How to pack your C18 HPLC Column

[sirdaniel]

Hello people,

I just got an idea of using some kind of online SPE or 2D-LC to pre-purify my cell extracts before LC-MS analysis.
I thought of using short self-packed C18 columns with big particle size (10µm) as they will get pretty wasted after a couple of samples.

Does anybody know if it's possible to pack your own short (4.6x 50 or 10 mm) HPLC column with particle size >10 µm with standard lab equipment (just a normal HPLC pump up to 300 bar, 1mL/min)

Thanx for any answer,

Daniel

[Vlad Orlovsky]

your pump needs to work at higher pressure and flow rate. You can use regular pump but you need a bigger reservoir for your slurry. Old prep column will work. You need to create 10-20% suspension of your silica in one solvent and then use a second one as a pusher. Working with bigger particles might be problematic if you pump cannot deliver at least 20 ml/min. Again all this is to achieve good symmetry and efficiency of packing, in your case it might not be critical and I would suggest to consider flash chromatography.
Another simple approach does not even require pump. It is usually used to pack prep guard column (10, 20 and 30 mm ID). You just prepare more concentrated slurry (25-40%), fill your column, let is settle, fill again, etc until your column filled with silica up to the top.

[sirdaniel]

Hello Vlad,
Thanks for your answer!
What would you recommend as a solvent for the slurry and what for the pusher (C18 silica)?

cheers,

Daniel

[Vlad Orlovsky]

use this guide lines:
http://www.kromasil.com/technical_suppo ... acking.php

Ideally you need to create 300-400 bar (4500-6000 psi). Problem is that in order to pack bigger particles you need to have significant capacity of the pump. We are using Haskel pumps to pack our columns:
http://www.flw.com/haskel/liquid-pump.htm