Biomedical subjects

L Varady

Publications and source records attributed to L Varady.

3 recordsLinked to original sources

PubMed1993

Fimbriated stationary phases for proteins.

This paper describes synthetic procedures for preparing fimbriated stationary phases on poly(styrene-divinylbenzene) (PS-DVB) packing materials. The synthesis consists of a five-step procedure in which the order in which the steps are carried out may be varied. These steps are (i) polymerization of monomers to form an amphiphilic copolymer or oligomer, (ii) adsorption of either monomers, or polymer onto the PS-DVB surface, (iii) solvent induction of functional group orientation at the PS-DVB-polymer interface and polymer-water interface, (iv) a cross-linking reaction that forms a hydrophilic surface layer, and (v) derivatization of the surface layer with stationary phase.

Chromatography, Affinity↗

PubMed1990

Flow-through particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography.

This paper reports a new technique for reducing resistance to stagnant mobile phase mass transfer without sacrificing high adsorbent capacity or necessitating extremely high pressure operation. The technique involves the flow of liquid through a porous chromatographic particle, and has thus been termed "perfusion chromatography". This is accomplished with 6000-8000 A pores which transect the particle. Data from electron microscopy, column efficiency, frontal analysis and theoretical modelling all suggest that mobile phase will flow through these large pores. In this manner, solutes enter the interior of the particles through a combination of convective and diffusional transport, with convection dominating for Peclet numbers greater than one. The implications of flow through particles on bandspreading, resolution and dynamic loading capacity are examined. It is shown that the rate of solute transport is strongly coupled to mobile phase velocity such that bandspreading, resolution of proteins and dynamic loading capacity are unaffected by increases in mobile phase velocity up to several thousand centimeters per hour. The surface area of this very large-pore diameter material is enhanced by using a network of smaller, 500-1500 A interconnecting pores between the throughpores. Scanning electron micrographs show that the pore network is continuous and that no point in the matrix is more than 5000-10,000 A from a through-pore. As a consequence, diffusional path lengths are minimized and the large porous particles take on the transport characteristics of much smaller particles but with a fraction of the pressure drop. Capacity and resolution studies show that these materials bind and separate an amount of protein equivalent to that of conventional high-performance liquid chromatography as well as low performance agarose-based media at greater than 10-100 times higher mobile phase velocity with no loss in resolution.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Peptides as affinity surfaces for protein purification.

The tetrapeptide GPRP was previously shown to be an effective affinity ligand for fibrinogen when immobilized to Fractogel (Kuyas et al., 1990). The authors synthesized the GPRP peptide directly onto an aminefunctionalized POROS chromatographic resin to demonstrate the effectiveness of this approach for generating perfusive affinity media. Fibrinogen from plasma bound to an NH2-GPRP-POROS column under 50 mM phosphate buffer, 0.15 M NaCl, pH 7 at 15 ml/min flow rate. The bound fibrinogen showed weak clotting activity when eluted with 20 mM acetate buffer, pH 4. The peptide column did not bind denatured fibrinogen. The dynamic binding capacity of the column by frontal analysis was 10.2 mg/ml column volume. The total analysis time was under 5 min. Similarly, the CAQCHTVEK peptide of cytochrome c with heme group covalently attached to the SH groups of the two cysteines is known to bind to albumins (Adams et al., 1989). A simplified peptide analogue, GAQGHTVEK, was synthesized directlyon POROS resin. Under 20 mM MES, pH 6, albumin from human serum bound specifically to this peptide column and eluted with a salt gradient at 0.2 M NaCl, 20 mM MES (2-[N-Morpholino]ethane sulfonic acid), pH 6. The dynamic binding capacity of human serum albumin by frontal analysis was 19 mg/ml column volume. Thus, this column can purify albumin from human serum under non-denaturing conditions.

Chromatography, Affinity↗