Method and device for extracting an analyte
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 columns characterized by low backpressure.
1. A pipette tip column for extracting an analyte 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 extending across the open channel, wherein the bottom frit is located at 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 or the bottom frit material is polyetheretherketone, vinyl polymer or polyvinyl chloride, wherein the top frit, bottom frit, and column body define an extraction media chamber; and
iv) a packed bed of hydrated gel resin positioned inside the extraction media chamber, wherein the gel resin is agarose or sepharose, wherein the bed is not tightly packed, and wherein said pipette tip column is characterized by a back pressure in the range of about 0.1 psi to 1.5 psi when an aqueous solution is passed through the column at a flow rate of 1 mL/min.
2. The pipette tip column of claim 1 wherein the gel resin is further comprised of an affinity binding group having an affinity for a functional, active, native or non-denatured protein analyte, and wherein the affinity binding group is selected from the group consisting of Protein A, Protein G, Protein L and an immobilized metal.
3. The pipette tip column of claim 1 , wherein the bed of gel resin has a volume of between about 0.1 μL and 1000 μL.
4. The pipette tip column of claim 1 , wherein the upper end of the column body is in operative engagement with a pump for aspirating fluid through the lower end of the column body, wherein said pump is a pipettor, a syringe or a syringe pump.
5. A multiplexed extraction system comprised of:
a plurality of pipette tip columns of claim 1 ; and
at least one pump in operative engagement with the pipette tip columns.
6. The multiplexed extraction system of claim 5 wherein the multiplexed extraction system is further comprised of:
a controller for controlling the pumps; and
a computer operating said controller, wherein the computer can be programmed to control the movement of the pumps through the controller.
7. A method for extracting an analyte from a sample solution comprising the steps of:
i) introducing a sample solution containing an analyte into the pipette tip column of claim 1 , whereby at least some fraction of the analyte is adsorbed to the gel resin positioned inside the pipette tip column;
ii) substantially evacuating the sample solution from the pipette tip column;
iii) introducing a desorption solvent into the pipette tip column, whereby at least some fraction of the bound analyte is desorbed from the pipette tip column into the desorption solvent; and
iv) eluting the desorption solvent containing the desorbed analyte from the pipette tip column.
8. The method of claim 7 , wherein an optional wash step is performed between steps (ii) and (iii).
9. The method of claim 8 , wherein the upper end of the pipette tip column body is in operative engagement with a pump and wherein the sample solution, the wash solution or the desorption solution is aspirated and discharged through the lower end of the pipette tip column at least once.
10. The method of claim 9 wherein the method is performed on a plurality of pipette tip columns operated in parallel.
11. The method of claim 10 wherein the pump is a multichannel pipettor or a liquid handing system.
12. A pipette tip column for extracting an analyte 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 extending across the open channel, wherein the bottom frit is located at 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 or the bottom frit material is selected from a group consisting of polyamide, polycarbonate, cellulose, nitrocellulose, cellulose acetate, polyvinylidine difluoride, polytetrafluoroethylene, polypropylene, polysulfone, metal, glass, polyetheretherketone, polyvinyl chloride, vinyl polymer, nylon and polyester, wherein the top frit, bottom frit, and column body define an extraction media chamber; and
iv) a packed bed of hydrated gel resin positioned inside the extraction media chamber, wherein the gel resin is comprised of dextran or a dextran copolymer, wherein the bed is not tightly packed, and wherein said pipette tip column is characterized by a back pressure in the range of about 0.1 psi to 1.5 psi when an aqueous solution is passed through the column at a flow rate of 1 mL/min.
13. The pipette tip column of claim 12 , wherein the bed of gel resin has a volume of between about 0.1 μL and 1000 μL.
14. The pipette tip column of claim 12 , wherein the upper end of the column body is in operative engagement with a pump for aspirating fluid through the lower end of the column body, wherein said pump is a pipettor, a syringe or a syringe pump.
15. A multiplexed extraction system comprised of:
a plurality of pipette tip columns of claim 12 ; and
at least one pump in operative engagement with the pipette tip columns.
16. The multiplexed extraction system of claim 15 wherein the multiplexed extraction system is further comprised of:
a controller for controlling the pumps; and
a computer operating said controller, wherein the computer can be programmed to control the movement of the pumps through the controller.
17. A method for extracting an analyte from a sample solution comprising the steps of:
i) introducing a sample solution containing an analyte into the pipette tip column of claim 12 , whereby at least some fraction of the analyte is adsorbed to the gel resin positioned inside the pipette tip column;
ii) substantially evacuating the sample solution from the pipette tip column;
iii) introducing a desorption solvent into the pipette tip column, whereby at least some fraction of the bound analyte is desorbed from the pipette tip column into the desorption solvent; and
iv) eluting the desorption solvent containing the desorbed analyte from the pipette tip column.
18. The method of claim 17 , wherein an optional wash step is performed between steps (ii) and (iii).
19. The method of claim 18 , wherein the upper end of the pipette tip column body is in operative engagement with a pump and wherein the sample solution, the wash solution or the desorption solution is aspirated and discharged through the lower end of the pipette tip column.
20. The method of claim 19 wherein the method is performed on a plurality of pipette tip columns operated in parallel.
21. The method of claim 20 wherein the pump is a multichannel pipettor or a liquid handing system.