Turid Ellingsen

Monosized stationary phases for chromatography

Abstract Polymer particles with a highly monodisperse particle size distribution were produced by a two-step microsuspension method. This process is based on the activation of monosized polymer seed particles by the introduction of a low-molecular-weight material, which leads to a large increase in the monomer swelling capacity of the seed particles. The versatility of the process allows the preparation of polymer monosized compact or macroporous particles of predetermined particle size in the range 1–100 μm and with application of a wide selection of polymeric materials. Underivatized, rigid, porous particles were developed for size-exclusion chromatography in organic solvents. The uniform packing that may be achieved with monosized particles has resulted in chromatographic columns with unusual efficiency and separation capacity. By coating the particle surface with a hydrophilic cross-linked polymer, supports for aqueous phase ion-exchange and size-exclusion chromatography may be produced.

Hydrophilic Monodisperse Particles as Solid-Phase Material in Immunoassays: Comparison of Shell-and-Core Particles with Compact Particles

Hydrophilic monodisperse shell-and-core particles with a density of 1.07 were superior to heavier compact particles as a solid-phase material for immunoassays. The shell-and-core particles formed a semistable suspension for 24 h and were easily collected by centrifugation. The hydroxyl groups of the particles were activated with two sulfonyl chlorides. The most reactive one, tresyl chloride, gave rapid chemical coupling of antibodies, whereas tosyl chloride favored a rapid hydrophobic adsorption which was followed by slow chemical coupling. The solid-phase sheep antirabbit IgG made was used as a separation agent in several immunoassays and gave solid-phase primary antibodies by immunoadsorption of rabbit antibodies.

Thermodynamics of Swelling of Polymer, Oligomer and Polymer-Oligomer Particles. Preparation and Application of Monodisperse Polymer Particles.

The treatment of the equilibrium distribution in various polymer-monomer or polymer-mixed solvent systems has most often been based upon the classical Flory-Huggins lattice theory and the extension of this theory by Morton to include the interfacial energy. Further developments of these theories have included the swelling of polymer particles with mixtures of different monomers, systems in which the continuous phase (water in the case of aqueous dispersions) is present in the monomer and polymer phases, and the swelling of oligomer and oligomer-polymer particles. Ugelstad et al. have shown experimentally and theoretically that oligomer particles and oligomerpolymer particles in aqueous dispersions are capable of absorbing monomer to an extent that are orders of magnitude higher than for pure polymer particles. This has led to development of emulsion polymerization procedures which allow preparation of highly monosized large particles in the range of 1–50 μm in diameter. Such particles have up to now found industrial application in liquid chromatography. The particles are furthermore used as standards for blood cell counters. Hydrophilic monosized particles prepared by the new methods are utilized in different immunoassays where they have been found to possess advantageous properties. The monodisperse particles have also founded the basis for the preparation of monosized magnetizable microspheres which have been applied for cell separation.

Excess antibody immunoassay for rat glandular kallikrein. Monosized polymer particles as the preferred solid phase material

The development of an excess antibody assay for rat glandular kallikrein is described. This assay permits immunological determination of kallikrein as well as a simultaneous specific measurement of kallikrein enzymatic activity. The assay is based on coupling of immunopurified anti-kallikrein immunoglobulin to a solid phase. In a first incubation step, kallikrein was bound to the immobilized antibody. Determination of kallikrein was subsequently done in a second incubation step; immunologically by addition of iodinated anti-kallikrein antibody, or enzymatically by a kallikrein substrate. Enzymatic quantification could also be followed by immunological measurements on the same sample. Comparison of Sepharose, cellulose, and acrylate based polymer particles proved the latter to be the best matrix in this assay. The main advantage of the polymer particles was the low non-specific binding of labelled antibody.

Biomedical Applications of Monodisperse Magnetic Polymer Particles

The method developed by Ugelstad and coworkers1-3 for preparation of monosized polymer particles allows preparation of particles of any size from 1 to 100 µm with standard deviation in diameter ~ 1%. Also the method allows preparation of particles from a large number of monomers. Particles with highly crosslinked polymer of high mechanical strength, porous macroreticular particles and core and shell particles are prepared. The application of the porous macroreticular particles in fast protein liquid chromatography (FPLC)4 as marketed by Pharmacia is already well established and has in many cases led to very significant improvements in analysis and separation of complex protein mixtures.