Bio-rad Laboratories, Inc.

Jia-Li Liao

Stellan Hjerten

Christopher J Siebert

Chromatographic beads with pores large enough to serve as through-channels for fast diffusion and a substantial absence of pores of smaller diameters that would cause band broadening due to slow diffusive transport, are formed by suspension polymerization of an aqueous solution of a monomer mixture that includes a total monomer (combination of polymer backbone monomer and crosslinking agent) concentration of 2% to 50% by weight relative to the aqueous solution and a crosslinking agent in an amount such that the mole ratio of crosslinking agent to total monomer is from about 0.1 to about 0.7. The suspension polymerization process involves forming a suspension of aqueous droplets of the monomer mixture in an organic phase.

Large-pore chromatographic beads prepared by suspension polymerization

Peak resolution in reversed-phase electrochromatography is improved in either of two alternative ways. The first is by a stepwise increase in the concentration of acetonitrile or other equivalent buffer constituent or modifier after the sample has been loaded, and the second is the inclusion of a surfactant in the mobile phase.

Methods of improving peak resolution in reversed-phase

A chromatography column with a continuous solid bed spanning the cross section of the column, the bed containing channels large enough for hydrodynamic flow, is prepared by polymerizing a mixture of monomers and ammonium sulfate in an aqueous solution. The monomers include a monofunctional monomer such as a vinyl, allyl, acrylic, or methacrylic compound, and a polyfunctional monomer (i.e., a crosslinker), the total monomer concentration being in the range of 10% to 20% by weight, the mole ratio of crosslinker to total monomer being in the range of 0.3 to 0.4, and the ammonium sulfate having a concentration in the range of 0.4 M to 0.8 M. Functional groups to impart specialized separation capabilities, notably anion and cation exchange, can be included in the monomer mixture.

Chromatography columns with continuous beds formed in situ from aqueous

Chromatographic media that demonstrate an artificially created recognition of any preselected molecular species with only can affinity-type adsorption are formed by polymerization from solutions of non-ionizable monomers with a quantity of the species contained in the solution, followed by removal of the species by conventional washing. The media are of particular utility and interest when used to achieve affinity binding of proteins. Uses of the media include chromatography and the general isolation and removal of the adsorbed species.

Selective recognition of solutes in chromatographic media by

Chromatographic media that demonstrate an artificially created recognition of any preselected molecular species with only an affinity-type adsorption are formed by polymerization from solutions of non-ionizable monomers with a quantity of the species contained in the solution, followed by removal of the species by conventional washing. The media are of particular utility and interest when used to achieve affinity binding of proteins. Uses of the media include chromatography and the general isolation and removal of the adsorbed species.

A separation medium for reversed-phase chromatography is prepared by polymerizing an aqueous vinyl monomer mixture to which a hydrophobic alkyl methacrylate with 3 or more carbon atoms in the alkyl group and a detergent have been added. The further inclusion of a vinyl monomer with a charged group in the reaction mixture produces a separation medium useful for reversed-phase electrochromatography. Performance of the polymerization in a capillary produces a continuous bed inside the capillary. An alternative for non-capillary separations, such as those conducted in larger diameter tubing, is the preparation of the medium in a vessel outside the tubing, followed by granulation of the polymer and placement of the granules in the tubing.

One-step preparation of separation media for reversed-phase

Macromolecular species in a liquid sample are chromatographically separated in a separation medium formed by polymerization of monomers in an aqueous solution with a sufficient amount of crosslinking agent to cause aggregation and precipitation of the polymer chains. The medium is either formed in the column in which chromatography is to take place as a continuous although channeled bed, or in a separate reaction vessel and then transferred to the column in comminuted form as a packed bed. Improvements in the performance of the bed are achieved by compression of the bed.

Hydrophobic polymers from water-soluble monomers and their use as

The present invention provides new methods for the concentration of ionic solutes, particularly ampholytes, such as, for example, peptides, proteins and nucleic acids. The methods are based on the fact that the electrophoretic migration velocities of solutes decrease upon a decrease in the absolute value of the zeta-potential of a solute or the pore size of the electrophoresis medium, and upon an increase in the cross-section of the electrophoresis chamber, the viscosity of the electrophoresis medium, or the electrical conductivity of the electrophoresis medium. When applied to capillary electrophoresis, the methods described herein permit concentration of a solution of solutes in the same capillary tube as is used for the electrophoretic analysis. Alternatively, however, the sample can be withdrawn from the capillary tube following concentration of the solution of solutes and processed by techniques other than high-performance capillary electrophoresis (HPCE).

Concentration of biological samples on a microliter scale and analysis

Electrophoresis is performed in buffers which exhibit both substantial buffering capacity and low electrical conductivity, permitting the separations to be performed at high field strengths without loss of resolution. Four classes of buffering agents are cited as examples:

Electrophoresis in low conductivity buffers

Highly compressible chromatographic stationary phase particles such as agarose beads are made rigid to a degree suitable for use in HPLC, and nonporous to proteins by one of two procedures. The first involves shrinking the beads with the use of an organic solvent in which the agarose bead will neither dissolve nor swell to collapse the porosity, followed by crosslinking the bead surfaces inside the collapsed pores to fix the pores in their collapsed state. The second involves filling the pores (without shrinkage of the beads) with a polymerizable substance which grafts to the pore surface, and performing the graft polymerization. The invention also extends to rigid beads, which are rendered deformable to a limited degree by coating the surface with a polymer. Finally, porous rigid beads are rendered nonporous by polymerizing a polymerizable material inside the pores in the same manner as the porous compressible beads.

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Uppsala, Sweden

Jia-Li Liao