EP0324539A2
Expired / Lapsed
EP
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Improved capillary gel electrophoresis columns
Assignee: Northeastern University
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Filing: 1989-01-05
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Granted: —
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Expires: 2009-01-05
Abstract
Abstract An improved microcapillary column for high performance electrophoresis includes a microcapillary, a hydrophilic polymer within a gel of crosslinked polyacrylamide polymerized in the tube, and preferably, a thin layer of connecting material covalently bonded to the inner surface of the microcapillary wall and to the polymeric gel. The microcapillary is prepared by first covalently bonding a suitable bifunctional reagent to the inner surface of the microcapillary wall, and then causing a mixture of the hydrophilic polymer, monomer, crosslinking agent, and polymerization catalyst to react in the bore of the microcapillary to form a hydrophilic polymer-containing gel matrix which is covalently bonded to the microcapillary wall via the bifunctional reagent. In electrophoresis, this improved gel-containing microcapillary can provide peak efficiencies in excess of 100,000 theoretical plates within separation times of less than thirty minutes, permits trace level determinations of molecular weights, and permits electrophoretic operation at fields of 1000 V/cm or higher, resulting in extremely high resolution separations.
Inventors
- 👤 Barry L. Karger
- 👤 Aharon S. Cohen
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 03:12
CPC Classifications
G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; G01N27/44704; G01N27/44717; G01N27/44721; G01N27/44747
Claims Analysis
Total Claims: 46
Claim 1
Independent Claim
41 words
A gel-containing microcapillary column for high precision and high performance electrophoresis, comprising: a microcapillary having an interior cavity and a wall with an inner surface; a crosslinked polymeric gel in said interior cavity; and a hydrophilic polymer in said polymeric gel.
Claim 2
Dependent Claim
41 words
A gel-containing microcapillary column for high precision and high performance electrophoresis, comprising: a microcapillary having an interior cavity and a wall with an inner surface; a crosslinked polymeric gel in said interior cavity; and a hydrophilic polymer in said polymeric gel.
Claim 3
Dependent Claim
40 words
The microcapillary column of claim 1, further comprising: a layer of connecting material between said inner surface of said wall and said polymeric gel, said connecting material being covalently bonded to said microcapillary inner surface and to said polymeric gel.
Claim 4
Dependent Claim
50 words
The microcapillary of claim 2 wherein said connecting material is derived from a bifunctional reagent which possesses a reactive silane functional group capable of reacting with functional groups on said microcapillary inner surface, and a vinyl group capable of reacting with the monomer molecules which polymerize to form said gel.
Claim 5
Dependent Claim
23 words
The microcapillary of claim 3 wherein said bifunctional reagent is selected from the group consisting of 3-Methacryloxypropyltrimethoxysilane, 3-Methacryloxypropyldimethylethoxysilane, vinyltriacetoxysilane, vinyltri(β-methoxyethoxy)silane, vinyltrichlorosilane, and methylvinyldichlorosilane.
Claim 6
Dependent Claim
19 words
The microcapillary column of claim 1 wherein said microcapillary is made of a silica-based material, alumina, beryllia, or TEFLON.
Claim 7
Dependent Claim
13 words
The microcapillary of claim 5 wherein said microcapillary is made of fused silica.
Claim 8
Dependent Claim
17 words
The microcapillary of claim 1 wherein said microcapillary has an internal diameter between 10 and 2000 micrometers.
Claim 9
Dependent Claim
17 words
The microcapillary of claim 1 wherein said microcapillary has a wall thickness in the range 24-400 micrometers.
Claim 10
Dependent Claim
21 words
The microcapillary of claim 1 wherein said polymeric gel further comprises a copolymer of acrylamide and at least one crosslinking agent.
Claim 11
Dependent Claim
22 words
The microcapillary of claim 9 wherein said crosslinking agent is selected from the group consisting of N,N′-methylenebisacrylamide, N,N′-(1,2-dihydroxyethylene)-bisacrylamide, N,N′-diallyltartardiamide, N,N′-cystamine-bisacrylamide, and N-acryloyltris(hydroxymethyl)aminomethane.
