tailing problem

<p><span>Hello to everyone.?m a new member.Working for a Pharmaceutical company as a QS specialist. ? dont have any method development experince.For my master tesis im transfering an HPLC assay method to UPLC and also im changing the mobile phase from ACN/H2O to MeOH/H2O because of the ACN crisis.Using Acquity BEH C18 column 100 mm x 2,1 x 1.7 micron.But my peak`s tailing so bad.it`s 1,5-1,6 and sometimes even 1,7.? tried to add some chemical to the mobile phase as TEA, Perchloric acid and Acetic acid.TEA doesnt work.With Acetic acid peak had a shoulder.The best one is PCA, in one injection i got 1,38 but another day it became 1,7! Please help me,it makes me mad i want a thin peak. </span></p><p></p><p><span>have a good day thanks a lot.</span></p><p></p><p><span>Handan </span></p>


  • sazimi

    dont worry just tell me what is your compound, your sample form & or any other info which could help me to develope your UPLC method for best peak symmetry & shape

  • lizh


    Method development is all about knowing as much as possible about the separation.

    We need more data. Original method (include everything you know) and new results, full details on the mobile phases used and chromatograms. Original flow rates and new flow rates. What was the original column - if you are seeing selectivity changes it may be that BEH was not the best choice. Did you explore the Waters selectivity chart?

    Also ensure while developing the method I would suggest using full loop mode as this is the most accurate and least complex, but ensure that you are not overlaoding your columns, use a 2 uL loop.

    Please please do not use old style pairing agents as a first choice before exploring other options. The great thing about the ACQUITY columns is pH stability and temperature stability so we can use this as a selectivity tools. Let's try and simplify the problem first. I am assuming the original method did not use TEA, so let's remove that first.

    Send the community more information and I am sure everyone will help. Also state your goals - is to simply swap with MeOH, maintain the same resolution, go faster, this alos helps decide.

  • dougm

    A couple questions and suggestions.

    1) Did you use the ACQUITY UPLC Columns Calculator to scale your HPLC method for your UPLC column? It is installed on the PC attached to the UPLC system. Please let us know

    2) If you took your HPLC method and replaced ACN with MeOH on your HPLC system/column, what is the peak shape? This will simply test the influence of MeOH on peak shape for your analyte(s).

    The reason I am asking these questions is that you are simultaneouly changing two things. Hence, there is no way tell what is causing your peak shape issues: 1) improperly scaled/transferred method; 2) use of MeOH as organic modifier.

    I agree with Liz, there is likely no need to add peak sharpening agents such as TEA or acids.


  • KWB

    Hello Handan,

    If the HPLC method did not suffer from tailing and used plain Acetonitrile and Water, then the change to Methanol is probably is behind the increase in tailing. Sticking with Acetonitrile may be the easiest thing to do. You may still be saving significantly in Acetonitrile use and waste disposal because of reduced run time and flow rate.

    If your compound has a high pKa, you can generally reduce tailing by going to a buffer which is 1 or more pH units above the pKa of the compound, forcing it into its unionized form. If this is the case, try ammonium bicarbonate pH'd appropriately. If you use this buffer, it should be made up every 24-48 hours.