Blank problems

<p>Dear users,

I’m running a gradient method from 2% Acetonitrile to 80% Acetonitrile than isocratic 80% Acetonitrile and back to 2% Acetonitrile in water. PDA 210 nm
normally my signal is going up until 80% ACN is reached and then flat and then going down until 2% ACN is reached.
But since a while a get a sort of hill in my chromatogram and not a continuous line. [file without TFA]
I’ve tried the following thing</p><p>Changed the solvent for fresh
Rinse the system with waters mix [IPA/MeOH/Water/1% formic acid] overnight
Changed the column [normal Acquity C18 column]
Replaced  the solvent inlet filters (the one’s in your flask)
Replaced the inlet filters of the pump
Replaced the zirconium mix valve after the pump layout
The Ryanodine valve was replaced
Some part of the injector where replaced

But still the problem exists, if I’m running the same method but than with 0,1% TFA in water and 0,1% TFA in Acetonitrile I get continuous line of my signal [file with TFA]
Anyone an idea how further?
Thanks for your support


  • ehenning

    Off subject, I hope Jeanne is doing well.  Every time I worked with her I learned something valuable and powerful.   She gave some of the best Waters courses I've ever had the privilege of attending. 

  • Eriksp

    The system is an UPLC (one of the first)  BSM,CM, PDA, SM with tower
    The column is an BEH C18 1.7 µm 2.1*50 mm column temperature 40°C
    ACN: Merck Lichosolv later changed to Uvasol and now Biosolve ULC/MC grade
    Water: Fisher UHPLC gradient water later changed to Biosolve ULC/MS grade
    Flowrate 0.8 ml/min
    Gradient start 2% ACN after 0,7 min gradient to 80% ACN in 4 min then back to 2% ACN 0.55 min total runtime 6.5 min
    0,1% TFA in water and 0,1% TFA in ACN are both from Biosolve ULC/MS grade

    In the past there was almost no difference between the blanks with or without TFA.
    Could it be due to algal growth ? and  what would be the best method to remove them?

  • Eriksp

    Hi Marc,
    Thanks for your support, although there are no peaks in my blanks the "hill" is not normal. This is also a bit of a problem with the integration of the chromatogram, since we are not able to use apex intergration methods due to QA/QC regime