Claim 12
Dependent Claim
21 words
The microcapillary of claim 1 wherein said hydrophilic polymer is present in said polymeric gel at a level of 1-40% weight/volume.
Claim 13
Dependent Claim
21 words
The microcapillary of claim 1 wherein said hydrophilic polymer is present in said polymeric gel at a level of 5-20% weight/volume.
Claim 14
Dependent Claim
19 words
The microcapillary of claim 1 wherein said hydrophilic polymer has an average molecular weight in the range 4000-500,000 Daltons.
Claim 15
Dependent Claim
19 words
The microcapillary of claim 1 wherein said hydrophilic polymer has an average molecular weight in the range 8000-200,000 Daltons.
Claim 16
Dependent Claim
12 words
The microcapillary of claim 1 wherein said hydrophilic polymer is polyethylene glycol.
Claim 17
Dependent Claim
19 words
The microcapillary of claim 15 wherein said polyethylene glycol has an average molecular weight in the range 4000-35,000 Daltons.
Claim 18
Dependent Claim
20 words
The microcapillary of claim 16 wherein said polyethylene glycol has an average molecular weight of at least approximately 8000 Daltons.
Claim 19
Dependent Claim
17 words
The microcapillary of claim 1 wherein said microcapillary possesses at least one cleanly and squarely cut end.
Claim 20
Independent Claim
137 words
A method for preparing a gel-containing microcapillary column for high performance high precision electrophoresis, comprising: contacting the interior surface of a silica-based microcapillary with a basic material; treating said microcapillary with a first solution including a bifunctional reagent, said reagent possessing a reactive silane functional group capable of reacting with silanol groups, and a vinyl functionality capable of polymerizing with monomers, to cause covalent bonding of said reagent to the inner surface of the wall of said microcapillary via said reactive silane; and treating said microcapillary with a second solution including at least one monomer, at least one crosslinking agent, and at least one free radical source, to cause polymerization, with formation of a crosslinked polymeric gel in the cavity of said microcapillary and covalent binding of said gel to said bifunctional reagent via said vinyl functionality.
Claim 21
Independent Claim
137 words
A method for preparing a gel-containing microcapillary column for high performance high precision electrophoresis, comprising: contacting the interior surface of a silica-based microcapillary with a basic material; treating said microcapillary with a first solution including a bifunctional reagent, said reagent possessing a reactive silane functional group capable of reacting with silanol groups, and a vinyl functionality capable of polymerizing with monomers, to cause covalent bonding of said reagent to the inner surface of the wall of said microcapillary via said reactive silane; and treating said microcapillary with a second solution including at least one monomer, at least one crosslinking agent, and at least one free radical source, to cause polymerization, with formation of a crosslinked polymeric gel in the cavity of said microcapillary and covalent binding of said gel to said bifunctional reagent via said vinyl functionality.
Claim 22
Dependent Claim
25 words
The method of claim 19 further comprising the step of heating said microcapillary at a temperature in excess of 100°C prior to said contacting step.
Claim 23
Dependent Claim
18 words
The method of claim 20 wherein said heating step is conducted at a temperature between 110 and 200°C.
Claim 24
Dependent Claim
43 words
The method of claim 19 wherein in said contacting step said basic material is ammonia gas and said contacting step further comprises flushing said microcapillary with said ammonia gas for a period of time sufficient to activate the inner surface of the microcapillary.
Claim 25
Dependent Claim
22 words
The method of claim 22 wherein said step of flushing with ammonia gas is conducted at a temperature in the range 20-35°C.
Claim 26
Dependent Claim
54 words
The method of claim 19 wherein in said contacting step the basic material is a solution of an alkali metal hydroxide and said contacting step further comprises filling said microcapillary with said solution and maintaining said solution in said microcapillary for a period of time sufficient to activate the interior surface of the microcapillary.
Claim 27
Dependent Claim
19 words
The method of claim 24 wherein said contacting step is carried out at a temperature in the range 20-35°C.
Claim 28
Dependent Claim
24 words
The method of claim 19 wherein said step of treating with a first solution including bifunctional reagent is conducted for at least one hour.
Claim 29
Dependent Claim
26 words
The method of claim 19 wherein said step of treating with a first solution including bifunctional reagent is conducted at a temperature in the range 20-35°C.
