Publications

A. Podgornik, M. Barut, J. Jančar, A. Štrancar, T. Tennikova

Analytical Chemistry, 1999, Vol. 71, No. 15, 2986-2991

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 chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is 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. It was shown that similar peak resolution can be provided in both gradient and isocratic modes.

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A. Podgornik, M. Barut, J. Jančar, A. Štrancar

Journal of Chromatography A, 848 (1999) 51–60

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.

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D. Josić, A. Štrancar

Ind. Eng. Chem. Res. 1999, 38, 333-342

For fast separation of biopolymers, recently developed media have become increasingly widespread. They consist either of membranes or of compact, porous disks and tubes, both called Convective Interaction Media (CIM). Separation can be carried out in every mode, e.g., ion-exchange, reversed-phase, hydrophobic-interaction, and affinity recognition. The units can be used for analytical as well as for preparative purposes. Such fast analytical units will allow separations within less than 10 s and can therefore be used for in-process analysis. The advantages and disadvantages of such analytical and preparative separations are discussed along with technical problems which have been solved.

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M. B. Tennikov, N. V. Gazdina, T. B. Tennikova, F. Švec

Journal of Chromatography A, 798 (1998) 55–64

The effect of porous structures of 2-mm thick diethylamine functionalized monolithic polymethacrylate discs on their chromatographic behavior in ion-exchange mode has been studied. Discs with small pores did not perform well because they exhibited high back pressure and substantial peak broadening. Discs characterized with pores larger than 1 000 nm did not provide good separations either because the time required for some protein molecules to traverse the length across the pore to reach the wall for adsorption/desorption process that is essential for the separation may be longer than their residence time within the matrix. Optimum pore size is centered at about 700 nm. Excellent separations have been achieved with these discs even at very steep gradients and high flow-rates which allow to shorten the separation times substantially.

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A. Štrancar, M. Barut, A. Podgornik, P. Koselj, D. Josić, A. Buchacher

LC-GC International – October 1998, 660-670

Separation supports based on convective interaction media (CIM) allow higher throughputs and separations; faster by one order of magnitude when compared with separation materials based on conventional porous particles (1). In the future, they may well play an important role in the production and quality control of diagnostic and therapeutic products based on large biomolecules. A major advantage of CIM is their use in control procedures during production processes — an important factor in satisfying the demands of regulatory authorities, both with regard to the registration of the product as a drug, and also in controlling the process in such a manner that immediate correction is possible should the process deviate from the prescribed path. The CIM supports can easily be scaled up because the larger units are made of the same material and can be used for fast separations on the preparative level. Additionally, these supports can be used for the ‘so-called’ conjoint liquid chromatography (CLC), by combining two or more discs with different ligand groups in the same housing. Finally, owing to their low back pressures and very fast response, CIM supports can be used as biosensors or bioreactors with immobilized enzymes.

A. Štrancar, P. Koselj, H. Schwinn, D. Josić

Analytical Chemistry, Vol. 68, No. 19, October 1, 1996

Production and downstream processing in biotechnology requires fast and accurate control of each step in the process. Improved techniques which can be validated are required in order to meet these demands. For these purposes, chromatographic units containing compact porous disks for fast separation of biopolymers were developed and investigated with regard to their performance and speed. The problems that have, in the past, arisen from the use of wide and flat separation units, such as membranes and disks, have chiefly been those of sample distribution and large void volumes before and behind the unit. Improvements in the construction of the cartridge have led to better performance of the compact porous disks and faster separation. Using these disks, three calibration standard proteins could be separated within less than 1 min by an anion-exchange, cation-exchange, and hydrophobic interaction mode. Such units can be used for in-process control in production and downstream processing of biopolymers, as was shown in experiments involving the purification of α1-antitrypsin and clotting factor IX and the immobilization of enzyme glucose oxidase on an epoxy-activated compact porous disk.

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B. G. Belenkii, V. G. Malt'sev

BioFeature, BioTechniques, 288, Vol. 18, No. 2 (1995)

In gradient chromatography for proteins migrating along the chromatographic column, the critical distance X0 has been shown to exist at which the separation of zones is at a maximum and band spreading is at a minimum. With steep gradients and small elution velocity, the column length may be reduced to the level of membrane thickness-about one millimeter. The peculiarities of this novel separation method for proteins, high-performance membrane chromatography (HPMC), are discussed and stepwise elution is shown to be especially effective. HPMC combines the advantages of membrane technology and high-performance liquid chromatography, and avoids their drawbacks.

T. B. Tennikova, B. G. Belenkii, F. Švec

Journal of Liquid Chromatography, 13(1), 63-70 (1990)

Basing on the fact that only short layers of a chromatographic column contribute to the separation in the interaction chromatography, 1 mm thick membranes from macroporous methacrylate polymer provided with functional groups were synthetized and used for protein separation. The chromatograms show that the separation is fully comparable with that experienced on a filled column but the advantage of a membrane is up to two orders of magnitude lower pressure during the process and very high loading reaching up to 40 g/m2. This recommends the high performance membrane chromatography also for large scale preparative separations.

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