High performance microcapillary gel electrophoresis

• US 4,997,537 A
• Filed: 09/12/1989
• Issued: 03/05/1991
• Est. Priority Date: 10/21/1986
• Status: Expired due to Term

• Current Assignee: Northeastern University
• Original Assignee: Northeastern University
• Inventors: Karger, Barry L., Cohen, Aharon
• Primary Examiner(s): Niebling, John F.
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US07/406,080
• Time in Patent Office: 539 Days
• Field of Search: 204/182.8, 204/180.1, 204/299 R
• US Class Current: 204/453
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture
• CPC Class Codes: G01N 27/44752 Controlling the zeta potential, e.g. by wall coatings

Abstract

A microcapillary column for high performance electrophoresis. A preferred column includes a microcapillary, a thin layer of coating material covalently bonded to the inner surface of the microcapillary wall, a thin layer of a hydrophilic polymer absorbed on the layer of coating material, and a gel comprising polyacrylamide polymerized in the tube, filling it. The gel-containing microcapillary is prepared by covalently bonding a layer of a suitable coating material to the inner surface of the microcapillary wall, applying a layer of hydrophilic polymer, and then causing a mixture of monomer with or without crosslinking agent, initiator, and polymerization catalyst to react in the bore of the microcapillary to form a polymeric matrix. In electrophoresis, the gel-containing microcapillary provides peak efficiencies in excess of 100,000 theoretical plates and in some instances over 1,000,000 theoretical plates within separation times of less than thirty minutes, permits trace level determinations of molecular weights, and permits electrophoretic operation at fields of 300 V/cm or higher, resulting in extremely high resolution separations.

Claims

1. A gel-containing microcapillary column high precision and high performance electrophoresis, comprising:

a microcapillary having an interior and a wall with an inner surface;

a layer of coating material covalently bonded to said inner surface of said wall;

a layer of hydrophilic polymer adsorbed on said layer of coating material; and

a polymeric gel filling said interior cavity.

2. The microcapillary of claim 1 wherein said microcapillary is made of fused silica.

3. The microcapillary of claim 1 wherein said hydrophilic polymer is polyethylene glycol.

4. The microcapillary of claim 1 wherein polymeric gel further comprises a copolymer of acrylamide and at least one crosslinking agent.

5. The microcapillary of claim 1 wherein said coating material originates as a bifunctional reagent selected from the group consisting of 3-Methacryloxypropyltrimethyoxysilane, 3-Methacryloxypropyldimethylethoxysilane, vinyltriacetoxysilane, vinyltri( -methoxyethoxy)silane, vinyltrichlorosilane, and methylvinyldichlorosilane.

6. A gel-containing microcapillary column for high precision high performance electrophoresis, comprising:

a silica microcapillary having an interior cavity, a wall having an inner surface, and an internal diameter between 10 and 200 micrometers;

a layer of coating material covalently bonded to said inner surface of said wall, said coating material being derived from 3-Methacryloxypropyl-trimethoxysilane or 3-Methacryloxypropyldimethylethoxysilane;

a layer of polyethylene glycol adsorbed on said layer of coating material; and

a gel comprising polyacrylamide filling said interior cavity.

7. The microcapillary of claim 6 wherein said gel is a copolymer of acrylamide monomer and N,N'"'"'-methylenebisacrylamide crosslinking agent.

8. A method of performing high resolution molecular sieving electrophoresis, comprising:

injecting an aliquot of a sample containing analytes to be separated onto a gel-containing microcapillary column comprising;

a microcapillary having an interior cavity and a wall with an inner surface;

a layer of coating material covalently bonded to said inner surface of said wall;

a layer of hydrophilic polymer on said layer of coating material; and

a polymeric gel filling said interior cavity;

applying an electric field of at least 100 volts/cm; and

instrumentally detecting and measuring the separated analytes sequentially.