Column life of Acquity UPLC BEH Shield RP18 2.1 mm x 100 mm, 1.7 µm

Lotus

<p> I am experiencing problems with pressure built up with the Acquity UPLC BEH Shiled RP18 2.1 mmx 100 µm.  The back pressure was doubled after 80 injections. Van guard column was used.</p><p></p><p></p><p>The mobile phase that I have using is 10 mM ammonium acetate in water and pure methanol.  The flow rate was held at 0.2ml/min. The back pressure was around 3300 psi at 0.2 ml/min when it was first installed.  The analysis was for water soluble vitamins in energy drinks and the runs are about 12 mins and the system was allowed to go back to intial mobile phase before and equilibrated before the next injection. All solvents used were filtered thru 0.22 µm and the methanol used was the Fisher Optima grade.  The sample preparations were direct dilution with the 10 mM ammoniumu acetate, filtered and injected.  The vitamin stock standards were prepared in PBS pH 2.0 and the final calibrants were diluted in 10 mM acetate.  The back pressure was 7200 psi after about 80 injections.</p><p></p><p>The retention times have all been very stable, except the back pressure gets very high. I have washed the column with volumes of water and methanol and can't seem to get the pressure down. I don't want to put the system under anything over 10,000 psi. I have installed a new column; the same back pressure built after about 80 injections.</p><p></p><p>Can anybody help?  Precipitation from sample impurities at inlet?  Bad batch of column packing?  Etc??</p><p></p><p>lotus</p>

atus

Hi,

Do you by any chance have Calcium ion is any of your solution?

Albert

Lotus

I don't think so. At least energy drinks that were analyzed didn't claim any on label. I can't see where the calcium ions may be introduced.

Thanks for asking.

Lotus

Lotus

Hi Albert,

I apologize for the previous response.  In fact, there are presence of calcium ions as one the water-solube vitamins is B5, some which are the in the form of calcium D-pantothenate.

How do you think that the presence of calcium ions may contribute to the back pressure increase?  Can you recommend a solution on how I can resolve the issue and get the pressure down without affecting the performance?

Thanks again.

Lotus

atus

Hi Lotus,

You have confirmed my hunch. You mentioned that the stock solutions were prepared in PBS. Calcium ion and phosphate ion should not be mixed in the same solution. They will form hydroxyapatite precipitate. That means you are injecting fine precipitate into the UPLC system and the column, and thus increase the back pressure in a predictable pattern. If you were using ion-exchange or SEC columns, the resolution might be affected. It may have less effect on resolution with reverse-phase columns.

If you switch to other compatible buffer, the issue will most likely go away.

Good luck.

Albert

Corrien

Try to premix the 10 mM ammonium acetate with methanol.

If you use only ammonium acetate the back pressure will built within a day.

Lotus

Thanks. Yes, that helped.  Issued resolved.

lotus

manoj

Hi friends, i am working on the LC-MS/MS method for one molecule on Acquity UPLC BEH Shield RP 18, 2.1 *50 mm, 1.7 µm colum with mobile phase acetonitrile: ammonium formate 2 mM (90:10v/v). The problem i am facing is of the inconsistent area ratio for the aqueous drug and internal standard (IS) mixture though the IS used is deuterated. I tried BEH column also, the same trend is being observed. Even on increasing buffer to 30 % the trend of fluctuation from same vial injection is being observed. Kindly suggest.

Lotus

I incline to look at the UPLC system first.  Have you checked for possible leaks in your UPLC system?  How about the autosampler? Are you using partial loop or full loop? I have found using full loop injection to be more consistent. Is there enough sample in your vial for your injections? Is the injector, needle or line blockage? Is the UPLC system equilibrated and stabilized before your injections?  Have you optimized your MS/MS conditions? How fast is your run?  Are you getting fairly good peak intensities? Do you know if the molecules you are working are stable under the conditions? How is the chromatography? Etc. 

Lotus

manoj

Dear Lotus,

Thanks for your response. Last day only while injections, sample pressure low error was observed. On physical observation, needle was found  removed, which was re-fixed. Two more things were done. First, Span window in MRM was reduced to 0.2 instead of pre-set 0.5. Resolution was increased to 14.5 instead of  pre-set 13 in MS tune anayser parameter. Problem is resolved now but i am getting high baseline in blank mobile phase injection after injections of aqueous higher concentration of drug and ISTD mixture  though no carry over effect is there ruling out the rinsing solution role.  System is primed thoroughly and stabilized before injections. Pls suggest.

Thanks.

Lotus

Make sure you are not using buffer for your priming solutions and needle wash.  Check manual for proper compositions for your weak and strong washes. Also, try to re-initilize on the tune page. Are you running a gradient?  Is the system is rinsed throughly and equilibrated before the next injection? Depending on what you are injecting and how many injections, dirty sample cone may give you noisy baseline also.

lotus