question on UPLC separation of protein digest

[snowflame]

Hi, I have a question on using UPLC to seperate protein digest. I was using a Waters ACQUITY UPLC system with the BEH C18 column. I noticed after about 200 injections of protein digest on the column, the column performance went bad. I have Waters tech support to look through my UPLC method and they don't think there is anything wrong. I also follow just normal protein digest procedure with trypsin. Waters thinks it's a column issue and recommend to wash the column completely with MeOH, which didn't do magic to me. I was wondering whether somebody else have seen the similar problem on the BEH C18 column.

Some details of the method:
Solvent A1: 0.05% TFA/water; Solvent B1: 0.045% TFA/Acetonitrile.
Column temp 60C.
Gradient: 2-37%B in 65min, 37-95% Bfor 7.5 min, then 2% B for 26min.
column was washed with ACN after use. MP was fresh every two weeks.

Thanks a lot.

[Uwe Neue]

There could be more than one reason for this problem. Tough to tell which.
Since the column appears to be dead, I suggest to run another one under the same conditions. If it also fails after about 200 injections, we have a reproducible problem to think about.

[unmgvar]

your application is at 60 temp.
this is the high up limit for the column
expect a shorten life time for it at the upper end,
if you wish to try and extend the column life time you need to make sure of several things

never turn flow off while oven is cooking it, always keep it under flow
try to have the column at 60 the least amount of time necessary, create a instrument method to run at the end of your sequence set that will at least bring temp down to a range of 25-30

[snowflame]

Thanks for the reply. I did see this problem for several of columns. The recommended column temp is 25-55 while Waters tech support seems OK with the column temp of 60. I make sure at the end of the run, I turn off the column heater and keep the flow. May there is something tricky about UPLC comparing to HPLC that I'm not aware of ?

[Uwe Neue]

OK, the problem is reproducible...

It could be the column temperature being at the borderline with the TFA mobile phase. How long do keep the columns running without injections, compared to the runs with injections? And do you keep TFA in your mobile phase when just purging the columns?

It could also be a consistent buildup of protein that you use to digest or buildup of incompletely digested sample protein. What do you do to remove the protein from your peptides?

[snowflame]

Thanks for the reply. I always wash the column with pure organic (ACN) and store the column in it, which is recommended by Waters. I suspect the problem is caused by protein build up too. In our digest procedure, we add trypsin in much excess and didn't remove extra undigested protein from the peptides. While in our gradient, we did wash it at high organic before equilibrating and run the next sample. Waters recommend to wash my bad column with pure MeOH overnight, which seems help a little bit, but not much. Any suggestions?

[unmgvar]

one thing possible to try and remove protein from the column head is to reverse it and wash with 100% ACN while injecting DMSO on it

but if in our case we would also see a pressure buildup due to this phenomena of protein build up

[snowflame]

I started to use the VanGuard Precolumn with the C 18 column when I noticed the problem in order to prevent the protein build up. I can't remember whether I see a pressure increase along with the problem. I think I'll try your suggestion with ACN back flush and DMSO. Ｔｈａｎｋｓ．

[snowflame]

It seems reversing the column and washing with 100% MeOH while injecting DMSO did the magic. The column performance is comparable to what it should be now, though the chromatogram of the peptide map doesn't look exactly the same. I guess the column need to be run in the right direction for a longer time since it's reversely washed. Thanks a lot!