Low Dead Volume Extraction Column Device

Abstract

The invention provides extraction columns for the purification of an analyte (e.g., a biological macromolecule, such as a peptide, protein or nucleic acid) from a sample solution, as well as methods for making and using such columns. The invention is characterized by the use of low dead volume columns, which is achieved in part by the use of low pore volume frits (e.g., membrane screens) to contain a bed of extraction media in the column. Low dead volume facilitates the elution of the captured analyte into a very small volume of desorption solution, allowing for the preparation of low volume samples containing relatively high concentrations of analyte.

1 . A pipette tip extraction column comprising:
 i) a column body having an open upper end for attachment to a pump, an open lower end for passing fluid into and out of the column body, and an open channel between the upper and lower end of the column body, wherein the column body comprises a pipette tip;   ii) a bottom frit bonded to and extending across the open channel, the bottom frit having a pore volume, and wherein the bottom frit is located at or near the open lower end of the column body;   iii) a top frit bonded to and extending across the open channel between the bottom frit and the open upper end of the column body, wherein the top frit and bottom frit are flexible, and wherein the top frit, bottom frit, and channel surface define an extraction media chamber; and   iv) a bed of gel resin extraction media positioned inside the extraction media chamber.   
     
     
         2 . The pipette tip extraction column of  claim 1 , wherein the top frit or the bottom frit is a membrane screen. 
     
     
         3 . The pipette tip extraction column of  claim 1 , wherein the gel resin is selected from the group consisting of agarose and sepharose. 
     
     
         4 . The pipette tip extraction column of  claim 1 , wherein the gel resin extraction media comprises an affinity binding group having an affinity for a biological molecule of interest. 
     
     
         5 . The pipette tip extraction column of  claim 4 , wherein the affinity binding group is selected from the group consisting of Protein A, Protein G, Protein L and an immobilized metal. 
     
     
         6 . The pipette tip extraction column of  claim 1 , wherein the upper end of the column body is attached to a pump for aspirating fluid through the lower end of the column body. 
     
     
         7 . The pipette tip extraction column of  claim 6 , wherein the pump is a pipettor or a syringe. 
     
     
         8 . A pipette tip extraction column comprising:
 i) a column body having an open upper end for attachment to a pump, an open lower end for passing fluid into and out of the column body, and an open channel between the upper and lower end of the column body, wherein the column body comprises a pipette tip;   ii) a bottom frit bonded to and extending across the open channel, the bottom frit having a pore volume, and wherein the bottom frit is located at or near the open lower end of the column body;   iii) a top frit bonded to and extending across the open channel between the bottom frit and the open upper end of the column body, wherein the top frit and bottom frit are flexible membrane screens, and wherein the top frit and the bottom frit are bonded to the column body by gluing or welding, and wherein the top frit, bottom frit, and channel surface define an extraction media chamber; and   iv) a bed of gel resin extraction media positioned inside the extraction media chamber.   
     
     
         9 . The pipette tip extraction column of  claim 8 , wherein the gel resin is selected from the group consisting of agarose and sepharose. 
     
     
         10 . The pipette tip extraction column of  claim 8 , wherein the gel resin extraction media comprises an affinity binding group having an affinity for a biological molecule of interest. 
     
     
         11 . The pipette tip extraction column of  claim 10 , wherein the affinity binding group is selected from the group consisting of Protein A, Protein G, Protein L and an immobilized metal. 
     
     
         12 . The pipette tip extraction column of  claim 8 , wherein the upper end of the column body is attached to a pump for aspirating fluid through the lower end of the column body. 
     
     
         13 . The pipette tip extraction column of  claim 12 , wherein the pump is a pipettor or a syringe. 
     
     
         14 . A pipette tip extraction column comprising:
 i) a column body having an open upper end for attachment to a pump, an open lower end for passing fluid into and out of the column body, and an open channel between the upper and lower end of the column body, wherein the column body comprises a pipette tip;   ii) a bottom frit extending across the open channel, the bottom frit having a pore volume, and wherein the bottom frit is located at or near the open lower end of the column body;   iii) a top frit extending across the open channel between the bottom frit and the open upper end of the column body, wherein the top frit and bottom frit are attached to the column body by a means selected from the group consisting of an annular pip, gluing, and welding, and wherein the top frit, bottom frit, and channel surface define an extraction media chamber; and   iv) a bed of gel resin extraction media positioned inside the extraction media chamber.   
     
     
         15 . The pipette tip extraction column of  claim 14 , wherein the top frit or the bottom frit is a membrane screen. 
     
     
         16 . The pipette tip extraction column of  claim 14 , wherein the gel resin is selected from the group consisting of agarose and sepharose. 
     
     
         17 . The pipette tip extraction column of  claim 14 , wherein the gel resin extraction media comprises an affinity binding group having an affinity for a biological molecule of interest. 
     
     
         18 . The pipette tip extraction column of  claim 17 , wherein the affinity binding group is selected from the group consisting of Protein A, Protein G, Protein L and an immobilized metal. 
     
     
         19 . The pipette tip extraction column of  claim 14 , wherein the upper end of the column body is attached to a pump for aspirating fluid through the lower end of the column body. 
     
     
         20 . The pipette tip extraction column of  claim 19 , wherein the pump is a pipettor or a syringe.