Packing long nLC columns - recommendations?

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botte
Phosphoserine Member
Phosphoserine Member
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Joined: Mon Apr 29, 2013 11:47 pm

Packing long nLC columns - recommendations?

Postby botte » Tue Sep 24, 2013 11:44 am

Dear all,

we are currently successfully packing our own nLC columns of 50 and 75 um ID with 3 um particles, up to 30 cm in length. We are using methanol slurries of the chromatography material and a homebuilt pressure apparatus. Now we would like to extend this to columns of 25-50 cm length. ideally with sub-2um particle size. Our focus is on regular proteomics fare, protein from complex mixtures, PTM work, SILAC, label-free profiling.

So my question is - is anybody out there successfully working with self-packed long columns, ideally with small particle size material? If so, any special tips/tricks/recommendations on protocol/solvents/materials? Any pressure devices that are commercially available that are helpful?

I would be grateful for any recommendations, but am sure many on this forum would also benefit!

daniswan
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Postby daniswan » Tue Sep 24, 2013 8:34 pm

We have done Reprosil 3um particles packed to 70cm length, that it easy, it just takes patience. We have also done Reprosil 1.9um particles packed to 30-40cm, this is more a luck based processes. Sometimes it packs super easy, and sometimes the particles seem to aggregate and it to take forever. Typically I pack in 100% methanol, or 70/30 methanol and IPA. Some people have said that making the slurry in chloroform helps, but I haven't tried this since I prefer not to spray chloroform all around. I also often use heat to speed up the packing process and let you pack at lower pressures. I put a heat block set to 60C next to the pressure bomb, curl up the column on the top of the heat block, tape to block, and then put another metal block on top (sandwiching the column in between the blocks).

Arne
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Commercial pressure bomb

Postby Arne » Wed Sep 25, 2013 7:21 am

There is a commercial pressure bomb available, but we do not use that particular one in our lab: http://www.westernfluids.net/Nanobaume.html

I have found chloroform to work well for packing small particle sizes (<2 um). Consider that you only use sub milliliter amounts, so it shouldn't be much of a health concern.

I think heating while packing is a great idea, Dani. If you can spare a column-heater from a mass spectrometer you could even pack the column in that.


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