Polymers for separation of biomolecules by capillary electrophoresis

Abstract

The invention provides uncharged water-soluble silica-adsorbing polymers for suppressing electroendoosmotic flow and to reduce analyte-wall interactions in capillary electrophoresis. In one aspect of the invention, one or more of such polymers are employed as components of a separation medium for the separation of biomolecules, such as polynucleotides, polysaccharides, proteins, and the like, by capillary electrophoresis. Generally, such polymers are characterized by (i) water solubility over the temperature range between about 20° C. to about 50° C., (ii) concentration in a separation medium in the range between about 0.001% to about 10% (weight/volume), (iii) molecular weight in the range of about 5×10 3 to about 1×10 6 daltons, and (iv) absence of charged groups in an aqueous medium having pH in the range of about 6 to about 9. In one embodiment, polymers of the invention are selected from the group consisting of polylactams, such as polyvinylpyrrolidone; N,N-disubstituted polyacrylamides; and N-substituted polyacrylamides. In accordance with the method of the invention, a sufficient amount of polymer adsorbs to the capillary surface to establish a zone of high viscosity that shields the analyte from the wall and impedes the movement of an electrical double layer under an electric field.

1 . A method of suppressing electroendoosmotic flow in capillary electrophoresis, the method comprising providing a separation medium containing one or more uncharged water-soluble silica-adsorbing polymers having (i) water solubility in a temperature range between about 20° C. and about 50° C., (ii) a concentration in the separation medium in a range between about 0.001% and about 10% (weight/volume), (iii) a molecular weight in the range between about 5×10 3  and about 1×10 6  daltons, and (iv) an absence of charged groups in an aqueous medium having a pH in the range between about 6 and about 9. 
     
     
         2 . The method of  claim 1  wherein said one or more uncharged water-soluble silica-adsorbing polymers are substantially non-hydroxylic. 
     
     
         3 . The method of  claim 2  wherein at least one of said one or more uncharged water-soluble silica-adsorbing polymer is a polylactam. 
     
     
         4 . The method of  claim 3  wherein at least one of said one or more uncharged water-soluble silica-adsorbing polymer is polyvinylpyrrolidone. 
     
     
         5 . The method of  claim 1  wherein at least one of said one or more uncharged water-soluble silica-adsorbing polymer is an N,N-disubstituted polyacrylamide or an N-substituted polyacrylamide, wherein said nitrogen substituents are selected from the group consisting of C 1  to C 3  alkyl; halo-substituted C 1  to C 3  alkyl; methoxy-substituted C 1  to C 3  alkyl; and hydroxyl-substituted C 1  to C 3  alkyl. 
     
     
         6 . The method of  claim 5  wherein said nitrogen substituents are selected from the group consisting of C 1  to C 3  alkyl; halo-substituted C 1  to C 3  alkyl; and methoxy-substituted C 1  to C 3  alkyl. 
     
     
         7 . The method of  claim 6  wherein said at least one of said one or more uncharged water-soluble silica-adsorbing polymer is poly(dimethylacrylamide). 
     
     
         8 . The method of  claim 7  wherein said electroendoosmotic flow is less than about 2×10 −5  cm 2 /sec-volts. 
     
     
         9 . The method of  claim 7  wherein said one or more uncharged water-soluble silica-adsorbing polymers provides a substantially linear relationship between number of theoretical plates and size of polynucleotide in the size range of between about 100 and about 500 nucleotides. 
     
     
         10 . A method of suppressing wall-analyte interaction in capillary electrophoresis, the method comprising providing a separation medium containing one or more uncharged water-soluble silica-adsorbing polymers having (i) water solubility in a temperature range between about 20° C. and about 50° C., (ii) a concentration in the separation medium in a range between about 0.001% and about 10% (weight/volume), (iii) a molecular weight in the range between about 5×10 3  and about 1×10 6  daltons, and (iv) an absence of charged groups in an aqueous medium having a pH in the range between about 6 and about 9. 
     
     
         11 . The method of  claim 10  wherein said one or more uncharged water-soluble silica-adsorbing polymers are substantially non-hydroxylic. 
     
     
         12 . The method of  claim 11  wherein at least one of said one or more uncharged water-soluble silica-adsorbing polymer is a polylactam. 
     
     
         13 . The method of  claim 12  wherein at least one of said one or more uncharged water-soluble silica-adsorbing polymer is polyvinylpyrrolidone. 
     
     
         14 . The method of  claim 10  wherein at least one of said one or more uncharged water-soluble silica-adsorbing polymer is an N,N-disubstituted polyacrylamide or an N-substituted polyacrylamide, wherein said nitrogen substituents are selected from the group consisting of C 1  to C 3  alkyl; halo-substituted C 1  to C 3  alkyl; methoxy-substituted C 1  to C 3  alkyl; and hydroxyl-substituted C 1  to C 3  alkyl. 
     
     
         15 . The method of  claim 14  wherein said nitrogen substituents are selected from the group consisting of C 1  to C 3  alkyl; halo-substituted C 1  to C 3  alkyl; and methoxy-substituted C 1  to C 3  alkyl. 
     
     
         16 . The method of  claim 15  wherein said at least one of said one or more uncharged water-soluble silica-adsorbing polymer is poly(dimethyacrylamide). 
     
     
         17 . The method of  claim 16  wherein said electroendoosmotic flow is less than about 2×10 −5  cm 2 /sec-volts. 
     
     
         18 . The method of  claim 16  wherein said one or more uncharged water-soluble silica-adsorbing polymers provides a substantially linear relationship between number of theoretical plates and size of polynucleotide in the size range of between about 100 and about 500 nucleotides. 
     
     
         19 . A composition for separating polynucleotides by capillary electrophoresis, the composition comprising:
 a charge-carrying component;   a sieving component; and   a surface interaction component consisting of one or more uncharged water-soluble silica-adsorbing polymers having (i) water solubility in a temperature range between about 20° C. and about 50° C., (ii) a concentration in the separation medium in a range between about 0.001% and about 10% (weight/volume), (iii) a molecular weight in the range between about 5×10 3  and about 1×10 6  daltons, and (iv) an absence of charged groups in an aqueous medium having a pH in the range between about 6 and about 9.   
     
     
         20 . The composition of  claim 19  having a viscosity less than about 5000 centipoise. 
     
     
         21 . A method of separating different-sized polynucleotides by elects electophoresis in an uncoated silica capillary, the method comprising the steps of
 providing an uncoated silica capillary having a first end and a second end; the uncoated silica capillary containing one or more uncharged water-soluble silica-adsorbing polymers having (i) water solubility in a temperature range between about 20° C. and about 50° C., (ii) a concentration in the separation medium in a range between about 0.001% and about 10% (weight/volume), (iii) a molecular weight in the range between about 5×10 3  and about 1×10 6  daltons, and (iv) an absence of charged groups in an aqueous medium having a pH in the range between about 6 and about 9;   loading a sample of different-sized polynucleotide in the uncoated silica capillary; and   applying an electrical field between the first and second ends of the uncoated silica capillary so that the different-sized polynucleotides in the sample migrate through the uncoated silica capillary.