Peak Splitting

<p><span>Hello, </span></p><p><span>I am having the issue of splitting peaks in a method for testing estrogen compounds. I am using the following method:</span></p><p></p><p><span>Column: 2.1X50mm BEH C18</span></p><p><span>Temp: 40C </span></p><p><span>Mobile Phase: Acetonitrile:Water 50:50</span></p><p><span>Flow: 0.75 mL/min</span></p><p><span>Pressure: <11,000 psi</span></p><p><span>Inj. : full loop (10uL)</span></p><p><span>Detection: PDA 205nm</span></p><p></p><p><span>I am seeing splitting occur after about 250 inj. I also tried a lower injection volume, but this did not help. The sample diluent is 0.9% saline and the highest sample concentration is 5ppm.</span></p><p></p><p><span>Any suggestions on the cause of the split peaks? I had a similar issue with the same method when I was using a sample diluent of 25% ethanol / 5% propylene glycol and the columns were lasting ~800 inj before split peaks. I was told that the likely cause was the PG. Now I am just using a saline solution and the column is only lasting for 250 inj. </span></p><p></p><p><span>Thanks,</span></p><p><span>Rob</span></p>


  • dougm


    On how many columns has this occurred? How long have you been running this method and has anything changed recently?

    Most importantly, have you contacted your local Chemistry Specialist?



  • RHuber


    This is the first column with using this sample diluent. I went through 4 columns with the 25% EtOH / 5% PG diluent and was informed that the issues were likely the PG contaminating the column. This method is not at extreme pH, temp, or pressure, so I would not expect the columns to have issues with this few of injections.

    I have not contacted the local Chemistry specialist as I am not sure who this would be. (I am in central MI)

  • lizh


    I would try a smaller injection. 10 uL seems a lot for a short column.

    The quickest is to go to PLNO - no loop change, though I always recommend FL just as you are using when developing a method. But here, choose a simple needle wash, as its isocratic you can use exactly the same 50/50 for the Weak and Strong needlewash, or whatever you are using right now - what is it.

    In the meantime I have asked some of our chemists to share a method I happen to know we are developing. I believe we had good success with HSS.

    Best regards,