Hydrophilic monolithic columns for separation of saccharide mixtures

Abstract

Polar, monolithic CEC columns having an amine and/or cyano functional stationary phase are used to separate complex mixtures of saccharides. Using the positive-ion mode of detection for neutral saccharides, detection conditions were optimized to low-femtomole sensitivities with the use of an ion trap mass spectrometer. The described column technology provides a universal system for separating a wide range of carbohydrates: mono- and oligosaccharides with the intact reducing end, as well as saccharide alditols.

1 - 12 . Cancelled  
     
     
         13 . A method of chromatographically separating a mixture of saccharides comprising the steps of 
 introducing a saccharide mixture to the column of claim  1 ;    inducing flow of a mobile phase through the column to produce a column effluent; and    detecting separated saccharides in the column effluent.    
     
     
         14 . The method of  claim 13  wherein flow of the mobile phase is induced by applying an electric field to the column.  
     
     
         15 . A method of chromatographically separating a mixture of saccharides comprising the steps of 
 introducing a saccharide mixture to a chromatographic column, wherein said column includes, as a stationary phase, a polyfunctional polyacrylamide gel, said polyacrylamide gel formed by polymerization of a monomer mixture, said mixture comprising acrylamide, bisacrylamide, a non-reactive filler compound for forming pores in the polyacrylamide gel, a polymerizable charge ligand of the formula RX wherein X is a functional group capable of maintaining a charge and R is an olefin functional group capable of free radical propagated polymerization and either a polymerizable cyano compound of the formula R′CN wherein CN is cyano and R′ is an olefin functional group capable of free radical propagated polymerization or a polymerizable amine of the formula R″N(R 1 )(R 2 ), wherein R 1  and R 2  are each independently either H or a C 1  to C 6  alkyl and R″ is an olefin functional group capable of free radical propagated polymerization;    inducing flow of a mobile phase through the column to produce a column effluent; and    detecting separated saccharides in the column effluent.    
     
     
         16 . The method of  claim 15  wherein the saccharide mixture contains monosaccharides, disaccharides, oligosaccharides or mixtures thereof.  
     
     
         17 . The method of  claim 15  wherein the charge ligand has a negative charge.  
     
     
         18 . The method of  claim 15  wherein the charge ligand has a positive charge.  
     
     
         19 . The method of  claim 15  wherein the saccharide mixture comprises saccharide anomers and the mobile phase comprises from about 80 vol % to about 99 vol % of acetonitrile.  
     
     
         20 . The method of  claim 15  wherein the column effluent is analyzed by mass spectroscopy.  
     
     
         21 . The method of  claim 15  wherein the amine is a primary amine.  
     
     
         22 . The method of  claim 15  wherein the filler compound is polyethylene glycol.  
     
     
         23 . The method of  claim 22  wherein the polyethylene glycol has a molecular weight from about 7,500 to about 20,000.  
     
     
         24 . The method of  claim 15  wherein the filler compound is from about 1% (w/v) to about 5% (w/v) of the monomer mixture.  
     
     
         25 . The method of  claim 15  wherein the flow of the mobile phase is induced by applying an electric field to the column.  
     
     
         26 . A method of chromatographically separating a mixture of saccharides comprising the steps of 
 introducing a saccharide mixture to a chromatographic column, wherein said column includes, as a stationary phase, a polyfunctional polyacrylamide gel, said polyacrylamide gel formed by polymerization of a monomer mixture, said mixture comprising acrylamide, bisacrylamide, a non-reactive filler compound for forming pores in the polyacrylamide gel, and either a polymerizable cyano compound of the formula R′CN wherein CN is cyano and R′ is an olefin functional group capable of free radical propagated polymerization or a polymerizable amine of the formula R″N(R 1 )(R 2 ), wherein R 1  and R 2  are each independently either H or a C 1  to C 6  alkyl and R″ is an olefin functional group capable of free radical propagated polymerization;    inducing flow of a mobile phase through the column to produce a column effluent; and    detecting separated saccharides in the column effluent.    
     
     
         27 . The method of  claim 26  wherein the saccharide mixture contains monosaccharides, disaccharides, oligosaccharides or mixtures thereof.  
     
     
         28 . The method of  claim 26  wherein the monomer mixture further comprises a polymerizable charge ligand of the formula RX wherein X is a functional group capable of maintaining a charge and R is an olefin functional group capable of free radical propagated polymerization.  
     
     
         29 . The method of  claim 28  wherein the charge ligand has a negative charge.  
     
     
         30 . The method of  claim 28  wherein the charge ligand has a positive charge.  
     
     
         31 . The method of  claim 28  wherein the flow of the mobile phase is induced by applying an electric field to the column.  
     
     
         32 . The method of  claim 26  wherein the saccharide mixture comprises saccharide anomers and the mobile phase comprises from about 80 vol % to about 99 vol % of acetonitrile.  
     
     
         33 . The method of  claim 26  wherein the column effluent is analyzed by mass spectroscopy.  
     
     
         34 . The method of  claim 26  wherein the amine is a primary amine.  
     
     
         35 . The method of  claim 26  wherein the filler compound is polyethylene glycol.  
     
     
         36 . The method of  claim 35  wherein the polyethylene glycol has a molecular weight from about 7,500 to about 20,000.  
     
     
         37 . The method of  claim 26  wherein the filler compound is from about 1% (w/v) to about 5% (w/v) of the monomer mixture.