolith C18, 10 x 0.46 cm i.d., mobile phase MeOH H20. 

An interesting idea was to use a monolith column to perform dual functions of online SPE and chromatographic separation. Because of the porous structure of a monolith column and its very low backpressure, plasma or diluted plasma can be directly injected. Plumb et al. (2001) used this approach to quantitate an isoquinoline drug and 3 -azido-3 -deoxy thymidine (AZT). Diluted plasma samples (plasma water 1 1) were injected directly into a Chromolith Speed ROD RP-18e column... 

Alnouti et al. (2005) used Symbiosis to measure propranolol and diclofenac in rat plasma. Twelve different SPE cartridges were screened. A C18 HD (2 x 10 mm inner diameter, Spark) was chosen because it provided the best recovery and peak shapes. When a Luna C18 (2.1 x 50 mm, 5 /an, Phenomenex) was used, the run time was 4 min it was 2 min when a monolithic Chromolith C18 (50 x 2.1 mm, Merck KgaA) column was used. 

Another approach is increasing throughput via a monolith analytical column. Vintiloiu et al. (2005) used a self-made RAM online SPE under turbulent flow conditions to measure rofecoxib, a cyclooxygenase-2 inhibitor, in rat plasma. They constructed a cartridge (0.76 x 50 mm) packed with LiChrosphere 60 RP-18 ADS particles (40 to 63 /an, Merck KgaA). The analytical column was a Chromolith Speed ROD (RP-18, 50 x 4.6 mm, Merck KgaA). The injection volume was... 

Phenyl (Cohesive Technologies), the polymer-based Oasis HLB (Waters), the Cyclone (Cohesive Technologies), and the porous graphitized carbon-based Hypercarb (ThermoHypersil, Cheshire, UK) Cohesive s 2300 system was the HTLC component. Merck s monolithic reversed-phased Chromolith Speed ROD (RP-C18 (50 x 4.6 mm) served as the analytical column. The Oasis HLB, Cyclone TFC, and Hypercarb yielded the best retention capacity and good elution efficiency and volume. Recovery was 42 to 94% with a sample volume of 10 mL. Run time was 14 min. LODs were 0.4 to 13 ng/L for most compounds. 

The liquid chromatograph Zarghi et al. used consisted of a Wellchrom K-1001 pump, Rheodyne 7125 injector, Eurochrom 2000 integrator, and K2600 fluorescence detector. The stationary phase was a Merck Chromolith Performance RP-18e column (100 x 4.6 mm). The mobile phase consisted of 0.01M dibasic sodium phosphate buffer and acetonitrile (60 40 v/v) adjusted to pH 3.5. The flow rate was 2 mL/min. The detector operated at an excitation wavelength of 240 nm and an emission wavelength of 340 nm. Letrozole served as the internal standard (IS). 

Another recent trend focused on supports in the shape of monolithic columns having the goal to benefit from the high permeability and the improved mass transfer characteristics of such structures. With this goal in mind, Lubda and Lindner [75] prepared enantioselective silica monolith columns with tert-butylcarbamoylquinine surface modification. A commercial sol-gel-derived Chromolith Performance Si (100 X 4.6 mm ID) monolith (1.9 tim macropore diameter, 12.5 nm mesopore... 

Column dimensions 4.6mm x 150mm, except for Chromolith RPige 4.6mm x 100mm. 

The absolute permeability (8 x 10 ° cm ) of currently available silica monoliths (Chromolith, Merck) is similar to that of columns packed with 9 jm particles [213], To increase efficiency, columns are usually packed with particles of 2, 3, and 5 jm, which possess absolute permeabilities of approximately 4x10 ", 9x10 ", and 2.5xl0" °cm, respectively. If we try to compare columns packed with 5 pm particles and monolithic columns with through-pores of an average size of 1.5 pm, we observe that the permeability of the monolithic columns is around three to four times larger than that of packed columns. 

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