Splitting Peak

<p><span>Hello, </span></p><p><span>I am running a test method for an API in 25% EtOH / 5% propylene glycol solution. I recently started having split peaks and assumed that there was a void somewhere. I removed the precolumn and the issue was gone. I ran a sample set and about 1/4 of the way through, the peaks started splitting again. I moved the column to a different position and tried again but with no luck. Does anyone have any suggestions on what to try? By the way, the instrument was serviced recently. </span></p><p></p><p><span>The method is 40% ACN: 60% water with a 50mm BEH C18 column at 40C. </span></p><p></p><p><span>Thanks,</span></p><p><span>Rob</span></p>


  • dougm


    First, is this a new method or is this an older method that suddenly is giving you problems?

    I believe that it's likely your sample matrix (25% EtOH / 5% propylene glycol solution). Why? The sample matrix (specifically propylene glycol) is likely chemically contaminating the packing material which causes peak shape deterioration. When you were using the VanGuard pre-column the packing material in that guard column was doing its job - protecting the packed bed of the UPLC column. The packing material inside the VanGuard pre-columns was getting contaminated. You removed the contaminated guard and the problem disappeared with it. Without the VanGuard present, the packed bed of the column was now taking the hit from the sample matrix.

    Want to check for sure? Prepare your API in mobile phase (or suitable clean solvent) and inject the same quantity under the same conditions onto a fresh VanGuard pre-column and UPLC column. Note its lifetime (or just stop after a sufficient period of time). Then re-do the experiment with the ethanol/PG sample solvent and see if you achieve the same number of injections (column lifetime).

    Let us know how that turns out.



  • Doug,

    Thank you for your response. I was worried that your answer is what I would get. This is a relatively old method in which that columns typically last ~2000 injections. The column that I am currently having the issue on only has ~700 injections, which is why I was trying to figure out what was going on.

    The reason for the matrix is that these samples are from drug release testing. The 25% EtOH / 5% propylene glycol is the receptor for the API being released from a transdermal formulation. I will probably look into changing this receptor since this is probably shortening the column life (however this is not going to be an quick change). Are there other things that I could do to improve the column life (e.g. strong solvent gradient, etc.)?



  • dougm

    Hi Rob,

    Are you open to any kind of sample preparation in order to remove the PG? If so, I can ask some of our sample prep experts for their opinion.

    However, before you do anything, can you confirm it is the sample matrix by injecting your API diluted in something other than PG? I would hate to steer you down the sample prep route if that is not the culprit.

    In the meantime, I'll bounce your problem off a few folks and get their thoughts.

    Hang in there!


  • Hi,

    Since in your sample preparation contains propylene glycol (PG), column might be giving peak split after few injection of sample solution.

    As per DOUG suggestion, try to find out whether peak split is due to PG. If the peak split is due to PG then try to remove PG before injecting into to HPLC.

    You can take about 1 gm of sodium sulphate anhydrous and 10 ml of sample solution in centrifuge tube, vortex for about 2 minutes and then centrifuge at high RPM

    (More than 4000 RPM , upto 15000 RPM is good). This might work. Pls try.