We recently installed a Hiplex Ca Duo Column from agilent for carbohydrate determination. It was working fine until a week ago, when the pressure of the column started to increase very slowly. As an example, on a period of 5 hours running, it went from 60 bar to 150 bar with no sign of stabilizing.
We have only run the column with water as mobile phase and at the specified temperature (80°C). Samples were mostly in deionized water, however we used some MIBK to extract some compounds from the samples before running them through the column.
We have tried to reverse flush it using water as indicated by the manufacturer, and pressure seems to stabilize at 78 bar (about 34 bar higher than before), but as soon as we put it back in normal flow, pressure starts increasing again.
We've checked the rest of the equipment for gas bubbles and compared system pressures (without column) from before and it all seems to be fine.
Any idea why this could happen and how could we correct it?
You may have some sort of fouling. Couple ideas:
1) Bacteria, fungi or algae can start to grow in your MP and or SP. To prevent, filter water through 0.2 um membrane and or lower pH of MP. You may need to make MP fresh daily. Don't leave your HPLC or column in water for extended periods; flush with aqueous MeOH if compatible.
2) There may be other components (e.g., polyphenolics) adsorbing to the SP especially at the front of the column. If this is the case and assuming the column is compatible, running a little NaOH through the column could aid in removal of said chemistries. If you do try this, ensure that you flush your system promptly with water so that no residual NaOH remains.
Hi,
A very low amount of methanol can be added to your mobile phase for preventing microbial growth at your routine method. 2% should be fine for this purpose. When it comes back to the question and problem you have, you may remove the frits from the inlet and outlet sides of the column using a proper wrench and put them to a degasser in hydroalcoholic solution at least for 1 hour. Be careful while removing the inlet and outlet frits and do not disturb the resin bed. Attach the frits and condition the column with your mobile phase and observe the pressure again.
Good Luck
Emir...
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