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 methods characterized by the elution of analyte in a small volume of liquid.
1. A method of extracting an analyte from a solution comprising the steps of:
a. passing a solution containing an analyte back and forth through a pipette tip column, wherein the pipette tip column is comprised of:
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 bonded to and extending across the open channel,
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, bottom frit, and column body define the extraction media chamber, wherein the top frit and the bottom frit are less than 350 microns thick, wherein the bottom frit is a membrane screen and the top frit is optionally a membrane screen, and wherein the membrane screen is selected from a group of materials consisting of nylon, polyester, polyamide, polycarbonate, cellulose, polyethylene, nitrocellulose, cellulose acetate, polypropylene, polysulfone, and glass, and
iv. a bed of extraction media having a bed volume, whereby analyte adsorbs to said extraction media; and
b. eluting the analyte by aspirating and discharging a desorption solvent through the pipette tip column, and wherein said analyte is an active protein.
2. The method of claim 1 , wherein the volume of desorption solvent is in the range of about 10% to about 300% of the bed volume.
3. The method of claim 1 , wherein step (a) is performed more than one time.
4. The method of claim 1 , wherein the bed volume is in the range of between 0.1 and 100 uL.
5. The method of claim 1 , wherein between step (a) and step (b), a quantity of wash solution is aspirated and discharged through the extraction media.
6. The method of claim 1 , wherein step (b) is performed more than one time.
7. The method of claim 1 , wherein the extraction media comprises an affinity binding agent.
8. The method of claim 1 , wherein the bed of extraction media comprises a packed bed of gel resin beads.
9. the method of claim 8 , wherein the gel resin beads are agarose beads or sepharose beads.
10. The method of claim 1 , wherein the bottom frit has a pore volume of 1 microliter or less.
11. The method of claim 1 , wherein the bottom frit is less than 200 microns thick.
12. The method 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.
13. The method of claim 12 , wherein the pump is a pipettor, a syringe, or a peristaltic pump.