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The growing demand from many areas of chemical science for increased throughput to save time and money has cascaded into many branches of analytical chemistry. In HPLC, elution times have been cut by decreasing the size of the packed particles in the columns. This has the effect of reducing the diffusion distances for the solutes but there is one major adverse effect that cannot be circumvented. As particle size is reduced, the column back pressure increases - it is inversely proportional to the square of the particle size. Most modern HPLC systems can cope with back pressures up to about 5000 psi (34.5 MPa), but this limit can be reached quickly. One solution has been the introduction of monolithic materials to the HPLC world. These are prepared from both silica-based and organic polymer-based materials and their great advantage is the absence of the interstitial voids that are found in particulate columns. In their place, there are various pores which are connected to form channels. The high degree of interconnectivity between the channels results in markedly lower back pressures and easily achievable high flow rates. The pores can be made relatively large, up to about 10 µm. However, to produce larger pore sizes in monolithics is difficult, according to a group of Japanese scientists, and this limits their use for very high throughput analyses. In addition, they argue that current production techniques for the in situ production of monolithic materials within the column tubing are complicated and give low reproducibility. So, this group has developed a novel monolithic material with large pore sizes which is easy to pack into HPLC columns. Takuya Kubo, Fuminori Watanabe, Kunimitsu Kaya and Ken Hosoya from Tohoku University prepared an ethylene-vinyl acetate copolymer using pentaerythritol as a pore template. The resulting product was extruded into a stick shape, cooled and washed to remove the templates. A spongy monolith with a porosity of 74% was produced and the pore sizes were greater than 10 µm. The diameter of the extrudate was controlled to 4.7 mm, slightly larger than the column internal diameter of 4.6 mm i.d. For packing, one end of the material was compressed with a thermal shrinkage tube. After soaking in water, the whole stick was pulled through the column until the non-shrunk portion filled it, then the shrunk portion was cut off. The spongy nature of the material ensured that it was packed effectively, with no void between the column wall and the monolith surface. In the crucial back pressure tests, the team found that the new material performed far better than common packed particle columns and a commercial octadecylsilica monolith, with back pressures less than 2 MPa for flow rates of up to 12 mm/s. However, there are other factors which contribute to the utility of the new material as an HPLC stationary phase. The monolithic material dissolved in non-polar solvents such as toluene and THF so is unsuitable for normal-phase separation. Conversely, the polymer is stable in polar solvents like water, acetonitrile and ethanol, so would be suited to reversed-phase separations. The permeability was higher than those of common HPLC columns and the hydrophobicity was similar to those of octadecylsilica monolithic columns. One drawback was that the adsorption capacity, measured with aqueous solutions of bisphenol A, was relatively low compared with 30-µm octadecylsilica and the commercial monolith. This was attributed to the absence of meso- and micropores which are found in the other materials. This weakness prevents the spongy monolith being used as an efficient HPLC column but it was demonstrated to be useful as a high-throughput preconcentration column in a column switching system, again using bisphenol A. Recoveries of more than 95% were achieved at flow rates of 6-10 ml/min, with high batch-to-batch and column-to-column reproducibilities. For the extraction of bisphenol A from simulated river water, complete recoveries were achieved. In an attempt to improve the adsorption capacity, Kubo modified the monolithic material by treatment with the crosslinking agent glycerol dimethacrylate. The appearance and elasticity of the material remained unchanged but the adsorption capacity was almost doubled. The modifications also increased the physical strength of the material. So, although the new spongy monolith is unsuitable for use as the main HPLC separation column, the researchers predict that it will find application as a high-throughput preconcentration medium in column-switching LC systems and in SPE. Future work will be carried out to optimise the properties, such as pore size, homogeneity and strength, and the effects of chemical modification will be more closely examined. Related links:
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