Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2002-12-12
2004-06-08
Foelak, Morton (Department: 1711)
Liquid purification or separation
Processes
Chromatography
C210S661000, C210S679000, C536S127000, C521S145000
Reexamination Certificate
active
06746608
ABSTRACT:
FIELD OF INVENTION
The invention relates to the use of fluorine-containing polymer adsorbent particles as a stationary phase for carrying out chromatographic separations.
BACKGROUND OF THE INVENTION
Support materials for use in high productivity liquid chromatography must be mechanically strong in order to withstand operation at high rates of flow under high pressures. Moreover, they must be stable over the wide range of pH to which such materials are subjected during normal operation and regeneration. The stability of the polymeric particles in their environment allows them to withstand degradation and decomposition. Physical properties of particular importance to chromatographic media are (1) sphericity of the particles, (2) high surface area; (3) high pore volume and availability; (4) wide range of pore diameters, and (5) wide range of particle diameters.
U.S. Pat. Nos. 5,438,129 and 5,625,054 disclose fluorinated silica for DNA separation.
Reverse phase chromatography involves the use of a relatively non-polar stationary phase in conjunction with a very polar mobile phase that is usually water. This technique is used to separate solutes of lower polarity. Reverse phase chromatography is usually performed using silica that is coated with an organic silane to provide hydrophobicity. However, the hydrophobised silica has a severe limitation in that it cannot be used at a pH greater than 8 and cannot be cleaned with concentrated caustic soda solutions without dissolving the particles.
SUMMARY OF THE INVENTION
The invention is directed to fluorinated particles having adsorbent properties for superior performance as the stationary phase for use in chromatographic separations. In particular, the fluorinated surface of such particles of the invention presents unusual and unexpected polarity that is beneficial in performing chromatographic separations such as that used for DNA.
Further, it has been found that one can place fluorinated coatings on substrates and the resultant composition will separate super-coiled DNA from a lysed plasmid containing RNA, proteins and endotoxins, e.g. when ion-pairing chromatography is employed. These substrates can be inert materials such as glass beads or silica or polymers. Any fluorinated surface will behave in a similar manner. These surfaces can be glass spheres, fluorinated films, porous fluorinated membranes, a porous monolith or any other suitable material.
One method of making the chromatographic medium is to synthesize a fluorinated chlorosilane and react that silane with the surface of a material such as silica. The resultant medium can then be used in an ion-pairing chromatography mode to adsorb and de-sorb supercoiled DNA.
The present invention provides uses for particles as a stationary phase in chromatographic techniques. Particles of the invention are particularly suited to use where the sample to be chromatographed is a macromolecule containing nucleotides, nucleosides or polypeptides, such as DNA, RNA or endotoxins.
More particularly, after a plasmid is lysed with caustic, the resultant mixture may contain DNA, RNA, endotoxin and proteins. If the mixture is allowed to flow through a bed of a fluorinated particles, the DNA components are adsorbed onto the polymer. If the polymer is then treated, e.g. with an aqueous solution of an ion-pairing agent at the appropriate pH, typically using an appropriate buffer, pure supercoiled DNA can be preferentially eluted.
Particular aspects of the invention are as follows:
1. The use of fluorinated surfaces to isolate DNA from a lysed plasmid.
2. The use of a fluorosilane on silica.
3. The use of a fluorosilane on glass beads.
4. The use of a fluorinated membrane.
5. The use of fluorinated materials to reduce endotoxin in DNA.
DESCRIPTION OF PREFERRED EMBODIMENTS
A preferred process for the preparation of porous spherical particles of fluorinated polymer adsorbent comprises the steps of:
(1) forming a water-insoluble solution of organic compounds comprising a monomer selected from C
2-4
alkylene glycol esters of a C
3-6
acrylic acid or divinyl benzene, a polyfluorinated vinyl monomer; a free radical initiator; and a water-insoluble, organic solvent-soluble porogenic material, the weight ratio of the comonomers to porogenic material being from 0.5:1 to 2:1;
(2) forming a dilute solution of a dispersing agent in water from which any oxygen has been purged with inert gas,
(3) with agitation and inert gas purging, rapidly dispersing the water-insoluble solution of organic compounds from step (1) into the dilute aqueous solution from step (2) and, as necessary, adjusting the temperature of the dispersion to 30-90° C. to initiate copolymerization of the monomers, the level of mixing energy being sufficient to disperse the water-insoluble solution of organic compounds in the solution from step (2) in the form of liquid droplets having an average diameter of no more than 10-300 &mgr;m, at least 90% of the droplets being within 40% above or below the average mean particle diameter,
(4) continuing the agitation and oxygen purging of the dispersion from step (3) for a time sufficient to effect complete copolymerization of the monomers and particulation of the droplets in the form of finely divided polymer particles by precipitation of the copolymer therein;
(5) separating the finely divided copolymer particles from the polymerization reaction medium;
(6) extracting the porogenic material from the separated copolymer particles of step (5) by washing the particles with inert organic solvent, thereby forming pores within the copolymer, and
(7) drying the porous copolymer particles.
Another preferred process for the preparation of porous spherical particles of fluorinated polymer adsorbent comprises the steps:
(1) forming a water-insoluble solution of organic compounds comprising (a) a monomer selected from C
2-4
alkylene glycol esters of a C
3-6
acrylic acid and a divinyl benzene, (b) a polyfluorinated vinyl monomer; (c) a monomer selected from acrylic acid, methacrylic acid and esters thereof; (d) a free radical initiator; and (e) a water-insoluble, organic solvent-soluble porogenic material, the weight ratio of comonomers (a) plus (b) plus (c) to the porogenic material being from 0.5:1 to 2:1,
(2) forming a dilute solution of a dispersing agent in water from which any oxygen has been purged with inert gas;
(3) with a agitation and inert gas purging rapidly dispersing the water-insoluble solution of organic compounds from step (1) into the dilute aqueous solution from step (2) and, as necessary, adjusting the temperature of the dispersion to 30-90° C. to initiate copolymerization of the monomers, the level of mixing energy being sufficient to disperse the water-insoluble solution of organic compounds in the solution from step (2) in the form of liquid droplets having an average diameter of no more than 10-300 &mgr;m, at least 90% of the droplets being within 40% above or below the average mean particle diameter,
(4) continuing the agitation and oxygen purging of the dispersion from step (3) for a time sufficient to effect complete copolymerization of the monomers and particulation of the droplets in the form of finely divided polymer particles by precipitation of the copolymer therein;
(5) separating the finely divided copolymer particles from the polymerization reaction medium,
(6) extracting the porogenic material from the separated copolymer particles of step (5) by washing the particles with inert organic solvent, thereby forming pores within the copolymer, and
(7) drying the porous copolymer particles.
High quality adsorbent fluoropolymer particles may be made by suspension polymerizaton with an aqueous solution containing a conventional dispersing agent. The basic components of the process are (1) the water-insoluble polymerization system, which is comprised mainly of a polyfluorinated monomer, two or more ethylenically unsaturated monomers and a free radical-initiating catalyst, and (2) the dispersion medium, which is a dilute aqueous solution containing a conventional dispersing agent. By water-
Foelak Morton
Prometic Biosciences Inc.
Saliwanchik Lloyd & Saliwanchik
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