Back pressure problem BEH phenyl column.
Answers
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It may seem that a UPLC is less forgiving of particles than HPLC and because column frit porosities and tubing inner diameters are smaller this is generally true. But it is also true that particles in the liquid stream will eventually clog any HPLC or UPLC system.
One explanation to your specific problem is that particulate from your mobile phase or samples may be clogging the inlet frit of the column.
But first you'll need to isolate the source of the pressure.
While flowing mobile phase, remove parts of the flow path (outlet tubing, detector, column, etc.) and monitor the pressure to confirm that the column is the source of the pressure increase. Clog-free tubing will typically only account for a few hundred of pounds/square inch (or less).
Some more questions:
What are your samples ?
Do you filter them ?
Do you use quality solvents & water ?
Do you employ good technique when you filter the solvent ?
Do you rinse the filtration flask with a small aliquot of filtrate and discard it before completely filtering ?
Do you rinse the mobile phase reservoir with a small aliquot of filtrate and discard it prior to filling ?
I generally do not recommend reversing flow to the column as a test/remedy, but in your case, if you have confirmed that the column is the source of the pressure, it is already unusable so there is no harm in trying.
Try the following:
1) Use the column that provides 14500 psi backpressure.
2) Disconnect and remove the column from the system.
3) Connect the normal outlet of the column to the column stabilizer tube (passive preheater) or active preheater.
4) Very important: Leave the normal inlet of the column disconnected and position it above a waste collection vessel.
5) Try a low flow rate (say 0.2 mL/min) at a 50:50 mix of your mobile phase for 15 minutes.
6) Monitor the pressure to see if it decreases (indicating that particles have been back flushed off of the inlet frit to waste).
7) If the pressure decreases re-install the column as you would normally.
If this works as a remedy it tells us that particles were indeed clogging the inlet of the column. It does not tell us where the particles originated nor will it prevent future occurrences. You will need to investigate more thoroughly.
Please let the community know how you make out.
Regards,
Rich
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Thanks Rich, sorry for the late reply, i was away for a while.
As per filtering, i followed exacly what you mentioned in the mail. Used HPLC grade ACN/ Water.
Then i isolated the source of pressure, it is the column (i followed your instructions)
Then reversed the column, flush it (as per your instructions),
When i reinstalled the column (correct direction) and tried again, pressure hadnt gone down.
Now column cannot be used, even at 0.2 ml.min flow pressure reaches more than 15000 psi.
waithing for a new colunmn to arrive, kind of worried about what to expect in future .
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Senaka,
You are taking the appropriate steps to safeguard against particulate contamination. If I were in your position, I would begin to look at sample prep.
1) What is your sample ?
2) Does it originate from a biological matrix (plasma, tissue, etc.) ?
3) If biological, are steps being taken to remove proteins prior to injection ?
3) Any chance that sample components can precipitate in the column ?
You are right to be concerned about what will happen with the new column. Since pressure problems have already presented themselves with 2 columns, it is reasonable to assume that the conditions that produce the elevated pressure have not been addressed and that your results will be no different with the 3rd column. Guard columns offer a way to extend the life of your columns. But you will want to evaluate the cost/benefit of guard column use/replacement versus additional sample cleanup.
Please keep the community informed of your progress.
Regards,
Rich
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