Janez Jančar

Isocratic separations on thin glycidyl methacrylate–ethylenedimethacrylate monoliths

In this work, the isocratic separation of oligonucleotides in the ion-exchange mode on thin glycidylmethacrylate–ethylenedimethacrylate (GMA–EDMA) monoliths in the form of commercially available CIM (Convective Interaction Media) disks is presented. It was found that isocratic separation occurs even on monoliths with a thickness of only 0.75 mm. Peak broadening of the components retained on the monolith is proportional to the retention time, which in turn is proportional to the thickness of the monolith. Peak height is inversely proportional to the retention time. From these results it can be concluded that the mechanism of the separation on such monoliths is similar to that in HPLC columns filled with conventional porous particles. The height equivalent to a theoretical plate of GMA–EDMA monoliths is calculated to be 18.0 μm. The capacity factor k′ depends, exponentially, on the salt concentration. The Z factor calculated from fitted equations increases linearly with the oligonucleotide’s length. It was also found that the difference between peak retention volume slightly increases with the flow-rate when the experiments are performed in the range from 0.5 to 7 ml/min. From the similarities between the isocratic separations on conventional columns and on thin GMA–EDMA monoliths it is reasonable to believe that separation based on a multiple adsorption/desorption process also occurs in thin monoliths.

Application of very short monolithic columns for separation of low and high molecular mass substances

ABSTRACT Convective Interaction Media® (CIM) disk monolithic columns are specific among the chromatographic columns because of their monolithic structure and extremely short column length. In this work, HETP values and Z factors for different groups of molecules—proteins, DNA, oligonucleotides, peptides, and organic acids on strong anion exchange CIM disk monolithic columns were determined. Results are discussed in terms of the molecule structures and applied to develop different approaches for successful separation of abovementioned group of molecules on these types of columns.

Design of monoliths through their mechanical properties

Chromatographic monoliths have several interesting properties making them attractive supports for analytics but also for purification, especially of large biomolecules and bioassemblies. Although many of monolith features were thoroughly investigated, there is no data available to predict how monolith mechanical properties affect its chromatographic performance. In this work, we investigated the effect of porosity, pore size and chemical modification on methacrylate monolith compression modulus. While a linear correlation between pore size and compression modulus was found, the effect of porosity was highly exponential. Through these correlations it was concluded that chemical modification affects monolith porosity without changing the monolith skeleton integrity. Mathematical model to describe the change of monolith permeability as a function of monolith compression modulus was derived and successfully validated for monoliths of different geometries and pore sizes. It enables the prediction of pressure drop increase due to monolith compressibility for any monolith structural characteristics, such as geometry, porosity, pore size or mobile phase properties like viscosity or flow rate, based solely on the data of compression modulus and structural data of non-compressed monolith. Furthermore, it enables simple determination of monolith pore size at which monolith compressibility is the smallest and the most robust performance is expected. Data of monolith compression modulus in combination with developed mathematical model can therefore be used for the prediction of monolith permeability during its implementation but also to accelerate the design of novel chromatographic monoliths with desired hydrodynamic properties for particular application.

Optimization of lytic phage manufacturing in bioreactor using monolithic supports

A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.