What are the possible causes of leakage in HPLC columns?

Hi all!

I just installed a Cortes C18 column on our lab's Alliance HPLC to condition it with 80:20Water/ACN at an injection volume of 1mL/min when the pressure (delta psi) rose and the connection between the tubing from the autosampler and the front of the column popped out and I notice leakage. What are the possible causes of leakage in HPLC columns? When I decrease the injection volume to zero, the pressure also decreases. I've tried to re-tightened the ferule strongly and increase the injection volume again back to 1 mL/min but still there is a leakage and the tubings still pops out when I resume pumping. what can I do on this matter to remove the leakage and to reduce the pressure when conditioning?

Hope to hear from you soon! Thank you very much!

Best regards,

Tin. :) 


  • MJS
    I presume you mean to say flow rate rather than injection volume.  Based on your description of the event, I presume you are also using PEEK tubing (flexible plastic with plastic nuts/fittings) rather than stainless steel with properly fitted ferrules since you describe it as popping out of place.  Issues like this are very common with brand new PEEK tubing as the ferrules can take a little while to seat properly and "grab" the tubing to keep it from popping out.

    I would start with ramping the flow rate up a bit more slowly than straight to 1 mL/min.  So, maybe 0.25 mL/min.  Is there a column temp setting?  If so, make sure that it is in the column chamber and sealed up so that it can equilibrate the temp too.  You generally never want to just jack up the flow rate to full, and especially if the column temp should be higher as it is very easy to exceed allowed pressures and trigger issues like this and overpressure alarms.  Let it equilibrate for a few minutes at the right temp, then maybe open the column heater and give it a little tightening. Close it up and bump the flow rate up a little higher.  Watch the pressure to see how high it is getting at the point that it pops off, if it does it again.
  • But we are using stainless steel tubings. There is no leakage when the flowrate is 0.35 mL/min but once I increase it to 0.4 mL/min, leakage starts to accumulate on the inlet of the column. 
  • MJS
    If you inspect the ferrule and tubing end, do you see anything resembling damage like scratches or warping?  The ferrule may be warped if it has been used a lot and people have over-tightened it from time to time to stop leakage like this and now it is just not sealing correctly anymore.

    What is your pressure sitting at when you are at 0.35 mL/min?  It may just need another 1/8th turn to tighten it down properly or be replaced, but at the risk of causing further warping like I comment on above.  When it starts leaking, does the pressure still remain high so it is a very slow leak or is it suddenly gushing and the pressure drops significantly?