Claim 30
Dependent Claim
26 words
The method of claim 19 further comprising the step of removing unreacted bifunctional reagent after the step of treating with a first solution including bifunctional reagent.
Claim 31
Dependent Claim
41 words
The method of claim 19 wherein in said step of treating with a second solution including monomer, crosslinking agent, and free radical source, said second solution is made up in the buffer to be employed in the electrophoresis, and is degassed.
Claim 32
Dependent Claim
38 words
The method of claim 19 further comprising the step of conducting said polymerization of said monomer, crosslinking agent, and free radical source in said microcapillary at the temperature to be employed for electrophoresis on the resulting gel-containing microcapillary.
Claim 33
Dependent Claim
84 words
The method of claim 19 wherein in said step of treating with a second solution of monomer, crosslinking agent, and free radical source, the concentrations of monomer and crosslinking agent in said second solution are predetermined and the concentration of free radical source is determined experimentally by varying the concentration of such free radical source in trial solutions and monitoring the polymerization reaction until a concentration of such free radical source is found which causes nearly complete polymerization to occur in approximately 45-60 minutes.
Claim 34
Dependent Claim
21 words
The method of claim 19 further comprising the final step of cutting an end of said microcapillary off cleanly and squarely.
Claim 35
Dependent Claim
14 words
The method of claim 19 wherein said second solution further includes a hydrophilic polymer.
Claim 36
Dependent Claim
21 words
The method of claim 33 wherein said hydrophilic polymer is present in said second solution at a level of 1-40% weight/volume.
Claim 37
Dependent Claim
21 words
The method of claim 34 wherein said hydrophilic polymer is present in said second solution at a level of 5-20% weight/volume.
Claim 38
Dependent Claim
19 words
The method of claim 33 wherein said hydrophilic polymer has an average molecular weight in the range 4000-500,000 Daltons.
Claim 39
Dependent Claim
19 words
The method of claim 36 wherein said hydrophilic polymer has an average molecular weight in the range 8000-200,000 Daltons.
Claim 40
Dependent Claim
12 words
The method of claim 33 wherein said hydrophilic polymer is polyethylene glycol.
Claim 41
Dependent Claim
19 words
The method of claim 38 wherein said polyethylene glycol has an average molecular weight in the range 4000-35,000 Daltons.
Claim 42
Dependent Claim
20 words
The method of claim 39 wherein said polyethylene glycol has an average molecular weight of at least approximately 8000 Daltons.
Claim 43
Dependent Claim
23 words
The method of claim 19 wherein said bifunctional reagent is selected from the group consisting of 3-Methacryloxypropyltrimethoxysilane, 3-Methacryloxypropyldimethylethoxysilane, vinyltriacetoxysilane, vinyltri(β-methoxyethoxy)silane, vinyltrichlorosilane, and methylvinyldichlorosilane.
Claim 44
Dependent Claim
21 words
The method of claim 19 wherein said solution of bifuncional reagent contains between 4% and 60% by volume of said reagent.
Claim 45
Dependent Claim
60 words
A method for performing high resolution molecular sieving electrphoresis, comprising: injecting onto a gel-containing microcapillary column of claim 1 an aliquot of a sample containing analytes to be separated; applying to said microcapillary an electric field of at least 400 V/cm, to cause separation of said analytes on said microcapillary column; and instrumentally detecting and measuring the separated analytes sequentially.
Claim 46
Dependent Claim
137 words
A method for determining the molecular weight of an analyte, comprising: injecting onto a gel-containing microcapillary column of claim 1 an aliquot of a solution containing several standard analytes of known molecular weight; applying to said microcapillary a preselected standard value electric field, said field being at least 400 V/cm, to cause separation of said standard analytes on said microcapillary column; instrumentally detecting each separated standard analyte; and measuring its mobility; plotting the log of the molecular weight for each standard analyte versus its mobility under the standard conditions to create a calibration plot; and electrophoretically analyzing a sample solution of analytes whose molecular weights are unknown on the same column under the same conditions, measuring the mobilities of analytes present in said sample solution, and determining the molecular weights of the analytes from the calibration plot.
References Cited (Backward Citations)
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