Continuous 3-phase Column
From Mswiki
Pack a continuous 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 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 (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 Baume (homemade, but also available commercial ly, e.g. the NANOBAUMETM assembly, cat # SP-300 from Western Analytical Products, Lake Elsinore, CA. http://www.westernanalytical.com), 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. replace the C18 tube with a tube of methanol and pass methanol through the column for a few minutes to prevent mixing with the next phase.
8.
prepare a slurry
of strong cation exchange (SCX) resin in methanol and pack 3-4 cm SCX into
column.
commonly used SCX materials:
1) Partisphere SCX, 5 micrometer (Whatman, Clifton, NJ)
2) polysulfoethyl-A, 5 micrometer, 200 anstrong material (polyLC INC.), good
for phosphopeptides.
9. replace the SCX tube with a tube of methanol and pass methanol through the column for a few minutes.
10. pack another 3cm C18 resin into column.
11. Equilibrate the column with buffer A at 150 ul/min for 20 min using a HPLC pump.
12. load sample and wash with buffer A.
sorry, the pressure bomb from Western Analytical is not the right one. check out
