D. Josić, Y.P. Lim, A. Štrancar, W. Reutter
Journal of Chromatography B, 662 (1994) 217-226
The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.
T. B. Tennikova, F. Švec
Journal of Chromatography A, Volume 646, Issue 2, 3 September 1993, Pages 279–288
High-performance membrane chromatography (HPMC) is a very effective chromatographic method in which all the mobile phase flows through the separation medium. The effects of process variables such as concentration of displacement agent, flow-rate and gradient slope on HPMC separations in the ion-exchange, hydrophobic interaction and reversed-phase modes were studied using model protein mixtures. The basic relationships characterizing column HPLC also apply in HPMC. Whereas the efficiency of the HPMC membrane does not depend on flow-rate, the resolution increases with increasing gradient volume. Separations obtained with a continuous linear gradient were used for the design of a stepwise gradient profile which decreases the consumption of both time and mobile phase in separations of proteins. According to calculations, the protein diffusivity enhanced by the convective flow through the membrane is about four orders of magnitude higher than the “free” diffusivity of the protein in the stagnant mobile phase located in the pores of a standard separation medium. This considerably speeds up the process and improves the efficiency of the separation.
D. Josić, J. Rauch, K. Löster, O. Baum, W. Reutter
Journal of Chromatography A, 590 (1992) 59-76
Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.
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.