Formulations for polyacrylamide matrices in electrokinetic and chromatographic methodologies

• US 5,470,916 A
• Filed: 06/03/1994
• Issued: 11/28/1995
• Est. Priority Date: 12/06/1991
• Status: Expired due to Term

• Current Assignee: Marcella Chiari, Pier G. Righetti
• Original Assignee: Marcella Chiari, Pier G. Righetti
• Inventors: Chiari, Marcella, Righetti, Pier G.
• Primary Examiner(s): Schofer, Joseph L.
• Assistant Examiner(s): Cheng, Wu C.
• Application Number:
  US08/244,608
• Time in Patent Office: 543 Days
• Field of Search: 526/304, 526/201, 522/49, 522/63, 522/67, 525/296
• US Class Current: 525/296
• CPC Class Codes: B01J 20/26 Synthetic macromolecular compounds
• CPC Class Codes: B01J 20/291 Gel sorbents
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: C08F 20/58 containing oxygen in addition to the carbonamido oxygen , e.g. N-methylolacrylamide, N-acryloylmorpholine
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture

Abstract

Matrices for electrokinetic and chromatographic separations, based on a unique class of mono- and disubstituted (on the amido nitrogen) acrylamides (e.g., acrylamido-N-ethoxy ethanol, acrylamido-N,N-diethoxy ethanol) off erthe following advantages: a) strong resistance to the alkaline hydrolysis (most zone separations occurring at basic pH values); b) a high hydrophilicity; and c) greater porosity, due to the larger M_r value of the monomers. Such matrices can be prepared as `chemically crosslinked` gels or as `physical` gels, the latter consisting of strings of monofunctional monomer in the absence of crosslinker, at concentrations above the entanglement threshold. It is moreover possible to further increment the gel porosity by polymerizing it in the presence of laterally aggregating agents (e.g., polyethylene glycol of M_r 6-20,000 Da), thus obtaining matrices which allow better resolution of large M_r proteins and of nucleic acid fragments; by polymerizing the matrix in the presence of a gradient of the laterally aggregating agent, it is possible to obtain a porosity gradient even at a constant percent of monomers in the gel. Finally, photopolymerization (e.g., with methylene blue and a redox couple, sodium toluene sulfinate and diphenyliodonium chloride and light), instead with the standard red/ox catalysts, prevents formation of N-oxides of amines present in the polymerizing solution.

Claims

1. A poly-(N-substituted)acrylamide matrix in the form of a gel, to be used in electrokinetic or chromatographic methods, obtained by polymerization of monomers having the formula (I):

##STR2## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides, said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

2. A poly-(N-substituted)acrylamide matrix according to claim 1, obtained by polymerization of N-(2-hydroxyethoxy)ethyl-acrylamide, N-(2-hydroxyethoxy)ethyl-methacrylamide, N,N-di(2-hydroxyethoxy)ethyl-acrylamide, or N,N-di(2-hydroxyethoxy)ethyl-methacrylamide, or by copolymerization thereof with N,N-dimethyl-acrylamide or N,N-dimethyl-methacrylamide.

3. A poly-(N-substituted)acrylamide matrix according to claim 1 wherein said hydrophilic polymer is a member selected from the group consisting of polyethylene glycol and polyvinylpyrrolidone.

4. A poly-(N-substituted)acrylamide matrix according to claim 1, obtained by photopolymerization or photocopolymerization of monomers having formula (I).

5. A poly-(N-substituted)acrylamide matrix according to claim 4, wherein said photopolymerization or photocopolymerization is carried out in the presence of riboflavin with a UV-A lamp of wattage >

50-W at a temperature >

50°

C.

6. A poly-(N-substituted)acrylamide matrix according to claim 4, wherein said photopolymerization or photocopolymerization is carried out in the presence of methylene blue, and sodium toluenesulfinate and diphenyliodonium chloride.

7. A poly-(N-substituted)acrylamide matrix according to claim 1, wherein said matrix is treated with one or more chemical scavengers to add to and thereby eliminate any double bonds of unreacted acrylamide.

8. A method for performing electrokinetic separation of a mixture of proteins, nucleic acid fragments, or both, said method comprising subjecting said mixture to electrokinetic separation conditions in a matrix obtained by polymerization of monomers having the formula:

##STR3## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides, said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

9. A capillary having an inner wall coated with a monomer having the formula:

##STR4## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, said monomer linked to said inner wall through bifunctional agents forming either --Si--O--Si-- bonds or direct --Si--C.tbd. bonds said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

10. A method for determining protein molecular sizes in a mixture of proteins by sodium dodecyl sulfate electrophoresis, said method comprising subjecting said mixture to electrophoresis in a matrix obtained by polymerization of monomers having the formula:

##STR5## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

11. A method for performing electrophoresis of a mixture of proteins, nucleic acid fragments, or both, said method comprising subjecting said mixture of electrophoretic separation conditions in a matrix obtained by polymerization of monomers having the formula:

##STR6## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

12. A method for purifying proteins in protein preparations containing at least one of pyrogens, nucleic acid fragments and viral particles, said method comprising isoelectrically focusing said proteins in isoelectric buffering membranes in multicompartment electrolyzers to remove any pyrogens, nucleic acid fragments or viral particles present in said protein preparations, said isoelectric buffering membranes comprised of a matrix obtained by polymerization of monomers having the formula:

##STR7## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

13. A method for preparing gel slabs for long-term storage prior to use in an electrokinetic separation, said gels having been obtained by polymerization of monomers having the formula:

##STR8## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides,

said method comprising either impregnating said slabs with a nonaqueous solvent or washing and drying said slabs said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

14. Chromatographic beads comprised of a polymer obtained by polymerization of monomers having the formula:

##STR9## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}--H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides, said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.

15. Chromatographic beads comprised of a core of plastic or glass coated with a polymer obtained by polymerization of monomers having the formula:

##STR10## in which R represents hydrogen or CH₃, while R₁ and R₂, independently, represent hydrogen or a group having the formula --{(CH₂)_n --O--(CH₂)_n --O}_m --H, where n=2 or 3 and m=1-5, with the proviso that one of R₁ and R₂ is different from hydrogen, or by copolymerization of the same monomers with other acrylamides or methacrylamides, said polymerization or copolymerization conducted in the presence of a hydrophilic polymer producing lateral chain aggregation.