UPLC High pressure problems

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
Hi,

Does anyone have any recommendations as to how to efficiently clean a potentially clogged UPLC detector Flow Cell?

Thanks
Are you sure its the flow cell that's causing the issue?

High pressure can be as a result of many things- temperature too low on Sample Manager or not activated at all, a blocked in line filter, blocked column head not to mention particulate matter embedded into any stage of the pump mechanism or injector rotary loops etc. Also, the sinker in your mobile phase container can get blocked, do you run buffered mobile phases? A faulty mixer or dirty mobile phase buffers/additives can also increase pressure. Is your flow rate constant and correct?

Try to isolate where the high pressure is coming from- disconnect the tubing from the mixer/pump valve selection right up to the detector, each stage putting typical flow through the system to check pressure. A high reading at the check mixer inlet suggests blocked check valves. Prime the lines A and B individually and check for constant flow. A long priming of about 20 minutes is often needed to clear air or blocakge from solvent lines.

Try to inject a zero volume injection and monitor pressure. If the problem is the flow cell, put all lines into first hot water (take off all sinkers and store in organic), prime up all lines for 10 mins each, then run hot water through the system (with the column disconnected, put a union it its place) for at least an hour, Repeat with Acetonitrile or Methanol, then put all lines (EXCEPT Seal Wash! Keep the seal wash line in your typical seal wash container) into a 30% Phosphoric Acid 70% HPLC Grade Water container, run at a flow of about 0.5 ml/min for at least 3-4 hours, then transfer all lines into 100% Water (again, except seal wash) and run at typical flow overnight. Check pH of eluent from waste line the next morning to ensure its pH 7. Set up system with fresh washes, mobile phase and a very long primimg/start up then try some diluent test injections. Good luck.
Thanks for the help.

It's definitely a blocked detector flow cell.
At the end of day on Friday I was running some trial work and noticed that my peaks were distorted and broad and not eluding at the expected RT. I went to investigate and noticed that I was leaking at the end of my column before the detector. I removed column, put on union and confirmed that there was no leaking or extremely high pressure all the way up to the inlet of the detector. Then no liquid was coming out the outlet of the detector.

I don't know if this was the right idea but I prepared a solution of ph8-10 in water, primed and tried to flow through the cell to dissolve anyhing stuck then tried running a nitric acid solution(ph2 about 1 drop). I tried reversing the outlet and inlet lines and all the time running flows of 0.001-0.005ml/min just to clear a path. But the line would just pop out of the detector.

We've had this happen before on this instrument and waters had to come in and replace the flow cell (very expensive) I've tried to since be diligent about cleaning after runs and not letting things sit in the flow cell by washing for an hour with 90:10 water methanol after all runs to clean out the flow cell for long term storage.

Again if I can clean it out somehow without calling waters that would be best
Do you run buffers in your mobile phase? Was there a run lately where the buffer had hit organic and possibly crashed out of solution and blocked detector cell. You could still try the wash I suggested in the first post to resolve this. It would take the guts of 2 days between prepping solutions, running acid through it, water overnight etc then prepping new solutions but it has always worked for me on any detector blockages on UPLC.
One more thing to add I'm willing to try the cleaning procedure you proposed but again the problem is that NOTHING comes out the outlet of the detector. Will the cleaning procedure work if I can't clear a path first?

Have you had this happen before?

Are there additional precautions to preparing mobile phase?

I work for a small company and they have a mix of HPLC and UPLC and I used to work for a much larger company where water and solvent quality was a big concern. Here they have a dI water system that produces about 13-15mohm water but I don't think it's filtered. No one filters mobile phase and they use the uplcs for hplc methods (high salt content etc). Again I'm used to cleaning but I need to try to
Implement stricter regiments if this is a continual problem.

I had this happen on another instrument (someone else had this problem of blocked flow cell of detector) and I think I got to it quick enough because I quickly tried reversing the flow path of the detector and putting the ph 8-10 water followed by ph 2 phosphoric acid through and once I got flow washed over lnight at 0.01 with water methanol 90:10 and that cleaned it up. But now I can't even get anything to come through the detector
It sounds like the quality of the solvents going through the systems isn't the best. Its always good practice to filter mobile phases before they are used in analysis but every company is different.
Because you are getting no outlet flow I suggest the hot water wash, just go straight to a hot water wash with typical flow for at least an hour and monitor the outlet (use a union, no column). Hot water usually dissolves any crystallized salt in the system. If you still get nothing after an hour, it may be time to ring Waters!
It does not sound at all like your solvent quality is the reason for the plugging problem you have described Your HPLC column acts as a super expensive filter and since the problem you have always occurs at the detector flow cell we can rule out the solvents as the main problem. *If the clogs occurred at the head of the column, then it would be injector related or precipitation (and solvent grade might play in role).

The reason is most likely failure to properly wash down the flow path after using buffers. You can not leave the system in buffers for any length of time, including overnight. A proper wash solution (the composition of which will depend entirely on your exact mobile phase composition) should include high quality water with some (not much) organic phase such as Methanol (e.g. 10 % MeOH / 90 % Water). Methanol is better than ACN for dissolving salt buffers and is a good choice for many simple wash solutions. When ever the HPLC is not running samples, it should be flushing down the flow path with a safe wash solution. In many case, you may be able to store the system overnight or on weekends with the same solution. Regular washing should eliminate the clogging issue.

- - You might want to go over some basic training in proper use (establish guidelines) with the users of these systems to insure they have training in how to both wash columns down after each analysis and flush down the system after each use. Keeping the flow path clean is essential to keeping these system working properly.

Sometime the plugged flow cells can be dissembled, the clogs lines replaced, then flushed and put back in service. Sometime the flow cells can be soaked in warm DH20 with some acid for a few days, then flushed and restored. Most plugged Waters brand flow cell do end up in the trash though.
Hi Geof325,

What is the particular Waters detector you speak of?

Probably a good idea is to consult the operator's manual for the exact detector involved, be it the TUV or PDA. These newer "light pipe" flow cells generally aren't able to be taken apart and cleaned, so as these other fellows recommend above (and likely as recommended in the manual(s)) a procedure for flushing the flow cell post-run must be done. For example, for the TUV:

http://www.waters.com/webassets/cms/sup ... 4733rb.pdf

refer to pp. 38 - 44. This guide is also helpful:

http://www.waters.com/webassets/cms/sup ... 1307rg.pdf

Best wishes moving forward!!
MattM
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