Automated group separations of complex mixtures with gel filtration pipette tips
A device for automated or manual group separations of complex biological mixtures and methods of use are described. The device is a gel filtration pipette tip having a size exclusion media therein and held in place by a filter at the distal end of the pipette tip and an optional removable or pierceable barrier at the proximal end of the pipette tip. The size exclusion media is wetted by aspirating a solvent from the bottom of the pipette tip and equilibrating the solvent with the size exclusion media. The solvent is then allowed to gravity drain from the pipette tip, allowing the size exclusion media to settle and reproducibly pack into a homogenous gel. Gel filtration separations can then be performed with the gelled size exclusion media.
1 . A method of preparing a gel filtration pipette tip, comprising:
a) attaching a filtration pipette tip having a hub at a proximal end to a pipetting aid, wherein said filtration pipette tip has a filter located at a distal delivery end and a dry size exclusion media above said filter and below said hub attached to said pipetting aid;
b) aspirating a solvent through said distal delivery end of said filtration pipette tip;
c) equilibrating said dry size exclusion media and said solvent to form a wetted size exclusion media in said filtration pipette tip; and
d) draining the excess solvent through said distal delivery end of said filtration pipette tip to form a gel filtration pipette tip, wherein said wetted size exclusion media settles during said draining and forms a homogenous gel filtration column.
2 . The method of claim 1 , wherein said pipetting aid is a hand-held pipettor, a syringe, or a robotic liquid handler that directly attaches to said filtration pipette tip or indirectly attaches thereto through the use of an adaptor or a top pipette tip.
3 . The method of claim 1 , wherein said attaching, aspirating, equilibrating, removing, and draining steps are performed on a robotic liquid handling platform by a robotic liquid handler.
4 . The method of claim 1 , wherein said filter is a screen or porous frit comprising stainless steel, porous polymeric material, porous glass, or porous ceramic.
5 . The method of claim 1 , wherein said dry size exclusion media comprises agarose, sepharose, polyacrylamide, dextran, dextran crosslinked with epichlorohydrin, polystyrene, polyacrylate, cellulose, or a combination thereof.
6 . The method of claim 1 , wherein said draining step comprises gravity flow or positive pressure.
7 . The method of claim 1 , wherein said filtration pipette tip has a barrier at or below said hub and said barrier is removed or pierced before use of said filtration pipette tip.
8 . A method of gel filtration comprising:
a) attaching a filtration pipette tip having a hub at a proximal end to a pipetting aid, wherein said filtration pipette tip has a filter located at a distal delivery end and a dry size exclusion media above said filter and below said hub, wherein said dry size exclusion media is loosely contained in said filtration pipette tip;
b) aspirating a solvent through said distal delivery end of said filtration pipette tip using said pipetting aid;
c) equilibrating said dry size exclusion media and said solvent in said filtration pipette tip to form a wetted size exclusion media;
d) draining excess solvent through said distal delivery end of said filtration pipette tip to form a gel filtration pipette tip, wherein said wetted size exclusion media settles during said draining and forms a homogenous gel filtration column;
e) introducing a sample solution to a top of said homogenous gel filtration column in said gel filtration pipette tip, wherein said sample solution comprises two or more analytes of different sizes;
f) loading said sample solution into said homogenous gel filtration column;
g) adding an elution solvent to said top of said homogenous gel filtration column,
h) flowing said elution solvent through said homogenous gel filtration column, wherein said homogenous gel filtration column separates said two or more analytes into two or more separated groups based on said different sizes; and,
i) eluting each of said two or more separated groups into its own collection well.
9 . The method of claim 8 , further comprising a step of analyzing each of said two or more separated groups using refractive index detector, UV-Vis detector, viscometer, multi-angle light scattering detector, gel electrophoresis and/or mass spectrometer.
10 . The method of claim 8 , wherein said pipetting aid is a hand-held pipettor, a syringe, or a robotic liquid handler that directly attaches to said filtration pipette tip or gel filtration pipette tip, or indirectly attaches thereto through an adaptor or top pipette tip.
11 . The method of claim 8 , wherein said pipetting aid is a robotic liquid handler and steps 7 a to 7 i ) are performed on a robotic liquid handling platform.
12 . The method of claim 8 , wherein said filter is a screen or porous frit comprising stainless steel, porous polymeric material, porous glass, or porous ceramic.
13 . The method of claim 8 , wherein said dry size exclusion media comprises agarose, sepharose, polyacrylamide, dextran, dextran crosslinked with epichlorohydrin, polystyrene, polyacrylate, cellulose, or a combination thereof.