Packing Micro Capillary HPLC Columns
First published April
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|This protocol was derived from notes taken during the 1991 lab portion of the Don Hunt MS/MS peptide sequence interpretation course at the University of Virginia|
Making the Frit
1.) Cut a 75 cm piece of 100 um I.D. fused silica
2.) Rinse with 2-propanol.
3.) Burn ½ cm at the end to make the frit visible.
4.) Tamp the burned end of the fused silica into the Lichrosorb Si60, 30 times.
5.) To sinter pass the new frit through the micro torch flame twice. It should look similar to this frit.
6.) Test the new frit by pushing hexane through the capillary at 1000 psi, watch to make sure the frit holds
Packing the Column
1.) Slurry 300 µl hexane and 300 mg C18 stationary phase.
2.) Sonicate the slurry to assure that the slurry is homogeneous.
3.) Stir the slurry with a micro stir bar (flea) after placing in the pressure vessel.
4.) Pack the column at 200 psi and stop the packing at 8 cm the final column will be approximately 12 cm.
6.) Switch the column to 2-propanol and let the remaining resin in the column continue to pack.
7.) Install column on the HPLC and run two blank gradients. Flow rate is 200µl/min pre split and the flow is spit 1:200.
Method Notes: The frit made by this method looks very much like the IntegraFrit™ sold by New Objective Inc. This type of column was state of the art in 1991 however one might wonder how well this blunt cut column would perform in comparison to a fritted column with a pointed emitter, see the PicoFrit ™ columns again from New Objective Inc. Many people now buy Pico Frit ™ capillaries and pack them with their resin of choice using a pressure vessel. My preferred column is a Pico Frit ™ column, 15 um tip for the LCQ ion trap and 8um tip for the Agilent XCT ion trap.
Materials: Used during the lab demonstration.
The torch used was from Microflame Inc.. The C18 resin was YMC gel ODS-A120-S10, this is a 10 um particle with 120Å pores. My favorite packing material is Vydac C18, 5 um, 300Å, I prefer to unpack a 4.6 mm column, not believing that the bulk material is of the same quality as the commercial column material. The frit material was Lichrosorb Si60.
Pressure Vessel Vendors
Proxeon: Sample and Column Loader
Mass Evolution, Inc. : Helium Pressure Cell
Links to External Capillary HPLC Packing Information
The MudPIT Page at Scripps. Has some information on the MudPIT technique as well as the hardware that they use for pulling and packing capillaries.
Links to Related IonSource Material
|Important Safety Information: Triflouroacetic acid, formic acid, heptaflouobutyric acid and acetic acid are all very caustic reagents. Acetonitrile, methanol, propanol and other solvents can present a health hazard use appropriate precautions. Consult the material safety data sheets (MSDS) that come with these reagents and get the permission of the safety officer at you company or institution before performing these experiments. Always wear the appropriate safety apparel; safety glasses, lab coat, and gloves. Capillaries can shoot from the pressure vessel if not secured properly, wear safety glasses and safety apparel. Refer to the safe operational guidelines supplied by the manufacturer of the pressure vessel before using. Use a fume hood when appropriate. Whenever dealing with the stationary phase (silica particles) use an appropriate hood since inhalation of these particles is a heath hazard. If you are not trained in laboratory safety you should not attempt these procedures. Read our disclaimer, follow the link at the bottom of this page.|