I have been using HPLC column, Phenomenex Luna NH2 (5 u, 100 x 2.0 mm) with a mobile phase: 50% acetonitrile/10 mM ammonium acetate/10 mM ammonium hydroxide, pH 9.4 and flow rate 400 uL/min for separation of pteridine-based compounds.
Now, the column seems to be clogged. It now cannot be operated at 100 uL/min of water. I have already checked individual tubing in the HPLC system and the tubes are all ok. Cleaning column was also done under a company guideline but it did not help.
If anyone has any experience on this type of column, please recommend or suggest.
Dear friend Pteridine is soluble in organic solvent so wash your column with 100% acetone or MeOH WHITH FLOW RATE 20uL/min to 100 gradually several times.
Reverse your column and run with methanol with variable flow from 0.2-1.5ml/min. You can also use hot water (5-10min.), but not longer time because it may dissolve your silica of your column.
gudluck
:)
I am agree with Robin Joshi also if your colum is suitable for remove the frit, you can wash the frit with nitric acid 30% and sonication. Good Luck!
Cony you have right but it is better to replace a new frit,
In addition, I suggest the use of universal HPLC Guard Columns from Phenomenex.
Thank you so much for all suggestions. Highly appreciated!
I have tried to wash the column as suggested but it did not help much. I still could not increase the flow rate, the pressure is still high. I sent the column to the company for further investigation. So, I am waiting for the result.
Although, the guard column was used and all reagents were filtrated with 0.2 micron membrane and degassed before use, I am a bit worried if there is any pH changing inside the column then generating salt formation.
Dear Suganya,
Did you solve this problem? I´m using the same column at similar conditions and I´m having the same issue. So far, I have cleaned the column according to manufacturer, but I haven´t been able to decrease the pressure on the column.
Could you please give me some advise?
Thanks in advance!
Dear Andres,
After sending the column to the company, it still had very high pressure. We then decided to open up the column to see the problem. We found that the column surface was very dirty, indicating that the guard column likely might not work well (it was such an old guard column used in our lab). And since the column had actually been used quite heavily under high pH condition, we then decided not to recover the column but bought a new one with a new security guard column. So far, everything works fine.
Dear Suganya,
Thank you very much for your answer. I've been using a new column and guard column, however, I always see an increase in the pressure although I clean the column carefully after each batch of runs. Have you experienced the same with your new column?
- Badges
- Science method