Posters

High performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic separation of plasmid DNAconformations. However, no study about the separation of small molecules was performed until now. In this work, we investigated the possibility of gradient and isocratic separations of small molecules with Convective Interaction Media (CIM) disks of different chemistries. We proved that it was possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed phase mode. Fairly good separation of four oligonucleotides could be achieved on the disk of 0.3 mm thickness. The effect of the gradient parameters on the resolution in the case of gradient mode was studied and compared with the separation under isocratic conditions.

It was shown that similar peak resolution can be achieved in both gradient and isocratic modes. In addition, it was found that the flow rate does not have a pronounced influence on the resolution in the flow rate range between 1 and 10 mL/min. However, it seems that the resolution with the flow rate even slightly increases as a consequence of the increased pore accessibility. In accordance with conventional particle HPLC columns, the resolution increases with the monolith thickness. On the other hand, the mobile phase composition has to be carefully adjusted to obtain optimal resolution, especially in the case of isocratic separations. Because of this feature, CIM monoliths seem to be competitive to other, commercially available stationary phases.

Organic acids are important metabolites of several biochemical pathways in microorganisms and as such they are frequent main or by-products in different bioprocesses. Consequently, a demand for their monitoring is often present. One of the most applied methods for organic acids determination is certainly HPLC using different separation mechanisms such as reversed-phase, ion-exchange or ion-exclusion chromatography, all based on separation under isocratic flow conditions. To achieve the isocratic separation, multiple steps of adsorption-desorption process are needed and therefore conventional chromatographic columns with long layer of separation material were considered as a necessary tool for achieving this effect.

Recently, it was shown that isocratic separation could also be performed on thin monolithic layers. The isocratic separations of plasmid DNA conformers (1), oligonucleotides (2, 3) and peptides (3) in the ion-exchange mode were demonstrated as well as isocratic reversed-phase separation of a mixture of steroids was obtained (3) all on thin GMA-EDMA monoliths commercially available under trademark CIM™ (Convective Interaction Media). The results indicated the possibility of applying CIM™ monolithic columns also for isocratic separation of some other small charged molecules. Since the average analysis time using CIM™ disk monolithic columns is up to a few minutes, these supports can be a material of choice for separation of organic acids.