Method and device for extracting an analyte

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 columns typically include a bed of extraction media positioned in the column between two frits. In some embodiments, the extraction columns employ modified pipette tips as column bodies. In some embodiments, the invention provides methods characterized by the use of verly slow flow rates.

1 . A method for passing liquid through a packed bed column comprising the steps of: 
 a. providing a packed-bed column comprising: 
 i. a column body having an open upper end, an open lower end, and an open channel between the upper and lower ends of the column body;  
 ii. a bottom frit attached to and extending across the open channel;  
 iii. a top frit attached to and extending across the open channel between the bottom frit and the open upper end of the column body, wherein the top frit, bottom frit, and column body define a media chamber; and  
 iv. a packed bed of media having a bed volume of less than about 100 uL positioned inside the media chamber; and  
   b. passing a liquid through the packed bed of media at a flow rate of between about 0.1 and about 4 mL/min.    
     
     
         2 . The method of  claim 1 , wherein the packed bed of media has a bed volume of less than about 25 uL.  
     
     
         3 . The method of  claim 1 , wherein the flow rate is between about 0.5 and 2 mL/min.  
     
     
         4 . The method of  claim 1 , wherein the column body comprises a luer adapter, a syringe or a pipette tip.  
     
     
         5 . The method of  claim 4 , wherein the upper end of the column body is attached to a pump for passing liquid through the lower end and the packed bed of media.  
     
     
         6 . The method of  claim 5 , wherein the pump is a pipettor or a syringe.  
     
     
         7 . The method of  claim 4 , wherein the column body comprises a pipette tip and the pump is a pipettor.  
     
     
         8 . The method of  claim 7 , wherein the pipette comprises an electrically driven actuator.  
     
     
         9 . The method of  claim 8 , wherein movement of the electrically driven actuator is controlled by a microprocessor.  
     
     
         10 . The method of  claim 9 , wherein the microprocessor is programmable.  
     
     
         11 . The method of  claim 9 , wherein the microprocessor is programmed to pass a substantially pre-selected volume of solution through the bed of media at a substantially pre-selected flow rate.  
     
     
         12 . The method of  claim 1 , wherein the solution is drawn and dispensed through the lower end and the packed bed of media.  
     
     
         13 . The method of  claim 1 , wherein the media is an extraction media.  
     
     
         14 . The method of  claim 13 , wherein the solution contains an analyte that adsorbs to the extraction media.  
     
     
         15 . The method of  claim 13 , wherein an analyte is bound to the extraction media and the solution is a desorption solution that elutes the analyte from the extraction media.  
     
     
         16 . The method of  claim 13 , wherein an analyte is bound to the extraction media and the solution is a wash solution.  
     
     
         17 . The method of  claim 14 ,  15  or  16 , wherein the analyte is a biomolecule.  
     
     
         18 . The method of  claim 17 , wherein the biomolecule is a protein.  
     
     
         19 . The method of  claim 13 , wherein the bed of extraction media comprises a packed bed of gel resin beads.  
     
     
         20 . The method of  claim 19 , wherein the gel resin beads are agarose beads or sepharose beads.  
     
     
         21 . A pipette comprising: 
 a. a body;    b. a microprocessor;    c. an electrically driven actuator disposed within the body, the actuator in communication with and controlled by the microprocessor;    d. a displacement assembly including a displacing piston moveable within one end of a displacement cylinder having a displacement chamber and having another end with an aperture, wherein said displacing piston is connected to and controlled by said actuator;    e. a pipette tip in communication with said aperture, wherein the microprocessor is programmable to cause movement of the piston in the cylinder at a rate that results in drawing a liquid into the pipette tip at a flow rate of between about 0.1 and about 4 mL/min when the tip is in communication with the liquid.    
     
     
         22 . The pipette of  claim 21 , wherein the pipette tip comprises: 
 a. a tip body having an open upper end, an open lower end, and an open channel between the upper and lower ends of the tip body;    b. a bottom frit bonded to and extending across the open channel;    c. a top frit bonded to and extending across the open channel between the bottom frit and the open upper end of the tip body, wherein the top frit, bottom frit, and column body define a media chamber; and    d. a bed of media positioned inside the media chamber.    
     
     
         23 . The pipette tip of  claim 22 , wherein the bed of media comprises a packed bed of chromatography or extraction media.  
     
     
         24 . The pipette tip of  claim 23 , wherein the media comprises gel resin beads.  
     
     
         25 . The pipette tip of  claim 24 , wherein the gel resin beads are agarose or sepharose.  
     
     
         26 . The pipette of  claim 21 , wherein the microprocessor is external to the body.  
     
     
         27 . The pipette of  claim 21 , wherein the flow rate is between about 0.5 and about 2 mL/min.