Chromotographic method and device in which a continuous...

Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...

Reexamination Certificate

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C210S656000, C210S198200, C210S502100

Reexamination Certificate

active

06290853

ABSTRACT:

REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/SE96/01508 filed Nov. 20, 1996, which was published as WO 97/19347.
The present invention relates to a chromatographic method for separating one or several organic substances in a liquid sample by passing said sample of substances through a chromatographic device and a chromatographic device for that separation. More precisely the invention relates to a separation method on a chromatographic device and the chromatographic device which contains as separating medium a continuous macroporous matrix. The separation medium is prepared by polymerization of a high internal phase emulsion system.
A common type of separation technique widely used in modern biotechnology is column chromatography. The chromatographic separation is carried out by forcing a liquid through a column packed with a matrix consisting of particulate beads. A sample which can be a mixture of e.g different proteins is introduced at the top of the column and then moves with the flow through the column. The proteins or substances will be retarded on the matrix in such a manner that proteins having different properties, e.g charge, size, hydrophobicity etc, will be retarded differently and therefore separated.
It is desirable that the separation should occur with fast kinetics and low band broadening in order to obtain a fast separation and to be able to well resolve the different substances from each other. To achieve this, the diffusive transport of the substances in and out of the beads has to be minimized, since this will cause band broadening and thus loss in resolution. The band broadening effects will increase with increasing flow rates and with increasing particle size. These effects have led the manufacturers of high performance chromatography media to reduce the size of the beads. However, small beads bring about other problems. Above all, packing with smaller beads gives a higher back pressure due to the narrowing of the convective flow channels in between the particles in a packed bed. To compensate for the increased back pressure a shorter column can be used which generally results in a lower separation capacity, or the column and fluid pumps can be redesigned to withstand higher pressure with accompanying costs. It is also difficult and expensive to prepare a perfectly packed column from particulate matrices.
To solve the problem with diffusive transport in and out of the beads it has been suggested to use a stationary phase shaped as a continuous porous plug. WO 90/07965 discloses a continous, coherent gel plug formed by bulk polymerization of monomers in such a way that the polymer chains adhere to each other in bundles with voids or channels formed between the bundles. In a later publication the inventor of this patent application states that this plug can not be used for chromatography, as the plug collapses when pressure is applied. Instead he recommend to compress the gel plug 10-15 fold. The compression would result in non-uniform channels in the plug and produce very high back pressures.
U.S. Pat. No. 5,334,310 relates to a continous macroporous polymer plug containing small pores having diameters less than about 200 nm and large pores with diameters greater than about 600 nm. The porous plug is produced by bulk polymerization of vinyl monomers in the presence of a porogen. A very high separation efficiency, easiness to prepare and versatility in the selection of monomer chemistry are advantages mentioned for the plug contained in a column. Due to the irregular structure of the pores in the plug and pores with rather small median size, also this plug results in separation at relatively high back pressures.
Therefore, there is a need for a further improved chromatographic separation method and a chromatographic device therefore.
Thus, the object of the present invention is to provide an improved chromatographic separating method and a chromatographic device with a separation medium without the disadvantages mentioned above.
A further object of the invention is to present a chromatographic separation method and a device comprising a medium with fast kinetics, with high efficiency, with good mechanical properties and with low back pressures.
It is yet another object of the invention to obtain a separating method and medium especially suitable for separating large biomolecules or aggregates.
The objects of the invention are achieved by the method of chromatographic separation and by the chromatographic device as claimed in the claims. According to the invention a chromatographic method for separating one or several organic substances in a liquid sample is obtained, in which said sample of substances is passed through a chromatographic device containing as separation medium at least one continuous macroporous matrix. The macroporous matrix comprises a cross-linked organic polymer prepared by polymerisation of a high internal phase emulsion system of a water-in-oil emulsion of organic monomers. The emulsion contains at least 75% by weight of water phase, and the polymerisation results in an open porous structure, to allow a convective flow to pass through the macroporous matrix, whereby said organic substances separate from each other and/or the liquid. The pores of the macroporous matrix can be unmodified, or surface modified in a manner,that the convective flow is not hampered.
Further according to the invention a chromatographic device is obtained which contains as separation medium at least one continuous macroporous matrix. The macroporous matrix comprises a cross-linked organic polymer prepared by polymerisation of a high internal phase emulsion system of a water-in-oil emulsion of organic monomers, said emulsion containing at least 75% by weight of water phase, said polymerisation results in an open porous structure, to allow a convective flow to pass through the macroporous matrix. The pores of the macroporous matrix can be unmodified, or surface modified in a manner,that the convective flow is not hampered.
It has been found that by preparing the macroporous matrix by polymerisation of the monomers in the form of a high internal phase emulsion, a method is obtained by which the organic substances separate from each other in the chromatographic device at low back pressures, with high theoretical plate numbers over a broad range of linear flow rates and that the mass transfer is driven by convective flow in the matrix pores.
The production of polymeric materials by polymerization of a water in oil emulsion having a high internal phase ratio of water to monomer have been known quite long. DE 1 160 616 and DE 1 494 024 disclose the production of porous polymers for isolation materials by polymerization of a high internal phase emulsion of water-in-oil type. The water content of the emulsion can be up to 98%.
EP 60 138 relates to cross-linked polymeric materials with low density and high absorbency. The material is produced by vinyl polymerization from a water-in-oil emulsion, where the emulsion contains at least 90% by weight of water. None of these patents relates to chromatography. EP 288 310 relates to a substrate comprising a porous cross-linked polymeric material filled with a gel material. The polymeric material is prepared by the high internal phase emulsion technique as described in EP 60 138. The substrate is used for peptide synthesis but chromatography is also mentioned as a possible field of use. However, the peptide synthesis is made on the gel, whereas the function of the cross-linked polymeric material is as a ridgid framework to enclose the gel. There is no disclosure or examples that the polymeric material could be used as stationary phase in chromatography. Less is there any information of the advantages of a plug of such a material as the stationary phase in column chromatography. On the contrary, the invention disclosed in EP 60 138 would not function in a method using the continuous matrix for chromatography, since the gel filled pores would not allow convective flow through the matrix. In EP 288 310 the subs

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