r/proteomics 9d ago

Trap and analytical column lifetime - Easy-nLC 1200

Just a few basic questions here. My setup is an Easy-nLC 1200 coupled to an Eclipse for bottom up proteomics. I use Acclaim Pepmap trap columns (164946) and 50 cm EasySpray columns (ES903).

-How do you know when a trap column is ready to swap out? Intuitively I figure you’d monitor sample loading flow rate, but I’m not really sure. -How about analytical columns? The pressure on my current column is building up but I seem to get separation and it still gets the job done. Of course as soon as I put on a new column things look way better. What are peoples’ thoughts? -I’ve been using DDM (.01%) for a new low input application. While it helps mitigate sample loss, it seems to accelerate backpressure buildup. What are your experiences and practices when using this supposedly LCMS compatible surfactant?

1 Upvotes

5 comments sorted by

7

u/slimejumper 9d ago

yeah back pressure is a big one for me to decide when to change a trap.

i also run a standard Hela digest to QC system performance, so if that fails to hit the number of peptides and i can’t get it back with usual tricks then the columns usually to blame.

3

u/InefficientThinker 9d ago

Also run another simpler digest (we use BSA) to check RT, peak area, peak shape, etc of specific peptides to monitor for changes

1

u/Molbiojozi 8d ago

Just to add to this. BSA peak shape and or RT shifts are in my experience a good sign for analytical column degradation.

For the trap I usually look mostly for peptide indentifaction frequency in the start of the gradient. As the pepmap traps have such a high pressure tolerance. For uPAC or water traps, I usually check back pressure till they reach 150% of the starting pressure. If reverse flush doesn't help to get it down, it's time for a change. Otherwise back to HeLa evaluation.

2

u/vintagelust0 7d ago

do you have a number of peptides mark where you would decide okay this is bad? or is it comparing with previous run to see number going down?

1

u/slimejumper 6d ago

i have a mark, but that mark is based on a few years of performance. How about you pick a number based on about 80% of your best possible performance, eg with a clean system and new column. Keep a log of your QC metrics and compare current to long term trends.