Split 3-phase Column
From Mswiki
Make a split 3-phase column
11/24/04 Meng-Qiu Dong
Make a column with a pulled tip
1. cut a piece of 100 micrometer ID, 365 micrometer OD fused silica tubing (part# 2000023, POLYMICRO TECHNOLOGIES, Phoenix, AZ) about 50 cm in length.
2. burn the center segment (2-3 cm) over an ethanol burner and wipe the blackened coating off the tubing with a methanol-drenched kimwipe.
3.
mount the tubing
into the laser puller (Model P-2000, SUTTER INSTRUMENT CO.) with the clear
segment in the path of the laser beam and use program 52 to pull the tip.
Program 52 (FIL-filament, VEL-velocity, DEL-delay, PUL-pull):
cycle1: HEAT=280, FIL=0, VEL=40, DEL=200, PUL=0
cycle2: HEAT=270, FIL=0, VEL=30, DEL=200, PUL=0
cycle3: HEAT=260, FIL=0, VEL=25, DEL=200, PUL=0
cycle4: HEAT=250, FIL=0, VEL=20, DEL=200, PUL=0
4.
dismount the
tubing and now you have two columns with pulled tips.
Pack the analytical column
5.
suspend a small
amount of reverse phase resin (Aqua or Polaris) in 100% methanol in a 1.5 ml
tube.
3 commonly used reverse phase packing materials:
1) Polaris C18-A, 5 micrometer, 18 anstrong (Metachem, Ventura, CA), good for
phosphopeptides.
2) Aqua,5 micrometer , C18 125 anstrong (part# 04A-4299, Phenomenex), for
analytical column and desalting column.
3) Aqua 3 micrometer, C18, 125 anstrong (part# 04A-4311, Phenomenex), for
analytical column only, better resolution and higher backpressure than the 5
micrometer cousin.
6. Using a pressure bomb to pack 7-10 cm reverse phase resin into the column with a pulled tip under 800 psi helium gas, (cat# UN1046, industrial grade, WestAir Gases & Equipment, Inc. San Diego, CA)). You may have to scratch the tip open if it’s not pulled open.
7. Equilibrate the column with buffer A at 150 ul/min for 20 min using a HPLC pump.
Make the desalting column
8. cut two pieces of 250 micrometer ID fused silica tubing (part# 160-2255-10, Agilent), about 20 cm each.
9.
assemble the two
pieces of tubing into a 2-micrometer filtered union (UpChurch Scientific, Oak
Harbor, WA). (I cut the sleeve on the filter side in half in order to see the
SCX phase when I pack it.)
A filtered union has:
1 Inline MicroFilter Body .006in thru (M-520-01)
1 Filter End Fitting 0.5 micrometer PEEK (M-120X) (use a new one each time.)
2 Micro Finger Tight PEEK 6-32 (F-125X)
2 MicroTight Sleeve Grn. .0155x.025 (F-185)
10. prepare a slurry of strong cation
exchange (SCX) resin in methanol and pack 3 cm SCX into the column.
commonly used SCX materials:
1) Partisphere SCX, 5 micrometer, 120 anstrong (Whatman, Clifton, NJ)
2) polysulfoethyl-A, 5 micrometer, 200 anstrong material (polyLC INC.), good
for phosphopeptides.
11. replace the SCX tube with a tube of methanol and pass methanol through the column for a few minutes.
12. pack 3cm of either 5 micrometer C18 material into the column.
13. equilibrate the column with buffer A at 200 ul/min for 20 min using a HPLC pump.
Loading and final assembly of the 3-phase column
14. load sample into the desalting column at less than 2 ml/min using the bomb (~200 psi)
15. wash the desalting column with buffer A at 200 ul/min for 20 min using a HPLC pump.
put the analytical column into the union and wash the now
completely assembled column with more buffer A before running MudPIT.
Tips for unclogging a column:
· blow hot air onto the column using a blow dryer
· run methanol through
About the pressure loading bomb
The ones in the Yates lab are home made. For commercial ly available ones, check out NextAdvance:
