Single Phase Column

Pack a single 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 materials in the Yates lab:
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 NANOBAUME^TM 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.        Equilibrate the column with buffer A at 150 ul/min for 20 min using a HPLC pump.

8.        load sample or store the column with tip submerged in buffer A.

sorry, the pressure bomb from Western Analytical is not the right one. check out

http://www.nextadvance.com/biology_laboratory_instruments_rockers_shakers/Next_Advance_pressure_injection_cell_bomb_loader.htm