Hi Junaid,

to minimize or even eliminate column bleeding , a simple procedure is to pump iso-propanol with 0.1% formic acid at room temperature for one hour through the column. For a column with 3 mm i.d., 0.5 mL/min flow-rate is appropriate. It is important that the column is not connected to the mass spectrometer to avoid contaminating the ion source. (source: LC-GC).

Regards

Markus

Thank you Markus for your response. However, can i apply this procedure for nano-LC column?

Junaid,

I have a few questions to help understand the issue - 1) what type of sample are you using; 2) how much of that same (volume if a fluid or mass if a lysate); 3) are you doing a direct inject (loading directly onto the nanoLC column) or using a trap column; 4) are you using a typical water / acetonitrile gradient with formic modification - to what % ACN are you running the gradient; 5) what are you doing to "clean" and re-equilibrate the column after the gradient.

Dear Andrew thanks for your response. 1) we have digested 1D gel bands using trypsin following common procedure ( Shevchenko et al. ‎2006 Nature Protocol).2) Typically 2-5uL is our injection volume. 3) we are injecting peptides using trap column and 4) then peptides are eluted using typical water acetonitrile gradient with 0.1% formic acid. 5)We are running blank with same gradient to clean the column.

Junaid, of course your response just raises more questions. Perhaps you can post a snapshot of the chromatogram that illustrates "bleeding". Can you clarify what you initially described - "column starts bleeding after a few dozen samples". Does this mean you've tried a more than one column (replacing the trap too?) and you repeatedly see degradation after ~40 injections? Do you see sample carryover in your blank runs? Have you stained the gel lane you're cutting and does the bleeding seem worse when injecting a digest from a darker stained band? The ideal load for your column is around 1 ug of digest, so you may be overloading, but that would be gel band specific I'd expect. Normally a in gel digest is pretty clean and your column should handle hundreds of injections. Other points to just verify - after the gradient you run up to ~75% ACN for ~5 min, your flowrate should be around 300 nL/min, you're using a matched pepmap100 trap column.

we shifted back to nano lc from analytical lc after some months and put back the previously used nano column (which was used for few dozen samples sometime ago). However we did not see any results. So we checked for flow. at that time we saw packing material coming out of the column suggesting column is damaged. We have seen similar problem with another nano column few times ago that after sometime their packing start coming out. However we are not sure why this is happening!

Yes our gradients runs upto ~75-80% ACN for ~5 min at 300nL/min flowrate

Seems what you're experiencing isn't a bleeding issue, but rather that the columns are damaged. The frits holding in the packing of a nano column are much more easily damaged than a larger bore column, so it is not too surprising that you're having an issue. Hard to say exactly why from afar, but I would suggest two considerations. 1) using a nano column generally means using a nano ESI sprayer, which are easily clogged. If you have experienced frequent tip clogging, which causes a buildup of pressure back on the end frit of the column, this may be your failure cause. 2) under normal flow you have a great deal of pressure built up across the column bed. When you go to stop flow, that pressure needs to be dissipated, ideally slowly out the distal end of the column. But you may be operating your pump in such a way that the line into the column is rapidly vented to drop pressure quickly. This can in time weaken the frits by having high pressure now pushing backward. Try to avoid such a condition if you can. Reach out to Thermo technical support for further guidance. Another point, it is best to run 100% acetonitrile through the nano column (avoid formic acid during storage) for 10 column volumes before removing and storing it.

Thank you so much Andrew for your kind help and suggestions.